Black cohosh research

Black cohosh: A potential remedy for breast cancer
Black cohosh- background:
Black cohosh (Cimicifugaracemosa) is an herbaceous
perennial plant. It’s a coarse woodland herb with large
compound leaves and a thick, knotted rhizome system (F.
Firenzuoli, 2007). Native to North America, the root and
rhizomes of black cohosh were used by Native American
women throughout their life for menstrual cramps,
difficult childbirth and complicated menopause, as well as
other conditions such as dysmenorrhea, colic and
rheumatism. Native Americans subsequently introduced
the herb to the American colonists, who used it for
women’s complaints, as well as illness such as bronchitis,
nervous disorders, inflammation and uterine disorders
(J.L. Mayo, 1998).
Today, black cohosh is widely used in various
pharmaceutical industrial preparations, often mixed with
other medicinal plants (F. Firenzuoli, 2007). It is widely
used in the U.S. and Europe to help alleviate menopausal
symptoms, such as hot flashes, sweats, irritability and
vaginal dryness.
The action of black cohosh is attributed to the synergy of
the entire profile of is active components (J.L. Mayo,
1998). The primary active constituent of the herb’s root is
believed to be the triterpene glycoside fraction, including

actein and cimicifungoside (B. Klinger, 2003). Other
potentially biologically active substances of the rhizome
include isoflavone (phytoestrogen) formononetin, the
triterpene glycosides 27- deoxyactein and racemoside, as
well as the aromatic acids ferulic acids and isoferulic acid.
In addition, cimicifungoside appears to affect the
hypothalamus- pituitary axis, resulting in reproductive
and nervous system effects, while the aromatic acids are
believed to be anti- inflammatory (J.L. Mayo, 1998). Other
components of the herb are cinnamic acid esters, sugars
and long- chain fatty acids (L.S. Einbond, 2004).
Breast cancer mechanisms- background:
The growth promoting effects on breast cancer cells are
attributed to estrogenic actions on estrogen receptor (ER)
alpha. Most breast cancers express estrogen receptors. In
these cases, estrogen is required for continued growth,
and in post- menopausal women the major source of
estradiol is through local conversion of circulating
androgens and estrone sulphate. Breast tissue can
convert circulating androgens through aromatase and 17-
beta- hydroxysteroid dehydrogenases and estrone
sulphate to estradiol through steroid sulphatase and 17-
beta- HSD. All these enzymes are highly expressed in
breast cancer tissue, such that the concentration of
estradiol in breast tumours is about 20- fold higher than

in the circulation. The reductive activity of 17- beta-
HSD1 that drives estrone to estradiol is dominant in
breast cancers, while oxidative 17- beta- HSD2 activity
that catalyses the reverse reaction predominates in
normal breast tissue. Steroid sulphatase mRNA
expression is correlated with breast cancer, and it has
been proposed that the sulphatase pathway is more
important than aromatase (S. Rice, 2007). Therefore, 17-
beta- HSDs act as molecular switches. Patients with
tumours with high 17- beta- HSD1 expression have
significantly shortened survival rates (J.M. Day, 2008).
In addition, the growth factor receptor kinase epidermal
growth factor receptor (EGFR) plays important roles in the
development, progression, aggressiveness and metastasis
of many tumours. EGFR (or Her1) and the related human
epidermal growth factor receptor Her2 have been proved
to be relevant for cancer too (Y. Qian, 2010).
Another factor affecting breast cancer potential are free
radicals and other reactive oxygen species (ROS) that are
constantly generated and cause damage to biomolecules.
This process is regulated by existence of antioxidants,
DNA repair systems and replacement of damaged lipids
and proteins. Scavenging of ROS can protect against
cancer. DNA is a significant target of oxidative stress,
because continuous oxidative damage contributes to
development of cancer. This may be due to effects on
cellular proliferation, prevention of apoptosis, damage to

DNA repair enzymes and damage to DNA polymerases
that can decrease fidelityof replication. Accordingly, if
direct damage to DNA bases caused by ROS contributes
to development of cancer, agents that reduce such
damage should decrease the risk of cancer development
(J.E. Burdette, 2002).
Another relevant factor is apoptosis, which is an essential
regulatory mechanism. An imbalance between mitosis
and apoptosis has pathologic implications and has been
associated with many autoimmune disorders, tumours
and viral infections. Caspases are the central executioners
of apoptosis, and one of their actions is the cleavage of
the intermediate filament cytokeratin 18 in epithelial
cells. There are increasing levels of caspases during
apoptotic cell deaths (K. Hostanska, 2004).
Tumour cell invasion is a complex process, which is
characterised by alterations in cellular attachment,
proteolytic and migratory activities. The invasive potential
of cancer cells is linked to their capacity to degrade
basement membrane and extracellular matrix to create a
path for migration. Metastasis is a complex multi- step
process, involving cell adhesion, invasion and motility.
Hence, interruption of one or more of these steps is one
approach of anti- metastatic therapy. Migration and
invasion are key functional activities in the progression of
early stage breast cancer into a more aggressive state.
Cellular growth independent from basement membrane,

