This document summarizes research analyzing the neurotrophic and cytotoxic activities of steroidal glycosides isolated from the plant Cynanchum otophyllum. Four C21 steroidal glycosides were tested on human cancer cell lines and normal fibroblast cells. Compound 2 showed selective cytotoxicity against colon cancer cells and inhibited cell proliferation. The compounds' effects on cell cycle and neurite growth in neuronal PC12 cells were also examined. The results suggest different substitutions at C-12 may influence activity against cancer cells, and compound 2 warrants further study for its anti-cancer effects.

1. Manuela Arbeláez Gómez
lll semestre
Biología molecular

2. Introduction
• Cynanchum otophyllum is genus of about 300
species in including some swallowwort, belonging to
the family Apocynacaeae, an endemic species in
China , is widely distributed in South west China .
The roots of the plant are traditionally used for
treating epilepsy, rheumatic pain, kidney weakness,
and muscle injury in Chinese folk medicine. The
main constituents in this herb were identified as
esteroidal glucosides with deoxysugars.

3. C21
• A steroid compund with structure based
on a 21-carbon (pregnane) skeleton that
acts as a hormone.
• In the Cynanchum otophyllum the
pregnane glycosides isolated from this
herb have attracted considerable attention
in recent years. The biological activities of
these steroidal glycosides, which possess
diferent diferent levels of glycosylation,
were further examined for their
neurotrophic activities in rat neuronal
PC12 cell model, as well as their cytotoxic
activities in human cancer cell lines and
normal fibroblast.

4. Objective
• Analize potential neurotrophic activity and cytotoxicity of selected
C21 steroidal glycosides from Cynanchum otophyllum.

5. Materials and methods
• Western blot is a laboratory
method used to detect specific
protein molecules from among a
mixture of proteins. This mixture
can include all of the proteins
associated with a particular tissue or
cell type. Western blots can also be
used to evaluate the size of a
protein of interest, and to measure
the amount of protein expression.
• Western blot analyses on cyclin D1
and cyclin E1 expressions in HCT-
116 cells treated with 2 for 24 h
were performed.

6. Citotoxicidad
• La citotoxicidad es la cualidad de
algunas células para ser tóxicas
frente a otras que están alteradas.
La citotoxicidad constituye uno de
los mecanismos efectores de ciertas
poblaciones celulares especializadas
del sistema inmunitario, consistente
en la capacidad para interaccionar
con otras células y destruirlas.
• Se evaluó con los compuestos
1–4 y el agente de control
positivo, cisplatino, se
evaluaron a partir de sus
actividades citotóxicas contra
cinco líneas celulares de cáncer
humano .

7. Cultivo MTT
• La prueba de MTT es una prueba colorimétrica cuyos resultados dependen
del metabolismo celular, es importante establecer el número de células que se
requiere sembrar para obtener una densidad óptica de 1 en un determinando
tiempo de cultivo.
• En el artículo se evaluaron cinco líneas celulares de cáncer humano (MDA-
MB-231, EC-109, K562, HCT-116 y A427) y fibroblastos humanos normales
(Hs27) usando el ensayo MTT después de 48 h de incubación.

8. Citometría de flujo
• La citometría de flujo es una
tecnología biofísica basada en la
utilización de luz láser, empleada
en el recuento y clasificación de
células según sus características
morfológicas, presencia de
biomarcadores, y en la ingeniería
de proteínas.
• Cell cycle of HCT-116 cells treated
with 2 for 24 or 48 h were
evaluated by flow cytometry.

9. Results
• A Immunoblotting was performed 4 times
using independently prepared cell lysates and
the representative blots were shown.
• The histograms showed the quantified
results of protein levels, which were adjusted
with corresponding β-actin protein levels and
expressed as fold of control (mean fold of
control + SD from four independent
experiments).

10. • El compuesto 2 mostró citotoxicidad selectiva
contra HCT-116 con valores de IC50 de 19,41
μM
• 3 mostró citotoxicidad contra todas las líneas
celulares de cáncer humano probadas con
valores de IC50 de 19–26 μM. Sin embargo, 2
(hasta 100 μM) no causaron efecto citotóxico
en fibroblastos de piel humana normal Hs27
• 3 indujeron la citotoxicidad de las células Hs27
(valor IC50 de 75.68 μM).
• Proliferación celular de HCT-116, que se
evaluó mediante el ensayo de incorporación de
3H-timidina, se inhibió en 2 de forma
dependiente de la concentración, con valores
de CI50 de 31,33 y 20,99 μM, después de 24 y
48 h de incubación.

12. • Neurite outgrowth-promoting activities of
compounds 1–4 incubated in PC12 cells for
72 h were assessed under a phase contrast
microscope.
• Neurite processes with a length equal to or
greater than the diameter of the neuron cell
body were scored as neurite-bearing cells.
• The ratio of the neurite-bearing cells to
total cells (with at least 100 cells
examined/view area; 5 viewing area/well)
was determined and expressed as a
percentage.
• Representative photos from two inde-
pendent experiments (with duplicate wells)
were shown

13. Li (2015) The four C21 steroidal glycosides
were identified by comparison of
experimental and literature
spectroscopic data and were
confirmed as caudatin 3-O-β-
cymaropyr- anoside
Greene (1976) The neurotrophic activities of the test
compounds were examined according
to an assay using PC12 cells as
reported
Dong (2018) properties, pregnane glycosides
isolated from this herb have attracted
considerable attention in recent years.
In our recent study, we have
successfully isolated several new C21
steroidal glycosides

14. CONCLUSSION
• Analysis of the structure-activity relationship shows that
different substitution group at C-12 may be responsible for
the differentiated activities in cancer cells.
• The compound 2 could inhibite the proliferation of colon cancer
cells and interfered the cell cycle.

15. Conclussions