teza doctorat

1. MINISTRY OF EDUCATION
UNIVERSITY OF ORADEA
FACULTY OF MEDICINE AND PHARMACY
DOCTORAL SCHOOL OF BIOMEDICAL SCIENCES
Doctoral field: PHARMACY
DIANA IOANA GAVRA
DOCTORAL THESIS
PHYSICO-CHEMICAL CHARACTERIZATION
AND EVALUATION OF THE THERAPEUTIC
POTENTIAL OF THE FRACTIONS EXTRACTED
FROM ENDEMIC PLANTS
Scientific coordinators:
Prof. univ. dr. Tünde Jurca
Prof univ. dr. Ildikó Bácskay
ORADEA
2022
Tarca Radu Catalin
Aprob acest document
18/10/2022 13:51:42 UTC+02

2. 2
Contents
Objectives and structure of the thesis ........................................................................................8
Introduction................................................................................................................................9
FIRST PART - LITERATURE STUDY.................................................................................10
I. Current state of knowledge...................................................................................................10
I.1. Structural characteristics of polyphenols.......................................................................12
I.2. Extraction of phenolic compounds................................................................................14
I.3. Determination of the total polyphenols and flavonoids content....................................15
I.4. Evaluation of antioxidant activity..................................................................................15
I.5. Pharmacological properties of polyphenols ..................................................................16
I.5.1. Fam. Asteraceae......................................................................................................16
I.5.2. Fam. Malvaceae......................................................................................................21
I.5.3. Fam. Rosaceae........................................................................................................22
I.5.4. Fam. Ericaceae........................................................................................................24
I.5.5. Fam. Hypericaceae .................................................................................................25
I.5.6. Fam. Lamiaceae......................................................................................................26
I.5.7. Fam. Canabaceae....................................................................................................29
I.5.8. Fam. Grossulariceae ...............................................................................................29
I.5.9. Fam. Urticaceae......................................................................................................30
I.5.10. Fam. Papaveraceae ...............................................................................................31
I.6. Alternative treatment for skin diseases..........................................................................32
I.6.1. Psoriasis prevalence....................................................................................................32
I.6.2 Psoriasis classification, pathogenesis and treatment ...................................................32
I.6.3. Alternative treatment for psoriasis .............................................................................34
I.6.4. Psoriasis evaluation on clinical trials..........................................................................35
I.7. Pharmaceutical forms used in the treatment of psoriasis ..............................................36
I.8. Partial conclusions.........................................................................................................38
SECOND PART - EXPERIMENTAL STUDY......................................................................39
II. Phytochemical screening of ethanolic extracts of Rosa species .........................................39
II.1. Introduction and objectives of the chapter ...................................................................39

3. 3
II.2. Materials and methods .................................................................................................41
II.2.1. Reagents and plant material ..................................................................................41
II.2.2. Preparation of the extracts.....................................................................................41
II.2.3. Total polyphenols content (TPC)..........................................................................42
II.2.4. Total flavonoid content (TFC) ..............................................................................42
II.2.5. Analysis of phenolic compounds by HPLC ..........................................................42
II.2.6. Determination of antioxidant capacity..................................................................43
II.3. Results..........................................................................................................................44
II.4. Discussion ....................................................................................................................55
II.5. Partial conclusions........................................................................................................57
III. The evaluation of the antimicrobial and antioxidant activity of the bioactive compounds of
Rosa x damascena Mill............................................................................................................58
III.1. Introduction and objectives of the chapter..................................................................58
III.2. Materials and methods................................................................................................58
III.2.1. Reagents...............................................................................................................58
III.2.2. Microscopic Examination of Rosa x damascena Mill. petals..............................58
III.2.3. Preparation of the extract.....................................................................................59
II.2.4. Statistical analysis .................................................................................................59
III.2.5. Antimicrobial activity..........................................................................................59
III.2.6. SOD-like activity .................................................................................................60
III.2.7. Cytotoxicity assays and cell culture.....................................................................60
III.3. Results.........................................................................................................................61
III.4. Discussion...................................................................................................................65
III.5. Partial conclusion........................................................................................................68
IV. In vitro and human pilot studies of different topical formulations containing Rosa species
for the treatment of psoriasis....................................................................................................69
IV.1. Introduction and objectives of the chapter..................................................................69
IV. 2. Materials and methods...............................................................................................70
IV.2.1. Preparation and characterization of dry extract...................................................70
IV.2.2. Formulation and Investigation of Self-Nano-Emulsifying Drug Delivery System
..........................................................................................................................................71

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IV.2.3. Formulation of Ointments containing lyophilized Rose extracts and Rose-
SNEDDS..........................................................................................................................72
IV.2.4. Texture analysis...................................................................................................73
IV.2.5. In vitro release Studies.........................................................................................73
IV.2.6. Superoxide Dismutase (SOD) Assay...................................................................74
IV.2.7. Cell viability study (MTT assay).........................................................................74
IV.2.8. Clinical study.......................................................................................................75
IV.3. Results.........................................................................................................................75
IV.3.1. Investigation of Self-Nano-Emulsifying Drug Delivery System.........................75
IV.3.2. Ointment formulation ..........................................................................................76
IV.3.4. Cell viability study (MTT assay).........................................................................77
IV.3.5. Texture analyzis...................................................................................................78
IV.3.6. In vitro release studies .........................................................................................78
IV.3.7. Superoxide dismutase activity of the topical formulations..................................80
IV.3.8. Clinical investigation...........................................................................................81
IV.4. Discussion...................................................................................................................93
IV. 5. Partial conclusions.....................................................................................................98
General conclusions.................................................................................................................99
The originality of the thesis ...................................................................................................100
Future perspectives ................................................................................................................101
References..............................................................................................................................102
Annexes..................................................................................................................................120
Annex 1. Decision of the research of Ethics Commission of Faculty of Medicine and
Pharmacy............................................................................................................................120
Annex 2. Application for approval of clinical research to SC Echo Laboratoare SRL.....121
Annex 3. Application for approval of clinical research to CMI Dr. Frățilă Simona .........123
Annex 4. Application for approval of clinical research to CMI Dr. Endres Laura............125
Annex 5. Protocol for clinical study ..................................................................................127

5. 5
ABBREVIATIONS
AA – ascorbic acid
ABTS – (2, 2 – azinobis – (3 – ethylbenzthiazoline – 6 – sulfonic acid)
AE – aqueous extract
BHT – a synthetic antioxidant
C3GE – cyanidin – 3 – glucoside chloride
CAE – caffeic acid equivalents
CAT – catalase
CUPRAC – Cupric Reducing Antioxidant Capacity
CY – cyaniding
DLQI – Dermatology Life Quality Index
DM – dry mass
DPPH – 2, 2 – diphenyl – 1 – picryl – hydrazyl – hydrate
DW – dry weight
E – ethanolic extract
EC – Equivalent cyanidin
FRAP – ferric reducing antioxidant power assay
FTC – ferric thiocyanate method
FW – fresh weight
GA – gallic acid
GAE – equivalent of gallic acid
GPP – Generalized Pustular Psoriasis
HAPX – hemoglobin ascorbate peroxidase activity inhibition assay
HAT – Hidrogen atoms transfer
HORAC – Hydroxyl radical antioxidant capacity
HPLC – high performance liquid chromatography
HPLC-MS - high performance liquid chromatography coupled with mass spectroscopy
HPTLC - high performance thin layer chromatography
IC50 – half-maximal inhibitory concentration
IPM – isopropyl myristate
LC-MS – Liquid chromatography-Mass spectrometry
MRSA – methicillin-resistant Staphylococcus aureus
MTT – (3 – (4, 5 – dimethylthiazol – 2 – yl) – 2, 5 – diphenyltetrazolium bromide)

6. 6
NPF – National Psoriasis Foundation - Psoriasis Score
ORAC – Oxygen Radical Antioxidant Capacity
PASI – Psoriasis Area and Severity Index
PGA – physician global assensment
Px – peroxidase activities
QE – equivalent quercetine
QoL – quality of life
RE – rutin equivalents
RU – rutin
SET – single electron transfer
SOD – superoxide dismutase
SNEDDS – Self-Nano-Emulsifying Drug Delivery System
TAC – total anthocyanin content
TE – trolox equivalents
TFC – total flavonoid content
TPC – Total polyphenol content

7. 7
List of published articles
1. Gavra D.I., Pallag A., Marian E., Vicaș L., Jurca T. A comparative study of the amount
of polyphenols, flavonoids and antioxidant capacity of Rosae caninae flos versus
cynosbati fructus. Annals of the University of Oradea, Fascicle: Environmental
Protection, vol. XXXI, 2018, p.23-30. B+
2. Gavra, D.I.; Marian, E.; Pallag, A.; Vicaș, L.G.; Lucaciu, R.L.; Micle, O.; Ionescu, C.;
Bacskay, I.; Hangan, A.C.; Sevastre, B.; et al. Phytochemical Screening and Biological
Activity of Ethanolic Extract of Rosa X damascena Mill. Cultivated in the Western
Region of Romania. Farmacia 2022, 70, 248–257. IF: 1.433
3. Gavra, D.I.; Endres, L.; Pető, Á.; Józsa, L.; Fehér, L.; Ujhelyi, Z.; Pallag, A.; Marian,
E.; Vicas L.G.; Ghitea T.; Muresan M.; Bácskay I.; Jurca T. In vitro and human pilot
studies of different topical formulations containing Rosa species for the treatment of
psoriasis. Molecules 2022, 27, 5499. IF: 4.148

8. 8
Objectives and structure of the thesis
The aim of this thesis was to characterize from a physico-chemical point of view and
to evaluate the therapeutic potential of fractions extracted from endemic plants in order to
develop new pharmaceutical preparations that can be used as prophylactic treatment or as
adjuvant treatment in various chronic conditions.
The main objectives of the study are: the extraction of polyphenolic compounds from
petals, the qualitative and quantitative determination of the phytochemicals of 11 Rosa sp., as
well as the antioxidant capacity of the biocompounds found in the alcoholic and lyophilized
extracts and the development of new pharmaceutical formulations using lyophilized extracts
from petals and the evaluation of their effectiveness.
This study is structured in two parts, namely: in the first part, is presented the current
state of knowledge regarding the chosen research topic, and in the second part of the study, the
original experimental part is presented.
Chapter I presents literature study regarding the extractions of phenolic compounds,
determination of the total polyphenols and flavonoids content, methods used for the evaluation
of antioxidant activity, pharmacological properties of polyphenols and structural
characteristics. In the same chapter are presented informations about the classification and
pathogenesis of psoriasis, the conventional and alternative treatment, about clinical trials and
formulations used in the treatment of psoriasis.
Starting with the second chapter, the experimental part is presented, which refers to the
presentation of the materials and methods used for the extraction of polyphenolic compounds
from petals of 11 Rose species, their qualitative and quantitative analysis and the determination
of antioxidant activity of the identified compounds.
In chapter III are presented the materials, methods and the preparation of the extract in
order to evaluate the antimicrobial and antioxidant activity of the bioactive compounds of Rosa
x damascena Mill.
Chapter IV reffers to the extensively analysis of 3 from 11 Rose sp. studied, regarding
the development of different topical formulations for the treatment of psoriasis and clinical
investigations to evaluate the effectiveness of the topical preparations.

