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Plant hydrolates – Antioxidant properties, chemical composition and

potential applications

Article  in  Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie · October 2021

DOI: 10.1016/j.biopha.2021.112033

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 Biomedicine & Pharmacotherapy 142 (2021) 112033

Contents lists available at ScienceDirect

Biomedicine & Pharmacotherapy

journal homepage: www.elsevier.com/locate/biopha

Plant hydrolates – Antioxidant properties, chemical composition and

potential applications
Karolina Jakubczyk a, *, Aleksandra Tuchowska b, Katarzyna Janda-Milczarek a
a
Department of Human Nutrition and Metabolomics, Pomeranian Medical University in Szczecin, 24 Broniewskiego Street, 71-460 Szczecin, Poland
b
Department of Studies in Aesthetic Dermatology, Pomeranian Medical University in Szczecin, 72, Powstańców Wielkopolskich Street, 70-111 Szczecin, Poland

A R T I C L E I N F O A B S T R A C T

Keywords: Hydrolates, are by-products of the hydrodistillation of plants. They consist of the distillation water in which very
Hydrolates small amounts of essential oils remain dispersed. Hydrosols are widely used in cosmetics. One of the greatest
Hydrosols challenges in skin care, whether it is healthy or affected by a pathological condition, is how to minimize
Antioxidant, flavonoids, polyphenols
oxidative stress. Extract also lend themselves to applications in the agri-food industry, to inhibit the development
Skin
Oxidative stress
of pathological microorganisms in food and to remove biofilms constituting a threat to public health in food,
pharmaceuticals and beauty products. Therefore, the aim of this study was to analyze the antioxidant potential of
hydrosols available in the cosmetics market, taking into account for the first time in scientific literature not only
plant species, but also origin (country, farming system, part of plant) and method of preservation. Antioxidant
activity, expressed as percentage inhibition of DPPH (1,1-diphenyl-2-picrylhydrazyl), ferric ion reducing anti­
oxidant power (FRAP) and content of polyphenolic compounds (Folin-Ciocalteu method), was determined in
seventeen hydrosols by spectrophotometric methods. Antioxidant potential was in the range of 4.43–39.87% of
DPPH radical inhibition and 1325.65–5794.38 µM Fe(II)/L. Total phenolic content (TPC) in the hydrosols
amounted to 9.33–44.23 mg GAE/L, while total flavonoid content (TFC) ranged from 1.48 to 14.82 mg rutin/L.
The hydrosols had a pH in the range of 3.31–5.42. Conclusions: Plant hydrosols appear to have a high antioxidant
potential, which depends on not only the plant species, but also its origin, part of the plant from which the
hydrosol was obtained and the preservation method used in the finished product.

1. Introduction protection is insufficient lead to oxidative stress.

One of the greatest challenges in skin care, whether it is healthy or
affected by a pathological condition, is how to minimize oxidative stress.
Oxidative stress is a phenomenon caused by oxygen free radicals, or
reactive oxygen species (ROS), i.e. highly reactive atoms or molecules
with one or more unpaired electrons in the outer shell. They are known
to cause oxidative modifications in each of the main cellular macro­
molecules: carbohydrates, lipids, proteins and DNA. Free radicals may
be produced in the body’s endogenous reactions, e.g. ATP production by
mitochondria, phagocytosis, β-oxidation of long-chain fatty acids, as
well as due to exogenous factors, such as environmental pollution,
stimulants (tobacco, alcohol, drugs), heavy or transition metals, indus­
trial solvents, certain foods (e.g. smoked meats, waste cooking oil and
fat) [1–4]. The damage caused by free radicals can be repaired by a class
of molecules generally referred to as antioxidants. However, situations
where excessive amounts of free radicals are produced and antioxidant
The fundamental changes that accompany chronological ageing
include alterations in gene expression, production of reactive oxygen
species by oxidative metabolism, decreased antioxidant defense, telo­
mere shortening, and defects in cellular DNA repair [5]. Skin, as the
body’s largest organ, is exposed to multiple factors causing oxidative
stress. The sources of free radicals in skin include both endogenous
processes taking place in skin cells, e.g. during melanogenesis, and
exogenous factors – mainly UV radiation and environmental pollution
[5,6]. Small amounts of reactive oxygen species may have a beneficial
effect on the body’s functioning. Moderate oxidative stress stimulates
the production of desirable compounds, such as cyclooxygenases and
lipoxygenases, which regulate the inflammatory process. On the other
hand, when produced in excess, free radicals damage skin cell structure
and function [7]. Accumulation of free radicals dysregulates signaling
pathways in cells, affecting cytokine release and leading to inflamma­
tory responses, as well as down-regulating collagen production and