as a prerequisite for migration and invasion is one of the
hallmarks of the metastatic phenotype. An invasiveness of
breast cancer cells is frequentlyassociated with absence
of estrogen receptors (K. Hostanska, 2007).
Black cohosh anti- breast cancer activity:
Extracts of black cohosh rhizomes have been recognised
as a rational choice for the treatment and prevention of
breast cancer. Ethanolic and isopropanolic extracts of
black cohosh rhizomes inhibited the growth of both
estrogen- dependent MCF-7 and estrogen- independent
MDA-MB-231 human breast cancer cells (K. Hostanska,
2007).
According to S. Rockwell and co, there is considerable
debate about whether black cohosh has estrogenic or
anti-estrogenic activities, since conflicting findings were
found. However, according to another research, C.
racemosa shows weak binding activity to estrogen
receptor, and shows no estrogenic activity in MCF-7 cells,
and no gene expression in estrogen-inducible cells, but
antagonises these activities. Black cohosh extract
significantly inhibits estrogen-induced proliferation of
MCF-7 cells adapted to estrogen-free medium. In
addition, the effect of E2 on proliferation and gene
expression in estrogen-inducible cells is antagonised by
whole extract of black cohosh. ER- cells are significantly

more sensitive than ER+ cells. There was dose-dependent
anti-proliferative action of black cohosh in breast cancer
cells, probably evoked by genomic (ER-mediated)and
non-ER-mediated mechanisms, because of the various
physic-chemical properties of individual components of
black cohosh. The spontaneous apoptotic rate of MCF-7
cells in comparison to MDA-MB-231 cells was higher.
This suggests that black cohosh cytotoxicityappears to
be explained in part by induction of apoptosis. MCF-7
cells, despite of their caspase-3 deficiency, show a high
responsiveness to black cohosh treatment. Therefore, the
apoptotic action of black cohosh must be mediated
through mechanisms other than its weak binding to ER (K.
Hostanska, 2004).
treatment
dose
(micrograms/
ml) survival (%) mcf-7 ic50 (micrograms/ml)
control 0 100 +/- 3.8
icr 38.7 97.5 +/- 2.3 128.5 +/- 4.9
77.4 95.2 +/- 3.4
154.8 89 +/- 4.2
ttg 0.1 96.9 +/- 1.9
1 93.5 +/- 1.5 >100
5 86.7 +/- 2.7
cae 0.1 94.3 +/- 1.9
1 90.9 +/- 2.4 ~25^a
5 83.3 +/- 3.7
treatment
dose
(micrograms/
ml)
control
0 survival (%) mda-mb231 ic50 (micrograms/ml)

Table 1: Effects of treatments by black cohosh extract,
TTGs and CAEs on proliferation of MCF-7 and MDA-MB-
231, reflected through the IC50 values obtained (L.S.
Einbond, 2004).
icr
38.7 100 +/- 6.5
77.4 104.3 +/- 1.2 92.5 +/- 3.5
154.8 89 +/- 2.7
ttg
0.1 90.9 +/- 4.1
1 103.9 +/- 0.8
5 102.6 +/- 1.1 96.3 +/- 5.3
cae
0.1 102.5 +/- 2.6
1 104.6 +/- 1.4
5 103.1 +/- 2.3 26.2 +/- 1.4
96.7 +/- 3.9

Table 2: Effect of iCR, TTG and CAE on tumor cell growth.
MCF-7 and MDA-MB 231 cells were treated for 24 h with
or without substances at the indicated concentrations.
Attached cells were stained with crystal violet and the

absorbance of the cell lysate was measured at 540 nm.
Data are expressed as mean±SD of triplicate wells from
two independent experiments. *p<0.05 vs. untreated
control.
In addition, another research claims that extracts of black
cohosh don’t bind to ER-alpha or beta (S. Rice, 2007).
MDA-MB-231 cells showed a higher sensitivity to the
cytotoxic effects of black cohosh than MCF-7 cells.
However, there were no differences in sensitivity of both
cell types to either triterpene glycosides or cinnamic acid
esters, according to IC50 values. Therefore, synergistic
action between the different plant compounds is likely (K.
Hostanska, 2007). In both MCF-7 and MDA cells, black
cohosh had no significant effect on the conversion of
androstenedione to estradiol at any dose, and only the
highest doses inhibited the conversion of estrone to
estradiol (S. Rice, 2007).
According to L.S. Einbond and co, the proliferation of ER+
and ER- human breast cancer cells was inhibited via
induction of apoptosis through activation of caspases.
Further, fractions of black cohosh enriched in triterpene
glycosides or cinnamic acid esters, inhibited cell growth
and induced apoptosis. An ethanolic extract inhibited the