9. 9
Introduction
During last decade was observed an increase of the interest for usage of medicinal plants
into the traditional medicine due to their content in active substances, which can represent an
alternative source for the development of new drugs. Active compounds of plant origin are
used worldwide for medicinal purposes, with the World Health Organization recognizing the
traditional use of medicinal plants in primary health care since 1992, and currently seeing a
significant increase in this use.
In maintaining human health, reactive oxygen species and free radicals play an
important role and when the equilibrium between the generating and scavenging of reactive
oxygen species and free radicals is destroyed, an oxidative stress may happen and it might lead
to extensive oxidative damage to important cellular biomolecules, such as proteins, DNA, and
lipids.
Polyphenols - flavonoid, lignans and polymeric lignans, stilbene, are a very large and
varied group of bioactive compounds, secondary metabolites of plants that act as a barrier
against ultraviolet radiation, oxidants and pathogens, which due to their therapeutic properties,
have in recent years aroused an interest in their scientific research, food production and
consumption.
Studies from scientific literature have shown that a high antioxidant intake is associated
with a lower risk of developing disorders such as cancer, cardiovascular disease, Parkinson,
different skin diseases etc. In the last years, several dietary and natural formulations that have
free radical scavenging capacity have obtained attention in treating many chronic diseases.
Despite of the strong radical scavenging activity of synthetic compounds, they usually have
side effects and the interest to find natural antioxidants, without undesirable side effects, has
increased. The antioxidative compounds, especially the phenolic ones found in plants, flowers,
fruits and seeds, have received attention for their potential role in the prevention of human
affections.
Plant materials obtained from medicinal herbs and flowers are very successful in the
pharmaceutical industry and offer new perspectives, suggesting important innovative
implications for existing clinical medicine.

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FIRST PART - LITERATURE STUDY
I. Current state of knowledge
For thousands of years, plants have been and continue to be an important resource in
medicine. Even today, the World Health Organization estimates that up to 80% of people still
rely on traditional remedies [1].
Medicinal plants and edible flowers are one of the main sources of new pharmaceutical
products. A whole range of nutritional supplements derived from herbs helps maintain health
and fight diseases. The role of medicinal plants in the prevention or control of diseases has
been attributed to the antioxidant properties of their constituents, generally called polyphenolic
compounds [2].
Different edible flowers as Centaurea cyanus L., Viola odorata L., Chrysanthemum
morifolium Ramat., Lavandula sp., Rosa sp., Calendula officinalis L. and other species
represent an interest for researchers and many species are deeper evaluated because of their
bioactive compounds .
Polyphenols (flavonoid, lignans and polymeric lignans) are phytochemical compounds
found in plants, coffee, vegetables, flowers, wine, fruits, tea. A large number of polyphenols
including phenolic acids, flavonoids, lignans, etc. have been identified [3]. These compounds
are secondary metabolites of plants and flowers that act as a barrier against UV radiation,
various oxidants and pathogens [4]. Based on therapeutic properties, in recent years, numerous
studies have focused on finding new sources, especially in the food and pharmaceutical
industry [5].
The dietary intake of polyphenols is estimated to be approximately 1 g of polyphenolic
compounds/day [6, 7]. The bio-availability of these bio-active compounds depends on the
process of extraction, gastrointestinal digestion, absorption and metabolism [8]. For absorption,
polyphenols are hydrolyzed by intestinal enzymes or microflora in the colon and then
conjugated in the intestinal cells and subsequently in the liver by methylation, sulfation or
glucuronidation [9]. Therefore, polyphenols are distributed in the body, accumulate in the
target tissue and induce biological properties; metabolites are mainly eliminated through bile
and urine.
Various studies in the literature have shown that these polyphenolic compounds have
rapid plasma absorption, with peak plasma concentrations within 2 – 3 hours after ingestion
[10]. Their biological activity has been proven by various properties: antioxidants, anti-allergic,
anti-inflammatory, antiviral and antimicrobial, anti-proliferative, anti-mutagenic, anti-

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carcinogenic, blocking free radicals, regulating cell cycle arrest, apoptosis; more interestingly,
polyphenols can modulate several important cell signaling pathways, such as kappa-B nuclear
factor, transcription factor for activation of proteins, extracellular signal-regulated protein
kinase, (phosphoinositide 3) kinase B/protein kinase (Akt), mitogen-activated protein kinases,
and nuclear factor 2 associated with erythroid nuclear factor 2 (Nrf2) [11].
The human body presents various endogenous and exogenous systems to defend against
the harmful action of free radicals. There are two main mechanisms by which the body acts,
which involves the action of antioxidant compounds: the first mechanism refers to enzymatic
protection (superoxidismutase – SOD – which catalyzes the reaction of dismutation of anions
superoxide to peroxide, catalase – CAT – which converts hydrogen peroxide into molecular
oxygen and water); a second mechanism refers to the action of compounds with antioxidant
capacity, without enzymatic activity, such as polyphenolic compounds, ascorbic acid,
carotenoids, etc. [12]. Polyphenols are the main compounds found in medicinal plants and have
the property of neutralizing free radicals. As epidemiological studies have shown that
polyphenols have beneficial effects on human health, in recent decades, research interest for
these compounds has increased [13]. Various herbal preparations, consumed in correct
combinations and quantity, captures free radicals before determining the appearance of
different diseases in the human body [14].
For this purpose, many plant and flower extracts are used in nutritional supplements.
Various researchers have prepared herbal supplements containing this type of compounds in
order to improve the quality of life of people with chronic diseases or to combat or prevent
many diseases. Researchers [14] selected several plant, flower and fruit products, such as
Vaccinium fructus, Hippophae Rhamnoides fructus, Salviae folium and Calendulae flos and
showed their antioxidant ability by quantifying the phenolic content using appropriate methods.
Polyphenols and their derivatives have biological and pharmacological properties, such
as hepatoprotective, diuretic, spasmolytic, antioxidant, anti-allergic and anti-cancerous
properties [15, 16]. It has been shown that a wide spectrum of diseases can be treated with the
help of herbs, flower and fruits which are found useful due to their therapeutic properties. Also,
about 35% of medicines are of plant origin. In most developing countries, the use of medicinal
plants in traditional medicine has brought about an improvement in the quality of life of people
[17].

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I.1. Structural characteristics of polyphenols
Polyphenols are a large and varied group of aromatic benzene ring compounds with one
or more hydroxyl groups, produced by plants mainly for protection against oxidative stress,
synthesized during plant development [18, 19] and in response to certain adverse conditions
(UV radiation, etc.) [4, 20].
Phenolic compounds can be classified into many categories depending on the number of their
phenolic rings and based on the structural elements that binds the rings to each other [21],
including simple phenols, phenolic acids, coumarins, flavonoids, stilbene, up to for
hydrolysable and condensed tannins, lignans and lignins.
Phenolic acids comprise two main branches: hydroxybenzoic acid derivatives
(protocatechic acid, gallic acid, p-hydroxybenzoic acid) and hydroxycinnamic acid derivatives
(caffeic acid, chlorogenic acid, p-coumaric acid, ferulic acid, synapic acid). Palm, kiwi, cherry,
apple, pear, chicory and coffee fruits are foods with high phenolic acids content [10]. Caffeic
acid, p-coumaric, vanillic, ferulic and protocatechic acids are present in almost all plants [20,
22].
Figure 1. Phenolic acids [23]
Flavonoids are the most abundant polyphenols in the human diet and over 4000 types
have been identified. These compounds have at least two phenol sub-units, and compounds
having three or more phenol sub-units called tannins (hydrolyzable and non-hydrolyzable).
Flavonoids are planar molecules, ubiquitous in plants, consisting of aromatic amino-acids,
phenylalanine, tyrosine and malonate [18, 19]. Flavonoids are the most abundant polyphenols
in the human diet and over 4000 types have been identified. These compounds have at least
two phenol sub-units, and compounds having three or more phenol sub-units, as hydrolyzable
or non-hyrolizable tannins. Flavonoids are planar compounds, present in plants, consisting of
aromatic amino-acids with aromatic structure as phenylalanine aminoacid, tyrosine and

13. 13
malonate aminoacids. The flavan nucleus is the basis of flavonoid structure and it has 15 carbon
atoms that are arranged in many rings (C6-C3-C6).
Figure 2. Flavoid structures [23]
Biocompounds as isoflavones, flavonones, flavonols, anthocyanins, flavanols and
flavonones are the six subclasses of flavonoids. In the family of red fruits (strawberry, cherry,
berries, etc.) we can found anthocyanins as malvidin, cyanidin compound, delfinidine or
pelargonidine. Flavonols, including quercetin, kaempferol and mirycetin, were mainly detected
in onions, leeks, broccoli and blueberries. Isoflavones are the most important dietary flavonoids
that include daidzein, genistein and glycytaine. Stilbenes appear in the human diet in small
quantities; resveratrol, one of the well-studied compounds in these groups, is largely detected
in grapes and red wine [3, 10, 24].

14. 14
Figure 3. The structures of sub-classes of flavonoids [23]
I.2. Extraction of phenolic compounds
Extraction of phenolic compounds from plant, flowers or fruit material is influenced by
their chemical nature, extraction time and drying conditions, as well as by the presence of
interfering substances. Solvent extraction is frequently used for the extraction of phenolic
compounds from plants due to their ease of use, efficiency and wide applicability. Extraction
depends on the type of solvent, the polarity of the solvent, the extraction time and temperature,
as well as the chemical composition of the samples.
A wide range of solvents, such as water, acetone, methanol, ethanol, N, N –
dimethylformamide (DMF) or their mixtures with water, have been studied for their extraction
efficiency due to polarity differences [25-27]. Hydrophilic polyphenols, including aglycones,
glycosides and oligomers, are extracted using water, polar organic solvents such as methanol,
ethanol, acetonitrile and acetone or their mixture with water. Polyphenols are more stable at
low pH, because the acidic medium helps the polyphenols to remain neutral, so they can be
easily extracted into organic solvents [6, 7]. The use of an alcoholic solution ensures a
satisfactory extraction.
Methanol, acetone and water are inefficient solvents for the total extraction of phenols
from vegetal products, because polyphenols are associated with other bio-molecules such as
proteins, polysaccharides, terpenes, chlorophylls, lipids and inorganic compounds. However,

15. 15
methanolic extracts seems to be better for the extraction of catechins, epicatechin and
epigalocatechin [28]. Aqueous mixtures of acetone are good solvents for polar polyphenols,
and the ballast substances remain in extracts [27]. The low solubility of polyphenols in absolute
organic solvents is due to the strong hydrogen bonds between polyphenols and proteins. The
increase of solubility by adding water to organic solvents is due to weakening of hydrogen
bonds in aqueous solutions [29].
I.3. Determination of the total polyphenols and flavonoids content
In the literature, a method used for determination of the total phenol content using
different solvents is the Folin-Ciocâlteu method. The Folin-Ciocâlteu reagent contains
molybdenum in a higher oxidation state (+6) and has a yellow color. Polyphenolic compounds
cause Mo6+
reduction at lower oxidation states (Mo4+
, Mo5+
) having a blue color [30].
Polyphenols can be determinated by various techniques, for example: nuclear magnetic
resonance spectroscopy, near-infrared reflection spectroscopy, high performance thin layer
chromatography (HPTLC), liquid chromatography coupled with mass spectroscopy,
electrophoresis high performance capillary and high performance liquid chromatography
(HPLC). In addition to the extraction with organic solvents, qualitative and quantitative
analysis techniques can be applied, as well as isolation and purification procedures for specific
results. Chromatographic techniques, such as thin layer chromatography (TLC), HPLC, gas
phase chromatography (GC) and later capillary electrophoresis (EC), column chromatography
(CC) over Sephadex LH-20 are used for final purification, because are obtained clear solutions
without residues [6, 7, 25].
Most of the results reported in the literature for the TFC were obtained using the AlCl3
colorimetric method. The principle on which the method is based is that AlCl3 forms stable
complexes with carbonyl groups (keto – C – 4 and/or with hydroxyl group C – 3 or C – 5) of
flavones and flavonols and with ortho-dihydroxyl groups of ring A or B of flavonoids, the
absorbance of which can be measured using a spectrophotometer at a corresponding
wavelength.
I.4. Evaluation of antioxidant activity
Many researches are focused on antioxidant activity, TFC and TPC of medicinal plants,
but there are still plants that have not been studied [31].
The methods used to determine the antioxidant capacity can be divided into two major
groups: SET-single electron transfer methods and HAT (Hidrogen atoms transfer) methods