* Corresponding author.
E-mail addresses: karjak@pum.edu.pl (K. Jakubczyk), tuchowska.a@gmail.com (A. Tuchowska), katarzyna.janda-milczarek@pum.edu.pl (K. Janda-Milczarek).

https://doi.org/10.1016/j.biopha.2021.112033
Received 29 June 2021; Received in revised form 1 August 2021; Accepted 7 August 2021
Available online 17 August 2021
0753-3322/© 2021 The Author(s). Published by Elsevier Masson SAS. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/).
K. Jakubczyk et al. Biomedicine & Pharmacotherapy 142 (2021) 112033

stimulating the synthesis and activation of matrix metalloproteinases
responsible for connective tissue degradation. All these processes
consequently lead to loss of skin elasticity and accelerate the formation
of lines and wrinkles [3,5]. ROS are also responsible for provoking the
senescence-associated secretory phenotype, which ultimately promotes
the progression of skin ageing [3]. Many dermatological diseases such as
psoriasis, vitiligo, atopic dermatitis, contact dermatitis also have a
free-radical basis. Free radicals damage cellular DNA, too, contributing
to the development of skin cancer. Moreover, ROS derived from envi­
ronmental pollutants can damage the outer layers of the epidermis,
which weakens the skin barrier and alters skin immunity [8].
Plants have been used to make medicinal preparations since time
immemorial. Different parts of the plant have been used for this purpose
(leaves, flowers, fruits, rhizomes, roots, stalks or bark), in the form of
extracts or mixes. In recent years, we can observe a growing interest
among scientists, manufacturers, as well as customers, in plant extracts
and phytochemicals in many cosmetic products [1,9]. Plant extracts
contain numerous bioactive compounds, such as alkaloids, terpenes and
polyphenols. Manufacturers are keen to include plant extracts in for­
mulations, due to their numerous and diverse properties, e.g. antioxi­
dant, anti-inflammatory, antimicrobial (including preservative effects),
softening the skin, reducing skin pigmentation, moisturizing and pro­
moting wound healing [9,10]. It has also been demonstrated that plant
preparations and diet exert anticarcinogenic effects, prevent skin dam­
age from exposure to UV radiation, promote healing and epidermal
regeneration, stimulate the production of collagen and elastin, alleviate
symptoms of skin diseases, such as psoriasis, rosacea, acne vulgaris, skin
allergies and atopic dermatitis [11–14]. One of the common forms of
incorporating plant material in cosmetics are hydrosols – products of
hydrodistillation of aromatic plants [15]. Hydrosols are obtained in the
process of extracting essential oils from aromatic plants. In particular,
they are made up of condensed water from the distillation process and
polar, oxygenated, odor-imparting, hydrophilic, volatile oil constituents
that form hydrogen bonds with water. They are a mixture of a variable
quantity of essential oil (usually less than 1 g/L) and volatile,
water-soluble, secondary metabolites [16–18]. Distillation water with
dissolved essential oil content can be found in the literature under
various labels, including: hydrosol, hydrolate, hydroflorate, plant aro­
matic waste, aromatic water, floral water, essential aromatic water [18,
19]. Hydrosols can be used in daily skin care routines as toners (to
restore the natural skin pH balance), due to the fact that they usually
have an acidic to neutral pH. They often constitute one of many in­
gredients of a formulation [20,21]. Therefore, the aim of this paper was
to analyze the antioxidant potential of hydrosols available in the cos­
metics market, taking into account for the first time in scientific litera­
ture not only plant species, but also origin of the material (country,
farming system, part of plant) and method of preservation.
2. Material and methods
2.1. Plant material
The material contained 17 of the most popular plant-derived hy­
drosols (from leaves, fruit, flowers) available on the cosmetics market in
Poland (West Pomeranian region). Hydrosols have also been divided
according to the origin, organic crops, part of the plant or the method of
conservation used (Table 1). Immediately after opening, the sample was
subjected to spectrophotometric analysis (without dilution).
2.2. Antioxidant activity by the DPPH methods
The antioxidant activity of samples was measured with the spectro­
photometric method using synthetic radical DPPH (2.2-diphenyl-1-pic­
rylhydrazyl, Sigma, Poznań, Poland) according to Brand-Williams et al.
and Pekkarinen et al. [22,23]. The spectral absorbance was immediately
measured at 518 nm (8453UV, AGILENT TECHNOLOGIES, Santa Clara,
USA). All assays were performed in triplicate. The results are shown in %
of DPPH radical inhibition.
Antioxidant potential (antioxidant activity, inhibition) of tested so­
lutions has been expressed by the percent of DPPH inhibition, using the
following formula:
%inhibition =
A0 − As
∗ 100
A0
where:
A0—absorbance of DPPH solution at 518 nm without tested sample
As—absorbance of DPPH solution at 518 nm with tested sample
2.3. The determination of the ferric ion reducing antioxidant power
(FRAP) method
The FRAP method, used to determine the total reduction potential,
which also means the antioxidant properties of tested ingredient, is
based on the ability of the test sample to reduce Fe3+ ions to Fe2+ ions.
The FRAP unit determines the ability to reduce 1 micromole Fe3+ to
Fe2+ according to Benzie and Strain [24,25]. Absorbance at 593 nm was
measured (8453UV, AGILENT TECHNOLOGIES, Santa Clara, USA). All
assays were performed in triplicate. The ferric ion reducing antioxidant
power was determined from the calibration curve using Fe(II)/L as the
reference standard (0, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900,
1000, 2000, 3000, 4000, 5000 µM Fe(II)/L).

