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Direct influence of rooibos-derived compound on rabbit ovarian functions

and their response to gonadotropins

Article  in  Biologia - Section Cellular and Molecular Biology · October 2015

DOI: 10.1515/biolog-2015-0163

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 Biologia 70/10: 1424—1428, 2015
Section Cellular and Molecular Biology
DOI: 10.1515/biolog-2015-0163

Direct influence of rooibos-derived compound on rabbit ovarian

functions and their response to gonadotropins

Aneta Štochmaľová1*, Attila Kádasi2, Radoslava Vlčková3, Drahomíra Sopková3,

Jozef Nitray4, Soňa Nitrayová5 & Alexander V. Sirotkin1,5*
1
Department of Zoology and Anthropology, Constantine the Philosopher University, Tr. A. Hlinku 1, SK-94974 Nitra,
Slovakia; e-mail: aneta.stochmalova@ukf.sk; asirotkin@ukf.sk
2
Faculty of Biotechnology and Food Science, Slovak University of Agriculture in Nitra, Tr. A. Hlinku 2, SK-94976 Nitra,
Slovakia
3
Institute of Physiology, University of Veterinary Medicine and Pharmacy, Komenského 73, SK-04181 Košice, Slovakia
4
Tekmar Slovensko Co., Ltd., Vinárska 26, SK-95141, Lužianky, Slovakia
5
Research Institute for Animal Production, NAFC Nitra, Hlohovecká 2, SK-95141 Lužianky, Slovakia

p y
Abstract: Rooibos is widely used in folk medicine, but its influence on reproductive processes has been studied insufficiently.
The aim of present study was to examine possible involvement of rooibos on the release of progesterone (P4), testosterone
co
(T) and insulin-like growth factor I (IGF-I), as well as its response to gonadotropin – follicle-stimulating and luteinizing
hormone (FSH+LH), the most known hormonal regulators for reproduction. Rabbit ovarian fragments were cultured in the
presence of rooibos (1, 10 and 100 µg/mL medium), FSH+LH (0.01, 0.1 and 1 IU/mL medium) and rooibos (10 µg/mL
medium) in combination with FSH+LH (0.01, 0.1 and 1 IU/mL medium). The release of hormones was evaluated by using
radioimmunoassay. It was observed that rooibos addition to culture medium stimulated P4 and inhibited T release. IGF-I
release was significantly stimulated by rooibos at all doses added. FSH+LH when given alone promoted IGF-I, but not P4
's
or T release. The addition of rooibos induced the suppressive effect of FSH+LH on P4 release, the stimulatory action of
FSH+LH on T output and reduced the response of IGF-I to FSH+LH treatment. These data suggest that rooibos is able
or

to directly affect the release of steroid (P4 and T) and peptide (IGF-I) hormones by rabbit ovarian cells and to modulate
their response to gonadotropins.
Key words: rooibos; gonadotropin; rabbit ovarian fragments; hormone release.
th

Abbreviations: FSH, follicle-stimulating hormone; IGF-I, insulin-like growth factor I; LH, luteinizing hormone; P4, pro-
gesterone; RIA, radioimmunoassay; ROS, reactive oxygen species; T, testosterone.
Au