activity of the cyclin D1 promoter and increased the
activity of the P21cipi1 promoter in the ER- human breast
cancer cells. Ethyl acetate fraction of black cohosh
inhibited growth of MCF-7 (ER+, Her2 low) cells and
induced cell cycle arrest at G1 after treatment with 30
microgram/ml, and the G2/M after treatment with 60
microgram/ml. This suggests that the fraction contains a
mixture of components with the more active or abundant
component causing G1 arrest, and the less active causing
G2/M arrest, or individual components in the same
fraction exert different effects at different concentrations.
Therefore, it is possible that at high concentrations the
fraction affects proteins that regulate later phases in the
cell cycle (L.S. Einbond, 2007).

Table 3: The effects of black cohosh extract and isolated
actein on proliferation of MCF-7 breast cancer cells
(F.Gaube, 2007).

Apoptosis, the essential regulatory mechanism of
programmed cell death, is responsible for the observed
inhibition of the proliferation of breast cancer cells by
isopropanolic extract (iCR). Apoptosis induced by iCR
involved cleavage of cytokeratin 18 and caspase
activation. However, in MCF-7 cells, black cohosh extract
also induced cell cycle arrest at G1 and G2/M, which
proposes as an additional pathway. Cinnamic acid esters
were the more potent inhibitor of proliferation and
apoptosis inducer in MCF-7 cells (K. Hostanska, 2007).
Black cohosh has an anti-proliferative effect, therefore
genes involved in proliferation control are significantly
over-represented. Transcripts related to cell cycle
regulation and DNA replication are regulated in a manner
supporting cell cycle arrest. Genes, whose products are
involved in the transition from G1 to S-phase, appear to
be down regulated, such as cyclins (CCNA2, CCNE2,
CCNF), cyclin-dependent kinase 2 (cdk2) and
transcription regulators (E2F2, PCNA, SKP2), whereas
transcription of inhibitory genes cyclin G2 (CCNG2),
GADD45A (growt arrest and DNA-damage-inducible,
alpha) and P21cip1 was increased. Elevated levels of
CCNG2, CCNB1TP1, FOXO3A, GADD45A and P21cip1
genes, as well as down-regulation of cyclin A2 (CCNA2)

and cdk2 provided evidence that cell cycle progression
might be additionally arrested at the G2/M-checkpoint.
The level of various DNA replication related genes (CDC6,
CDT1, FEN1, MCM2, MCM3, MCM4, pfs2, RFC3) was also
reduced, thereby suggesting a reduction in the replication
rate. In addition to regulation of genes involved in
proliferation control, there is also regulation of
apoptosis-linked genes in a pro-apoptotic manner. An
increase in apoptosis events also contributes to decrease
in cellular proliferation. In cells treated with black cohosh,
the transcript of apoptosis inhibitor surviving was down-
regulated, whereas genes coding for apoptosis-inducing
and supporting products were increased. FOXO3A,
GADD45A, GDF15 and P21cip1, whose mRNA levels
increased, are also connected with apoptosis in addition
to their role in cell cycle control. Transcript of tyrosyl-
tRNA-synthetase (YARS), whose secretion is liked to
apoptotic events, was up-regulated. Down-regulation of
lymphoid-specific helicase (HELLS), is associated with
apoptosis. The up-regulation of JNK1 and DDIT3 is
related to stress induced apoptosis. P8, IER3 and DDIT4,
whose transcript is strongly up-regulated, are expressed
under cellular stress, and are associated with both pro
and anti-apoptotic events. Over-expression of transcripts
involved with cellular stress is statistically significant after
treatment with black cohosh. 40 transcripts associated
with metabolic stress response, such as hypoxia,