16. 16
[32]. These tests are commonly used to measure the antioxidant capacity of the extracts, but
not a single test will reflect all the antioxidants present.
A schematic classification of the methods for determining the antioxidant capacity is given in
figure 4.
Figure 4. Methods used to determine the antioxidant capacity
I.5. Pharmacological properties of polyphenols
In order to study the beneficial contribution of polyphenols, in this section was
highlighted the relative concentration of the bioactive compounds of some plant extracts
reported in literature, the methods used for their detection and quantification, as well as their
antioxidant capacity. For this purpose, were selected plants with medicinal uses, frequently
used in various disorders.
I.5.1. Fam. Asteraceae
Arnica montana L. (Fam. Asteraceae) is strictly protected in several European countries
and, it is a medicinal plant of high commercial value. Alcoholic extract of the inflorescences is
traditionally used to treat damages and bruises [33]. The plant is rich in lactone sesquiterpenes,
phenolic acids, flavonoids and essential oils responsible for the pharmacological properties –
antioxidants, antiseptics, anti-inflammatories, antibacterials properties [33, 34]. Phenolic
compounds and flavonoids are among the main components of A. montana L., the total
polyphenol content and antioxidant activity of A. montana L. extracts have been reported in
several publications [35-37]. Studies have shown that the phenolic profile of A. montana L.
depends on the environmental conditions [38, 39]. Some data about A. montana L. from the
studies published in literature are reproduced in Table 1.
Artemisia absinthium L. (Asteraceae) is an aromatic-bitter plant that has anthelmintic
[40], antimycotic [41], antimicrobial [42] activity, antiseptic, diuretic and can be used in the

17. 17
treatment of leukemia and sclerosis [43, 44]. Antioxidant activity has been reported for A.
absinthium L. essential oil [41, 42] and for methanolic extract (by DPPH and hydroxyl radical
neutralization methods) [43, 44].
The methanolic extract from A. absinthium L. at was tested for antioxidant properties
using many test systems. By forced swimming tests and also, using tail suspension tests was
determined antidepressant activity.
Consumption of different types of edible flowers offers health benefits to the consumer,
as they represent a good source of phytochemicals, including phenolic compounds [45] that
can be used to prevent chronic degenerative diseases, such as diabetes, decline cognitive and
cardiovascular diseases, as well as different types of cancer [46, 47]. The literature data show
that Calendula officinalis L. (syn. Mariogold) and Centaurea cyanus L. are among the most
popular edible flowers.
Due to the content of flavonoid and phenolcarbonic acids, Achillea millefolium L. has
been used for many affections as gastrointestinal and spasmodic disorders, skin inflamations.
Among the many beneficial properties of this plant are cytotoxic, choleretic, antimicrobial and
anti-inflammatory activity [48].
Alghazeer R. et al. [49] studied TPC and TFC in methanolic and aqueous extracts from
Cynara scolymus L. rhizomes from Libya, rhizomes used in various popular medicines. The
root is used in adjuvant drugs with antihypertensive, antidiabetic and hypocholesterolemic
effect. The activity of neutralizing the free radicals of the extracts was investigated and
compared with the ascorbic acid. The antibacterial activity of extracts from C. scolymus L.
rhizomes, tested in vitro against a wide spectrum of microorganisms, may be related to their
TFC and TPC [50].
Antimicrobial activity may be attributed to the presence of phenolic compounds,
chlorogenic acid, caffeic acid, cinarine, and four flavonoids, luteolin – 7 – rutinoside,
cyarozide, apigenin – 7 – rutinoside [51]. A medicinal plant widespread in the cultivated flora
of Romania is Chrysanthemum parthenium L. whose active principles are sesquiterpene
lactones, essential oils, polyphenolic compounds. It is traditionally used for the treatment of
fever, headache, migraines, rheumatoid arthritis, stomach pain, tooth pain, insect bites and
infertility. It has multiple pharmacological properties, including anticancer, anti-inflammatory,
cardiotonic, antispasmodic and antioxidant properties [35, 52-55]. It was compared the
antioxidant activity of ethanolic extract of C. parthenium L. flowers from Bulgaria [31] and
from aerial parts of C.parthenium L. from Romania, and ethanolic extract from flowers had
higher values than aerial parts.

18. 18
A wide range of pharmacological properties is presented by silymarine isolated from
fruits and seeds of milk thistle (Silybum marianum L.), which is a mixture of three structural
components: silybinin, silyldianine and silychristine. Milk thistle is part of the Asteraceae
family [56] and has multiple pharmacological activities, including antioxidant,
hepatoprotective and anti-inflammatory, antibacterial, anti-allergic, antimutagenic, antiviral,
anti-neoplastic, anti-thrombotic, and vasodilating action [25, 57]. Researchers [58] have
suggested that silymarine can be used to prevent free radicals as a natural antioxidant dietary
supplement. The constituent flavonoids are: catechin, myrcetin, quercetin, naringin,
naringenin, resveratrol, rutin. Taxifoline is the only isomer of silymarine which is known for
its strong antioxidant activity [59]. DPPH was used to evaluate the ability of the individual
components of silymarine compared to the crude mixture of silymarine to act as free radical
capture agents by determining their EC50 (lower values indicate a higher radical absorption
power). The EC50 value determined for taxifoline was 32 µM. Isosilibine A (EC50=855 µM)
and B (EC50=813 µM) were the least active of the seven components in the free radical capture.
Silihristine and silydianine were moderately active, but more active than other tested isomers.
It was determined that taxifoline is the most effective antioxidant in ORAC assay with a Trolox
equivalent=2.43 and that it has a good antioxidant capacity determined by the HORAC
(Hydroxyl radical antioxidant capacity) method with a GAE=0.57. Other antioxidant assays
did not show significant differences between samples [60].
Table 1. Research results of plants from Asteraceae family
Scientific
name of the
plant
TPC, TFC
Antioxidant
activity
Observations References
Arnica
montana L.
TPC – 221.80 mM
GAE/g DW
TPC – 247.00 mM
GAE/g DW.
Not described
the dried plant product at room
temperature.
the dried plant product at
105°C.
[37]
TPC1 – 23.65±1.6 mg
GAE/g extract
TPC2 – 24.78±1.1 mg
GAE/g extract
TFC1 – 1.48±0.8 mg
RE/g extract.
TFC2 – 1.49±0.7 mg
RE/g extract
1 – IC50>200
mg/mL.
2 – IC50>200
mg/mL.
1 – methanolic extract from
leaves of multiple plants
2 – methanolic extract from
leaves of rooted plants.
[61]
3 wild
species of A.
Montana L.
(flowers,
leaves, roots,
rhizomes)
TPC as caffeic acid are
extracts of roots and
rhizomes – 7.7839 –
15.9098% w/w d.m.
obtained with solvent
ethanol 70 – 80% v/v
under reflux extraction
TFC as hyperoside was
obtained from the
Not described
Ethanolic extract (30 – 70%
v/v), by reflux.
[62]

19. 19
extraction of flowers
and leaves – 2.578–
2.7518% w/w d.m. with
solvent ethanol 50 –
70% v/v in conditions
of reflux.
A. absinthium
L (aerial
plants)
TPC=194.9 ± 9.7 mg
EGA/g extract.
TFC=12.4 ± 0.6 mg
QE/g extract.
DPPH: IC50 = 612
± 30.6 µg/mL.
Low nitrogen
oxide activity =
0.4 - 3.2 mg/mL.
IC50 was 1.77 ±
0.08 mg/mL.
Methanolic extract
The IC50 for H2O2 = 243 ±
12.15 µg/mL. Ascorbic acid:
IC50 = 21.4 ± 1.07 BHA: IC50
=52.0 ± 2.6 μg/mL. Tested
extract showed a good
chelating capacity of Fe2+
(IC50= 419 ± 20.95 μg/mL).
EDTA: IC50=
18 ± 0.05 μg/mL).
[63]
Calendula
officinalis L.
Lyophilized extract
TPC=151.62 ± 0.03 mg
GAE/100 g DW
TFC=266.62 mg
QE/100 g DW.
Alcoholic extract
TPC = 112.42 ± 0.01
mg GAE/100 g DW.
TFC = 42.12 ± 2.41 mg
QE/100 g DW
DPPH=4.17 ±
1.31%;
ABTS=6.68 ± 1.14
mmol TE/g DW
FRAP=15.01 ±
0.03 mmol TE/g
DW.
Other researchers found a
TPC= 15.12 mg GAE/g, and
TFC=5.13 mg rutin/g DW.
In the hydroalcoholic extract
(ethanol-water 50/50) of the
yellowish petals, TPC was
29.79 mg GA/g and 45.13 mg
GA/g using aqueous extract.
All extracts showed a strong
activity of DPPH radical
capture (27.31 - 96.35%).
[64]
[65]
[66]
[67]
Centaurea
cyanus L. and
Calendula
officinalis L.
TPC of Centaurea
cyanus L. (718.81 ±
1.12 mg GAE/100g
DW) is higher, but TFC
is higher in Calendula
officinalis L. (4.21 ±
1.05 mg QE/100g DW).
Calendula off. L.
FRAP=129.5 mg
TE/mL.
DPPH=41.70%
CUPRAC=1.56
mmol Trolox/100
g DW.
Centaurea cyanus
L.
FRAP=78.01 mg
TE/mL.
DPPH=83.42%
CUPRAC=1.86
mmol Trolox/100
g DW.
Centaurea cyanus L. extracts
registered a higher SOD-like
activity compared with
Calendula officinalis L.
Studies showed that phenolic
acids were more abundant in
the non-edible part of
Centaurea (5.5 ± 0.002 mg
spigenin–O–7–glucoside/g
extract) than in flowers (0.134
± 0.003 mg chlorogenic acid/g
extract).
[68]
[69]
Alchillea
millefolium
L.
TPC 58 – 64.5 mg
quercetin/100 grams of
leaves.
Phenolic acids were
more abundant in the
non-edible part of
Centaurea (5.5±0.002
mg spigenin–O–7–
glucoside/g extract)
than in flowers (0.134 ±
0.003 mg chlorogenic
acid/g extract.
DPPH=17.82–
18.31%.
Leaf extract of A. millefolium
L., using water/acetonitrile
(70/30) as solvents.
[70].
Not described.
DPPH=308.8
µmol/g TE
Inhibition of H2O2 generation
in the mitochondria of the heart
rat was 45%.
[71]

