This document discusses research into the effects of black cohosh (Cimicifuga racemosa) on breast cancer cells. It finds that extracts of black cohosh inhibit the growth of both estrogen-dependent and estrogen-independent breast cancer cell lines. The active compounds in black cohosh, such as actein and cinnamic acid esters, induce apoptosis in breast cancer cells through multiple pathways including interaction with Na-K-ATPase, HER2 receptors, and reduction of reactive oxygen species. Synergy between compounds contributes to the anti-cancer activity of black cohosh.

1. Black cohosh (Cimicifuga
racemosa): A potential remedy for
breast cancer
Black cohosh-A gift from Native Americans
Black cohosh(Cinicifuga racemosa) is a herbaceous perennial plant. Native to North
America, the root and rhizomes of black cohoshwere used by Native American women for
menstrual cramps, difficult childbirth and complicated menopause, as well as other
conditions likedysmeorrhea, colic and rheumatism. Native Americans subsequently
introduced the herb to the American colonists, who used it for women’s complaints, as well
as illness like bronchiitis, nervous disorders and inflammation. Today, black cohoshis
widely used in various pharmaceutical preparations. It is widely used in the U.S. and Europe
against menopausal symptoms, suchas hot flashes, sweats, irritability and vaginal dryness.
The action of black cohoshis attributed to synergy of its active components. The main active
compounds ofthe herb’s root are believed to be the triterpene glycosides (TTGs), including
actein and cimifungoside. Other possible biologically active compounds include the
isoflavone formononetin, the TTGs 27-deoxyactein and racemoside, as well as the aromatic
(CAEs), sugars and long-chain fatty acids.
One aim of this research was to examine the effects of the various black cohoshcompounds
on the growth of human breast cancer cells, including inhibition effect, cell cycle progression
and expression proteins involved in cell cycle control. Another aim of the research was to
identify new approaches to breast cancer prevention and therapy, leading to following
studies concerning the use of herbology in cancer treatment.
Cimicifuga racemosa structure of the triterpene glycoside actein:

2. Basic structure of cinnamic acid esters:
Methodology:
This research was library-based, and took place at the library of the University of East
London (on Stratford campus). It included the collection and analysis of data from 29
different scientific articles concerning the anti-breast-cancer activity and properties of black
cohosh(Cimicifuga racemosa) and its different compounds. Thearticles for the research
were obtained through PubMed and Google Scholar.
Results:
Ethanolic and isopropanolic extracts of black cohoshrhizomes inhibited the growth of both
estrogen-dependent MCF-7 and estrogen-independent MDA-MB human breast cancer cells.
Black cohoshalso showed no estrogenic activity in MCF-7, but antagonised these activities.
In addition, estrogen-receptor-negative (ER-) were significantly more sensitive than ER+
cells. There was dose-dependentanti-proliferative action of black cohoshin breast cancer
cells, probably evoked by genomic (ER and non-ER mediated) mechanisms, due to the
various properties of different components of the herb. Also, it was found that black cohosh
doesn’tbind to ER-alpha or beta. However, there were no differences in sensitivity of MCF-
7 and MDA-MB cell lines to either TTGs or CAEs, according to IC50 values. In both cell
types, the herb 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.
Black cohoshwas also found to initiate cytotoxicity through induction of apoptosis. An
ethanolic extract of the plant was found to inhibit the activity of cyclin D1 promoter and
increase the activity of P21cip1 promoter in ER- human breast cancer cells. Ethyl acetate
fraction of black cohoshinhibited growth of MCF-7 (ER+, Her2 low) cells and induced cell
cycle arrest at G1 after treatment with 30 micrograms/mL, and at 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, it is possible that at high concentrations the fraction affects proteins
regulating later phases in the cell cycle. CAEs turned out to be the more potent inhibitors of
proliferation and apoptosis inducers in MCF-7 cells.
Black cohoshhas as anti-proliferative effect, therefore genes involved in proliferation control
are significantly over-expressed. 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, appears to be down-regulated, such as cyclins
(CCNA2, CCNE2, CCNF), cdk2 and transcription regulators (E2F2, PCNA, SKP2), whereas