unfolded protein response in the endoplasmic reticulum
or starvation for amino acids or glucose, have been
identified. Transcript of HIF1-alpha/ARNT (HIF1) binds to
hypoxia-responsive elements, thereby regulating the
expression of hypoxia-response genes. VEGF, HMOX1,
BHLHB2, P21cip1 and DDIT4, whose transcript was also
up-regulated, are known to be direct target genes. A
hypoxia response pathwayvia mTOR including
inactivation of EIF4EBP1, and finally resulting in increased
mRNA translation is known to be inhibited by DDIT4. This
could explain the increase of EIF4EBP1 mRNA observed.
The increase of CCAAT, EPAS1, EGR1 and SESN2 mRNA
are also related to hypoxia. There is also regulation of
genes related to endoplasmic reticulum stress response
(UPR), which involves activation of 3 different pathways:
transcription of JNK1 was regulated, which is a target of
one UPR-pathway and its activation may lead to
apoptosis. Phosphorylation of eIF2-alpha is involved not
only in UPR, but also in response to hypoxia and other
cellular stresses. PERK, whose mRof the mRNA-level was
increased by black cohosh, is a kinase linking hypoxia
stress response and UPR to eIF2-alpha-phosphorylation,
whereas amino acid and glucose starvation response acts
via GCN2 kinase. As a result of eIF2-alpha-
phosphorylation, the translation of most mRNAs is
inhibited, but the translation of ATF4 is increased. There
is an up-regulation of the ATF4 gene, as well as various

ATF4-induced downstream target genes, like ASNS, ATF3,
CHOP, GADD45A, HERPUD1 and HSPA5. Gene products of
these transcripts are involved in cell survival and tumour-
genesis, as well as apoptotic events. Some processes of
protein turnover are affected by black cohosh extract. The
expression levels of various ubiquitin cycle-related genes
were influenced by black cohosh. Some of these
transcripts code for products involved with cell cycle
progression, and are regulated in a cell cycle arresting
manner. Inhibitory CCNB1IP1 is up regulated, while SKP2
and UHRF1 are down regulated (F. Gaube, 2007).

Table 4: Functional categories of genes regulated in MCF-
7 cells after 24 h incubation with black cohosh extract.
Genes were grouped in 5 large groups (Apoptosis,
Proliferation, General Growth, Signaling & Transport,
Metabolism), some consisting of subgroups. Genes that
are not clearly associated with these groups are
summarized in the categoryothers. The category stress
response contains genes also grouped into one of the 6
main classes. Each bar represents the number of genes
that were up- (dark) or downregulated (white) in the
respective group (K. Hostanska, 2007).

Motility is another propertyof malignant cells needed for
them to migrate from the primary site to a secondary
organ. Any alteration of this property would interrupt the
metastatic cascade. Also, iCR treated cells had a
moderately reduced motility. Low doses of triterpene
glycosides and cinnamic acid esters didn’t affect cell
migration. The mechanisms by which iCR (triterpene
glycosides and cinnamic acid esters) inhibit cell invasion
is not clear and needs further investigation. Cyclo-
oxygenase (cox)-2 enzyme plays a role in the metastatic
process of cancer. Hence, the inhibition mechanism of iCR
could be cox-2 related (K. Hostanska, 2007).
Triterpene glycosides and breast cancer:
Figure 1: Structure of actein (L.S. Einbond, 2008).

The growth inhibition activity of black cohosh extracts
appears to be related, in part, to their triterpene glycoside
composition (L.S. Einbond, 2008). Triterpene glycosides
are well known as a group of secondary metabolites,
typically found in plants. They possess a wide spectrum
of biological activities, including cytotoxic, haemolytic,
anti-fungal and antibacterial properties. This results from
their ability to form a complex with unsaturated sterols of
cellular membranes. The physiological effects of
glycosides are probably related to the certain structural
conformity between the glycosides and steroidal
hormones like glucocorticoids and their formation of a
complex with steroidal receptors (S.N. Kovalchuk, 2006).
The most potent cimicifuga component tested is actein,
which has an acetyl group at position c-25. Thus, the
acetyl group at this position enhances growth inhibitory
activity. MCF-7 cells transfected for Her2 are more
sensitive than the parental MCF-7 cells to growth
inhibitory effects of actein, indicating that that Her2 plays
a role in the action of actein. Treatment with actein alters
the distribution of actin filaments and induces apoptosis
in these cells. Treatment with actein induces a stress
response in human breast cancer cells, and the growth
inhibitory effect of black cohosh on these cells is thought
to be mainly due to the triterpene glycoside fraction,

