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GENERAL GYNECOLOGY
Phytochemical analyses and effects of Alchemilla mollis (Buser)
Rothm. and Alchemilla persica Rothm. in rat endometriosis model
Esra Ku
¨peli Akkol •Murside Ayse Demirel •Ozlem BahadırAcıkara •
Ipek Su
¨ntar •Burcin Ergene •Mert Ilhan •Serkan Ozbilgin •
Gulcin Saltan •Hikmet Keles¸ •Mehmet Tekin
Received: 15 October 2014 / Accepted: 12 February 2015
ÓSpringer-Verlag Berlin Heidelberg 2015
Abstract
Purpose The aim of the present study is to evaluate the
treatment potential of Alchemilla mollis (Buser) Rothm.
and Alchemilla persica Rothm. in the experimentally in-
duced endometriosis model in rats.
Methods Endometriosis was surgically induced in rats by
autotransplanting endometrial tissue to abdominal wall.
Thirty-six rats were randomly divided into six groups. The
groups were orally treated with the methanol:water (80:20)
extracts of aerial parts and roots of A. mollis and A. persica.
Buserelin acetate (20 mg) was used as the reference drug.
The phytochemical contents of the most active extracts
were determined by high performance liquid
chromatography.
Results The cystic formation was determined to be sig-
nificantly decreased with the aerial part extract of A. mollis.
A reduction in the endometrioma was also determined for
the aerial part extract of A. persica group. However, sig-
nificant reduction on the levels of cytokine were recorded
for the A. mollis aerial part extract group. Therefore, the
phytochemical contents of the aerial part extracts of A.
mollis. and A. persica were analyzed.
Conclusion The results of the present study revealed that
the aerial part extracts of A. mollis and A. persica could be
beneficial in the treatment of endometriosis.
Keywords Alchemilla mollis Alchemilla persica
Cytokine Endometriosis Phenolic constituent Rat
Rosaceae
Introduction
Alchemilla L. genus (Rosaceae) represented by nearly 100
species all around the world [1] has medicinal properties in
traditional medicine worldwide such as antiinflammatory,
antiseptic, sedative, haemostatic, wound healing, expecto-
rant, diuretic, astringent and, furthermore, is used for the
treatment of atherosclerosis, diabetes [2,3] and against
menorrhagia [4]. In Bulgarian folk medicine Alchemilla
species are used against acute diarrhoea, dysmenorrhoea
and menorrhagia; on the other hand, in Turkish folk
medicine they are used as diuretic, laxative, tonic and for
wound healing [5–9]. From this genus, Alchemilla vulgaris
L. (A. xanthochlora Rothm.), known as lady’s mantle,
bear’s foot or lion’s foot, is the species that is described in
European Pharmacopoeia. The medicinal part of the plant
is the aerial parts collected in flowering season. Lady’s
mantle is traditionally used for menopausal complaints,
dysmenorrhea, gastrointestinal disorders, and as a gargle
for mouth and throat inflammation internally as well as for
E. Ku
¨peli Akkol (&)I. Su
¨ntar M. Ilhan
Department of Pharmacognosy, Faculty of Pharmacy, Gazi
University, Etiler, 06330 Ankara, Turkey
e-mail: akkolesra@gmail.com; esrak@gazi.edu.tr
M. A. Demirel
Laboratory Animals Breeding and Experimental Researches
Center, Faculty of Pharmacy, Gazi University, Etiler,
06330 Ankara, Turkey
O. BahadırAcıkara B. Ergene S. Ozbilgin G. Saltan
Department of Pharmacognosy, Faculty of Pharmacy, Ankara
University, 06100 Tandog
˘an-Ankara, Turkey
H. Keles¸
Department of Pathology, Faculty of Veterinary Medicine,
Afyon Kocatepe University, 03200 Afyonkarahisar, Turkey
M. Tekin
Department of Biology, Faculty of Science, Cumhuriyet
University, 58140 Sivas, Turkey
123
Arch Gynecol Obstet
DOI 10.1007/s00404-015-3665-6
ulcers, eczema, skin rashes and as an additive in baths for
the treatment of lower-abdominal ailments externally and
usage against diarrhoea is approved by Commission E [10].
