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Файл:Ординатура / Хирургия / Библиотека им академика М.И. Перельмана / Книга_5607_Библиотеки_им_академика_М_И_Перельмана
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In Vitro Potential Anticancer Effects of Ursolic Acid
Cell Line Cytotoxicity Evaluation Effects References
Method (Dose and
Incubation Time)
HepG2, Hep3B, Huh7, and MTT assay (2, 4 and 8 Induces Yan et al.,
HA22T cell lines, L-02 cell μmol/l UA for 48 h) apoptosis 2010
(human normal liver cell
line)
BGC-803 cell and MTT assay (10–60 μM for Inhibits Wang et
hepatocellular carcinoma 12, 24, 36 and 48 h) proliferation, al., 2013
cell line H22 Induces
SW480 and LoVo Human MTT assay (20 and 40 μM Inhibits Weng et
colon cancer cell lines for 48 h) FITC-annexin V/ proliferation, al., 2014
GBC-SD and SGC-996 MTT assay (40–70 μmol/l Antitumoral Nam and
(human cell lines) for 24, 48 or 72 h) effects Kim, 2013
DNA fragmentation (2, 4
and 8 μmol/l UA for 48 h)
PI (20 and 40 μM for 48 h) Induces
FITC-annexin V (40–70
μmol/l for 36 h)
Biomarkers as Targeted Herbal Drug Discovery
apoptosis
apoptosis
SW480 (human-colon MTT assay (1–32 μM for Induces Lin et al.,
adenocarcinoma cells) 24 h) DNA fragmentation apoptosis 2014
(1–8 μM for 24 h)
U937 (human leukemia NBT reduction assay (30 Differentiation- Park et al.,
cells) μmol/l for 4 days) inducing agent 2013
for leukemia
therapy
PC-3, DU145, LNCaP MTT assay (5–80 μM for Induces Kim et al.,
(prostate cancer cells), Raw 24 h) DAPI (30 μM for apoptosis 2011
264.7 (leukemic monocyte- 24 h)
macrophage cells) HEK293
(human embryonic kidney
293)
MDA-MB-231, human MTT assay (5–100 μM for Induces Shin and
breast cancer cell line 24 or 48 h) apoptosis Park, 2013
FACS (40 μM for 24 or
48 h)
LNCaP, PC3, DU145, MTT assay (30 μM for Induces Zheng et
A549, MCF7, HCT116 and 24 h) apoptosis al., 2013
HeLa cells
T24 and BIU-87 lines MTT assay (219 μM for Induces Zheng et
(human bladder cancer cell) 72 h) FACS/PI (219 μM apoptosis al., 2012
for 48 h)
75 Ursolic Acid: A Pentacyclic Triterpene from Plants in Nanomedicine
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(Continued)
Cell Line Cytotoxicity Evaluation Effects References
Method (Dose and
Incubation Time)
T24 (human bladder cancer MTT assay (6.25–400 μg/ Contributes Xavier et
cell) mL for 24 h; 50, 100 and to growth al., 2013
200 μg/mL for 24 and 48 inhibition and
h) apoptosis
FACS/PI (50, 100 and 200
μg/mL for 24 h)
HCT15 and CO115 (human TUNEL assay (4 μM for Induces Limami et
colon carcinoma-derived 48 h) apoptosis, al., 2012
cells)
PI (4 μM for 48 h)
Autophagy
modulation
HT-29 (human colorectal) DNA fragmentation (25 Induces Shin et al.,
DU145 (human prostate
μM for 48 h) apoptosis 2012
carcinoma cells)
PC3 and DU145 cells MTT assay (10–40 μM for Induces Huang et
24 h) DNA fragmentation apoptosis al., 2011
(30 and 40 μM for 24 h)
PI (30 and 40 μM for 24 h)
HNBE (human normal lung MTT assay (2, 4, 8 and 16
cells) A549, H3255, and μmol/l for 48 h)
Calu-6 (lung cancer cells)
LDH assay (2, 4, 8 and 16
μmol/l for 48 h)
Inhibition Limami et
of cell al., 2011
proliferation,
invasion, and
migration
DNA fragmentation (2, 4,
8 and 16 μmol/l for 48 h)
HT-29 and HCT116 (human DNA fragmentation (20 Induces Messner et
colorectal cell line) and 30 μM for 24 and 48 apoptosis al., 2011
h)
DAPI (30 μM for 48 h)
HUVECs (isolation and XTT assay (3.125–50 μM Inhibits Bari et al.,
culture of human umbilical for 24 and 48 h) endothelial 2017
vein endothelial cells)
Annexin V-FITC/PI (6.25
and 12.5 μM for 6, 12, 18,
24 and 48 h)
DAPI: 4′,6-Diamidino-2-phenylindole; FACS: Fluorescence-activated cell sorting; FITC: Fluorescein
isothiocyanate; GBC-SD: Cell line human; HeLa: Cell line human (epitheloid cervix carcinoma);
HNBE: Normal human bronchial epithelial cells; HUVEC: Human umbilical vein endothelial cells;
LDH: Lactate dehydrogenase; LoVo: Cells line human colon; MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide; NBT: Nitroblue tetrazolium; PI: Propidium iodide; TUNEL: Transferase
dUTP nick end labeling; UA: Ursolic acid; XTT: 2,3-bis- (2-methoxy-4-nitro-5-sulfophenyl)-2Htetrazolium-5-carboxanilida.