Introduction This is accompanied by cell cycle arrest and induction

Rooibos (Aspalathus linearis) is used to prevent and to
treat several kinds of diseases in folk medicine (Inanami
et al. 1995; Na et al. 2004; Gilani et al. 2006). It is
a shrub-like leguminous bush native to the Cedarberg
Mountains in the Western Cape region of South Africa
(McKay & Blumberg 2007). Recently, rooibos biological
effects have attracted more scientific and public atten-
tion due to its potential in several diseases. Rooibos is
commonly used for treating cardiac arrhythmias, colic,
diarrhea (Duke et al. 2002), asthma (McKay & Blum-
berg 2007) and hypertension (Marnewick et al. 2011). It
contains abundant flavonoids (Shimoi et al. 1996) with
a potent antioxidative activity (Yoshikawa et al. 1990),
whilst rooibos polyphenols, such as aspalathin, nothofa-
gin, quercetin, rutin, isoquercitrin, orientin, luteolin,
vitexin and chrysoeriol (Bramati et al. 2002) exhibit
cytotoxicity towards various human tumour cell lines.
of apoptosis (Roy & Nicholson 2000). The antioxidant
properties of rooibos have been confirmed both in vitro
and in vivo (Joubert et al. 2004; Marnewick et al. 2005;
Nikolova et al. 2007).
Reactive oxygen species (ROS) are a double-edged
sword – they serve as key signal molecules in physio-
logical processes, but they also have a role in patholog-
ical processes involving the female reproductive tract.
ROS affect multiple physiological processes from oocyte
maturation to fertilization, embryo development and
pregnancy (Agarwal et al. 2005). Rooibos showed a
tendency to decrease the levels of ROS and lipid per-
oxidation (Awonyi et al. 2012). Rooibos antioxidants
and phytoestrogens may affect female reproduction
(Opuwari 2013). Treatment with rooibos positively af-
fected egg production and prolonged the productive pe-
riod of aged Japanese quail (Coturnix coturnix japon-
ica) (Juráni et al. 2008). Awonyi et al. (2012) demon-