A. vulgaris has been used as tonic, stomachic, diuretic,
haemostatic, astringent, against menstrual pains as well as
catarrh in Italian folk medicine [11,12]; as antidiabetic, to
relieve stomach and intestine pain, for the treatment of
inflammation, asthma and obesity in Jordan folk medicine
[13,14]; for the treatment of menstruation ailments and
headache in Bosnia and Herzegovina [15] and at the
southwest of Serbia [16]; as astringent (against bleeding
and diarrhoea) and anti-inflammatory agent in Central
Europe [17] and in some regions of Israel [18]; for the
healing of skin diseases in traditional Arab herbal medicine
[19] and southeast of Europe and west of Balkans [12,20,
21]. According to the ethnoveterinary records, leaves of A.
vulgaris are used against hormonal imbalances of horses in
Canada [22] and against reproductive disorders in Italy
[23].
The ethnobotanical studies on the other species of
Alchemilla revealed that the leaves of Alchemilla pseu-
docartalinica Juz. are used as constipant, diuretic and
tonic in the eastern region of Turkey [24]. In Spain,
decoction of Alchemilla alpina L. is drunk after meals as
digestive [25]. There are also records about the use of
decoction of its aerial parts as intestinal anti-inflamma-
tory and for kidney stones [26]. Decoction prepared from
Alchemilla arvensis (L.)Scop.isusedasdiuretics[27].
It was reported that tincture prepared using leaves of
Alchemilla mollis (Buser) Rothm. is also used against
menorrhagia [4].
Alchemilla species were reported to possess various
biological activities such as angioprotective, anticarcino-
genic, antioxidant, wound healing, antimicrobial and anti-
influenza [28–33] and were shown to have highly-con-
densed tannin content flavonoids, proanthocyanidins and
triterpenic compounds [17,29,34–42].
As reported above the literature, much attention has
been focused on the biological activity studies and phyto-
chemical analyses of the Alchemilla genus plants. Due to
the ethnobotanical usage of the plant regarding the gy-
naecological diseases, the present study was designed to
evaluate the potential effect of A. mollis and A. persica on
the treatment of endometriosis.
Materials and methods
Plant material
The aerial parts and roots of A. mollis were collected from
Sivas, Sarıyar village and identified by Mehmet Tekin from
Cumhuriyet University Faculty of Science. A voucher
specimen was deposited in Cumhuriyet University Faculty
of Science Herbarium (CUFH 1344). A. persica was col-
lected from Erzincan, Kop Passage and identified by Prof.
Dr. Hayri Duman from Department of Biological Sciences,
Faculty of Art and Sciences, Gazi University. Voucher
specimen was kept in the herbarium of Ankara University,
Faculty of Pharmacy (AEF 25896).
Preparation of the extracts
Aerial parts and roots of the plants were separated, then
dried and powdered plant materials were extracted with
methanol:water (80:20) mixture by continuous stirring at
room temperature for 8 h. After filtration, extract was
concentrated to dryness under reduced pressure and low
temperature (40–50 °C) on a rotary evaporator to give
crude extract. Extracts were weighed and yield percentages
were calculated as 26.09, 25.99, 19.90, 14.47 % for A.
mollis aerial parts and roots, and A. persica aerial parts and
roots, respectively.
