Section SOCIOLOGY AND HEALTHCARE

PHENOLIC PROFILE AND ANTIOXIDANT ACTIVITY OF

A SEMPERVIVUM RUTHENICUM KOCH ETHANOLIC
EXTRACT

Assoc. Prof. Drd. Sebastian Mihai1

Dr. Antoanela Popescu1
1
Drd.
1
Dr. Marius Daniel Radu
Dr. Victoria Badea1
1

ABSTRACT
The current goal of this study is a phytochemical analysis of a Sempervivum
ruthenicum Koch ethanolic extract, in order to find potential medical applications.
The ethanolic extract was prepared from the dried leaves of the plant by
maceration in absolute ethanol. Total phenolic content was assessed with a
slightly modified Folin-Ciocalteu method. A HPLC analysis was conducted and
the total antioxidant activity of the extract was assessed with a DPPH method. The
total phenolic content of the extract was calculated at 1.0344 ± 0.0237 mg/mL.
After conducting the HPLC analysis on the ethanolic extract, phenolic acids,
flavonoids and glycosides were identified and quantified. The most prevalent
phenolic acids in the extracts were gallic and ellagic acids, with concentrations of
0.5137 ± 0.0110 mg/mL, respectively 0.3139 ±0.0046 mg/mL. The glycoside
astragalin is also present in relatively high concentrations, of 0.0929 ± 0.0049
mg/mL. The EC value for the ethanolic extract was 4.6112 ± 0.08 mg/mL. These
results suggest a good scavenging ability of the extract, which is due to the
abundance of phenolic compounds found within. The ethanolic extract of
Sempervivum ruthenicum Koch harvested from the Dobrogea Region of Romania
has a complex phenolic profile with high levels of gallic and ellagic acids, and a
strong anti-oxidant activity which can be exploited both in traditional medicine
and in phytotherapeutic preparations.

Keywords: anti-oxidant, chromatography, DPPH, phenolics, Sempervivum

INTRODUCTION
Since the beginning of medicine, natural remedies were used for therapeutic
purposes for a large spectrum of ailments. These remedies were used empirically,
with uncertainty regarding the exact composition of the plant material used or the
extracts obtained by processing. However, with the progress of analytical
techniques and technologies, laboratories all over the world began screening
known medicinal plants for bioactive molecules that can be used for treating
diseases [1]. Antibiotics and antioxidants are currently being used in combating
pathological agents such as bacteria and fungi. Such compounds have been
derived from natural sources and are the only line of defense against infections
[2], [3]. Screening for natural bioactive molecules derived from plants is one of

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the leading trends in pharmaceutical research, due to the diversity and complexity
of natural products.
Sempervivum ruthenicum ember of the
Crassulaceae family, is a rare plant, growing on harsh rocky terrain. It has a radial
disposition, having a short stem, numerous leaves placed in helicoidal position
around the stem. Flowering occurs after a average of 3-4 years. The main stem
extends upwards forming a leafy thin stem which holds the flowers. The rosettes
produce offsets on stolons that can vary in length and are firmly attached to the

survival and it can be theorized that the bioactive molecules it produces may be of
use in pharmaceutical research. However, the literature on this plant is limited to
mostly traditional remedies and old phytochemical studies [5], [6], [7], [8].
The current goal of this study is a phytochemical analysis of a Sempervivum
ruthenicum Koch ethanolic extract, to find potential medical applications.
MATERIALS AND METHODS
Biological material
The plant species selected for this study was Sempervivum ruthenicum Koch,
a rare plant found in the rocky terrain of south-eastern region of Romania. The