20. 20
Not described.
ABTS=97.40% for
ethanolic extract
from flowers;
ABTS=55.76% for
aqueous extract of
seeds.
DPPH= 91.03%
for ethanolic
extract of flowers
ethanolic extract
of seeds (79.94%).
Aqueous and ethanol extracts
of the flowers, leaves and seeds
of A. millefolium L.
[72]
Aqueous extract
(flowers, leaves and
seeds)
TPC= 74, 134, 78 mg
QE/g and 18.82, 19.30,
20.25 mg pyrocatechol
equivalent/g DW.
Ethanolic extract
(flowers, leaves and
seeds)
TPC=128, 70 and 126
mg QE/g DW and
18.34, 19.78 and 18.82
mg pyrocatechol
equivalent/g DW.
Hydrogen
peroxide
scavenging
capacity: 17.75–
40.63% for 100 μg
extract were
obtained.
At 100 μg/mL, A.
millefolium L.
extracts inhibited
90.31-92.09%
lipid peroxidation
of
linoleic acid
emulsion.
Flavonoids/g of aqueous
flower extract were
determined: 52 μg rutin, 24 μg
resveratrol, 2 μg morin, 54 μg
myricetin, 529 μg naringin, 12
μg naringenin and 673 μg total
flavonoids. From one gram of
aqueous leaf extract were
determined 979 μg rutin, 53 μg
resveratrol, 1797 μg naringin,
11 μg quercetin and 2840 μg
total flavonoids.
[7]
Cynara
scolymus L.
Methanolic extract:
TPC=45.11 mg GAE/g
DW TFC=37.00 mg
rutin/g.
Aqueous extract:
TPC=37.79 mg GAE/g
DW TFC=15.51 mg
rutin/g DW and IC50 =
66.3 µg/mL,
respectively.
Methanolic
extract:
IC50 = 17.77
μg/mL
Aqueous extract:
IC50 = 66.3 µg/mL
Isolated flavonoids exhibited
the highest free radical
neutralization activity (IC50 =
13.33 µg/mL).
[49]
FTPC=14.16 mg/g
DW (for bracts) and
9.06 mg/g DW (for
heart).
TFC=9.85 mg/g DW
(for bracts).
TFC=5.91 mg/g DW
(for heart).
Not described.
The inner and outer part of
artichoke contain a small
amount of bound phenolic
compounds (5.35 and 4.2 mg/g
DW, respectively.
Artichoke extract contains 8.1
mg tannic acid/g DW.
[73]
[74]
C.
parthenium
L.
TPC = 152.8 ± 0.8 mg
GAE/g essential oil.
DPPH = 73.8 ±
1.3%
HPLC: synapic (3.86 ± 0.1
mg/g DW) and ferulic acid
(2.59 ± 0.1 mg/g DW).
Essential oil of T. parthenium
L. with Iranian origin contains
camphor (43.97%),
chrysanthenyl acetate
(12.46%), farnesol (7.54%),
and fatty acids: palmitic acid
(57.27%) and myristic acid
(14.7%).
[75]
TPC=3.48 ± 0.17 g
GAE/100 g plant
product;
DPPH: IC50
(μg/mL): 149.76 ±
6.23;
HPLC-MS revealed the
presence of phenolic acids
(gentisic acid, caffeic acid and
[76]

21. 21
TFC=1.27 ± 0.07 g
RE/100 g plant product.
Caffeic acid
derivatives=1.30 ± 0.11
g CAE/100 g plant
product.
SNP: 29.85 μmol
GA/g plant
product, EPR:
106.717 μmol
GA/g plant
product.
chlorogenic acid), in
concentrations lower than 0.2
μg/g plant product and
flavonoid aglycones:
quercetin, in the highest
quantity (27.61 ± 0.39 μg/g
plant product), apigenin (9.71
± 0.18 μg/g plant product).
I.5.2. Fam. Malvaceae
The use of plants from the Malvaceae family for herbal therapy is very common in the
Middle East, one of the examples being Althaea species. Althaea officinalis L. is native to Asia,
Europe and the United States. Althaea rosea L. is a popular garden plant, native to China, South
Europe, the Middle East, the Near East, the Mediterranean Sea and Central Asia [77]. Studies
presented in literature have shown that Althaea officinalis L. has antimicrobial, anti-
inflammatory, immuno-modulatory effects, is used for the demulcent and soothing, anti-tussive
and many other pharmacological effects. Althaea rosea L. had numerous pharmacological
effects, such as antimicrobials, antiestrogens, cytotoxic and immunomodulatory,
cardiovascular and is also used to prevent urolithiasis [77].
Dudek M. et al. [78] investigated the distribution of phenolic acids in the flowers of
Althaea rosea L. variety black using the methanolic and methanolic-aqueous extracts of entire
flowers, petals and calyx of Althaea rosea L. and found that the plant contains cinnamic acids
(ferulic, p-coumaric, caffeic), benzoic acids (p-hydroxybenzoic, vanillic, syringic) and p-
hydroxyphenylacetic acid. P-coumaric, syringic and p-hydroxybenzoic acids were detected in
almost all fractions. In the petals were found almost all phenolic acids detected (except caffeic
acid in methanolic extract, syringic and p-hydroxyphenylacetic acids in methanolic-aqueous
extract). In calyx, vanillic and p–hydroxyphenylacetic acids were not found. The total content
of phenolic acids in flowers was 60 mg%, in petals 120 mg% and 30 mg% in calyx. Phenolic
acids (p-hydroxybenzoic, p-coumaric, ferulic, syringic) dominated in both extracts examined
(methanolic and methanolic-aqueous) and they may contribute to the estrogenic activity of this
plant [78].
Was reported in literature by Hărmănescu M. et al. [37] using GA as the reference
substance: for dry extract at normal temperature, 52.20 mM GAE/g and 63.40 mM GAE/g for
dry extract at 105 °C.
Of the polyphenols, flavonoids are the main secondary metabolites that characterize the
genus Tiliae [79]. Natural antioxidants (flavonoids and polyphenols) are known to have an
significant role in the cells as protecting them against free radicals, so antioxidant activity

22. 22
consists of anticancer activities, pro-apoptotic, DNA-damaging, anti-angiogenic and
immunostimulatory activity [80]. An ethanolic extract of the flowers of a species of Tiliae
showed a selective anti-proliferative action on a lymphoma cell line called BW 5147, and one
of its main compounds, rutin, showed antioxidant activity [81]. An extract from T. cordata
Mill. has shown anti-proliferative action on BW 5147 cells [82].
Tiliae species have been used in Asia, Europe and America to treat anxiety and to treat
colds and inflammation. Species reactive to oxygen (ROS) (hydrogen peroxide (H2O2) and
superoxide anion (O2
.
) are involved in the proliferation/death of balance cells in lymphocytes.
Brizi M. et al. [83] compared the effect of an aqueous (AE) and ethanolic (E) extract
from Tilia x viridis on the proliferation of tumor and normal murine A lymphocytes stimulated
in relation to antioxidant activities such as peroxidase activities (Px), catalase (CAT) and SOD
involved in H2O2 modulation. Both extracts showed anti-proliferative action on both types of
lymphocytes, but ethanolic extract was more selective on inhibition of tumor lymphocytes
(EC50 = 50 ± 4 µg/mL) and of normal lymphocytes (EC50 = 323 ± 20 µg/mL). This action was
related to high polyphenols (150 ± 10 mg GAE/g DW) and high SOD activity. Due to TPC,
the extracts could be a source of antioxidant compounds that contribute to a selective anti-
proliferative action on tumor cells, which act by modulating H2O2 levels.
I.5.3. Fam. Rosaceae
Particular interest among the endemic species containing phytochemicals are various
medicinal and culinary plants, that have antioxidant capabilities and may have many benefits
for human health could be used to product raw materials or other natural preparations.[84]. The
literature mentions numerous plants that can be used in the treatment and prophylaxis of
diabetes and complications of diabetes [85]. People with diabetes have an increased risk of
cardiovascular disease 2 to 6 times higher [86], and the Agrimonia species could have a
beneficial effect on diabetes.
Agrimonia eupatoria L. is the most common Agrimonia species in Europe, and the
aerial parts are used in the form of infusion, decoction or tincture.
Kubínová R. et al. [87] studied 5 species of Agrimonia collected in the Plant Medicine
Center of Masaryk University in July 2008 and estimated the highest flavonoids content (3.5
mg Q/g dry extract) in a methanolic extract from flowers of the studied species. For TPC and
TFC the following results were reported: polyphenolic compounds (%) expressed as caffeic
acid, between 1.03 – 1.73, respectively flavonoids (%) expressed as rutoside, between 8.03 –
11.81. Higher values of polyphenol content were obtained by researchers [87] in aqueous

23. 23
extracts: A. procera – 104.8 ± 0.5 GA/g dry plant, A. leucantha – 90.1 ± 6.3 mg GA/g dry plant,
A. japonica – 88.6 ± 2.3 mg GA/g dry plant, A.eupatoria – 72.4 ± 3.8 GA/g dry plant. The
aqueous extract of Agrimonia procera works as an excellent antioxidant (86.7% of DPPH
reduction).
To enhance the therapeutic effect of Agrimonia species, they have been associated with
other extracts from selective plants, which the literature mentions as having a hypoglycemic
effect, such as Lythri salicariae herba, Myrtilii folium, for their content in flavonoids and
tannins (catechic and galenic), the content in flavonoids, Phaseoli fructus - for isoflavones,
soluble silicates and chromium salts [88].
The genus Crataegus is the largest genus in the subfamily Maloideae of the Rosaceae
family, comprising 265 species, generally known as hawthorn [89]. Crataegus monogyna Jacq.
is a spongy European shrub widely used as a sedative, diuretic, anti-inflammatory and
cardiotonic [90, 91] which is prescribed by the European Pharmacopoeia and recommended by
the World Health Organization. There are several reports on antioxidant capacity and phenolic
compounds present in several hawthorn species, including C. monogyna Jacq., studies being
conducted with HPLC-MS [90, 92].
Simirgiotis M.J. [93] studied the fruits and aerial parts of Crataegus monogyna Jacq.
(German Peumo) from Re-Re, Región del Bio-Bio, Chile, harvested in May 2011. Quantitative
analysis showed a TPC in fruit of C. monogyna Jacq. of 28.30 ± 0.02 mg GAE/g DW, and in
aerial parts 114.38 ± 1.62 mg GAE/g DW. The TFC was found in the aerial parts (64.9 ± 0.00
mg QE/g DW), compared to fruits (8.77 ± 0.00 mg QE/g DW). Alcoholic extracts from fruits
and leaves of Crataegus monogyna Jacq. were evaluated for antioxidant power by DPPH and
FRAP methods. Thus, fruits and aerial parts had a high antioxidant capacity proven by the
DPPH method (IC50 = 61 ± 0.01 and 3.34 ± 0.38 μg/mL respectively) and by the FRAP method
(85.65 ± 0.09 and 95.05 ± 0.15 μmol TE/g). C. monogyna Jacq. fruits from Chile showed an
antioxidant activity, a higher TPC and TFC than the fruits harvested from Portugal, as a result
of Barreira J.C.M. et al., 2013: DPPH (15 ± 1% scavenging activity at 100 µg/mL),
respectively 83 ± 2 and 51 ± 14 mg GAE.
Methanolic extracts of Crataegus monogyna Jacq. from Tunisia were studied by
researchers [94] and it was established that the main source of total polyphenols and flavonoids
is bark, where 123.35 mg GAE/100 g DW and 198.53 mg RE/100 g DW were found. In the
model system of β-carotene/linoleic acid, the extract from the fruit peel showed a relative
antioxidant activity (82.23%), the DPPH value expressed by IC50 reported being 750 μg/mL.
The antioxidant capacity for extracts from different parts of Crataegus fruit is very varied, from