HPLC analysis
HPLC analyses were carried out using Agilent LC 1260
chromatograph (Agilent Technologies, Darmstadt, Ger-
many). The diode array detector (DAD) was set at wave-
length of 254 nm and peak areas were integrated
automatically using Agilent ChemStation Software. Sepa-
ration was carried out using a ACE 5 C18
(250 mm 94.6 mm; 5 lm) column. The method which
was used in our previous study [43] was modified and
optimized as given below. The mobile phase was com-
posed of acetonitrile (A) and 0.2 % phosphoric acid
(B) using gradient elution: initial A:B (8:92, v/v), in 10th
min A:B (18:82, v/v), in 20th min. A:B (20:80, v/v), in 30th
min (25:75, v/v), in 35th min. A:B (30:70 v/v), in 40th A:B
(100:0, v/v). This was followed isocratic flow A:B (100:0,
v/v) to 45th min. The flow rate was 0.5 ml/min, column
temperature was maintained at 35 °C. The sample injection
volume was 10 ll. Caffeic acid (1), orientin (2), rutin (3),
hyperoside (4), isoquercitrin (5), luteolin-7-glycoside (6),
ferulic acid (7), myricetin (8) and agenin (9) purchased
from Sigma used as standards. Standard stock solutions
were prepared as 1 mg/ml. Compounds were weighed in
10 ml volumetric flask, dissolved in methanol:water
(80:20) mixture and adjusted to the final volume separately.
Different concentration levels (0.005, 0.01, 0.02, 0.03,
0.04, 0.05 mg/ml) for isoquercitrin and (0.001, 0.002,
0.004, 0.006, 0.008, 0.01 mg/ml) for hyperoside were
prepared by diluting the stock solution. Triplicate 10 ll
injections were performed for each standard solution. Peak
area of each solution was plotted against the concentration
to obtain the calibration curves.
Arch Gynecol Obstet
123
The identification of compounds in extracts was carried
out using the comparison of retention time and UV spec-
trum obtained from the analysis of single compounds.
Furthermore, combined injection of single compound with
extract was used to confirm the compound presence.
Standard HPLC validation procedures were used to de-
termine the limits of detection and quantification (LOD and
LOQ), respectively. The LOD and LOQ were established at
signal to noise ratios (S/N) of 3 and 10, respectively. The
LOD and LOQ concentrations were verified experimentally
by repeating each analysis six times.
European Pharmacopoeia test
Chlorogenic acid and caffeic acid presence of the A. mollis
and A. persica extracts were tested according to the method
described in European Pharmacopoeia 6.0 using thin layer
chromatography. 1.0 mg of caffeic acid R and 1.0 mg of
chlorogenic acid R dissolved in 10 ml of methanol R, were
used as reference solutions. Mobile phase was made up
anhydrous formic acid R, water R, ethyl acetate R
(8:8:84 V/V/V) mixture. Detection was performed under
ultraviolet light at 365 nm [44].
In vivo biological activity studies
Animals
Six-week-old female, intact, 36 Sprague–Dawley rats
weighing 200–250 g were housed in polysulfone cages at
21–24 °C, at 40–45 % humidity, and light-controlled (12 h
light/12 h dark) conditions at Faculty of Pharmacy, Gazi
University (Ankara, Turkey). Rats were provided from
Laboratory Experimental Animals, Kobay, Turkey, and
were quarantined for at least 2 weeks. Animals were
maintained in accordance with the directions of Guide for
the Care and Use of Laboratory Animals. All experimental
processes were achieved at Laboratory Animals Breeding
and Experimental Researches Center, Faculty of Pharmacy,
Gazi University. Animals were fed ad libitum water from
polysulfone bottles and food. Estrous cycle stage was fol-
lowed by daily assessment of vaginal cytology and rats
exhibiting regular 4–5 day estrous cycles were used in
endometriosis model.
Surgical induction of rat endometriosis model
The rats at the proestrus stage of the estrous cycle were
used in the study. The endometriosis model in the rats was
induced due to the method described by Vernon and Wil-
son [45].
Phase I: Animals were anesthetized with an intramus-
cular administration medetomidin (0.5 mg/kg; Domitor
Ò
,
Orion Pharmos, Pharmaceutical Pfizer Animal
Health, Finland) and ketamine hydrochloride 10 %
(100 mg/kg; Ketasol
Ò
, Interhas, Ankara, Turkey). For
laparotomy, the rats were lined in supine position, their
abdominal hair was shaved and disinfection of abdomen
was provided with iodine. A midline incision of 3 cm was
performedbythescalpelblade. Subcutaneous and muscle
layer were separated and abdominal cavity was achieved.