Table 1
Characteristics of plant hydrosols.
Plant Species Part of plant Origin Farming system Preservatives

Lavender (a) Lavandula angustifolia Flower Bulgaria – –

Damascus rose (a) Rosa damascena Flower Bulgaria – –
Camomile Chamomilla recutita Flower Bulgaria – –
Lemon balm Mellisa officinalis Leaves Bulgaria – –
Mint Mentha piperita Leaves Bulgaria – –
Juniper Juniperus communis Fruit France – –
Tea tree Melaleuca alternifolia Leaves France – Potassium Sorbate, Citric Acid
Witch hazel (a) Hamamelis virginiana Leaves France Organic Potassium sorbate, Sodium benzoate, Citric acid
Green tea Camellia sinensis Leaves France Organic Potassium sorbate, Sodium benzoate, Citric acid
Linden tree Tilia cordata Flower France Organic Potassium sorbate, Sodium benzoate, Citric acid
Prickly pear Prickly pear Fruit France Organic Potassium sorbate, Sodium benzoate, Citric acid
Linden tree Tilia platyphyllos Flower France Organic Radish Root ferment filtrate
Wild lavender (b) Lavandula angustifolia Flower France Organic Radish Root ferment filtrate
Rosemary Rosmarinus officinalis Flower France Organic Radish Root ferment filtrate
Witch hazel (b) Hamamelis virginiana Leaves France Organic Radish Root ferment filtrate
Damascus rose (b) Rosa damascena Flower France Organic Radish Root ferment filtrate
Roman chamomile Anthemis nobilis Flower France Organic Radish Root ferment filtrate