rather than the isoferulic (cinnamic acid ester) content.
Since the colonies were smaller on average in the actein-
treated cells, actein appears to decrease the rate of cell
proliferation. Therefore, actein appears to have cytostatic
as well as cytotoxic activity. MDA-MB-453 cells, which are
ER- and Her2+, were the most sensitive of the cells to
actein. Treatment of MCF-7 or MDA-MB-453 cells with
actein altered their cell structure, since the actin filaments
around the cell nuclei and the nuclei itself appeared
doughnut-shaped. Aggregation of actin around the
nucleus occurs in response to cell stress (L.S. Einbond,
2008). Actein induces cell cycle arrest at G1. Cyclin D1
plays a critical role in mediating the transition from G1 to
S and is over-expressed in 50-60% of human breast
carcinomas, and also over-expressed in several human
breast cancer cell lines. Treatment of MCF-7 cells with 40
microgram/ml of actein for 3 or 10 hours resulted in a
partial decrease, and treatment for 24 hours caused a
marked decrease in cellular levels of cyclin D1, when
compared to untreated cells. After treatment with 40
microgram/ml for 24 hours, there was a complete loss of
the protein. Normal mammary epithelial cells do not
express cyclin D1. Cyclin D1 binds to and activates the
cyclin-dependent kinases cdk4 and cdk6. The resulting
complexes phosphorylate and inactivate pRb, thereby
preventing pRb from inhibiting the transcription factor
E2F, thus allowing the cells to progress from G1 to S. The

ability of actein to arrest cells in G1 may be due to the
decreased expression of cyclin D1 and cdk4 and the
increased expression of P21cip1, which results in a
decrease in the level of the hyper phosphorylated form of
pRb. The level of epidermal growth factor (EGFR), which is
over-expressed in various cancers, was not significantly
affected by actein treatment. Nor was there a consistent
effect of actein on the phosphorylated and activated form
of the EGFR (p-EGFR), but there was a significant decrease
with the 40 micrograms/ml dose at 24 hours. The EtoAc
fraction exhibited the greatest growth inhibitory activity.
This fraction inhibited growth of both the ER+ MCF-7 and
ER-/Her2+ MDA-MB-453 human breast cancer cell lines,
with IC50 values of 18 micrograms/ml and 10
micrograms/ml, respectively. Actein turned out to be the
most potent compound. It decreased the level of cyclin
D1 mRNA within 3 hours of treatment, and significantly
reduced the level at 24 hour, suggesting an effect at the
level of transcription. The level of the EGFR was not
altered after treatment with actein, nor was there a
consistent effect on the level of the phosphorylated form
of EGFR (p-EGFR), which reflected its state of activation.
Thus, the EGFR does not appear to be a direct target for
actein (L.S. Einbond, 2004). There are several potential
mediators of the growth inhibitory effect of actein,
including increased expression of ATF3 and DDIT3, and
decreased expression of the cell cycle related genes cyclin

E2 and cell division cycle 25A. The increased expression
of the transcription factor ATF3 may be of particular
interest, since it can repress the transcription of several
survival genes. The number of genes affected increases
with the dose of actein and the duration of exposure.
Actein alters the expression of 5 genes: DDIT4, WIPI49,
HERPUD1, SLC7A11 and LETMD1, which are involved in
cellular responses to diverse stresses, including DNA
damage. The effect of actein on expression of genes
related to the integrated stress response are not limited
to the MDA-MB-453 cell line, since treatment of the ER+
MCF-7 cell line with actein also induced increased
expression of genes involved in calcium metabolism and
the Na-K-ATPase effects calcium metabolism. Hence, the
ability of actein to inhibit the activity of the Na-K-ATPase
and activate related down-stream pathways is possible.
Na-K-ATPase mediates many stress responses and
proliferation pathways that are affected by actein.
Therefore, actein subsequently phosphorylates
downstream proteins. Inhibition of the enzyme has been
shown to be related to a compound’s ability to interact
with the enzyme’s lipid-rich environment. Actein may act
through interaction with the Na-K-ATPase promoters in
the cell membrane and induction of clustering of ATPase
with neighbouring proteins in micro-domains. GRP78,
which is expressed on the cell surface and is involved in
endocytosis of Na-K-ATPase in cells, is also activated by

actein and may therefore be instrumental in actein-
mediated ATPase inhibition (L.S. Einbond, 2008).
Other triterpene glycosides potentially affecting
proliferation of breast cancer cells are 23-epi-
deoxyactein, cimifungoside and cimiracemoside A, that
inhibit MCF-7 cell proliferation, and cimiracemoside G
which is cytotoxic to human oral squamous cell
carcinoma, but not to normal human fibroblasts (R.L.
Ruhlen, 2008). Also, cimiracemoside A and B both reduce
in dose-dependent manner the single-stranded DNA
breaks induced by menandione in breast cancer cells (J.E.
Burdette, 2002).
Cinnamic acid esters and breast cancer:
Figure 2: Basic structure of a cinnamic acid ester (L.S.
Einbond, 2008).
Some cinnamic acid derivatives are also potential
inhibitors of EGFR and Her2. The IC50 value for inhibition

