Keywords |
| Ginger Pinellia, SGC7901 cell, Flow cytometry |
Introduction |
| Banxia, also known as sanyebanxia, banyuelian, sanbutiao,
dibadou, shoutian, shuiyu and yangyan, is the dried tuber
of Pinellia ternata (Thunb.) Breit. in the genus Pinellia of
the family Araceae, which was originally recorded in the
Shen Nong's Herbal Classic. It is pungent, warm, toxic,
and enters the spleen, stomach and lung meridians, with
dampness-eliminating, phlegm-reducing, adverse qilowering,
vomiting-arresting, lump-dissolving and massdissipating
effects. Major active constituent of Pinellia
tubers is volatile oil; besides, it also contains alkaloids
that have similar pharmacological actions to coniine and
nicotine, and substances with similar skin irritating effect
to protoanemonin [1, 13, 10]. |
| Traditional pharmacological actions of Pinellia ternata
include dampness elimination, phlegm reduction, adverse
qi lowering, vomiting arrest, lump dissolution and mass
dissipation; as well as external treatment of acute mastitis,
acute & chronic suppurative otitis media and other
diseases. Due to differences in extraction technology,
medication regimen and compatibility, Pinellia ternata
may exhibit different pharmacological effects. Modern
pharmacological studies have shown that Pinellia ternata
also has anti-tumor [14, 3, 11] anti-fertility, hypolipidemic,
hepatoprotective and coronary heart disease curing [4, 7]. |
| Crude Pinellia ternata is toxic, which may cause vomiting
and even death in severe cases. Extraction of Pinellia
ternata with a variety of materials can effectively weaken and eliminate its toxicity. In the thousands of years of
clinical application history of Pinellia ternata, in order to
fully exert its clear clinical efficacy while reducing toxic
side effects, numerous physicians and scholars have made
extensive attempts to explore the extraction technology
of Pinellia ternata from various perspectives, so as to
achieve the best attenuated synergistic effect. This study
optimizes the extraction process of ginger Pinellia, and
studies its anti-gastric cancer effect. |
Materials |
| Instruments |
| 751G UV-Vis spectrophotometer (Shanghai Analytical
Instrument Factory); CO-150 CO2 incubator (NBS,
USA); inverted fluorescent microscope (OLYMPUS,
Japan); FACSAria Flow Cytometer (BD, USA); ELX 800
microplate reader (BioTEK, USA). |
| Drugs, Reagents and Cells |
| Pinellia ternata and licorice were purchased from Bozhou
medicine market, which were identified as the dried tubers
of Araceae plant Pinellia ternata (Thunb.) Breit. and the
roots and rhizomes of Leguminosae plant Glycyrrhiza
uralensis Fisch., respectively. Alum was purchased from
a pharmacy. Quality of the three drugs was all in line with
the current Chinese Pharmacopoeia requirements. RPMI
1640 was product of Gibco; FBS was product of Hangzhou
Shijiqing Bioengineering Materials Company; and MTT,
DMSO and PI were products of Nanjing Keygen Biotech
Company. Human gastric cancer SGC7901 cells were
purchased from China Medical University. |
Methods and Results |
| Sample Preparation |
| Nine aliquots of 200 g of uniformly sized Pinellia ternata
were soaked separately in water (with level 3 cm higher than Pinellia ternata) until completely moist, then taken
out, shaken to remove surface water, and set aside for
orthogonal experiment. |
| Orthogonal Factors and Levels |
| During the extraction of Pinellia ternata, licorice
concentrating volume, lime water addition and extraction
temperature had comparatively great influences on the
extraction efficiency. On the basis of single factor design,
orthogonal experiment was conducted according to L9(3)3
array selecting the above three factors by determining the
monoammonium glycyrrhizinate content in each extract
using UV spectrophotometry and HPLC as the index, as
shown in Table 1. |
| Orthogonal experiment showed that during the extraction
of ginger Pinellia, the degrees of influence of factors on
ginger Pinellia extraction efficiency were: B>A>C, i.e. lime
water addition>licorice concentrating volume>extraction
temperature in descending order. Based on the orthogonal
results and range analysis, optimal extraction process of
ginger Pinellia was identified as: A3B3C3, i.e., licorice
concentrating volume of 300 ml, lime water addition of
80, and extraction temperature of 50°C, as shown in Table
2. |
| Cell cultivation |
| Gastric cancer SGC-7901 cells were seeded in culture
flasks with RPMI 1640 medium containing 10% FBS,
penicillin (100 mL/L) and streptomycin (1 mg/ml),
cultured statically in a 37°C, 5% CO2 incubator, and
digested and passaged with 0.25% trypsin. Cells passaged
three times were used in the experiment. |
| SGC-7901 Cell Growth Inhibition Experiment by MTT Assay |
| After trypsinization for 2 min, exponential phase cells were
prepared into a 5 × 104 cells/ml cell suspension, seeded in
96-well plates at 180 μl per well, cultured for 24 h, and then added with different concentrations (1g/L, 2 g/L, 3 g/L) of
ginger Pinellia ethanol extracts. Five replicate wells were
set up for each concentration. Meanwhile, control group
was added with an equivalent volume of culture medium.