After right uterine corn was resected, 15 mm piece from
tissue was taken by microscissor. This section was lon-
gitudinally opened and endometrium layer was dissected
from myometrium. A 5 95 mm fragment of endometrial
tissue was autotransplantedwithUSP4/0polyglactin
(Lactasorb PGLA
Ò
, Orhan Boz, Turkey) into abdominal
wall. The muscle layers of abdomen were closed with
USP 3/0 polyglactin (Lactasorb PGLA
Ò
, OrhanBoz,
Turkey). For the recovery from anesthesia, atipamezole
(0.5 mg/kg; Antisedan
Ò
, Orion Pharmos, Pharmaceuti-
cal Pfizer Animal Health, Finland) was applied
subcutaneously.
Phase II: Twenty-eight days after the first operation, rats
were done to determine a second laparotomy under the
same anesthesia procedure. Endometriotic foci and adhe-
sion areas were evaluated. The endometrial foci areas were
measured the size of their length, width, height by mi-
crometer. For calculation was used an ellipsoid volume
formula (p/6 9length 9width 9height). Intraabdominal
adhesions were classified according to the Blauer’s scoring
system (Table 1)[46].
The double blinded examiner collected the data. The
abdomen was closed with the same procedure as stated
above. Thirty-six rats were randomly divided into six
groups to be control group, reference group and treatment
groups. According to groups treatments were applied
3 days after identifying groups.
Treatment procedure
Control group (n=6): Sodium carboxymethylcellulose in
distilled water 5 % (CMC 5 %, 2 ml/rat/day) was given by
oral gavage.
Table 1 The presence of intraabdominal adhesion according to
Blauer’s scoring system
Score The presence of adhesion
0 No adhesion
1 Thin adhesions
2 Thick adhesion in one area
3 Widespread thick adhesions
4 Adhesions of the internal organs to the abdominal wall
Arch Gynecol Obstet
123
Extract groups (n=6, each): All of the extracts were
prepared in 100 mg/kg doses in 0.5 % CMC suspension in
distilled water and given daily by oral gavage.
Buserelin group (n=6): Buserelin acetate (Receptal
Ò
,
Intervet International GmbH, Germany) (20 mg) was ad-
ministrated daily subcutaneous to the rats.
Phase III: All rats were killed at the end of the treatment
procedure. Endometriotic foci areas and intraabdominal
adhesions were again evaluated and compared with previ-
ous findings.
Histopathological investigation
The samples were fixed in 10 % neutral formol saline,
embedded in paraffin, cut into 5 lm fine sections, stained
with hematoxylin-eosine (HE) and Masson’s trichrome
(MT) and examined under a light microscope (Nicon
Eclipse Ci attached Kameram
Ò
Digital Image Analyze
System). In histopathological analyses peritoneal adhe-
sions, epithelial invasions between the muscle fibers,
cyst-like structures and epithelial cells of these struc-
tures, vascularized granulation tissue, inflammatory cells,
extravase erythrocytes and hemosiderosis were scored
as mild, moderate and severe according to their severity
in all specimens and then the group averages were
calculated.
The detection of TNF-a, VEGF, IL-6 levels
in peritoneal fluid
Rat’s peritoneal fluid was collected to detect tumor
necrosis factor-a(TNF-a), vascular endothelial growth
factor (VEGF), interleukin-6 (IL-6) levels in second la-
parotomy and after sacrifice. The obtained results were
compared as pretreatment and posttreatment values and
statistically evaluated. These cytokine levels were quanti-
tatively evaluated using a commercially available enzyme-
linked immunosorbent assay kit [TNF-a, IL-6 (Bio
Source
Ò
International, Nivelles, Belgium; catalog numbers
MBS453189 and MBS701221) and VEGF (eBioscience
Ò
,
Austria; catalog number: BMS626/2)] according to the
manufacturer’s instructions. Peritoneal fluid was re-col-
lected and the same procedure was applied after the ani-
mals were killed and consequently pre- and post-treatment
results were statistically compared.