24. 24
5.44 to 8.88 expressed as mM Trolox/100 g dry weight to 5.68 – 9.12 mM AA/100 g dry weight.
In the alcoholic extracts, the FRAP method showed that the antioxidant activity decreases in
the following order: shell> pulp> seed, the results being in accordance with DPPH values.
Other analyzed fruits are those of the Rosa canina L. plant of the Rosaceae family
(Cynosbati fructus), due to their content in compounds with potential therapeutic activity. In
the fruits of Rosa canina L. a high content of ascorbic acid, phenols and flavonoids have been
reported, compounds that confer antioxidant, antimutagenic and anticarcinogenic activity [95].
Gavra D.I. et al. [96], analyzed Cynosbati fructus and Rosa canina flos, cultivated and
collected from Bihor County, Romania and evaluated TPC and TFC. The highest amount of
polyphenols was found in the alcoholic flower extract extract (618.07 mg GAE/mL), and the
greatest value for flavonoids was found in the dried fruits (5.75 mg QE/mL). Using FRAP and
CUPRAC methods, the methanolic extracts from the flowers of Rosa canina L. showed
antioxidant activity: FRAP (232.52 ± 0.15 mg TE/mL), CUPRAC (2.89 ± 0.03 mM Trolox).
Regarding TPC some data from the literature were reported: Nowak et al. [97] (990 mg
GAE/100g DW), Fattahi et al. [98] (199 mg GAE/100g DW) and Duda-Chodak et al. [99]
(110 mg GAE/100g DW).
I.5.4. Fam. Ericaceae
In order to prevent the complications of diabetes, numerous medicinal plants that can
be used as adjuvants, such as Vaccinium myrtillus L., have been mentioned in the literature
because of their high level of anthocyanins and phenolic compounds. Thus, bean pericarp is a
component of herbal mixtures and remedies used in diabetes herbal medicine. It can also be
applied in laryngology, where it is used for antiseptic, anti-inflammatory and astringent
properties [100]. The various herbal preparations, taken in sufficient doses and correct
combinations, neutralize free radicals before they lead to the appearance of diseases in the
body.
The total amount and proportion of different classes of phenolic compounds in the
varieties of berries may be different. Wild and cultivated species of berries as "Elliot sp.",
"Bluecrop sp.", "Duke sp." from Romania were compared to determine TPC, total
anthocyanins, TFC, antioxidant activity which was evaluated using ABTS, FRAP, DPPH and
an atom transfer of hydrogen method, ORAC. Was found a TPC of 424.84 - 819.12 mg gallic
acid equivalents/100 g fresh weight, TFC of 84.33-112.5 mg quercetin equivalent/100 g fresh
weight and total anthocyanins between 100.58 – 300.02 C3GE/100g fresh weight.

25. 25
In Vaccinium myrtillus L., petunidin – 3 – glucoside and delfinidin – 3 – glucoside are
the most important anthocyanins found, and in Vaccinium corymbosum L., peonidin – 3 –
galactoside is the major anthocyanin. Except the ORAC test, all the antioxidant activity values
obtained were correlated with the TPC. Wild blueberries had a higher polyphenol content and
higher antioxidant activity compared to cultivated ones.
Researchers [101] analyzed and revealed that in Turkey, wild Vaccinium sp. obtained
a greater antioxidant activity than the cultivated ones. V. myrtillus L. had a TPC (11.538 –
20.742 mg GAE/g DW), TFC (1.182 – 2.676 mg QE/g DW) and anthocyanins (3.305 – 11.473
mg Cyn/g DW) higher compared to V. corymbosum L. In wild species the values of CUPRAC,
FRAP and DPPH were high.
Researchers [64] studied the lyophilized and the alcoholic extract of blueberries fruits
and obtained a TPC of 343.93 ± 0.01, respectively 812.13 ± 0.02 mg GAE/100 DW, a TFC of
125.82 ± 8.92 using alcoholic extracts and 72.80 ± 7.61 mg quercetin equivalent/100 g dry
weight in lyophilized extract. Alcohol samples exhibit the best antioxidant capacity
demonstrated by the DPPH test (34.02 ± 2.01%), the anthocyanin content of 284.8 ± 17.2 mg
cyanidin/100 g DW and the TPC of 812.13 ± 0.02 mg GAE/100 g DW.
I.5.5. Fam. Hypericaceae
The flowering branches of Hypericum perforatum L., used as a folk remedy in the
treatment of various diseases, were analyzed by Fathi H. and Ebrahimzadeh M.A. [102], which
determined the antioxidant activity of the extract using different in vitro testing systems. The
IC50 for DPPH radical capture activity was 96.0 ± 3.7 µg/mL, the percentage of inhibition being
increased with increasing extract concentration (IC50 was 21.1 ± 1.8 µg/mL). The extract of H.
perforatum L. showed a strong neutralization activity of nitric oxide (6.25 and 100 µg/mL) and
a good reduction power comparable to vitamin C (p> 0.05). The capacity of the chelating
extract of Fe2+
was found to be very low. H. perforatum L. contains a large amount of TPC,
determined by the Folin-Ciocâlteu method (505.7 ± 18 mg GAE) and a high TFC, determined
by the colorimetric method with AlCl3 (23.8 ± 1.6 mg QE), which gives it good antioxidant
activity. The plant product has anti-inflammatory activity and activity at the Central Nervous
System level, these can be correlated with the absorption capacity of the nitric oxide.
Corciovă A. et al. [103] determined from the alcoholic extract of Hyperici herba from
Romania TPC (47.73 mg GA/g DW) and TFC (3.5 mg chlorogenic acid/g DW). Gioti E.M. et
al. [104] analyzed the aerial parts of Hypericum peroforatum L. plant grown in Greece and
determined a content of 257 ± 4 mg GAE/g DW. The best antioxidant activity has the ethanolic

26. 26
extract (60%) of the leaves and flowers, comparable to BHT. Zou Y. et al. [105] evaluated the
antioxidant activity by the DPPH method with an IC50 value of 10.63 μg/mL and identified
flavonoids as kaempferol, luteolin, myricetin and quercetin. Hyperosides (hyperins) and rutin
usually dominate among H. perforatum plant glycosides followed by quercitrin and
isoquercitrin [105, 106].
I.5.6. Fam. Lamiaceae
Plants of the Lamiaceae family are widely used in the cosmetic, food and medical
industries. In addition to volatile oils, plants also contain polyphenols, compounds with
biological activities useful in the prevention and treatment of many disorders [107].
A number of plants in Romania widely used as natural food additives or for health
promotion in traditional medicine have antioxidant activity [108]. Oregano (Origanum vulgare
L.) is part of the Lamiaceae family, originally from the Mediterranean region and Melissa
officinalis L. also from the Lamiaceae family are used in traditional medicine [84]. Spiridon I.
et al. [108], 2011 reported for Origanum vulgare L. a TPC of 67.8 mg GA/g extract and TFC
43.6 mg RU/g extract. Values in the literature show that different species of Origanum are a
natural source of phenolic compounds with high antioxidant activity. Benchikha N. et al. [109]
analyzed the alcoholic extracts from the leaves of two Origanum species from Algeria and
determined values for TPC (266.86 and 194.78 mg GAE/g extract) and antioxidant activity;
DPPH (IC50 = 1.37 and IC50 = 1.53 mg/L) for the majorana species L., respectively, vulgare
L., respectively for the essential oil of Origanum vulgare L. (IC50 = 15.360 mg/L).
Ocimum basilicum L., also from the Lamiaceae family is an aromatic plant with
medicinal properties, traditionally used in the treatment of headaches, cough, constipation,
warts, worms and kidney diseases [110]. The extracts of O. basilicum L. contain polyphenolic
compounds, vitamins and essential oils that have insecticidal, nematicide, fungal,
antimicrobial, also it have anti-inflammatory properties [111-114].
In the extract of O. basilicum L. from Romania, TPC (175.57 mg GAE/g DW), TFC
(6.72 ± 0.19 mg RE/g DW) and caffeic acid derivatives (12.11 ± 0.39 mg CAE/g DW). The
antioxidant activity of ethanolic extract of O. basilicum L. was evaluated using DPPH method
(IC50 = 124.95 ± 4.46 µg/ml), TEAC - 25.69 ± 2.96 µmol Trolox/mg DW), HAPX – 18.84 ±
1.12 % [115].
Rosmarinus officinalis L. (Lamiaceae) is a continuous edible green shrub in the
Mediterranean area, producing an essential oil with antimicrobial effect. Ursolic, oleanolic
acids and micromeric acids are compounds responsible for their anti-inflammatory effects

27. 27
[116]. Hărmănescu M. et al. [37] determined the TPC (348.0 mM GAE/g) of Rosmarinus
officinalis L., and other authors determined from the rosemary extract TFC of 448.4 mg
catechin/100 g DW [12].
Melissa officinalis L. is used to treat headaches, gastrointestinal disorders, nervousness
and rheumatism [117]. Melissa off. L. essential oils have antimicrobial properties and a strong
ability to protect against lipid peroxidation [117, 118].
Atanassova M. et al. [119] studied the composition in bio-active compounds and the
antioxidant capacity of Melissa off. L., harvested from Bulgaria and have TPC (48.86 mg
GAE/100g DW), TFC (45.06 mg CE/100 g DW), and by DPPH assay evaluated antioxidant
capacity (IC50 = 10.87 mL/L methanolic extract).
Lavandula officinalis L., known as medicinal lavender, true lavender, or common
lavender, also known as Lavandula angustifolia Mill. L. is a plant with beneficial properties
for humans. Bioactive compounds as anthocyanins, tannins, phytosterols, also, minerals,
coumarin and essential oil with important therapeutic effects, phenolic acids and herniarin were
found in Lavandula off. L.[120].
Natural products obtained fom lavender flowers are used in headaches due to the
analgesic effect, in depressions or anxiety due to the sedative effect on central nervous system
have sedative and analgesic properties [121]. Studies on animals using the lavender extract
showed that it prevented dementia that was caused by Alzheimer's disease [122]. Lavandula
off. L. essential oil has antibacterial activity at doses of 4.0 – 9.0 mg/mL [123]. A study from
scientific literature that was carried out on 65 bacterial strains, confirmed the antimicrobial
properties of lavender, having a better efficacy against gram-positive strains [124].
The vegetable product of Lavandulae sp. it is used for the spasmolytic, carminative,
stomach, diuretic properties and is used today as a light sedative and cholagogue in different
phytopharmaceuticals [125, 126].
Salvia species are generally known for their multiple pharmacological effects. Each
species contains a large amount of flavonoids and tannins (caffeic acid, chlorogenic acid,
ellagic acid, gallic acid) [127]. Rosmarinic acid and its derivatives are compounds responsible
for the antioxidant activities of some Salvia species and for astringent, anti-inflammatory,
antibacterial and antiviral activities. Some studies examined the in vitro anti-proliferative
activity of crude methanolic extracts from six Salvia species and found that they can be used
as potential anti-tumor agents [128, 129].
Jurca T. et al. [64], 2016 compared two types of leaf extracts from Salvia off. L.,
collected from Romania: the lyophilized and the alcoholic extract. For the lyophilized extract