2
K. Jakubczyk et al. Biomedicine & Pharmacotherapy 142 (2021) 112033

2.4. The determination of the total polyphenols content (TPC)
Determination of polyphenols was performed according to ISO
14502-1; Singleton and Rossi method using the Folin-Ciocalteu reagent
[26]. Absorbance at 765 nm was measured (8453UV, AGILENT TECH­
NOLOGIES, Santa Clara, USA). All assays were performed in triplicate.
The content of polyphenols was determined from the calibration curve
using gallic acid (GAE) as the reference standard (0, 10, 20, 30, 40, 50,
75, 100, 200 mg/L of gallic acid).
2.5. The determination of the total flavonoids content (TFC)
concentrations of flavonoids were used in the plotting of the standard
calibration curve. The content of flavonoids was determined from the
calibration curve using rutin equivalent as the reference standard
(0–120 mg/L of rutin equivalent). Absorbance at 510 nm was measured
(8453UV, Agilent Technologies, Santa Clara, USA). All assays were
performed in triplicate.
2.6. The determination of pH
The pH of hydrosols was determined by a pH meter (SCHOTT In­
struments; SI Analytics Mainz, Mainz, Germany).

Determination of total flavonoids content was performed according

to the Pękal and Pyrzynska and Hu methods [27,28]. Different

Table 2
Antioxidant potential (DPPH. FRAP) and biochemical composition in plant hydrosol. * p ≤ 0.05 between particular subgroup of hydrosol (Lavender (a) - 1, Damascus
rose (a) - 2, Camomile - 3, Lemon balm - 4, Mint - 5, Juniper - 6, Tea tree - 7, Witch hazel (a) - 8, Green tea - 9, Linden tree - 10, Prickly pear - 11, Linden tree - 12, Wild
lavender (b) - 13, Rosemary - 14, Witch hazel (b) - 15, Damascus rose (b) - 16, Roman chamomile - 17.
Plant FRAP DPPH TPC TFC pH

[µM Fe (II)/L] [%] [mg/L] [mg/L]