of Her2 kinase is higher than that observed for EGFR
kinase. However, they have the same trends of inhibitory
effect. It is evident that there is also a reasonable
correlation between the EGFR and Her2 inhibitory
activities. Thus, this isn’t surprising considering the high
sequence homology of the catalytic domains of these 2
kinases (Y. Qian, 2010). A study of the cytotoxic activity
of cinnamic acid derivatives also showed that they have
an anti-proliferative activity without toxicity towards non-
cancer cells, in general (M. Cardenas, 2006). Methyl
caffeate, caffeic acid, ferulic acid, fukinolic acid,
cimicifugic acid A and cimiracemate B are all cinnamic
acid derivatives that contain a phenol group para to the
conjugated ethylene side chain. It is thought that the
hydrogen on the phenol can be abstracted easily by a
radical, resulting in formation of phenoxyradical. The
unpaired electron can delocalize across the entire
molecule, hence stability. Among the compounds found
to have antioxidant activity, those with the greatest
number of phenolic groups tended to be the most
effective free radical scavengers. The presence of a
second phenolic group allows formation of a catechol,
which is important for antioxidant behaviour. Methyl
caffeate has the highest potencyin protecting DNA
against single-strand cleavage with breast cancer cells.
The conversion of the acid group to the methoxy group
decreases the polarity and might facilitate the transport

of this compound across the cell membrane, where it can
scavenge free radicals. Ferulic acid and caffeic acid also
have a dose-dependent reduction in DNA damage. The
caffeic and ferulic acids protect DNA by reducing reactive
oxygen species (ROS) (J.E. Burdette, 2002).
Phytoestrogens and breast cancer:
Black cohosh also contains phytoestrogens (flavonoids)
that were also shown to inhibit growth of breast cancer
cells through breast cancer resistance protein (BCRP) in
some types of cancer cells. They can also inhibit both
aromatase and 17-beta-HSD1, although their IC50s are
generally low (S. Rice, 2007). 17-beta-HSD inhibitors
consisting of a non-steroid core and devoid of a residual
steroidogenic activity, like phytoestrogens, have potential
for inhibiting cancer proliferation. The biosynthesis of
phytoestrogens proceeds via cinnamic acid or related
phenolic acids, like caffeic or ferulic acid that are also
present in black cohosh. Many flavonoids have been
shown to be potent inhibitors of type 1, 3 and 5 17-beta-
HSD. Flavonoids can inhibit both the oxidative and
reductive reactions catalysed by 17-beta-HSD at low
micro-molar concentrations (K. Kristan, 2006). Flavonoids
with a hydroxyl group in position 7 of the A-ring, which
mimics the D-ring of steroids, inhibit 17-beta-HSD1.
Unfortunately, they are not useful as therapeutic

inhibitors, as they are often estrogenic, or aren’t specific
for 17-beta-HSD1, having inhibitory effects on other
steroidal enzymes and receptors (J.M. Day, 2003). Some
flavonoids also produce moderately active compounds in
MCF-7 cell lines, but only ones containing a chlorine or
bromine atom (M. Cardenas, 2006).
Black cohosh and hepatotoxicity:
Recently, the European medicines agency has proposed a
causal relationship for acute liver disease, observed under
treatment with black cohosh (R. Teschke, 2008). To
identify herbal remedy as being responsible for
hepatotoxicity, there should be a clear demonstration of a
temporal relationship between consumption of the
product and development of the illness (D. Joy, 2008).
According to one research, this was not the case for black
cohosh in general (R. Teschke, 2008). Another research
by F. Firenzuoli and co also claims that black cohosh has
a very good safety profile, as confirmed in a review of
more than 3800 women. However, according to that same
research, there were also 42 cases of suspected
hepatotoxic reactions in patients consuming the herb,
which gives it a potential connection with hepatotoxicity.
Also, according to a research by D. Joy and co, there have
been several case reports of hepatotoxic effects of
varying severity associated with black cohosh. 2 reviews

have found it to be well tolerated when taken up to 6
months. According to E.T. Enbom and co, the safety of
black cohosh has been evaluated during several clinical
trials, resulting in the conclusion the adverse effects
secondary to black cohosh consumption are relatively
common, including liver damage. The extent of
hepatotoxic effects of black cohosh extract varies and
depends on gender, age, duration of treatment and
underlying liver pathology (E.T. Enbom, 2014).
Also, genetic polymorphisms have a strong influence on
drug metabolism and may increase the risk of
hepatotoxicity (F. Firenzuoli, 2007). It is also important to
remember that since the liver is central to metabolic
disposition of all drugs, drug-induced hepatic injury is a
potential complication of nearly every medication.
However, the mechanisms of liver injury by black cohosh
remain unclear.
Black cohosh contains a mixture of alkaloids, tannins and
terpenoids, and diterpenoids that have been shown in
animal models to result in liver injury, either by reactive
metabolites, or by an autoimmune mechanism (P.W.
Whiting, 2002). The mechanism of hepatotoxicity due to
black cohosh is probably idiosyncratic, and has identical
presentation to troxis necrosis that occurs during
autoimmune hepatitis (F. Firenzuoli, 2007). Another study
demonstrated formation of immunologic synapses
between hepatocytes and lymphocytes, and by