After culturing in 5% CO2 incubator for additional 24 h,
48 h and 72 h, 30 μl of MTT solution (5g/L) was added
to each well on the third day, and the cultivation was
continued for 4 more h. Then supernatant was discarded,
each well was added with 150 μl of DMSO, and shaken for
10 min. Absorbance of each well was measured at 490 nm
with microplate reader, and inhibition rate was calculated
for cells in each group. |
 |
| Inhibition rate = (A value of negative control group - A
value of treatment group) / A value of negative control
group × 100%. |
| MTT assay results showed that different doses of ginger
Pinellia ethanol extracts (1 g/L, 2 g/L, 3 g/L) could all
inhibit the proliferation of SGC7901 cells after acting
for 24 ~ 72 h. Moreover, the anti-proliferative effect of
the ginger Pinellia ethanol extracts became increasingly
evident with the increase of drug concentration and
prolongation of incubation time. At test doses, cell viability
declined continuously with increasing dose; and at the
same concentration, cell viability declined continuously
over time. In particular, after 72 h of drug intervention,
SGC7901 cell inhibition rate reached 88.6% in the high
dose group, which was statistically different from the
control group, as shown Figure 1. |
| Effect of Ginger Pinellia Ethanol Extracts on Cell Cycle
Distribution of SGC-7901 Cells |
| Logarithmic phase SGC-7901 cells were trypsinized,
prepared into a 5 × 104 cell suspension, and cultured under
37°C, 5% CO2 and saturated humidity conditions for 24
h. Then cells were collected, and added with different
concentrations (1 g/L, 2 g/L, 3 g/L) of ginger Pinellia
ethanol extracts and treated for 72 h. Five replicate wells
were set up for each group. Next, cells were collected,
prepared into single cell suspension, and centrifuged
at 2,000 r/min for 5 min. Afterwards, supernatant was discarded, and the remaining cells were washed 3 times
with PBS, and fixed in 70% precooled ethanol at 4°C for
12 h, then washed with PBS twice, and PI stained in an
ice bath for 30 min. Finally, changes in the cell cycle after
drug intervention were analyzed by flow cytometry. |
| Flow cytometry results revealed that the ginger Pinellia
ethanol extracts had very significant effects on cell cycle
distribution of SGC-7901 cells. Compared with the control
group, with the increasing concentration of ginger Pinellia
ethanol extracts, the proportion of S phase SGC-7901 cells
increased from 18.46% in the control group to 36.35%,
while the proportion of G2/ M phase cells decreased
significantly from 29.27% in the control group to 11.93%.
This suggests that the ethanol extracts of ginger Pinellia
can arrest SGC-7901 cells in S phase. Compared with
the control group, G2/M phase cells decreased, while the
percentage of S phase cells increased, presenting a marked
time-dose-response relationship, as shown Table 3. |
| Morphological Changes in SGC7901 Cells after 72 h of
Intervention with Ginger Pinellia Ethanol Extracts |
| Logarithmic phase SGC-7901 cells were seeded in 6-well
plates at 5 × 104/ml for growth on slides. Well growing,
rapidly dividing, morphologically normal cells without
cytoplasmic granules were collected, and cultured with
medium containing ginger Pinellia ethanol extract
(concentration of 3 g/L) for 24 h, then observed under an
inverted microscope. |
| At high magnification, after culturing with medium
containing ginger Pinellia ethanol extract (concentration
of 3 g/L) for 24 h, the SGC-7901 cells in the control group:
were grown firmly adherent, which were bright, clear,
refractive and fully stretched, with intact membrane and
uniform cytoplasmic distribution. In comparison, cells in
the ginger Pinellia ethanol extract groups: were rounded,
with unclear boundary. Secretory granules increased,
cellular refraction decreased, and cellular glycogen
lowered. Organelle swelling and liquefaction degeneration
of cytoplasm were present. In addition, mitochondria
decreased, with only residual organelle fragments visible. |
 |
| Figure 1: Effect of ginger Pinellia ethanol extracts on proliferation of SGC-7901 cells |
 |
Discussion |
| Traditional Chinese medicine Pinellia ternata, as an
antiemetic, plays an important role in clinical practice in
China [1]. |
| In the Chinese Pharmacopoeia 2010 edition, four extract
varieties of Pinellia ternata are included, they are:
unprocessed Pinellia, liquorice processed Pinellia, ginger
processed Pinellia, and alum processed Pinellia. Extraction
procedure of ginger processed Pinellia is as follows: soak
clean Pinellia ternata in water until completely moist,
then remove; decoct ginger slices, add alum and Pinellia
ternata and boil out, then remove and air dry, or air dry
half and dry; or slice and dry [2]. |