3. transcription of inhibitory genes cyclin G2 (CCNG2), GADD45A (growth arrest and DNA-
damage-inducible) and P21cip1 was increased.
Since actein was found to alter the expression of genes involved in calcium metabolism and
the Na-K-ATPase affects calcium metabolism, the ability of actein to inhibit the activity of
the ATPase and activate related down-stream pathways is possible. Na-K-ATPase mediates
many stress responses and proliferation pathways that are affected by actein. Actein may act
through interaction with the Na-K-ATPase promoters in the cell membrane and induction of
clustering of ATPasewith neighbouring proteins in micro-domains.
Some CAEs are also potential inhibitors of EGFR and Her2 proteins, which have a proven
connection with breast cancer. It had also been shown that CAEs have selective anti-
proliferative activity towards cancer cells. Among the compounds isolated from black
cohoshwith anti-oxidant behaviour, methyl caffeate has the highest potency in protecting
DNA against single-strand cleavage. Also, both caffeic and ferulic acids protectDNA by
reducing reactive oxygen species.
Functional categoriesof genesregulatedinMCF-7cellsafter24 h incubationwithblackcohoshextract.Geneswere
groupedin5 large groups(Apoptosis,Proliferation,General Growth,Signaling& Transport,Metabolism),some
consistingof subgroups.Genesthatare notclearlyassociatedwiththese groupsare summarizedinthe category
others.The categorystressresponse containsgenesalsogroupedintoone of the 6 mainclasses.Each bar represents
the numberof genesthat were up- (dark) ordownregulated(white) inthe respective group.

4. . Effectof iCR, TTG and CAE on tumor cell growth.MCF-7 and MDA-MB 231 cellswere treatedfor24 h withor
withoutsubstancesatthe indicatedconcentrations.Attachedcellswerestainedwithcrystal violetandthe
absorbance of the cell lysate wasmeasuredat540 nm.Data are expressedasmean±SDof triplicate wellsfromtwo
independentexperiments.*p<0.05 vs.untreatedcontrol.

5. Discussion:
From the aspectof general function of black cohosh, the herb seems to have anti-
cancer properties, since both ethanolic and isopropanolic extracts of black cohosh
rhizomes inhibit growth of both estrogen-dependent MCF-7 and estrogen-
independent MDA-MB cell lines. In addition, MDA-MB-231 cells showed a
higher sensitivity to the cytotoxic effects of black cohoshthan MCF-7 cells.
It also seems, for this reason, that the activity of black cohoshon breast cancer
cells is mainly non- estrogenic. This is also supported by the fact that there was no
significant effect on the conversion of estrone to estradiol.
It is far more likely that black cohosh, especially its actein fraction, causes
cytotoxic effects on breast cancer cells through apoptosis induction through the
Her2 receptor, as well as the Na-K-ATPase enzyme. Action via binding to Her2 is
likely, since the Her2+ MDA-MB cell lines are more sensitive to the cytotoxic
effect than MCF-7 (Her2 low) cells. Also, MCF-7 cells transfected for Her2 are
more sensitive than normal MCF-7 cells to actein.
Actein also seems to act via activation of Na-K-ATPase, due to actein’s effect on
calcium metabolism related genes, which are also related to ATPase activation.
This makes sense also due to actein’s role in alteration of actin filaments’
distribution, through which apoptosis is induces. Inhibition of ATPase has also
been shown to be related to a compound’s lipid-affinity, and actein has steroid-like
sterol composition, that makes it lipophilic enough for this role.
Unlike TTGs, CAEs do seem to inhibit EGFR, in addition to Her2. It most likely
acts through reduction of DNA damage by protection from reactive oxygen
species.
Synergy between different black cohoshcompounds is likely, since it has been
shown to inhibit breast cancer through several pathways. Probably the binding of
actein to ATPase makes the cell membrane more penetrable through structure
alteration, which allows CAE access. Then, the CAEs can act against the reactive
oxygen species, and also inhibit EGFRs. By binding to ATPase and Her2, actein
also has anti-cancer activity in its own right, but also increases the effectiveness of
CAEs.
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