proliferation,
Inducer of
endothelial cell
death
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Biomarkers as Targeted Herbal Drug Discovery
repair materials for treating efficaciously bone defects (Rezwan et al., 2006).
Excellent biocompatibility, osteoconductivity, and bioactivity should be exhibited by optimal bone repair materials (Kim et al., 2016; Chen et al., 2018).
UA is rarely employed for bone tissue regeneration as the few studies were
performed in the eld of bone tissue repair. Most importantly, osteoblast differentiation also involves the Smad signaling pathway. The release prole of UA
from the suitable incorporated dosage form remarkably increased the alkaline
phosphatase (ALP) activity, osteogenic differentiation-related gene type I
collagen, runt-related transcription factor 2 expression, and osteoblast-associated protein expression. Moreover, the results of micro-CT images, observations
from histomorphological data demonstrated that the UA in a suitable dosage
form can improve new bone formation ability. Therefore, the UA can be used as
novel bone tissue engineering materials by incorporating into a suitable matrix.
Nanotechnology has emerged as an efficient tool to address issues of solubility,
stability, and oral bioavailability (Valdes et al., 2014). Indeed, a nanosystembased delivery of drugs, photosensitizers, biomolecules, phytochemicals,
and other compounds of interest presents numerous advantages to traditional
delivery methods, including facilitated transport across biological barriers,
enhanced bioavailability when water solubility is poor, targeted delivery,
protection from biological and/or environmental degradation (particularly
for sensitive compounds) and controlled release (Morales et al., 2015; Li et
al., 2015). Recent improvements in therapeutic efficiency through the use
of nanotechnology have gained attention due to enhanced phytochemical
delivery to tumors and cancer cells (Gao et al., 2014; Bertrand et al., 2014;
Amiji, 2006; Wang et al., 2014).
In oncology, numerous potential benets, such as protecting the entrapped
therapeutic drug from degradation, reducing toxicity to normal cells, modi-
fying the pharmacokinetics and tissue distribution prole to increase drug
distribution in the tumor are offered by targeted drug delivery systems.
Preventing the side effects of clinical formulations for improving solubility;
and increasing cellular uptake and internalization in cancer cells (Bertrand
et al., 2014). Several delivery nanosystems like liposomes, nanoemulsions,
micelles, solid lipid nanoparticles (SLNs), and nanostructured lipid carriers
(NLCs), etc., have been used to enhance the physicochemical nature of
phytochemicals. These are the most commonly used nanosystems and can
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be administered orally, intravenously, intraperitoneally, or transdermally,
among other routes (Pattni et al., 2015).
In vesicles formed by nonionic surfactants (niosomes) and phospholipids
(liposomes), the different regions (i.e., aqueous medium, interface, and
hydrocarbon chains) allow interactions with a wide variety of substrates
(Uchegbuk and Florence, 1995; Sandoval et al., 2015). For example, electrostatically charged species would bind to the interface, whereas hydrophobic
substrates would locate inside the bilayer. Specifically, many advantages for
drug delivery, include good biocompatibility, biodegradability, low toxicity,
and a controlled release of the entrapped drug are represented by liposomes
(Han et al., 1997) (Figure 4.2).
Basic structure of liposomes.
Regarding UA incorporation into the liposomes Han et al. was the first
who studied the effect of triterpenes on membrane fluidity, with 1,2-dipalmitoyl-sn-glycerol-3-phosphocholine (DPPC) liposomes. The study
wants to find that UA has a strong condensing effect on the liposomal
membrane not only in the liquid-crystalline state but also in the crystalline state. UA displays fewer effects in decreasing the fluidity of the
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liquid-crystalline liposomal membrane as well as higher membrane-stabilizing effects are comparable with another triterpenes and natural products
(Both et al., 2002).
In another study, injection formed liposomes with a low concentration of
UA (3 mM) was incorporated into a gel of Carbopol 981. This study reported
that UA incorporation into liposomes increased both the ceramide content of
cultured normal human epidermal keratinocytes and the collagen content of
cultured normal human dermal broblasts (Caldeira et al., 2013).