Statistical analysis
The results were expressed as the mean ±SD Dunnett’s
test was used to determine the significance of differences
between groups. In order to compare two groups Student’s
ttest was used [47].
Results
The endometrial foci areas were measured by using a
digital micrometer in order to determine the treatment ca-
pacity of the aerial part and root extracts of A. mollis and A.
persica. The cystic formation was determined to be de-
creased from 101.35 to 11.87 mm
3
by the oral application
of the aerial part extract of A. mollis to the rats. A reduction
in the endometrioma was also determined for the aerial part
extract of A. persica administered group. The other extract
groups did not show any significant difference between
pre- and post-treatment (Table 2).
A remission in the adhesion was also determined for the
A. mollis aerial part extract treated group (Table 3).
Numerous peritoneal adhesion and invasions were detected
at the implantation sites (Fig. 1). Superficially or profound
invasions between the muscle fibers were seen. En-
dometriotic lesions were formed with different degree in
groups. Sometimes a few epithelial cell islands with fibrous
stroma, but mainly, one or more encapsulated cyst-like
structures lined by epithelial cells at luminal surface were
observed.
These structures were circularly arranged by a vascu-
larized granulation tissue from bottom and sometimes
contained extravasated erythrocytes or hemosiderin filled
macrophages. The most severe endometriotic lesions were
seen in the control group. The severity of lesions were
reduced in the root extracts of A. persica and A. mollis
administered groups, aerial part extracts of A. persica and
A. mollis and reference groups, respectively. The findings
were visualized with microscopical figures (Fig. 2).
Results of the cytokine level analyses showed that sig-
nificant reduction on the levels of TNF-a, VEGF and IL-6
were recorded for the A. mollis aerial part extract group
with the values of 7.04 ±1.62, 17.83 ±6.92 and
34.75 ±14.91, respectively. On the other hand, the rest of
the extracts did not show a significant reduction on the
cytokine levels tested (Table 4).
According to the European Pharmacopoeia lady’s
mantle fingerprint analysis should allow to observe
chlorogenic and caffeic acids presence. In current study
chlorogenic and caffeic acids were investigated according
to the method described in pharmacopoeia in A. mollis and
A. persica aerial part and root extracts. However, neither
chlorogenic nor caffeic acids were detected. HPLC analysis
of the tested extracts were also performed to investigate
their chemical composition using some standards displayed
in Fig. 3.
Seven different concentrations were prepared to estab-
lish the calibration curves for each standard. Both
calibration curves showed good linearity responses with
high correlation coefficients (Table 5). LOD and LOQ
Arch Gynecol Obstet
123
concentrations were experimentally verified by six injec-
tions of hyperoside and isoquercetin and the results were
given in Table 5. Among the tested compounds hyperoside
and isoquercetin were identified in both species aerial parts
and quantified as 0.236 ±0.002, 0.622 ±0.003 and
0.223 ±0.001, 0.534 ±0.001 mg in 100 mg plant mate-
rial, respectively for A. mollis and A. persica. As shown in
Fig. 4a, b both A. mollis and A. persica extracts were
similar with little differences. UV absorbances of the re-
maining peaks suggested that both species are probably
containing many phenolics.
Discussion
In the present study, the activity potential of A. mollis and
A. persica, both of which are growing wild in Turkey was
assessed on the basis of ethnobotanical data by using sur-
gical induced-endometriosis model in rats. A. vulgaris,
another species of Alchemilla genus was reported to have
beneficial effects in the treatment of gynecological dis-
eases, and is known to be frequently used by the women
living in rural areas for this purpose [10–12,48]. The re-
sults of the present study reveal the promising effects of the
aerial parts of A. mollis and A. persica.