| In this study, the degrees of influence of licorice
concentrating volume, lime water addition and extraction
temperature on ginger Pinellia extraction efficiency are
given full consideration. The influence of each level
of each factor on ginger Pinellia extraction efficiency
is determined through orthogonal experiment. The
experimental results show that the degrees of influence
of factors on ginger Pinellia extraction results are lime
water addition>licorice concentrating volume>extraction
temperature in descending order. Based on the orthogonal
results and range analysis, the optimal extraction process
of ginger Pinellia is identified as licorice concentrating
volume of 300 ml, lime water addition of 80, and extraction
temperature of 50°C. In addition to the toxicity to mucous
membranes, Pinellia ternata mainly acts on organs of
liver, intestines and kidneys; besides, it also has strong
toxicity and teratogenicity to embryos. After extraction,
Pinellia ternata's mucous membrane irritating effect
decreases significantly, which has thus gained broader
clinical application. |
| Cell cycle refers to the process of cell proliferation in
which cells produce colonies that resemble themselves
through division. The period from the beginning of one
cell division to the beginning of the next is called a cell
cycle, which is divided into four phases: G1 phase, the
phase before DNA synthesis; S phase, DNA synthesis
phase; G2 phase, the phase after DNA synthesis; and M
phase, mitotic phase [12]. According to modern oncology
studies, an increasing number of tumors cause excessive
cell growth and reproduction by unbalancing the cell
cycle, thereby resulting in dissimilation [5, 8, 9]. |
| In the experiment on anti-proliferative activity of ginger
Pinellia on SGC7901 cells, different doses of ginger Pinellia
ethanol extracts (1 g/L, 2 g/L, 3 g/L) can all inhibit the
proliferation of SGC7901 cells after acting for 24 ~ 72 h.
Moreover, the anti-proliferative effect becomes increasingly
evident with the increase of drug concentration and
prolongation of incubation time. At test doses, cell viability
declines continuously with increasing dose. |
| In addition, ginger Pinellia ethanol extracts also have very
significant effects on cell cycle distribution of SGC-7901
cells. Compared with the control group, with the increasing
concentration of ginger Pinellia ethanol extracts, the
proportion of S phase SGC-7901 cells increases from
18.46% in the control group to 36.35%, while the
proportion of G2/M phase cells decreases significantly from 29.27% in the control group to 11.93%. This suggests
that the ethanol extracts of ginger Pinellia can arrest SGC-
7901 cells in S phase. Clearly, ginger Pinellia ethanol
extracts can achieve the therapeutic goal of suppressing
tumor activity by affecting cell cycle progression. |
References |
- Cao LJ, Han C, Wang SH. A study on extraction process of Pinelliaternata. Tianjin Pharmacy 2011; 23: 56-57.
- Chinese Pharmacopoeia 2010 edition Vol. I: 111-112.
- Fitzgerald TL, Bradley CJ, Dahman B, Zervos EE. Gastrointestinal Malignancies: When Does Race Matter. Am CollSurg 2009; 209: 645-652.
- GUI MW, WEI PK, LU Y. Effects of XiaotanSanjie Decoction-containing serum on proliferation and apoptosis of human gastric cancer cells MKN-45. Journal of Chinese Integrative Medicine 2010; 8: 250-255.
- Hartwell LH, Kastan MB. Cell cycle control and cancer. Science 1994; 266: 1821-1824.
- Mao ZC, Peng ZS. Progress on research of Pinelliaternata. Jiangxi Science2002; 20: 42-46.
- Mijatovic T, Jungwirth U, Heffeter P, Hoda MAR, Dornetshuber R, Kiss R, Berger W. The Na+/K+-ATPase is the Achilles Heel of multi-drug-resistant cancer cells. Cancer Lett 2009; 282: 30-34.
- Ngeow J, Tan IB, Choo SP. Targeted therapies in the treatment of gastric cancer. Asia Pac J ClinOncol 2011; 7: 224-235.
- Sherr CJ. Cancer cell cycle. Science1996; 274: 1672-1677.
- Stark T, Hofmann T. Structures, sensory activity, and dose/response functions of 2,5-diketopiperazines in roasted cocoa nibs (Theobroma cacao). Journal of Agricultural and Food Chemistry 2006;53: 7222-7231
- Tsuburaya A, Hayashi T, Arai T,Asaka M. Current status of treatment for elderly patients with gastric cancer. GanTo Kagaku Ryoho 2010; 37: 2817-2822.
- Williams GH, Stoeber K. The cell cycle and cancer. J Pathol 2012; 226: 352-355.
- Xu JK, Zhang TL, Yi GQ, Xu Y, Wu HH, Pei YH. Isolation and structure elucidation of chemical constituents from Pinelliaternata. Journal of Shenyang Pharmaceutical University 2010; 27: 429-434.
- Xu XY. Clinical observation on treating Chroniccervistis by Banxia. Chinese Journal of the Practical Chinese with Modern Medicine2001; 1: 22-25.
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