accumulation of lipid peroxidation products. Clinical data
showed that discontinuation of black cohosh ingestion
has stopped this process and led to recovery (E.T. Enbom,
2014). Also, it has been hypothesizes that the
hepatotoxic effect could be due to inhibition of CYP3A4, a
cytochrome p450 enzyme responsible for metabolism of
several drugs in the liver. This could have particular
implications regarding drug efficacyand toxicity. Serious
adverse interactions may result from ingestion of drugs
that are CYP3A4 substrates (D.Joy, 2008).
However, natural and synthetic estrogens can alter liver
physiology, and it has been reported that black cohosh
extracts and isolated compounds do not possess
estrogenic activity. But black cohosh also contains several
catechols, like caffeic acid, fukinolic acid and cimicifugic
acid A and B. That could be of significant concern in
toxicology, because they could be activated metabolically
or chemically, to electrophilic quinones. This could lead
to formation of reactive oxygen species. However, black
cohosh did not show any effect on lipids, glucose, insulin
and fibrinogen (F. Firenzuoli, 2007).
Factors that could contribute to toxicity also include
mistaken identity of the plant, wrong part of the plant
being used, incorrect storage, contamination during
preparation, pesticides and heavy metals, and
inconsistency in nomenclature and labelling of the final
product (D. Joy, 2008).

Discussion:
First of all, it is important to mention that there wasn’t
enough consistent data regarding the IC50 values of each
component of black cohosh, or the whole herb extract, to
draw statistical analysis through 2- way ANOVA or X^2
test from a few sets of information collected from
different researches, neither was there enough data
regarding, for instance, the number of genes expressed
while treatment with different concentrations of black
cohosh components, or quantitative sets of data
regarding the same type of cell line (for each line). So, for
this reason, ANOVA or X^2 could not be performed as
desired, since the sets of data obtained from all the
articles analysed were too few and different from one
another.
From the aspect of the function of black cohosh extract in
general, this herb seems to have anti-carcinogenic
properties, since both ethanolic and isopropanolic
extracts of black cohosh rhizomes inhibit growth of both
estrogen-dependent MCF-7 and various estrogen-
independent MDA-MB human breast cancer cell lines.
There is also evidence that MDA-MB-231 showed a
higher sensitivity to the cytotoxic effects of black cohosh
than MCF-7 cells. However, there were no differences in

sensitivity of both cell types to either triterpene
glycosides or cinnamic acid esters, according to IC50
values.
It also seems most likely that the activity of black cohosh
on breast cancer cells is largely non-estrogenic, except
for a weak binding of flavonoid phytoestrogens to
estrogen receptors. This assumption is supported by the
fact that black cohosh showed no estrogenic activity in
MCF-7 cells, or on gene expression in those cells, but
rather antagonising activity. Also, in both MCF-7 and
MDA cells, black cohosh had no significant effect on the
conversion of androstenedione to estradiol at any dose,
and only the highest doses inhibited the conversion of
estrone to estradiol.
It is far more likely that black cohosh, especially its actein
part of the triterpene glycoside fraction, causes cytotoxic
effects on breast cancer cells through mediated apoptosis
induced through the Her2 receptor, and also the Na-K-
ATPase membrane-bound enzyme. Activity through
binding to Her2 is likely, since the Her2+ MDA-MB cell
lines were far more sensitive to the cytotoxic effect than
their parental MCF-7 cells to growth inhibitory effects of
actein, indicating that Her2 plays a role in the action of
actein.
Actein seems to act through activation of Na-K-ATPase,
since actein alters genes involved in calcium metabolism