28. 28
they found: TPC (258.87 ± 0.01 mg GAE/100 g DW), TFC (427.23 ± 9.42 mg QE/100 g DW)
and antioxidant capacity was measured by DPPH (5.61 ± 1.83%), ABTS (67.84 ± 11.78 mmol
TE/g DW), FRAP (61.07 ± 0.01mmol TE/g DW). For alcoholic sage extract were found: TPC
– 963.01 ± 0.01 mg gallic acid equivalent/100 g dry weight, TFC – 267.42 ± 8.21 mg quercetin
equivalent/100 g dry weight, and antioxidant capacity was measured by DPPH (46.94 ±
3.56%), ABTS (244.12 ± 23.42 mmol TE/g DW), FRAP (177.62 ± 0.11 mmol TE/g DW).
Due to the complex chemical composition, Mentha sp. has anti-inflammatory,
antimicrobial, spasmodic, carminative, antioxidant properties. Mint is used in traditional
medicine and in the cosmetic and food industry for the production of various sweets and
beverages [130, 131].
Two species native to Mentha: M. viridis L. and M. longifolia L. from Cluj area,
Romania were studied by Benedec D. et al, 2013 to determine TPC and antioxidant capacity.
The extract of M. viridis L. (246.7 ± 0.47 mg GAE/g DW) showed the best results regarding
the TPC, followed by M. longifolia L. (219.2 ± 0.97 mg GAE/g DW).
The extract of M. viridis L. contains the highest amount of flavonoids (9.41 ± 0.08 mg
RE/g DW) compared to M. longifolia L. (6.75 ± 0.09 mg RE/g DW). The best value for
phenolic acids was determined in the extract of M. viridis L. (43.80 ± 1.39). The effect of free
radical capture of M. longifolia L. at a concentration of 0.4 mg plant product extract/mL was
25.31%, followed by the extract of M. viridis L. (18.34%) at the same concentration, compared
to BHT (94.77 ± 0.64 %) at the same concentration (0.4 mg/mL). Although TPC of M. viridis
L. is larger than M. longifolia L., it has a lower antioxidant capacity.
In another study [103]was determined the flavonoids, polyphenolic acids and TPC with
potential therapeutic effects of the natural products commonly used as traditional treatments in
Romania. They investigated different parts from the medicinal plants that are used in different
conditions, such as: in dentistry for various infections and inflammatory disorders (Salvia
officinalis L.- Lamiaceae family); gastrointestinal affections, damages and inflammatory
diseases (Achillea millefolium L.-Asteraceae family), cardiovascular diseases and anxiety
(Crataegus monogyna Jacq.- Rosaceae family), insomnia, colds, infections and inflammation
(Chamomilla recutita L.- Asteraceae family) respiratory infections (Tilia cordata Mill.-
Tiliaceae family), anxiety, sores, pustules, acalculuous gallbladder disease (Hypericum
perforatum L.- Hypericaceae family).

29. 29
I.5.7. Fam. Canabaceae
Another species, Robinia pseudoacacia L., commonly named false acacia or black
acacia, belongs to the Fabaceae family, is widespread as a wild species and cultivated in
temperate areas around the world. False acacia flowers contain volatile compounds, flavonoids,
proteins, robinins, polysaccharides and some microelements [132]. In 2000, flavonoids of
acacia ethanolic extracts were extracted from acacia, including isoflavonoids (isovestitol,
isomucronulatol), flavones (acacetin) [133, 134]. Recently, many investigations have referred
to the antioxidant properties of different nutritional products [135]. Using HPLC analysis,
Marinas C.I. et al. [136] identified catechin (0.925 μg/mL alcoholic extract), rutin (0.831
μg/mL alcoholic extract), resveratrol (0.664 μg/mL alcoholic extract) and quercetin (0.456
μg/mL alcoholic extract) in leaf extract and, catechin (0.127 μg/mL alcoholic extract),
epicatechin (0.239 μg/mL alcoholic extract) and rutin (0.231 μg/mL alcoholic extract) in the
seed extract. Alcoholic extracts of Robinia pseudoacacia L. presents antimicrobial activity that
was evaluated using Gram-positive and negative strains, also for Candida sp. Researchers
showed that flowers of R. pseudoacacia L. has high levels of polyphenols and strong
antioxidant properties. The same study showed that topical formulations with false acacia can
exhibit the antioxidant activity in order to reduce cellular stress and it can be used as a natural
formulation for skin aging [137].
I.5.8. Fam. Grossulariceae
Compounds contained in Ribes nigrum L. fruits and leaves can act preventively and
therapeutically on the human body [138]. Ribes nigrum L. is a shrub that grows in temperate
areas [139]. Black currant fruits contain polyphenols that have antioxidant, antimicrobial,
antiviral and antibacterial properties [140]. Polyphenols protect and support many functions of
organs and systems, especially the digestive system [141], the nervous system and the
circulatory system [142]. Contained in black currant leaves, quercetin derivatives have
antimicrobial, anti-inflammatory, antiviral, anti-toxic, antiseptic and antioxidant effects, so it
is assumed that the leaves can be used as an adjuvant in the treatment of cancer [143-146].
Cytosolic phospholipase A2α (cPLA2α) is one of the potential targets for anti-inflammatory
drugs, as this enzyme plays a key role in the inflammatory processes observed in health
problems, such as asthma, allergic reactions, arthritis and neuronal diseases. Arnold E. et al.,
2015 [147], inhibited cPLA2α with 43 methanolic extracts from polyphenol-rich medicinal
plants. The extract of Ribes nigrum L. was the strongest inhibitor of cPLA2α (IC50=27.7

30. 30
μg/mL), having the highest TPC (131.25±7.15 GAE mg/g extract). The EC50 value for DPPH
radical reduction was 13.36±0.6 μg/mL extract.
I.5.9. Fam. Urticaceae
In the tissues of various spices and herbs with therapeutic were found constituents that
have antioxidant and antimicrobial activity. Antioxidants may scavenge and neutralize the
harmful and pathological effect of free radicals [148].
Urtica dioica L. has beneficial properties, which is why its extract has been used for
hundreds of years in traditional world medicine to treat various conditions: rash, digestive
problems, joint pain and anemia [149]. The extract of U. dioica L. made with ethyl acetate
contains flavonoids and alkaloids, phenols, saponins and tannins [150]. Taraxacum officinale
L. has a high concentration of phytoconstituents in stem, root and flower, such as saponins,
flavonoids, alkaloids and phenols [25].
Antibacterial and antioxidant activities of extracts from Urtica dioica L.and Taraxacum
officinale L. using ethyl acetate as a solvent, were compared by Kassim Ghaima K. et al. [151].
The results showed that the extract from U. dioica L. was more effective than Taraxacum off.
L. on the bacterial strains and was found an inhibition area of 24 mm against B. Cereus, which
was also the highest one; A. hydrophilaa had the highest resistance. U. dioica L. had a large
area of inhibition comparing S. typhi (22 mm) and the highest TPC (48.3 mg GAE/g DW).
Through qualitative phytochemical screening, were identified flavonoids, glycosides and
phenols. Kassim Ghaima K. et al. [151], conducted a comparative study to verify antioxidant
activity, using α-Tocopherol as standard by the ferric thiocyanate method (FTC). Urtica dioica
L. had a greater activity (76% lipid peroxidation) in inhibiting linoleic acid emulsion, than
Taraxacum off. L. (44%) and α – tocopherol (65%). Other research has shown, using the ferric
thiocyanate method, that aqueous extract of U. dioica L. exhibited effective antioxidant activity
at all doses (50 – 250 μg) [152]. Low antioxidant activity of Taraxacum off. L. may be due to
the presence of active radical scavenging compounds such as luteolin and luteolin – 7 – o –
glycoside in other parts of plants, such as flowers and roots, more than in leaves [153].
The antioxidant, hepatoprotective and anthelmintic activities of the methanolic extract
from the leaves of Urtica dioica L. were investigated by Manjir Sarma Kataki et al. [154].
Significant hepatoprotective effect and maximum hepatoprotection was observed at the dose
of 400 mg/kg. Using the extract as a pre-treatment of animals at all doses (100, 200 and 400
mg/kg) resulted a significant decrease in malonildehyde (MDA) level, as well as a significant
increase in superoxide dismutase (SOD) level, indicating lipid inhibition, peroxidation and

31. 31
improvement of the enzyme antioxidant defense system. The anthelmintic activity of the
methanolic extract was investigated using adult worms (Pheretima posthuma), and the results
revealed an increase of the anthelmintic activity that is dose dependent of the extract at
concentrations of 25, 50 and 100 mg/mL [154].
The highest TPC was found by Semih Otles and Buket Yalcin [155] in the leaves of
Urtica dioica L. in the Aegean region: 1941.00 ± 15.06 mg GAE/g DW followed by the extract
from the roots of Urtica dioica L. in the Black Sea: 1020.16 ± 69.40 GAE/g DW. Using the
DPPH test, it was found that in the root sample, the highest antioxidant activity had Urtica
dioica L. in the Marmara region: 370.27 ± 0.11 mg GAE/g DW. The thermal process used for
drying the plant could be the reason for the lower antioxidant activity compared to the fresh
vegetable product.
I.5.10. Fam. Papaveraceae
Maria Laura Colombo and Enrico Bosisio [156] investigated the pharmacological
activity of the species Chelidonium majus L. (syn. Celadine) and identified the antiviral, anti-
tumor and antimicrobial activities, as well as the presence of flavonoids and phenolic acids.
Jakovljevic Z.D. et al. [157] investigated the TPC, TFC and antioxidant activity of
Chelidonium majus L. extracts in different phenological stages (rosette stage, initial flowering
stage, complete flowering stage and the stage of fruit formation). For each phase, five extracts
were obtained with different solvents: water, methanol, acetone, ethyl acetate and petroleum
ether. The TPC determined by the Folin-Ciocâlteu method and the highest values were obtained
for the rosette stage (60.96 mg GA/g extract). Of the solvents used, methanol has been shown
to be most effective for the extraction of phenolic compounds from Ch. Majus L. The highest
flavonoid concentration was found for the initial flowering stage (291.58 mg RU/g extract),
harvested in May, and acetone and ethyl acetate were the most suitable solvents for flavonoid
extraction. Antioxidant activity was determined in vitro using DPPH reagent, and the highest
antioxidant activity was the plant product in the rosette stage (50.72 mg/mL extract).
Hărmănescu M. et al. [37] determined TPC in seventeen samples of Ch. Majus L., dried
under normal conditions and at 105o
C for 4 hours. They used Folin-Ciocâlteu reagent, and the
values obtained were: 105.40 mM GAE/g for drying under normal conditions, respectively
159.60 mM GAE/g for drying at 105°C. High values were also obtained by Papuc C. et al.
[158] using alcoholic extracts of Ch. Majus L. The TPC expressed as mg gallic acid
equivalent/100 mL extract and TFC expressed as mg cyanidin equivalent/ 100 mL extract were
determined in the flower extract (381.7 ± 23.2 and 299.5 ± 18.4), while the lowest