* Corresponding authors

c 2015 Institute of Molecular Biology, Slovak Academy of Sciences
Influence of rooibos-derived compound on rabbit ovarian functions
strated that rooibos improved rat sperm quality, pro-
tected sperm against oxidative damage, increased an-
tioxidant enzyme activities and increased the levels of
glutathione in oxidative stress-induced rats. These ob-
servations suggest that fermented rooibos could be an
excellent adjuvant support in the therapy of infertil-
ity and can generally be used as a supportive therapy
in cases where oxidative stress is involved. It has been
shown that phytoestrogens can act on the hypothala-
mus and pituitary gland to inhibit gonadotropin se-
cretion and may disrupt normal reproductive function
(McGarvey et al. 2013).
Thus, it is possible that rooibos can also indirectly
influence ovarian function affecting release of ovarian
hormones. The most known regulators of reproduction
are follicle-stimulating hormone (FSH) and luteinizing
hormone (LH) exerting their effect on steroidogenesis
as well as growth and proliferation of ovarian granulosa
cells through divergent intracellular signalling pathways
(Gonzalez-Robayna et al. 2000; Sirotkin et al. 2003;
Kayampili & Menon 2004; Wang & Roy 2009). Dif-
ferent flavonoids presented in rooibos may affect both
basal and FSH-stimulated progesterone synthesis by
cultured rat (Nejaty et al. 2001) and human (White-
head & Lacey 2003) ovarian granulosa cells. Ovarian
cells produce steroid hormones including progesterone
(P4) and testosterone (T), precursors for the biosynthe-
sis of estrogen, a known stimulators of ovarian functions
and regulators of embryonic development and mam-
's
mary gland functions (Hagan et al. 2009; Sirotkin 2014).
Insulin-like growth factor I (IGF-I) represents an im-
or
portant regulator of proliferation, apoptosis and of se-
cretory and generative functions of mammalian ovarian
cells (Schams et al. 1999). The direct action of rooibos
th
on basal P4 release by ovarian cells in other species
(rabbit) cultured with and without gonadotropin has
not been studied. The action of rooibos in ovarian T
Au
and IGF-I and their response to gonadotropin remained
unknown until now.
The aim of our in vitro study was to examine pos-
sible direct rooibos effect on steroid (P4 and T) and
peptide (IGF-I) release by isolated rabbit ovarian tis-
sue and the response of this tissue to gonadotropins
(FSH+LH).
Material and methods
Isolation and culture of rabbit ovarian fragments
Ovaries were obtained from noncyclic rabbits of hybrid
line New Zealand White about 3.5–4 months of age in
the Research Institute for Animal Production in Nitra.
Ovaries were obtained after slaughter at a local abattoir
and transported to the laboratory in containers at 4 ◦C.
They were washed several times in 95% alcohol, sterile
physiological solution (0.9% NaCl) and dissected in 8–16
fragments using multiblade knife (approximately 2–3 mm
size, weight 2–3 mg). Immediately after section, these frag-
ments (1 fragment per a well) were incubated in culture
24 well plates (Beckton Dikinson 1 mL/well) with 1 mL of
sterile culture medium DMEM/F12 1 : 1 (BioWhittakerTM ,
Verviers, Belgium) supplemented with 10% foetal bovine
1425
serum (BioWhittakerTM ) and 1% antibiotic-antimycotics
(Sigma, St. Louis, MO, USA).
Rooibos molecules were extracted from rooibos powder
(Rooibos Factory, Clanwilliam, South Africa) by dimethyl-
sulfoxide (DMSO; 1 mg of rooibos powder in 50 µL DMSO
during 24 h). Thereafter this extract was dissolved in cul-
ture medium immediately before its addition to the cells,
such that the final concentration of DMSO did not exceed
0.001%. Previous studies did not reveal substantial effects
of 0.001% DMSO on ovarian cells viability and hormones se-
cretion (not shown). The fragments were incubated with or
without rooibos extract at the doses of 0, 1, 10, 100 µg/mL
at 37 ◦C, 5% CO2 . Controls represented ovarian fragments
cultured without rooibos treatment and incubation medium
(with or without 0.001% DMSO) cultured without ovarian
tissue and treatments (blank assay).
In the 2nd series of experiments fragments were in-
cubated with and without FSH+LH (Pluset, Barcelona,
Spain). Active substances of this gonadotropin preparation
represented 500 IU of FSH and 500 IU of LH. The prepa-
rations were dissolved in culture medium and added to the
culture plates immediately before the experiment. FSH+LH
y
was added to the culture medium at concentrations of 0.01,
0.1 and 1 IU/mL alone and in combination with rooibos at
dose 10 µg/mL. The ovarian fragments were cultured for
p
48 h.
co After cultivation of fragments the culture medium from
plate wells was taken from wells plates by syringe and stored
at –70 ◦C until used for radioimmunoassay (RIA). All exper-
iments were carried out with the approval of a local ethical
committee in accordance with Slovak and European Union
regulations concerning animal experiments.
Immunoassay
Concentrations of P4, T and IGF-I were determined
in duplicate in incubation medium by RIA. Hormones
were assayed using RIA kits according to the manu-
facturer’s instructions (P4, T – Izotop, Budapest, Hun-
gary; IGF-I – DRG Instruments GmbH, Marburg, Ger-
many). Antiserum against P4 cross-reacted less than 13%
to 17-hydroxyprogesterone, 0.03% to pregnenolone, 0.07%
to cortisol, 0.6% to corticosterone, 0.08% to 11-dezoxy-
17-hydroxycorticosterone, 0.01% to testosterone, 0.003%
to estriol, 0.001% to 5-dihydrotestosterone, 17-estradiol
and dehydroepiandrosterone. Sensitivity of the assay was
0.44 ± 0.12 nmol/L, intra- and inter-assay coefficients of
variation did not exceed 10.2% and 11.8%, respectively. The
sensitivity of the T assay was 0.087 nmol/L. Intra- and
inter-assay coefficients of variation did not exceed 6.72%
and 9.63%, respectively. The cross-reactivity of antiserum
against T was less than 35% to 5α-dihydrotestosterone, less
than 0.8% to 5β-dihydrotestosterone and less than 0.01% to
17β-estradiol and cortisol. The cross-reactivity of antiserum
against IGF-I was non-detectable (less than 0.0001%) to
IGF-II, insulin, proinsulin and growth hormone. The max-
imal intra- and inter-assay coefficients of variation were
3.4% and 8.2%, respectively. Sensitivity of the assay was
0.8 ng/mL. All RIAs were validated for use of the samples
of culture medium by dilution tests.
Statistical analyses
The data shown are means of values obtained in experi-
ment using separate pools of ovaries obtained from 6–10
animals. Each experimental group was represented by six
culture wells. Assays of hormone concentration in incuba-
tion medium were performed in duplicates. The values of
1426 A. Štochmaľová et al.