Caldeira et al. proposed the pH-sensitive liposomes containing UA. This
was prepared with the lipid hydration method in a total lipid concentration
of 20 mM using the same amount of dioleoyl-phosphatidyl-ethanolamine
and cholesteryl-hemisuccinate and di-stearoyl-phosphatidyl-ethanolaminepolyethyleneglycol (PEG) 2000 in very low concentration. All materials
should be dissolved in chloroform, with a UA equivalent at 0.1% or 0.05%
(w/v). Then the whole solution will be added to the lipid solution. Approximately 88% of the vesicles were < 300 nm, had almost neutral surface
charges, showed no effects on stability, and had a UA entrapment of 0.77 ±
0.01 mg/mL which proves that UA can be successfully incorporated into it
(Yang et al., 2014).
A recent study of Yang et al. reported the antitumor effects of a
folate-targeted UA stealth liposome prepared by the thin lm dispersed
hydration method. The lipid compositions were soybean phosphatidylcholine/CHOL/monomethoxy polyethylene glycol 2000-distearoylphosphatidyl-ethanolamine and soybean phosphatidylcholine/CHOL/
mPEG-DSPE2000/folate-PEG cholesteryl hemisuccinate, with a 1: 20
(w/w) UA to lipid ratio. The lipid suspension was then extruded to produce
unilamellar vesicles. Liposome characterizations were similar in both
cases, with mean size distributions (150160 nm), potentials (–23.15 and
–21.24 mV), and UA entrapment efciencies (86.7 and 88.9%) (Qian et
al., 2015). The result determines that UA can be successfully incorporated
into these matrixes.
In vitro and in vivo models both have been used to assess the anti-inflammatory, anti-proliferative, and pro-apoptotic effects of triterpenoids in relation
to potential anticancer activities.
The effect of pH-sensitive liposome-UA on breast and prostate cancer
cell line viabilities were also studied by MTT assays, revealing that IC50
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values obtained after liposome-UA treatment (48 h) were signicantly lower
than IC50 values obtained after free-form UA treatment of MDA-MB-231
cancer cells (Yang et al., 2014).
Regarding in vivo studies, Yang et al. evaluated PEGylated liposome and
folate-receptor-targeted liposome antitumor efcacies using the human KB
tumor xenograft model in female Balb/c nude mice. The obtained results
demonstrated that free-form UA did not decrease tumor growth, which was
in contrast to mice treated with folate-receptor-targeted-liposome-UA, which
resulted in a 55% reduction in tumor volume compared with PBS-treated
mice (Qian et al., 2015).
Qian et al. recently examined the safety and activity of UA liposomes
against tumors in 20 subjects of age 18–75, in whom the presence of advanced
solid tumors had already conrmed by cytological or histological data. All
subjects received intravenously administered UA liposomes at doses of 56,
2
74, and 98 mg/m
for 14 consecutive days over a 21-day time period. All
the subjects were evaluated at the tolerability and toxicity scale. The results
demonstrating the safety prole of UA liposome treatment particularly for
subjects with advanced solid tumors. Indeed, 60% of patients achieved stable
disease status after two treatment cycles (Wang et al., 2013).
Wang et al. evaluated the toxicity and single-dose PKs of intravenous UA
liposomes. All subjects received a single-dose of UA liposomes (11, 22, 37, 56,
74, 98, and 130 mg/m
2
) administered as a 4 h intravenous infusion. The clinical
data reported, for the rst time, that the UA liposome had manageable toxicities, with a maximum tolerated a dose of 98 mg/m
2
encountered at 74, 98, and 130 mg/m
, and consisted of hepatotoxicity and
2
. Dose-limiting toxicity was
diarrhea. The single-dose pharmacokinetic parameters revealed a linear relationship between Cmax, AUC024 h (Merisko-Liversidge et al., 2003).
For years the use of nanocarrier-mediated drug delivery systems into the
improvement of the therapeutic activity and safety of drugs. Formulations for poorly water-soluble drugs like nanoliposome is a promising
approach (Zhu et al., 2013). The new technologies for the encapsulation
and delivery of bioactive agents are nanoliposome, or submicron bilayer
lipid vesicle. The immense list of bioactive material ranging from pharmaceuticals to cosmetics and nutraceuticals can be incorporated into
nanoliposomes. Due to the nanosize along with their biocompatibility
and biodegradability, their, nanoliposomes have demonstrated their
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Biomarkers as Targeted Herbal Drug Discovery
potential applications in numerous fields. By improving the solubility,
bioavailability, in vitro, and in vivo stability, along with the prevention
of unwanted interactions with other molecules, nanoliposomes are able
to enhance the performance of bioactive agents. Cell-specific targeting is
an another advantage of nanoliposomes, which is a prerequisite to attain
drug concentrations, required for obtaining maximum therapeutic efficacy
in the target site while minimizing adverse effects on healthy cells and
tissues. It is used mainly in cosmetics, food technology, and agriculture
and also including nano-therapy like diagnosis, cancer therapy and gene
delivery (Figure 4.3).