mean SD mean SD mean SD mean SD mean SD

1 2, 3, 5, 7, 8, 9, 2, 3, 4, 5, 6, 7, 8, 9, 2, 5, 7, 8, 9, 10, 11, 4, 7, 8, 9, 10, 11, 2, 3, 4, 5, 6, 7, 8, 9,
Lavender (a) 2 336.24 58.55 4.43 0.36 9.93 0.59 10.13 61.53 3.36 0.01
10, 11, 12, 13, 14, 15, 16, 10, 11, 12, 13, 14, 15, 12, 13, 14, 15, 16, 17 12, 13, 14, 15, 16 10, 11, 12, 13, 14, 15, 16,
* *
17 16, 17 17
* * *
Damascus rose 5 794.381, 3, 4, 5, 6, 7,
245.06 12.011, 3, 4, 5, 6, 7, 8, 0.30 25.211, 3, 4, 5, 6, 7, 8,
1.63 10.034, 7, 8, 9, 10, 11, 2.57 3.8651, 3, 4, 5, 6, 7, 8, 0.01
(a)2 8, 9, 10, 12, 13, 14, 15, 16, 9, 10, 11, 12, 13, 14, 15, 9, 10, 11, 12, 13, 14, 15, 12, 13, 14, 15, 16
* 9, 10, 11, 12, 13, 14, 15,
17 16, 17 16, 17 16, 17
* * * *
Camomile3 1 325.651, 2, 4, 6, 7, 8,
23.52 6.221, 2, 4, 6, 7, 8, 11, 0.30 9.332, 5, 7, 8, 9, 10, 11, 0.53 5.764, 6, 7, 8, 9, 10,
35.79 3.641, 2, 4, 5, 6, 7, 8, 9, 0.03
9, 10, 11, 12, 13, 14, 15, 12, 13, 14, 15, 16, 17 12, 13, 14, 15, 16, 17 11, 12, 13, 14, 15, 16 10, 11, 12, 13, 14, 15, 16,
* * *
16, 17 17
* *
Lemon balm4 2 152.682, 3, 5, 7, 8, 9,
184.46 7.391, 2, 3, 7, 11, 12,
0.60 10.98, 2, 5, 7, 8, 9, 10,
0.42 8.831, 2, 3, 5, 6, 7, 8,
43.99 3.341, 2, 3, 5, 6, 7, 8, 9, 0.01
10, 11, 12, 15 13, 14, 15, 16, 17 11, 12, 13, 14, 15, 16, 9, 10, 11, 12, 13, 14, 15, 10, 11, 12, 13, 14, 15, 16,
* *
17 16, 17 17
* * *
Mint5 1 555.911, 2, 4, 6, 7, 8,
105.09 6.941, 2, 6, 7, 11, 12,
0.11 12.841, 2, 3, 4, 6, 7, 8,
1.27 10.184, 7, 8, 9, 10, 11, 5.37 3.401, 2, 3, 4, 6, 7, 8, 9, 0.01
9, 10, 11, 12, 13, 14, 15, 13, 14, 15, 16, 17 9, 10, 11, 12, 13, 14, 15, 12, 13, 14, 15, 16 10, 11, 12, 13, 14, 15, 16,
* *
16, 17 16, 17 17
* * *
Juniper6 2 292.982, 3, 5, 7, 8, 9,
207.33 8.191, 2, 3, 5, 7, 9, 10, 0.67 10.832, 5, 7, 8, 9, 10,
0.84 9.973, 4, 7, 8, 9, 10,
31.37 3.281, 2, 3, 4, 5, 7, 8, 9, 0.01
10, 11, 12, 13, 15, 16 12, 13, 14, 15, 16, 17 11, 12, 13, 14, 15, 16, 11, 12, 13, 14, 15, 16 10, 11, 12, 13, 14, 15, 16,
* * *
17 17
* *
Tea tree7 4 088.021, 2, 3, 4, 5, 6,
147.74 5.561, 2, 3, 4, 5, 6, 8, 9, 0.17 21.841, 2, 3, 4, 5, 6, 8,
0.01 5.451, 2, 3, 4, 5, 6, 8,
29.87 4.711, 2, 3, 4, 5, 6, 8, 9, 0.03
8, 9, 10, 11, 12, 13, 14, 15, 10, 11, 12, 13, 14, 15, 10, 11, 12, 13, 14, 15, 16, 9, 10, 11, 12, 13, 14, 15, 10, 11, 12, 13, 14, 15, 16,
16, 17 16, 17 17 16, 17 17
* * * * *
Witch hazel 4 601.541, 2, 3, 4, 5, 6,
45.55 7.891, 2, 3, 4, 11, 12,
0.26 15.751, 2, 3, 4, 5, 6, 7,
0.35 2.241, 2, 3, 4, 5, 6, 7,
1.91 3.521, 2, 3, 4, 5, 6, 7, 9, 0.01
(a)8 7, 11, 12, 13, 14, 15, 16, 13, 14, 15, 16, 17
* 9, 11, 12, 13, 14, 15, 16, 9, 10, 12, 13, 14, 15, 16, 10, 11, 12, 13, 14, 15, 16,
17 17 17 17
* * * *
Green tea9 4 815.231, 2, 3, 4, 5, 6,
47.11 6.891, 2, 6, 7, 11, 12,
0.12 20.631, 2, 3, 4, 5, 6, 8,
0.26 1.481, 2, 3, 4, 5, 6, 7,