blank control were subtracted from the specific values de-

termined by RIA in cell-conditioned medium to exclude
any non-specific background (less than 10% of total values).
The rates of hormone secretion were calculated per mg tis-
sue per day. Significant differences between the experiments
were evaluated by using one-way ANOVA by using statisti-
cal software Sigma Plot 11.0 (Systat Software, Inc., GmbH,
Erkhart, Germany). Differences between groups in hormone
release were evaluated by t-test using the Sigma Plot 11.0
(Systat Software, Inc.). Differences from control groups were
considered as statistically significant at P < 0.05.

Results and discussion

Rooibos affects hormones release

In the 1st series of experiments, RIA detected accu-
mulation of P4, T and IGF-I in medium conditioned
by cultured ovarian fragments. Our results showed that
rooibos significantly stimulated P4 release at the lowest
dose (1 µg/mL) added (Fig. 1a) and inhibited T release
at 10 µg/mL and 100 µg/mL doses added (Fig. 1b). The

y
IGF-I release was significantly stimulated by rooibos at
all doses added (Fig. 1c).

p
Rooibos can modify FSH+LH effects on hormones re-
lease
The 2nd series of experiments documented that FSH+
co
LH, when added alone, did not substantially affect P4
(Fig. 2a) or T (Fig. 2b) release, but it significantly pro-
moted IGF-I output when added at dose 0.01 IU/mL. In
's
the presence of rooibos, LH+FSH inhibited P4 output
(at doses 0.1 and 1.0 IU/mL; Fig. 2a), but promoted T
or

(at doses 0.01 and 0.1 IU/mL; Fig. 2b) and IGF-I (at
dose 1.0 IU/mL; Fig. 2c) release.
th

Discussion concerning the rooibos influence

The results of our in-vitro experiments demonstrated
the direct influence of rooibos on the release of steroid
Au