during its flowering period (30th of June 20th of July). The plant was positively
identified at the Faculty of Pharmacy of Constanta, The Botanical Department,
where a voucher specimen of the plant is stored. The harvested plant material was
washed with double distilled water, dried and weight. The plant was dried for 6
months at a constant temperature of 20 oC ± 2 oC and a constant relative humidity
of 50% (±10%). After drying the plant material was weight and the total moisture
content was calculated [9]. The leaves were separated from the remaining plant
material (roots, stems, flowers) and were ground to a fine powder using a mortar
and pestle. The powder was then passed through a no. 7 sieve and was stored at
room temperature for further use.
Chemicals and reagents
All chemicals, reagents and standards used for this study were purchased
from Sigma-Aldrich (Munich, Germany), Chromadex (Wesel, Germany), and
Extrasynthese (Lyon, France). All chemicals, reagents and standards were of
analytical quality.
Preparation of the extract
The ethanolic extract was prepared from the dried leaves of the plant by
maceration in absolute ethanol, as proposed by Sukhdev et al. [10]. Ten grams (10
g) of powdered dry leaves were macerated in 100 g absolute ethyl alcohol for 14
days, shaking the maceration vial manually at 8-hour intervals for 1 minute. The
crude extract was then filtered through Whatman no. 42 papers until the final
product was a clear, dark green liquid. The final extract was then placed in a
sterilized glass vial and stored at -8o C for later use.
Total phenolic content

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To determine the total phenolic content of the extract, the method employed
by Shirazi et al. [11] was used, with minor modifications consisting of using a
pyrogallol standard. The standard curve of the pyrogallol solution was drawn by
preparing 6 dilutions of this compound in methyl alcohol (0.05, 0.1, 0.5, 1.0, 2.5,

-Ciocalteu reagent (Sigma-Aldrich GMBH,

Munich, Germany), allowing the mixture to react for 6 minutes. After the reaction

water were added to the reaction mixture. The absorbance of the samples was
recorded after 90 minutes, at 760 nm, using a UV-Vis UV-6300PC (VWR)
spectrophotometer. The same procedure was followed for the plant extract that
had a concentration of 10% (m/m). The total phenolic content was calculated as
pyrogallol equivalents (mgPIR/mL). All experiments were run in triplicate, and
the results are expressed as mean ± SD.
Analysis by HPLC
To identify and quantify the bioactive compounds in the plant extract, a
standardized HPLC method for determining phenolic compounds was used,
described in the USP 30-NF25 Pharmacopoeia [9].
The equipment used included am Agilient 1200 chromatogram with
quaternary pump, DAD, thermostat, degas system and autosampler. The
chromatographic column employed was a C18 Zorbax XDB 250 mm X 4.6 mm; 5

employed a linear gradient as follows: 10% B for 13 minutes, 22% B for 1 minute,
40% B for 3 minutes and 10% B for 1 minute. The column temperature was 35 oC

elution time was 20 minutes. Detection was carried out using the DAD system at
310 nm, 335 nm and 360 nm, simultaneously. Standards used included: E-
resveratrol, Z-resveratrol, caffeic acid, chlorogenic acid, cinnamic acid, ellagic
acid, vanillin, gallic acid, ferulic acid, astragalin, isorhamnetin, kaempferol,
scutellarin, rutin and quercetin. The ethanolic extract vas injected 4 times (each

Anti-oxidant activity
The ability of scavenging the free radical 2,2-diphenyl-1- picrylhydrazyl
(DPPH) was assayed according to the method proposed by Ravichandran et al.
[12] with minor modifications. The DPPH 4% stock solution was prepared using
absolute methanol and was stored in the dark. Seven dilutions of the plant extract
were prepared in concentrations of 100, 50, 25, 12.5, 10, 5, and respectively 1

DPPH stock solution. The mixture was left to stand in the dark for 30 minutes,
and the absorbance was measured at 517 nm using a UV-Vis UV-6300PC (VWR)

negative control. The scavenging ability of the extract dilutions were calculated
using the following equation:

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(1)

where A is the absorbance measured at 517 nm. The EC50 (efficient

concentration) is the concentration necessary to inhibit 50% of the DPPH free
radical and was calculated by linear interpolation from the obtained results [13],
[14]. All experiments were run in triplicate and the results are expressed as mean
± SD.
Statistical analysis
The data has been subjected to analysis of variance (ANOVA) using IBM
SPSS Statistics 17 (IBM IBM, Armonk, New York, NY, USA)
RESULTS AND DISCUSSION
Total phenolic content and analysis by HPLC
The total phenolic content of the extract was calculated at 1.0344 ± 0.0237
mg/mL.
After conducting the HPLC analysis on the ethanolic extract, phenolic acids,
flavonoids and glycosides were identified and quantified. The phenolic acids
quantified included gallic acid, chlorogenic acid, caffeic acid, ferulic acid, ellagic
acid and cinnamic acid. All these compounds were identified according to their
retention times and quantified using standard curves.
Other bioactive compounds were also identified, such as kaempferol,
astragalin, quercetin and isorhamnetin, however, only the former and second
compounds could be quantified. The results of the HPLC analysis can be observed
in Table 1.

Table 1. HPLC quantification of bioactive compounds

Polyphenolic acids (mg/mL) Flavonols Glycosides
Injection (mg/mL) (mg/mL)
no. Gallic Chlorogenic Caffeic Ferulic Cinnamic Ellagic Kaempferol Astragalin
Acid Acid Acid Acid Acid Acid
I1
0.5009 0.0028 0.0071 0.0048 0.0037 0.31176 0.0177 0.0929
I2
0.5251 0.0026 0.0058 0.0045 0.0038 0.31934 0.0240 0.0883
I3
0.5083 0.0030 0.0064 0.0048 0.0025 0.30882 0.0160 0.0896
I4
0.5204 0.0032 0.0050 0.0046 0.0030 0.31594 0.0142 0.1010
Average
0.5137 0.0029 0.0061 0.0047 0.0033 0.31396 0.0180 0.0929
SD
0.0110 0.0002 0.0008 0.0001 0.0006 0.00462 0.0042 0.0049
The compounds quantified by the HPLC analysis account for 92.37% of the
total phenolic content of the extract. Given this result, it can be assumed that de
difference can be attributed to phenolic bioactive compounds with different
molecular structures that have not yet been identified.

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The most prevalent phenolic acids in the extracts were gallic and ellagic
acids, with concentrations of 0.5137 ± 0.0110 mg/mL, respectively 0.3139
±0.0046 mg/mL. The glycoside astragalin is also present in relatively high
concentrations, of 0.0929 ± 0.0049 mg/mL. Gallic acid accounts for 49.66% of the
total phenolic content of the extract, and ellagic acid for 30.34%, making both
acids the main bioactive constituents of the ethanolic extract of Sempervivum
ruthenicum Koch.
A similar study was conducted by Stojkovic et al. [15] on the plant species
Sempervivum tectorum Koch, used in Southern Serbia traditional medicine. The
authors used a crude leaf extract and identified several glycosides such as

Also, the authors identified several free organic acids such as maleic acid,
ascorbic acid, citric acid, fumaric acid, succinic acid and oxalic acid, all of which
were not identified in the present study. In contrast, the concentration of astragalin
/g, which is significantly higher.
Anti-oxidant activity
The total scavenging ability of the dilutions was calculated, and the results
can be viewed in Table 2. The EC value for the ethanolic extract was 4.6112 ±
0.08 mg/mL. These results suggest a good scavenging ability of the extract, which
is due to the abundance of phenolic compounds found within.

Table 2. Antioxidant activity after the DPPH assay

Concentration of extract Total DPPH scavenging ability (%) EC50 (mg/mL)
(mg/mL)
100 95.6531±0.1 4.6112±0.08

50 90.4098±0.42

25 78.8070±0.21

12.5 68.5775±0.2

10 62.9418±0.35

5 50.7876±0.62

1 23.5733±0.86

CONCLUSION
Sempervivum ruthenicum Koch is considered a rare plant species in Romania,
and it possesses a unique biochemical composition which cand be exploited. The
ethanolic extract of Sempervivum ruthenicum Koch harvested from the Dobrogea
Region of Romania has a complex phenolic profile with high levels of gallic and
ellagic acids, and a strong anti-oxidant activity which can be exploited both in
traditional medicine and in phytotherapeutic preparations.

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