32. 32
concentrations were recorded in extract from the strains (52.7 ± 4.2 mg, respectively 36.75 ±
2.9).
I.6. Alternative treatment for skin diseases
Psoriasis is the major autoimmune skin disorder characterized by deregulated epithelial
cells proliferation and chronic dermatitis [159] and it has many clinical phenotypes that results
from the interaction of many factors as: genetic, environmental and immunological. Pathogenic
mechanisms and effective therapies of psoriasis were elucidated after a lot of work on clinical
and basic research.
I.6.1. Psoriasis prevalence
In Western countries the population is affected by this skin disease more and more with
prevalence rates that are influenced by genetics, age, nutrition, geographic location [160].
Studies from literature showed that psoriasis is a very commonly disease, were analyzed 46
studies regarding the prevalence of psoriasis and in seven of them was showed the incidence
of psoriasis in general population [161]. Results showed that in adults prevalence ranged from
0.91% to 8.5% compared to children, where the prevalence ranged from 0% to 2.1% where
most affected were the persons with age between 30–39 years and 60 years. Depending on the
country, the prevalence is different, having a geographical model that shows less prevalence in
the countries that are closer to equator, compared with those that are more distant from equator,
that also correlates the beneficial properties of UV radiation exposure and the amelioration of
the disease [162]. In the countries from Europe it ranges from 0.73% to 2.9%, which could
compare to the values of the Unites States (0.7% – 2.6%). The lower prevalence is in Africa,
Latin America and Asia which showed a prevalence with values between 0 – 0.5%. Studies,
also showed that both genders, female and male, are affected by psoriasis, equally [163, 164].
I.6.2 Psoriasis classification, pathogenesis and treatment
Main forms of psoriasis were identified by International Psoriasis Council: plaque-type
psoriasis, guttate psoriasis, generalized pustular psoriasis (GPP) and erythroderma, and many
further subphenotypes depending on the distribution (may be localized or widespread),
localization (flexural, on scalp, may appear on palms, nail or soles), depending on size (small
or large) and thickness (thin or thick) of plaques, if appears early or late and disease activity, if
psoriasis is active or it is stable [165].
This disease is characterized by erythema and irritation. Regarding the pathological
point of view, it is chronic dermatitis, which has rapid uncontrolled epithelial cell proliferation
on the surface, and hyperemia, and dense lymphocytic-infiltration on the corium side. Psoriasis

33. 33
can start in any stage of the life and persists for a long period of time with permanent or periodic
eruptions [166].
Different comorbidities as psoriatic arthritis, crohn's disease, cancer, metabolic
syndrome, type 2 diabetes mellitus, increased cardiovascular risk, depression, and decreased
quality of life are associated with psoriasis [167]. Smoking is also listed among the risk factors
that play a role in the occurrence of psoriasis [168]. 80% of psoriasis cases are represented by
the plaque type, and in contrast is the erythrodermic type with a percentage of only 1 – 2%. In
adults the most common type of psoriasis is the pustular type (5%). Psoriasis has three major
forms such as mild, moderate, and severe that depends on the covered area and severity of the
symptoms. Regarding the mild form, 5 – 10% of total area is covered, while moderate form,
covers area between 10 and 20% and severe form covers more than 20% of the body area. The
exact etiology of psoriasis is yet unknown but different risk factors like genetic factors, immune
system, and environmental factors have been identified to develop psoriasis like genetic
factors, immune system, and environmental factors. It was observed that 30% of people
suffering from psoriasis have psoriasis family history. Pathogenesis of psoriasis is well
understood [169]. Helper T – cell (Th) 1/Th17 cells infiltration takes place in epithelial tissue,
which triggers macrophages and dermal dendritic cells to secrete different cytokines to mediate
inflammation and abnormal keratinocyte proliferation. Infiltration of activated CD4+ and
CD8+ T – cells are responsible for the psoriatic plaque formation. Infiltration of CD4+ T-cells
takes place in the dermis while CD8+ T – cells infiltrate into the epidermis. Development of
lesions in psoriasis is due to the cytokines and chemokines released by T – lymphocytes.
Keratinocyte hyper proliferation with parakeratosis and elongation or rete ridges, increased
blood vessels synthesis, inflammatory cells infiltration like T lymphocytes, macrophages,
neutrophils, dendritic cells, and presence of micro abscesses are the major histological features
of psoriasis [169].
An important role in the pathogenesis and etiology of psoriasis is represented by diet.
Some researchers show that psoriasis symptoms are reduced by vegetarian diets, low energy
diets, and fasting periods. The metabolism of polyunsaturated fatty acids and suppress the
inflammatory response are affected by these diets. Various combined therapies, oral inositol,
intravenous omega-3 fatty acids, and vitamin D are effective in psoriasis. Low-calories diet,
cyclosporine, thiazolidinedione, retinoids, fish oil, and ultraviolet B phototherapy are found
effective in clinical trials [170]. Because of this disease causes some patients have physical and
psychological problems and it has been observed that thoughts of suicide and self-harm are
more in psoriasis patients compared to normal persons. Scalp psoriasis is very distressing and

34. 34
patients suffer from psychological problems. It is not contagious disease but some people report
that friends and colleagues consider that psoriasis is transmitted by shaking hands or skin
contact. This thing makes the patient socially isolated and he suffers from depression [171].
Treatments as phototherapy and/or systemic medications are needed to treat moderate and
severe forms of psoriasis. Psoriasis can be treated by exposure to narrowband ultraviolet B
(NB-UVB) radiation. Radiation spectrum having wavelength b/w 311 and 312 nm is known as
NBUVB and this is more beneficial and effective to treat psoriasis [172]. Due to
antiproliferative, immunosuppressant and anti-inflammatory characteristics, UV radiation
induces regression or controlling the evolution of dermatitis [173, 174]. Capsaicin,
glycyrrhetinic acid preparation is applied on affected parts of the body. Some studies suggest
that psoriasis causes a nutritional deficiency in sufferers. The outbreak of psoriasis increases
with an increase in stress and anxiety. Although modern medicines including cyclosporine,
acitretin, apremilast, methotrexate, secukinumab, adalimumab, ixekizumab, etanercept,
infliximab, and ustekinumab are effective but they have some side effects [175, 176].
Medicinal plants are safe and efficacious [177]. In developing countries, most people rely on
herbal medicine due to their easy availability, low cost, more efficacies, and not well-reported
side effects.
I.6.3. Alternative treatment for psoriasis
Polyphenols present in plants are effective in the treatment of psoriasis due to
significantly high-antioxidant activity. Nitric acid level and hydroxyl or free radical present in
the blood of patients with psoriasis is reduced by antioxidants that have also antiproliferative
and anti-inflammatory properties and they have the ability to inhibit calgranulins A and B genes
that are involved in the inflammatory process.
Plants can be used as symptomatic treatment of psoriasis and also suppress the disease
for a period. Clinical efficacy in vivo/vitro has been demonstrated for Glycyrrhiza uralensis,
Aloe vera [115, 178], Memecylon malabaricum [179], Capsicum annuum, Psoralea corylifolia,
and Mahonia aquifolium, different parts of vegetal herbs as Matricariae flos, Calendulae flos,
Hamamelidis folium, Quercus cortex, Violae tricolor, Salviae folium, Echinaceae herba,
Dulcamarae stipites, Arnicae flos, Symphyti rhizome, Melaleuca hypericifolia, Melissae
folium, Momordica charantia, Azadirachta indica, Arctium lappa and Caesalpinia bonduc
[180].

35. 35
I.6.4. Psoriasis evaluation on clinical trials
Regarding the clinical practice, global evaluation of psoriasis progress and its effect on
the quality of life of patients are applied to assess the severity of the disease and the
effectiveness of the treatment. In human clinical trials are needed more validated objectives
and instruments. Many instruments have been expanded and continue to be expanded to
provide an evaluation of the degree of the lesions [181]. Because a lesion’s impact on patients
lives varies among patients, there has been growing recognition of the need to measure the
quality of life impact of the disease along with the severity of the lesions. Redness, thickness,
and scaliness are the main characteristics of the lesions and assure a means on evaluating the
degree of psoriasis. PASI score is the gold standard for estimation of large psoriasis [182]. It
measure the damage of redness, induration, and desquamation of the lesions (each parameter
have a scale from 0–4), evaluated by the affected area. Even if PASI score is a very useful
measure, it has the disadvantage to show less accuracy for small affected areas. For localized
psoriasis plaques, target lesion evaluations are generally performed that also measure redness,
thickness, and desquamation of the target plaques.
Another measure used in psoriasis clinical trials is the physician global assensment
(PGA). General evaluation can be used for large psoriazis as well as for localized plaques.
There are two main forms of evaluation: a static one, that measures the medical impression of
the psoriasis at a single point, and a dynamic one, in which the doctor appreciates the global
progress from the initial value. In clinical trials for the treatment for psoriasis, to assess the
yield of the drug is necessary a predetermined primary endpoint, which must prove that more
patients accomplish clinically meaningful success in using the drug treatment than the placebo
[183].
Another important psoriasis measurement instruments are being developed. The
physician assess the redness, induration and scaliness of the psoriasis lesion, using a scale from
none to mild, moderate or severe. The percentage of affected area is also evaluated in categories
of 0% affected area, <10%, 10 – 29%, 30 – 49%, 50 – 69%, 70 – 89%, 90 – 100%. By
combining the percentage of affected areas with the character of the plaques, this affection can
be classified into one of eight categories on a scale from clear to very severe. This method
indicates a good correlation with the global assessment of the physician and PASI score and
provides a better reliability than PASI [184].
Another tool is the National Psoriasis Foundation - Psoriasis Score (NPFS) that have
many subdomains: induration and current and baseline body surface area, physician global
evaluation, patient global evaluation, and patient evaluation of itch [185, 186]. To help improve

36. 36
the reliability of the induration score, the tool utilises a standard reference card with levels that
increase at intervals of 0.25 mm. Biopsies and photographs are two other quantitative ways to
evaluate psoriasis.
A major component of the evaluation of psoriasis is the examination of the quality of
life that doesn`t measure in a directly manner the impact of a drug on disease, but it measure
the impact of the psoriasis and the capacity of treatment to ameliorate patients lives [187, 188].
Quality of life assesments are very important because the main goal of the therapy is to increase
the quality of patients lives.
For the same purpouse are used nonspecific assesments as the Medical Outcome Survey
Short Form 36, also, the Euro QoL, and utility measures estimate in general patients’ quality
of life [189, 190]. Another specific tools, the Dermatology Life Quality Index (DLQI), also the
Skindex, are methods that focus on the aspects of quality of life on psoriatic patients [191, 192].
To compare psoriasis to other medical diseases or to show the impact of it, the SF – 36 is a
great or the greatest measure [193]. In psoriasis investigations to evaluate the quality of life of
patients related to the skin affection the most used evaluation index is DLQI that is a
questionnaire consisting of 10 questions that cover many domains gouped as: symptoms and
feelings, close relationships, daily activities, work or school, leisure and trouble with psoriasis
treatment. The answers are evaluated from 0 which means that quality of life is not affected,
to 3, which means very much affected. DLQI index gives a range between 0 – 30 and is
interpreted as the lower scores the better quality of life of patient.
I.7. Pharmaceutical forms used in the treatment of psoriasis
For the treatment of psoriasis there are several treatments that can be used topically.
Most of them are approved or have been clinically used to treat psoriasis. The interest for the
future investigations for psoriasis treatment has grown due to the valuable results obtained in
the preliminary clinical evaluations. Recently, was developed a combination of drugs and photo
therapies, which include UVB and UVA, also a combination of two drugs used in psoriasis
treatment showed higher efficience. Most of the drugs used in psoriasis are developed as
ointments, creams, lotions or hydrogels using conventional vehicles, but some novel carriers
as liposomes, microemulsions are being invetigated to improve efficiency of topical therapies.
Due to the epidermal hyperproliferation the penetration of skin may be difficult and
many factors may improve skin penetration as drug-loaded nanometer-sized vehicles that are
small particles which can result in a stronger occlusive effect due to membrane formation. An
important mechanism is the interaction of lipids and surfactants in nanometer-sized vesicles