Thus, for the first time development of UA nanoliposomes (UANL)
were happening in the People’s Republic of China. Because due to
bypassing the stomach by nanoparticles the bioavailability of UANL is
considered to be improved at intravenous (IV) administration. Till date,
a wide range of preclinical studies have been completed in China (Li,
unpublished data, 2005). A further unpublished study has demonstrated
the induced minimal toxic effects even with the long-term application
by UAN L.
There are some studies which revealed the inhibition of the growth of
various human cancer cells and nude mice xenografts by the nanoliposomeencapsulated UA. That the concept of the first entry of UANL into the
stomach and intestines, followed by the rapid declination of its concentrations is suggested by tissue distribution experiments in mice. Conversely,
the hepatic concentration of UA increases rapidly and exceeded the
concentrations in the stomach and intestines at 4 hours after IV injection.
The results demonstrated that UANL delivers UA to the liver, where it
accumulates. Consequently, the drug disposition behavior changes in vivo,
and the toxic and side effects of UA on other tissues is decreased (unpublished data). The antitumor activities and the minimal toxic effects of UA t
were observed in preclinical studies and promoted the human clinical trials
of UAN L.
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Basic structure of nanoliposome.
In Phase I, a nanoliposome study by Zhu et al., the UA nanoliposome
exhibited a relatively linear pharmacokinetic behavior at dose levels between
37 and 98 mg/m
2
. Furthermore, there was no such evidence of drug accumulation with repeated doses of UANL was observed, and the intravenous
infusion in patients with advanced tumors and by healthy volunteers was
well tolerated (Alvarado et al., 2015).
Generally, nanoemulsions ranges from 10 to 1,000 nm are a system of
colloidal particles in the submicron size range which enacting as a transporter of drug molecules. The surface of these carriers is amorphous and
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Biomarkers as Targeted Herbal Drug Discovery
lipophilic with a negative charge and with the solid spheres. To enhance
site-specificity, magnetic nanoparticles can be used. They may enhance
the therapeutic effectivity of the drug and customize the adverse effect
and toxic reactions as a drug delivery system. The treatment of infection
of the reticuloendothelial system (RES), enzyme replacement therapy in
the liver, treatment of cancer, and vaccination are included as major applications. An emulsion a biphasic system, is a thermodynamically unstable
system, can be stabilized by the addition of an emulsifying agent (emulgent or emulsifier), where one phase is dispersed into the other in the form
of minute droplets ranging in diameter from 0.1 to 100 m. The dispersed
phase is also known as the internal phase or the discontinuous phase while
the outer phase is called dispersion medium, external phase, or continuous
phase. The intermediate or interphase is an emulsifying agent. The terminology nanoemulsion’ is fine oil/water or water/oil dispersion also refers
to a mini-emulsion which stabilized by an interfacial film of surfactant
molecule having droplet size range 20,600 nm. Because of small size, of
nanoemulsions are responsible for the transparent nature of it. There are
three types of nanoemulsion as per their formulation technique:
1. Oil in water nanoemulsion: oil/nonaqueous phase are dispersed in
the continuous aqueous phase;
2. Water in oil nanoemulsion: water droplets/aqueous phase are
dispersed in the continuous oil phase; and
3. Bicontinuousnanoemulsions (Figure 4.4).
Alvarado et al. developed nanoemulsions using natural or synthetic UA
mixtures. The nanoemulsion composition was obtained from pseudoternary phase diagrams composed of castor oil (oil phase, 20%), a 4:1
ratio mixture of Labrasol (surfactant), and Transcutol P (co-surfactant),
propylene-glycol (aqueous phase, 20%) and OA/UA mixtures (0.2%).
The sizes of droplets of nano-emulsions (NE) were 200.95 nm (p.i.
0.25) and 139.70 nm (p.i. 0.18), respectively which formed with a
natural or synthetic UA mixture, ultimately shows great activity (Zhou
et al., 2009).
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The basic structure of nanoemulsion droplets.
The existence of nanoparticles is only on the nanometer scale (i.e., below
100 nm in at least one dimension). The physical properties demonstrated by
them are uniformity, conductance, or special optical properties. These are the
most desirable properties in materials science and biology. Various nanoparticle drug delivery systems have been explored, including nanoparticles,
nanospheres (NSs), nanocapsules (NCs), SLNs, and polymeric nanoparticles
(Figure 4.5).
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