21.95 3.721, 2, 3, 4, 5, 6, 7, 8, 0.01
7, 11, 12, 13, 14, 15, 16, 13, 14, 15, 16, 17 10, 11, 12, 13, 14, 15, 16, 8, 10, 12, 13, 14, 15, 16, 10, 11, 12, 13, 14, 15, 16,
*
17 17 17 17
* * * *
Linden tree10 4 750.731, 2, 3, 4, 5, 6,
86.05 6.561, 2, 6, 7, 8, 11, 12, 0.09 16.301, 2, 3, 4, 5, 6, 7,
0.18 4.681, 2, 3, 4, 5, 6, 7,
66.81 3.601, 2, 3, 4, 5, 6, 7, 8, 0.03
7, 8, 9, 11, 12, 13, 14, 15, 13, 14, 15, 16, 17 9, 12, 13, 14, 15, 16, 17 8, 9, 11, 12, 13, 14, 15, 9, 11, 12, 13, 14, 15, 16,
* *
16, 17 16, 17 17
* * *
Prickly pear11 5 565.981, 3, 4, 5, 6, 7,
270.60 8.681, 2, 3, 4, 5, 7, 9,
0.31 18.021, 2, 3, 4, 5, 6, 7,
0.58 1.741, 2, 3, 4, 5, 6, 7,
8.98 3.4851, 2, 3, 4, 5, 6, 7, 0.01
10, 12, 13, 14, 15, 16, 17 10, 12, 13, 14, 15, 16, 8, 9, 12, 13, 14, 15, 16, 10, 12, 13, 14, 15, 16, 8, 9, 10, 12, 13, 14, 15,
*
17 17 17 16, 17
* * * *
Linden tree12 1 818.761, 2, 3, 4, 5, 6,
7.27 28.231, 2, 3, 4, 5, 6, 7, 0.64 37.181, 2, 3, 4, 5, 6, 7,
0.44 14.871, 2, 3, 4, 5, 6, 7, 24.92 5.421, 2, 3, 4, 5, 6, 7, 8, 0.03
7, 8, 9, 10, 11 8, 9, 10, 11, 13, 14, 15, 8, 9, 10, 11, 13, 14, 17 8, 9, 10, 11, 13, 14, 15, 9, 10, 11, 13, 14, 15, 16,
* *
16, 17 16, 17 17
* * *
Wild lavender 2 019.101, 2, 3, 4, 5, 6,
37.44 39.871, 2, 3, 4, 5, 6, 7, 0.25 41.321, 2, 3, 4, 5, 6, 7,
1.89 12.621, 2, 3, 4, 5, 6, 7, 25.73 4.321, 2, 3, 4, 5, 6, 7, 8, 0.03
(b)13 7, 8, 9, 10, 11, 12, 14, 15, 8, 9, 10, 11, 12, 14, 15, 8, 9, 10, 11, 12, 15, 16, 8, 9, 10, 11, 12, 15, 16, 9, 10, 11, 12, 14, 15, 16,
16, 17 16, 17 17 17 17
* * * * *
Rosemary14 2 060.831, 2, 3, 5, 6, 7,
1.77 33.531, 2, 3, 4, 5, 6, 7, 0.49 40.241, 2, 3, 4, 5, 6, 7,
1.19 12.971, 2, 3, 4, 5, 6, 7, 22.44 4.431, 2, 3, 4, 5, 6, 7, 8, 0.01
8, 9, 10, 11 8, 9, 10, 11, 12, 14, 15, 8, 9, 10, 11, 12, 13, 14, 8, 9, 10, 11, 12, 17 9, 10, 11, 12, 13, 14, 15,
* *
17 15, 16, 17 16, 17
* * *
Witch hazel 1 851.091, 2, 3, 4, 5, 6,
11.64 30.861, 2, 3, 4, 5, 6, 7, 0.34 36.941, 2, 3, 4, 5, 6, 7,
0.15 13.601, 2, 3, 4, 5, 6, 7, 6.68 3.311, 2, 3, 4, 5, 6, 7, 8, 0.01
(b)15 7, 8, 9, 10, 11
* 8, 9, 10, 11, 12, 13, 14, 8, 9, 10, 11, 13, 14, 17
* 8, 9, 10, 11, 12, 13, 17
* 9, 10, 11, 12, 13, 14, 16,
16, 17 17
* *
Damascus rose 2 031.201, 2, 3, 5, 6, 7,
23.99 32.541, 2, 3, 4, 5, 6, 7, 1.63 37.371, 2, 3, 4, 5, 6, 7,
0.06 13.581, 2, 3, 4, 5, 6, 7, 32.58 4.171, 2, 3, 4, 5, 6, 7, 8, 0.03
(b)16 8, 9, 10, 11
* 8, 9, 10, 11, 12, 13, 15, 8, 9, 10, 11, 13, 14, 17
* 8, 9, 10, 11, 12, 13, 17
* 9, 10, 11, 12, 13, 14, 15,
17 17
* *
Roman 2 071.561, 2, 3, 5, 7, 8,
26.35 36.601, 2, 3, 4, 5, 6, 7, 0.53 44.231, 2, 3, 4, 5, 6, 7,
0.95 10.314, 7, 8, 9, 10, 11, 68.36 4.561, 2, 3, 4, 5, 6, 7, 8, 0.01
chamomile17 9, 10, 11
* 8, 9, 10, 11, 12, 13, 14, 8, 9, 10, 11, 12, 13, 14, 12, 13, 14, 15, 16
* 9, 10, 11, 12, 13, 14, 15,
15, 16 15, 16 16
* * *