Previous studies showed that A. vulgaris has astringent
and anticarcinogenic properties [12,49]. Moreover, A.
vulgaris was reported to possess inhibitory activity on in-
tercellular matrix degrading proteases [28], antioxidant
[30] and angioprotective effects [28–31]. In vivo studies
have shown that the wound healing activity of A. vulgaris
is associated with promitotic activity in epithelial cells and
myofibroblasts. Clinically this activity of lady’s mantle was
also proved on healthy patients [50]. A. vulgaris is also
reported to show inhibitory activity of pancreatic lipase in
the study of Slanc et al. [51]. Regarding the biological
activities of other Alchemilla species, Barbour et al. [52]
proved the antimicrobial activity of the water extract of A.
diademata against Staphylococcus aureus.A. mollis ex-
hibited potent free radical scavenging [32] and anti-in-
fluenza activity [33]. Antioxidant activity of A. persica was
Table 2 Comparison of the
pre-treatment and the post-
treatment endometriotic implant
volumes
Bold values indicate statistical
significant
p\0.05; ** p\0.01;
*** p\0.001; SD standard
deviation
Material Parts used Volume of endometrioma (mm
3
)±SD
Pre-treatment Post-treatment
Control – 105.92 ±15.62 96.74 ±17.82
Alchemilla mollis Aerial parts 101.35 ±10.26 11.87 ±2.32**
Roots 95.16 ±18.44 89.68 ±12.24
Alchemilla persica Aerial parts 103.30 ±17.53 42.56 ±8.59*
Roots 92.78 ±13.78 80.52 ±13.46
Reference – 98.65 ±12.47 0.0±0.0***
Table 3 Intraabdominal adhesion scores of the endometriotic
implants
Material Parts used Adhesion scores ±SD
Pre-treatment Post-treatment
Control – 1.9 ±0.5 2.7 ±0.7
Alchemilla mollis Aerial parts 1.3 ±0.7 0.4±0.3**
Roots 2.9 ±0.8 2.3 ±0.2
Alchemilla persica Aerial parts 1.7 ±0.6 1.4 ±0.3
Roots 3.1 ±0.8 3.8 ±0.9
Reference – 2.2 ±0.5 0.0±0.0***
Bold values indicate statistical significant
*p\0.05; ** p\0.01; *** p\0.001; SD standard deviation
Fig. 1 a Established endometriotic lesion consisting of endometrial
tissue and cysts 28 days after induction in control group; bpersistent
lesion was observed in control group; cAlchemilla mollis-aerial part
extract group (prior to treatment of endometriotic lesion) with
adhesion score of 4; dAlchemilla mollis-aerial part extract group
(after the treatment) with no measurable endometriotic tissue and no
adhesion formation in implantation site
Arch Gynecol Obstet
123
proved by DPPH radical scavenging assay and measure-
ment of malondialdehyde (MDA) levels [53]. The in vitro
study of [1]onA. erythropoda, A. ikizdereensis, A. orit-
urcica and A. trabzonica showed that these species had
apoptotic and necrotic effects on HeLa cells. In the study of
Nikolova et al., A. jumrukczalica Pawl. which is an en-
demic species in Bulgaria showed significant antioxidant
activity. In the same study, phenolic content of this species
has also been investigated and the results have revealed
that the antioxidant activity and phenolic content were
correlated [54].