and the Na-K-ATPase affects calcium metabolism.
Therefore, the ability of actein to inhibit Na-K-ATPase
activity and activate related down-stream pathways is
possible. This makes sense, also because actein alters the
distribution of actin filaments and induces apoptosis
through that mechanism, and calcium is a known factor in
the function of actin as a cell-skeleton compound.
Inhibition of Na-K-ATPase has been also shown to be
related to a compound’s ability to interact with the
enzyme’s lipid-rich environment, and actein, as a
tritepene glycoside, does possess some steroid-like lipid
conformation. Actein’s inhibition of ATPase is relatively
weak, so a promoter-dependent mode of action is
suggested. Actein may act through interaction with the
Na-K-ATPase promoters in the cell membrane and
induction of ATPase clustering with neighbouring proteins
in micro-domains. Also, GRP78, which is expressed on
the cell surface and is involved in endocytosis of ATPase
in cells, is also activated by actein and may therefore be
instrumental in actein-mediated ATPase inhibition.
However, the level of epidermal growth factor (EGFR, or
Her1), which is over-expressed in various cancers, was
not significantly affected by actein treatment. Nor was
there a consistent effect of actein on the phosphorylated
and activated form of EGFR (p-EGFR), except for a
significant decrease with the 40 micrograms/ml dose for

24 hours. Therefore, the EGFR does not appear to be a
direct target for actein.
It appears that the main cytotoxic mechanism through
which the breast cancer cells’ proliferation decrease is
apoptosis-related, via activation of caspases. Fractions of
black cohosh enriched in triterpene glycosides or
cinnamic acid esters inhibited cell growth and induced
apoptosis. An ethanolic extract inhibite the activity of the
cyclin promoter and increased the activity of the P21cip1
promoter in the ER- human breast cancer cells. Ethyl
acetate fraction of black cohosh inhibited growth of MCF-
7 cells and induced cell cycle arrest at G1 after treatment
with 30 micrograms/ml, and G2/M after treatment with
60 micrograms/ml. This suggests that the fraction
contains a mixture of components, with the more
active/abundant component causing G1 arrest, and the
less active causing G2/M arrest. Therefore, I tis possible
that at high concentrations the fraction affects proteins
that regulate later cell cycle phases. I tis possible,
according to this data that the triterpene glycoside
fraction works as a limiting factor, hence over a certain
concentration there is no added advantage of the
concentration increase, but under a certain
concentration- the other fraction, cinnamic acid esters,
becomes more influential and potent (especially if the
receptors for triterpene glycosides are limited in number).
Therefore, cinnamic acid esters also seemed to be more

potent in MCF-7 cell cytotoxicity, which could also
indicate an additional pathway for cinnamic acid esters
function.
As expected, genes involved in proliferation control are
significantly over-represented in cells treated with black
cohosh. Transcripts related to cell cycle regulation and
DNA replication are regulated in a manner supporting cell
cycle arrest: genes whose products are involved in
transition from G1 to S-phase are down-regulated
(cyclins, cdk2, transcription regulators), while
transcription of inhibitory genes (cyclin G2, GADD45A and
P21cpi1) are increased. The level of DNA replication
genes was also reduced, hence the probability of
regulation of pro-apoptotic genes (like surviving or tRNA
synthetase).
Unlike triterpene glycosides, some cinnamic acid esters
do seem to have inhibitory effect on EGFR, in addition to
Her2, and they have a high and selective anti-proliferative
activity against cancer cells, due to this fact. This is
probably the alternative anti-cancer pathway
demonstrated against MCF-7 cell proliferation. Cinnamic
acid esters have anti-oxidative properties against
formation of carcinoma also by conversion of the acid
group to methoxygroup. This protects DNA against
single-strand cleavage by decreasing the polarity and
might initiate the transport of the compound across the
cell membrane, where it can scavenge free radicals.

Methyl caffeate was found to have the highest potency via
this pathway, although also ferulic acid and caffeic acid
have a dose-dependent reduction in DNA damage, by
protecting DNA through reduction of reactive oxygen
species.
According to a few researches, synergistic action between
the different compounds of black cohosh is likely. This
makes sense, since the herb has been shown to inhibit
breast cancer cell proliferation through more than one
pathway. It is probably likely that by binding to the Na-K-
ATPase enzyme, actein makes the cell membrane more
penetrable through alteration of its structure (due to its
steroid-like sterol composition), which allows more
access for the cinnamic acid esters. After entering the
cell, the cinnamic acid esters can perform their activity
against reactive oxygen species, and prevent DNA
damage by free radicals. Entering the cell also enables the
cinnamic acid esters to inhibit EGFRs, an attribute that
actein and other triterpene glycosides lack. While
triterpene glycosides are lipophilic, cinnamic acid esters
seem to be hydrophilic, so are therefore more dependent
on triterpene glycosides as agents for cell penetration. By
binding to ATPase and Her2, actein also has anti-cancer
activities in its own right, mainly of apoptosis induction
and also cytostatic activity, but also increases the
effectiveness of cinnamic acid esters and their anti-
oxidative activities.

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