hormones and IGF-I by ovarian fragments. These ob-

servations represent the first demonstration of the in-
volvement of rooibos in direct control on ovarian P4,
T and IGF-I release by rabbit ovarian fragments. P4,
T and IGF-I play an important role in control of
ovarian folliculogenesis, oogenesis, atresia, proliferation
and apoptosis (Schams et al. 1999; Hagan et al. 2009;
Sirotkin 2014), therefore the influence of rooibos on
these hormone-dependent processes might be hypoth-
esised. Our observations do not allow to identify bioac-
tive components of rooibos responsible for these ef-
fects on rabbit ovarian hormones. Probably it is not
quercetin, daidzein and biochanin because Nejaty et al. Fig. 1. The influence of rooibos on the release of progesterone
(2001) and Whitehead & Lacey (2003) reported an in- (a), testosterone (b) and insulin-like growth factor I (c) by
cultured rabbit ovarian fragments. Data from RIA. Values are
hibitory, but not stimulatory (as in our experiments) means ± SEM. A indicates significant (p < 0.05) differences
effects of these rooibos isoflavones on P4 production between rooibos-treated (1, 10 or 100 µg/mL) and control
by cultured human granulosa cells. It is more probable (0 µg/mL) rabbit ovarian fragments.
that the effects of rooibos observed in our experiments
are due to other isoflavones. Alternatively, the different
effect of rooibos on rabbit and human ovarian cells can of ovarian cell apoptosis (Fang et al. 2006). Further-
be proposed. more, IGF-I is a potent stimulator of ovarian P4 release
IGF-I is a known promoter of ovarian cell prolifera- (Schams et al. 1999; Sirotkin 2014). It is possible that
tion, oocyte maturation, follicullogenesis and inhibitor rooibos can affect these ovarian functions via promotion
Influence of rooibos-derived compound on rabbit ovarian functions
's
or
th
Au
Fig. 2. The influence of FSH+LH when added alone and in the
presence of rooibos on the release of progesterone (a), testosterone
(b) and insulin-like growth factor I (c) by cultured rabbit ovarian
fragments. Data from RIA. Values are means ± SEM. A indi-
cates significant (p < 0.05) effect of rooibos (differences between
the corresponding groups of ovarian fragments cultured with and
without rooibos), whereas B indicates significant (p < 0.05) ef-
fect of FSH+LH; differences between ovarian fragments cultured
with FSH+LH (0.01, 0.1 and 1 IU/mL) and without FSH+LH
(0 IU/mL).
of IGF-I release, which in turn stimulates P4 output.
Ovarian cell luteinisation is associated with increase in
1427
P4 and decrease in ovarian T as well as estradiol out-
put (Sirotkin 2014). It is thus possible that rooibos,
via promotion of ovarian IGF-I release, can stimulate
ovarian cell luteinisation and change the related P4/T
rate.
Our observations are in line with previous knowl-
edge (Sirotkin 2014; Sirotkin & Harath 2014) concern-
ing the regulation of ovarian P4, T and IGF-I release
by both gonadotropins and phytoestrogens in rooi-
bos. These results correspond with Opuwari & Mon-
sees (2015) who found an inhibitory effect of rooibos
on T production in Leydig cell cultures. Furthermore,
our results demonstrate that rooibos can affect not only
basal hormone release, but also the response of ovarian
cells to gonadotropins. Addition of rooibos induced the
suppressive effect of FSH+LH on P4 release and the
stimulatory action of FSH+LH on T output. Moreover,
it reduced the response of IGF-I to FSH+LH treat-
ment: without rooibos FSH+LH promoted the IGF-I
release at dose 0.01 IU/mL, whilst in the presence of
y
rooibos it had done that effect at much higher dose
(1.0 IU/mL). These observations suggest that rooibos
p
can induce both stimulatory and inhibitory action of
gonadotropins on ovarian hormones release. Since FSH
co
and LH are considered to be the main hormonal reg-
ulators of ovarian functions – release of hormones and
hormone-dependent ovarian follicullogenesis and ooge-
nesis (Sirotkin 2014), the influence of rooibos on these
gonadotropin-dependent processes might be proposed.
The mechanisms, biological and practical significance