Literature survey on the secondary metabolites showed
that Alchemilla species have phenolic and triterpenic
compounds [17,37,38,40,42]. Especially A. vulgaris was
shown to have highly-condensed tannin content flavonoids
and proanthocyanidins [17,29,34–36,39,41]. Quercetin-
3-O-a-L-arabinopyranoside, quercetin-3-O-b-D-glucopyra-
noside, quercetin-3-O-b-D-rutinoside, quercetin-3-O-a-D-
arabinofuranoside, 3-O-kaempferol-600-O-(p-coumaroyl)-b-
D-glucopyranoside were isolated from A. vulgaris [11]. On
the other hand, the major constituents of the volatile oil of
A. vulgaris was determined as cis-3-hexenol and linalool
Fig. 2 Histopathological view of endometriosis model in the test
materials administered animals. Original magnification was 94 and
the scale bars represent 100 lm. aControl group; mild endometriotic
lesions, a new grow out of endometriotic mass to abdominal cavity
with hemorrhage, HE; bAlchemilla mollis-aerial part extract
administered group; a cyst-like structures, invasion of epithelial
cells, HE; cAlchemilla mollis-root extract administered group; a cyst-
like structures, profound invasion of epithelial cells between the
muscle fibers, hemosiderosis in the vascularized granulation tissue,
HE; dAlchemilla persica-aerial part extract administered group; a
cyst-like structures, vascularized granulation tissue, HE; emoderate
endometriotic lesions, Alchemilla persica-root extract administered
group; a cyst-like structure, vascularized granulation tissue, HE;
fReference group; severe endometriotic lesions, a cyst-like structure,
vascularized granulation tissue, HE; gAlchemilla mollis-aerial part
extract administered group; invasion of epithelial cells between the
vascularized granulation tissue, MT. Arrow pointed abbreviation. AC
abdominal cavity; Lcyst lumen; ecf epithelial cell fold; hhemorrhage;
hs hemosiderin; mf muscle fiber; vgt vascular granulation tissue; ce
cyst epithel
Table 4 Peritoneal TNF-a, VEGF and IL-6 levels before and after treatment in all groups
Material Parts used TNF-alevel (pg/ml) ±SD VEGF level (pg/ml) ±SD IL-6 level (pg/ml) ±SD
Pre-treatment Post-treatment Pre-treatment Post-treatment Pre-treatment Post-treatment
Control – 8.16 ±1.29 9.02 ±1.66 18.44 ±5.27 20.34 ±6.56 57.34 ±22.10 52.43 ±18.31
Alchemilla mollis Aerial parts 9.92 ±1.73 7.04 ±1.62* 20.49 ±9.42 14.83 ±6.92** 55.47 ±19.38 34.75 ±14.91**
Roots 8.63 ±1.91 9.31 ±1.22 19.48 ±7.08 19.25 ±5.54 48.21 ±12.35 50.62 ±18.17
Alchemilla persica Aerial parts 8.86 ±2.11 7.92 ±1.57 19.90 ±8.14 18.84 ±7.42 50.31 ±17.19 45.77 ±17.61
Roots 9.67 ±1.85 9.26 ±1.70 20.34 ±9.42 21.76 ±8.09 51.02 ±18.41 50.17 ±12.45
Reference – 7.85 ±1.63 4.36 ±0.41*** 15.36 ±4.17 9.22 ±3.28*** 39.05 ±10.86 25.12 ±7.30***
Bold values indicate statistical significant
*p\0.05; ** p\0.01; *** p\0.001; SD standard deviation
Arch Gynecol Obstet
123
0 5 10 15 20 25 30 35 40
0
50
100
150
200
250
300
350
400
DAD1 A, Sig=254,4 Ref=off (D:\ALCHEMILLA HPLC 2014\ALCHEMILLASTD 2013-12-26 17-29-16\KARISIM-9MADDE.D)
1
2
3
4
5
6
78
9
Fig. 3 Chromatogram of the standard mixture; caffeic acid (1), orientin (2), rutin (3), hyperoside (4), isoquercitrin (5), luteolin-7-glycoside (6),
ferulic acid (7), myricetin (8) and apigenin (9)
Table 5 Linearity and LOD-LOQ results of the standards
Standards Retention times Calibration curves r
2
LOD (lg/ml) LOQ (lg/ml)
Hyperoside 25.482 Y=36,835.882X-2.411 0.9997 3.16 ±0.28 5.49 ±0.21
Isoquercetin 26.194 Y=43,331.562X-57.915 0.991 9.16 ±0.36 14.76 ±0.27
0 5 10 15 20 25 30 35 40
0
25
50
75
100
125
150
175
DAD1 A, Sig=254,4 Ref=off (D:\ALCHEMI... HPLC 2014\STD+EKSTRE\ALCHEMILLA9 2013-12-18 17-15-37\AMHERBA3.D)
4
5
0 5 10 15 20 25 30 35 40
0
50
100
150
200
250
DAD1 A, Sig=254,4 Ref=off (D:\ALCHEMI... HPLC 2014\STD+EKSTRE\ALCHEMILLA9 2013-12-18 17-15-37\APHERBA3.D)