37. 37
with skin lipids. Also, to encapsulate antipsoriatic drugs for topical treatment, solid lipid
nanoparticles, micelles and nanostructured lipid carriers can be used to improve drug delivery
into this skin affection. New physical methods are investigated as iontophoresis, lasers and
electroporation to obtain an effective and safe therapy for psoriatic patients.
Phototherapy, topical, oral or injectable drugs are the therapies used for psoriasis
treatment. Topical drugs used for psoriasis comprise corticosteroids, topical retinoids,
Anthralin, Calcineurin inhibitors etc. Topical corticosteroids are the first-line therapy, but
chronic use or overuse of strong corticosteroids may have side effects and it can thin the skin
and eventually stop working over time [194].
Different natural therapies were developed and investigated to improve the quality life
of psoriatic patients. Natural plants or flowers with medicinal properties as Aloe barbadensis
Mill., Azadiracta indica, Nigella sativa Linn., Psoralia corylifolia Linn., Silybum marianum
and others, were incorporated as extracts in different pharmaceutical forms and were used as
an auxiliary treatment in psoriasis. Natural plants and flowers are a rich source of bioactive
compounds that confers great antioxidant, antiinflamatory, antiseptic, antitumor,
immunomodulatory activity.
A study with ethanolic extract of Nigella sativa L. seeds was reported in the literature
which revealed a substantial differentiation of the epidermis in its degree of orthokeratosis
compared to placebo group and indicated an effect comparable to the positive control which
was tazarotene gel. The extract showed adequate anti-proliferative properties, so the study
supported the use of the extract as a traditional therapy for psoriatic patients with a reduction
in thickness compared to the control group [195].
Due to their medicinal benefits, Psoralea corylifolia contains babchi oil, that was
analyzed by researchers in a microemulsion gel-based system for psoriasis treatment and
showed a better penetration of oil in the skin and also had a great in-vivo antiinflammatory
activity [196].

38. 38
I.8. Partial conclusions
Polyphenols play an important role in improving the quality of life of people by
prophylaxis of the onset of diseases or preventing complications of various chronic diseases
such as diabetes, skin diseases, cardiovascular diseases, neoplasms, etc.
A variety of plants with therapeutic effect were studied, highlighting their bio-active
compounds.
The most important bioactive compounds are: phenolic acids, flavonoids, anthocyanins,
lignans, tannins, with a predominant antioxidant, antimicrobial, immunostimulatory and anti-
inflammatory action.
In most studies, the Folin-Ciocâlteu test, respectively the colorimetric method using
AlCl3, are used as methods to determine the TPC and TFC and, for the evaluation of antioxidant
activity, the most used methods are DPPH, FRAP, CUPRAC, ABTS, HAPX, ORAC, β –
carotene/linoleic acid assay and ferrous ion chelating activity.
Pharmaceutical preparations with extracts from plants or flowers could improve
psoriasis episodes by reducing dryness, itching, scaling and inflammation of the skin, compared
to conventional products, which with long-term administration can produce various side
reactions or even reduce the therapeutic effect.

39. 39
SECOND PART - EXPERIMENTAL STUDY
II. Phytochemical screening of ethanolic extracts of Rosa species
II.1. Introduction and objectives of the chapter
Phenolic compounds, including phenolic acids, flavonoids, anthocyanins and
carotenoids, are natural antioxidants that are present in all parts of a plant (bark, stem, leaf,
fruit, root, flower and seed), which help reduce the risk of various diseases by delaying or
inhibiting oxidation. Among plant tissues, flower petals have a high content of flavonoids and
are known as a potential source of natural antioxidants.
Medicinal plants are used as alternative products, not only in traditional medicine, but
also in a number of food and pharmaceutical products due to their nutritional value and
bioactivity. Various species of the Rosaceae family have a great therapeutic importance due to
their use in different food preparations and in various pharmaceutical preparations.
Taxonomically, roses belong to the family Rosaceae and the genus Rosa, which comprises
almost 200 species distributed worldwide, in North America, Europe, Asia, and the Middle
East.
Rosa x damascena Mill. is an ornamental plant, a perennial shrub that can reach a height
of up to 2 meters, with large, colorful flowers and pinnate leaves, composed of 5 – 7 leaflets.
It originates in the Middle East, it is part of the genus Rosa, family Rosaceae, a family with
over 2500 species, cultivated for its proven pharmacological properties, as ornamental plants
or for its well-known perfume effect. This plant is cultivated in Iran and Greece and around the
world for its essential oil and rose water that have therapeutic properties [197].
Rosa x canina L. is well known for its high phenolic content. These compounds are
known to have antioxidant, antimutagenic and anticarcinogenic effects. Polyphenolic
compounds are potential antioxidant substances and protective agents against the development
of human diseases. That's why rose flowers, leaves, roots, branches and fruits have been studied
and used for thousands of years for their medicinal benefits. Rosehip teas have mild laxative
and diuretic tendencies and help regulate the menstrual cycle, while leaf and petal teas are
soothing to the skin and can help heal rashes and abrasions. Hydroalcoholic extracts represent
the best way to obtain preparations enriched with phenolic compounds, while infusions are the
most commonly used for regular consumption [198].
Rosa x centifolia L. is a widespread shrubby rose that grows up to 1.5 – 2 meters in
height and it’s cultivated as an ornamental plant throughout India, but it is especially cultivated
in Grasse for its fragrance. Leaves are imperipinnate, having 5-7 leaflets grayish green in

40. 40
colour, with 5 – 7 ovate leaflets and green colour. Flowers may varying in colour, but it is
usually pink, with many petals.
It has medicinal properties and it is used in asthma, hypertension and bronchitis. Data
from scientific literature show their chemical analysis for their bio-active principles [199].
The species Rosa x hybrida L. is an artificial category that includes modern roses.
Among modern roses, the hybrids represent the largest class and that grows up to 1 – 2 m height
with single, and well-shaped flowers that have high spiralling centres at the end of the strong
and long stems; petals are shiny and sturdy, buds are pointed; leaves are large and glossy [200].
A very large number of hybrid tea varieties have been introduced by breeders over the years.
This category also includes the analyzed roses purchased from rose growers as Rosa x cairo
L., Rosa x ambassador L., Rosa x monica L., Rosa x Edward leone L., Rosa x eroica L., Rosa
x black magic L., Rosa x mr. lincoln L., Rosa x emerad’or L. which has a high concentration
of polyphenols and good antioxidant activity. There are no data in scientific literature regarding
this species. These roses were created by crossing two types of roses.
Many scientific studies involved different uses of seeds, petals, flowers and fruits of the
genus Rosa at different stages of maturity, and there were also comparative studies between
biochemical compounds and their antioxidant properties.
In the research of Rosa medicinal plants, there are various reports in the literature
regarding the chemical composition and few descriptions regarding their bioactivity or the
mechanism of action to be explored. Currently, there is still much work that can be done in
terms of developing new products with Rosa species.
The objectives of this chapter are to present the chemical composition, the total content
of polyphenols, flavonoids, the presentation of the antioxidant capacity and the evaluation of
the results in order to choose the species with the best phytochemical profile and the highest
antioxidant activity for extensive analysis in order to develop new medicinal products.

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II.2. Materials and methods
II.2.1. Reagents and plant material
All chemicals and reagents used in this study have a high degree of purity. Sodium
carbonate and gallic acid were provided by Fluka, Switzerland; all the other reagents used are
from Sigma Aldrich, Germany. Twice-distilled water was obtained using a Milli-Q system
(Millipore, Bedford, MA, USA). Rosa x damascena Mill. was purchased from S.C. Green-E
Concept S.R.L., Sanicolau Roman 477, Bihor, Romania in 2019 and 2020 and the other species
were purchased from rose growers from Oradea, Bihor County and identification and
authentication of the specimens was done by Prof. Dr. Pallag Annamaria, Department of
Pharmacy, Faculty of Medicine and Pharmacy, University of Oradea.
II.2.2. Preparation of the extracts
The petals were dried at 40° C, for 120 minutes using an UTD – 1295 Laboratory oven
50 L. The preparation method used to obtain the alcoholic extract solution was maceration,
method of extraction with alcohol at a temperature of 20° C. Over 10 g petals of Rosa sp. 100
mL of 70% ethanol were added and left at 20° C, for 7 days. All subsequent determinations
were performed in triplicate.
A – fresh petals B – dried petals C – preparing ethanolic extract
Figure 5. Fresh and dried petals of Rosa x damascena Mill. in laboratory oven

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II.2.3. Total polyphenols content (TPC)
TPC was determined using Folin-Ciocâlteu method, a widely used colorimetric method,
and the total polyphenols content was calculated as gallic acid equivalents/100 g dry vegetable
product (mg GAE/100 DW). Over 0.1 mL of alcoholic extract solution they were added 0.2
mL of dilute Folin-Ciocâlteu reagent (1:10), 1 mL of 20% Na2CO3; the resulted mixture was
incubated for 1.5 hours, at room temperature, in the dark, then the absorbance was read on a
spectrophotometer UV-VIS (Shimadzu UV-VIS 1700 PharmaSpec, Shimadzu Corp., Kyoto,
Japan), at 765 nm. The determination of the total polyphenols content was done using a
calibration curve [201].
II.2.4. Total flavonoid content (TFC)
TFC was performed by the colorimetric method, using AlCl3, which forms a complex
with the carbonyl groups of flavonoids. The absorbances of the samples were read with the
UV-VIS spectrophotometer at a wavelength of 415 nm, using a blank solution as a reference.
Results were expressed as mg quercetin equivalent (QE)/100 g DW, using a calibration curve.
Over 1 mL of alcoholic extract, 0.3 mL of 5% NaNO2 was added, stirred and allowed to stand
for 5 minutes; then, 0.3 mL of 10% AlCl3 was added, stirred and allowed to stand for 6 minutes,
and 2 mL NaOH were added, stirring vigorously [202].
II.2.5. Analysis of phenolic compounds by HPLC
A new LC – MS method described in literature [203] was used to identify polyphenols
in extracts of Rosa sp. The chromatographic separation was performed using an analytical
column (Zorbax SB-C18, 100 mm x 3.0 mm i.d., 3.5 µm) with a mixture of methanol: 0.1%
acetic acid (v/v) as mobile phase and a binary gradient (start 3% methanol, at 3 min 8%
methanol, at 8.5 min 20% methanol, keep 20% methanol until 10 min then rebalance column
with 3% methanol). The flow rate was 1 mL/min and the injection volume was 5 μL. The
qualitative detection of the compounds was performed on MS mode (SIM-MS). The MS
system operated using an electrospray ion source in negative mode (capillary +3000 V,
nebulizer 60 psi (nitrogen), dry gas nitrogen at 12 L/min, dry gas temperature 360ºC). The MS
signal was used only for qualitative analysis based on specific mass spectra of each polyphenol.
The MS spectra obtained from a standard solution of polyphenols were integrated in a mass
spectra library. Later, the MS traces/spectra of the analyzed samples were compared to spectra
from library, which allows positive identification of compounds, based on spectral match. The
UV trace was used for quantification of identified compounds from MS detection. The
quantification of a compound previously detected in MS mode was made in UV at 330 nm for