of the effect of rooibos on the responsibility of ovarian
fragments to gonadotropins require further studies. It is
possible that IGF-I, the release of which is promoted by
rooibos, can stimulate the production of gonadotropin
receptors and post-receptor production of cAMP medi-
ating gonadotropin action on ovarian cells (Sirotkin et
al. 2003; Sirotkin 2014). The action of phytoestrogens
of rooibos on steroid receptors, involved in control of
numerous ovarian functions including those after gen-
eration of the gonadotropin receptors (Sirotkin 2014),
should not be excluded, too.
Conclusion
Taken together, our study provides the first evidence of
possible stimulatory effect of rooibos on the release of
P4 by rabbit ovaries. Furthermore, it is the first demon-
stration of the up-regulation of ovarian IGF-I and
down-regulation of ovarian T release by rooibos. The
mechanism of rooibos effect on ovarian cells requires
further both the in-vitro and in-vivo studies. Never-
theless, our present observations suggest that rooibos
can directly regulate the release of ovarian steroid hor-
mones and IGF-I, the key regulators of reproductive
functions, as well as the response of ovarian cells to
gonadotropins. These potential reproductive effects of
rooibos are to be taken into account by consumption of
rooibos-containing drinks, by application of rooibos as
a medicine, as well as by possible application of rooibos
for control of reproductive processes in animal produc-
tion and medicine.
1428
Acknowledgements
The authors thank to Ž. Kuklová and K. Tóthová (Research
Institute for Animal Production in Nitra-Lužianky) for tech-
nical assistance. This publication was written during real-
ization of the project “REPROPLANT” No. APVV-0854-
11, “NANOREPRO” No. APVV-4040-11, “ZDRAVIE” No.
26220220176 supported by the Operational Programme Re-
search and Development funded from the European Re-
gional Development Fund and VEGA 1/0613/13.
References
Agarwal A., Sajal G. & Sharma R.K. 2005. Role of oxidative
stress in female reproduction. Reprod. Biol. Endocrinol. 3:
28.
Awonyi D.O., Aboua Y.G., Marnewick J. & Brooks N. 2012. The
effects of rooibos (Aspalathus linearis), green tea (Camellia
sinensis) and commercial rooibos and green tea supplements
on epididymal sperm in oxidative stress-induced rats. Phy-
tother. Res. 26: 1231–1239.
Bramati L., Aquilano F. & Pietta P. 2002. Unfermented rooibos
tea: quantitative characterization of flavonoids by HPLC-UV
and determination of the total antioxidant activity. J. Agric.
Food Chem. 51: 72–74.
Duke J.A., Bogenschutz-Godwin M.J., Ducelliar J. & Duke
P.A.K. 2002. Handbook of Medicinal Herbs, 2nd Edn., CRC
Press, Boca Raton.
Fang J., Zhou Q., Shi X.L. & Jiang B.H. 2006. Luteolin inhibits
insulin-like growth factor 1 receptor signaling in prostate can-
cer cells. Carcenogenesis 28: 713–723.
Gilani A.H., Shah A.J., Ghayur M.N. & Majeed K. 2005. Phar-
macological basis for the use of tumeric in gastrointestinal
and respiratory disorders. Life Sci. 76: 3089–3105.
's
Gonzales-Robayna I.J., Falender A.E., Ochsner S., Firestone G.L.
& Richards J.S. 2000. Follicle-stimulating hormone (FSH)
stimulates phosphorylation and activation of protein kinase
or
B (PKB/Akt) and serum and glucocorticoid-induced kinase
(Sgk): evidence for A kinase-independent signaling by FSH
in granulosa cells. Mol. Endocrinol. 14: 1283–1300.
Hagan C.R., Faivre E.J. & Lange C.A. 2009. Scaffolding actions
th
of membrane-associated progesterone receptors. Steroids 74:
568–572.
Inanami O., Asanuma T., Inukai N., Jin T., Shimokawa S., Kasai
N., Nakano M., Sato F. & Kuwabara M. 1995. The suppres-
Au
sion of age-related accumulation of lipis peroxides in rat brain
by administration of Rooibos tea (Aspalathus linearis). Neu-
rosci Lett. 196: 85–88.
Joubert E., Winterton P., Britz T.J. & Ferreira D. 2004. Super-
oxide anion and α,α-diphenyl-β-picrylhydrazyl radical scav-
enging capacity of rooibos (Aspalathus linearis) aqueous ex-
tracts, crude phenolic fractions, tannin and flavonoids. Food
Res. Int. 37: 133–138.
Juráni M., Lamošová D., Máčajová M., Košťál Ľ., Joubert E. &