4
5
A
B
Fig. 4 Chromatogram of the A. mollis (a) and A. persica (b) aerial part extracts
Arch Gynecol Obstet
123
[17]. Previous studies on other Alchemilla species have
revealed that a flavonoid glycoside, miquelianin, was de-
tected for A. coriacea, A. filicaulis,A. glabra and A. mollis
[39]. Besides those, hyperoside, isoquercetin, cis- and
trans-tiliroside, sinocrassoside D2 and rhodiolgin were
isolated from A. mollis [8,32]. A. procerrima, A. stricta,
A.hirtipedicellata and A. sericata were investigated for
their flavonoid content and orientin, vitexin, rutin, hyper-
oside, isoquercetin and quercitrin were identified [55]. Five
triterpenes were purified namely, ursolic acid, 2-a-hy-
droxyursolic acid, tormentic acid, euscophic acid, and
oleanolic acid from the aerial parts of A. faeroensis [56–
58]. Investigations on A. alpina and A. vulgaris demon-
strated the presence of the same compounds [42]. In the
present study, hyperoside and isoquercetin were identified
in the aerial parts of A. mollis and A. persica, which were in
accord with the previous findings.
Endometriosis is strongly associated with pelvic pain
and dysmenorrhea. The severity of the pain caused by the
lesion could be related to the subperitoneal nerves [59]. In
endometriosis, insensitivity to antiproliferative signals via
progesterone and cyclin-dependent kinase inhibitors is also
evident. This demonstrates an alternative antiproliferative
signaling pathway to that mediated by progesterone. This
literature reported that the benefit of A. vulgaris used in
treatment of endometriosis may be attributed to known
effects on the signaling pathways associated with the
pathogenesis of endometriosis, hence demonstrating anti-
inflammatory, anti-proliferative, analgesic, antispasmodic
and antioxidant effects on endometrial cells. A. vulgaris
shows antioxidant activity, inhibitory effects on leuko-
trienes as well as antiproliferative effects when tested in
human cancer cell lines and mouse models [49].
Cytokines including TNF-a, VEGF and IL-6 are
essential parameters in the pathogenesis of endometriosis
[60,61]. Indeed, the level of cytokines was reported to be
high in the peritoneal fluid of the women with en-
dometriosis [62]. Thus, we also aimed to evaluate the
peritoneal TNF-a, VEGF and IL-6 levels of the animals in
all groups. Low cytokine levels were detected in the ref-
erence and the aerial part-extract of A. mollis treated
groups. However, A. persica treated group did not show
reduced cytokine values although demonstrated remission
in endometrial implants, which suggested that cytokines
might not directly involved in the effects or that maybe A.
mollis and A. persica have different ways of action.
In the previous studies, it was reported that phenolic
compounds not only produced a statistically significant
reduction in the volume and weight of endometriosis-like
lesions [63–66] but they also inhibited the proliferation of
the endometrial cancer cells [67]. According to the current
study results, the effects of A. mollis and A. persica could
be partly attributed to their strong antioxidant activity and
their phenolic constituents. However, the other constituents
present in A. mollis and A. persica could contribute the
activity. Further phytochemical analyses should be war-
ranted for the determination of the other active
components.
Conclusion
The results of the present study revealed that the aerial part
extracts of A. mollis and A. persica could be beneficial in
the treatment of endometriosis. This is the first study to
report the beneficial effects of the aerial part-extracts of A.
mollis and A. persica in the treatment of endometriosis.
Conflict of interest None.
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