Greksák M. 2008. Effect of rooibos tea (Aspalthus linearis) on
Japanese quail growth, egg production and plasma metabo-
lites. Br. Poult. Sci. 49: 55–64.
Kayampilly P.P. & Menon K. 2004. Inhibition of extracellular
signal-regulated protein kinase-2 phosphorylation by dihy-
drotestoterone reduces follicle stimulating hormone mediated
cyclin D2 messenger ribonucleic acid expression in rat gran-
ulosa cells. Endocrinology 145: 1786–1793.
Marnewick J., Joubert E., Joseph S., Swanevelder S., Swart P.
& Gelderbolm W. 2005. Inhibition of tumour promotor in
mouse skin by extracts of rooibos (Aspalathus linearis) and
honeybush (Cyclopia intermedia), unique Southern African
herbal teas. Cancer Lett. 224: 193–202.
View publication stats
A. Štochmaľová et al.
Marnewick J.L., Rautenbach F., Venter I., Neethling H., Black-
hurst D.M., Wolmarans P. & Macharia M. 2011. Effects of
rooibos (Aspalathus linearis) on oxidative stress and bio-
chemical parameters in adults at risk for cardiovascular dis-
ease. J. Ethnopharmacol. 133: 46–52.
McGarvey C., Cates P.S., Brooks A.N., Swanson I.A., Milligan
S.R., Coen C.W. & O’Byrne K.T. 2013. Phytoestrogens and
gonadotropin-releasing hormone pulse generator activity and
pituitary luteinizing hormone release in the rat. Endocrinol-
ogy 142: 1202–1208.
McKay D.L. & Blumberg J.B. 2007. A review of the bioactiv-
ity of South African teas: rooibos (Aspalathus linearis) and
honeybush (Cyclopia intermedia). Phytother. Res. 21: 1–16.
Na H.K., Mossanda K.S., Lee J.Y. & Surh Y.J. 2004. Inhibition
of phorbol ester-induced COX-2 expression by some edible
African plants. Biofactors 2: 149–153.
Nakano M., Ioth Y., Mizuno T. & Nakashima H. 1997. Polysac-
charide from Aspalathus linearis with strong anti-HIV activ-
ity. Biosci. Biotechnol. Biochem. 61: 71–267.
Nejaty H., Lacey M. & Whitehead S.A. 2001. Differing effects of
endocrine-disrupting chemicals on basal and FSH-stimulated
progesterone production in rat granulosa-luteal cells. Exp.
Biol. Med. 226: 570–576.
Nikolova V., Petrova S., Petkova V., Pavlova A. & Georgieva
T. 2007. Antioxidant effects of rooibos tea on workers oc-
y
cupationally exposed to lead. Toxicol. Lett. 172 (Suppl.):
120–121.
p
Opuwari C.S. 2013. Effect of tea and herbal infusions on
mammalian reproduction and fertility. PhD Thesis, Fac-
ulty of Science University of Western Cape, South Africa;
co http://etd.uwc.ac.za/xmlui/handle/11394/3015.
Opuwari C.S. & Monsees T.K. 2015. Reduced testosterone pro-
duction in TM3 Leydig cells treated with Aspalathus linearis
(rooibos) or Camellia sinensis (tea). Andrologia 47: 52–58.
Roy S. & Nicholson D.W. 2000. Cross-talk in cell death signaling.
J. Exp. Med. 8: 21–25.
Schams D., Berishaa B., Kosmanna M., Einspaniera R. & Amsel-
gruberg W.M. 1999. Possible role of growth hormone (IGFs),
and IGF-binding proteins in the regulation of ovarian func-
tion in large farm animal. Domest. Anim. Endocrinol. 17:
279–285.
Sirotkin A.V. 2014. Regulators of Ovarian Functions. New York,
Nova Science Publishers, Inc., 194 pp.
Sirotkin A.V., Florkovičová I., Makarevich A.V., Schaeffer H.J.,
Kotwica J., Marnet P.G. & Sanislo P. 2003. Oxytocin me-
diates some effects of insulin-like growth factor-I on porcine
ovarian follicles. J. Reprod. Dev. 49: 141–149.
Sirotkin A.V. & Harrath A.H. 2014. Phytoestrogens and their
effects. Eur. J. Pharmacol. 741: 230–236.
Wang C. & Roy S.K. 2009. Expression of bone morphogenetic
protein receptor (BMPR) during perinatal ovary development
and primordial folicle formation in tle hamster: possible reg-
ulation by FSH. Endocrinology 150: 1886–1896.
Whitehead S.A. & Lacey M. 2003. Phytoestrogens inhibit aro-
matase but not 17β-hydroxysteroid dehydrogenase (HSD)
type 1 in human granulosa-luteal cells: evidence for FSH in-
duction of 17β-HSD. Hum. Reprod. 18: 487–494.
Yoshikawa T., Naito Y., Oyamada H., Ueda S., Tanigawa T.,
Takemura T., Sugino S. & Kondo M. 1990. Scavenging ef-
fects of Aspalanthus linearis (Rooibos tea) on active oxygen
species. Adv. Exp. Med. Biol. 264: 171–174.
Received August 26, 2015
Accepted October 22, 2015