3
K. Jakubczyk et al. Biomedicine & Pharmacotherapy 142 (2021) 112033

2.7. Statistical analysis
In all the experiments, three samples were analyzed and all the as­
says were carried out at least in triplicate. The statistical analysis was
performed using Stat Soft Statistica 13.0 and Microsoft Excel 2017. The
results are expressed as mean values and standard deviation (SD). To
assess the differences between examined parameters, one way analysis
of variance (ANOVA) with Tukey’s post-hoc test was used. Differences
were considered significant at p ≤ 0.05.
3. Results
The antioxidant activity of hydrosols from different plant materials
was evaluated according to the total content of polyphenols and flavo­
noids, redox potential (FRAP) and DPPH radical scavenging potential
(Table 2). The analysis also accounted for the origin of material (coun­
try, farming system), part of the plant and preservation method
(Table 3). Statistically significant differences are presented in Tables 2
and 3.
Total phenolic contents (TPC) in the hydrosols amounted to
9.33–44.23 mg GAE/L (Table 2), in relation to the gallic acid standard
curve. The highest total phenolic content was observed in hydrosols with
radish root ferment (p < 0.05), irrespective of the plant species (Linden
tree - TPC - 37.18, Wild lavender (b) - 41.32, Rosemary TPC - 40.24,
Roman chamomile – 44,23 mg GAE/L), and in Damascus Rose (a) –
25,21, Tea Tree – 21, 84 and Green Tea hydrosols – 20. 63 (Table 2). This
parameter was also affected by other factors, including origin – higher
amounts of polyphenols were found in hydrosols from France, as well as
those made from flowers (p < 0.05) and those originating from organic
farming (Table 3).
Total flavonoid contents (TFC) ranged from 1.48 to 14.82 mg rutin/
L, in relation to the standard curve. Hydrosols preserved with radish root
ferment, irrespective of the plant variety, had the highest content of
these compounds (p < 0.05) (Table 3). Part of the plant emerged as
another important factor – the highest levels of flavonoids were
observed in flowers, followed by leaves, and the lowest in fruits
(p < 0.05) (Table 3).
It was demonstrated that plant hydrosols had differing antioxidant
properties. The percentage of DPPH radical scavenging amounted to
4.43–39.87%. In this case, too, plant hydrosols with the ferment showed
the strongest antioxidant activity, irrespective of the plant species (Ta­
bles 2, 3). Hydrosols made from flowers, originating from France, grown
organically, and above all those preserved naturally using radish root
ferment have a significantly higher antioxidant potential expressed as
percentage of DPPH radical inhibition (p < 0.05) (Table 3).
Redox potential, expressed in µM Fe(II)/L, was varied and fell in the
range of 1325.65–5794.38 µM Fe(II)/L. The highest reducing power was
observed in the Damascus Rose (a) and Green Tea hydrosols. Factors
with a significant impact on redox potential included part of the plant,
specifically fruit, as well as chemical preservation (p < 0.05).
In our study, the pH of plant hydrosols was in the range of 3.31–5.42.
With respect to pH, no significant differences were noted for parameters
related to plant origin (country, type of farming) (Table 2). Higher
values were observed in the case of extraction from flowers and bio­
logical preservation (p < 0.05) (Table 3). Differences were also found
between respective plant species. The highest values (>4) were reported
for Tea Tree, Linden Tree, Wild Lavender (b), Rosemary, Damascus Rose
(b), Roman Chamomile (Table 2).
In the case of duplicate hydrolates obtained from the same plant,
their composition and properties were mainly influenced by the method
of their conservation. In the case of Witch hazel, Lavender and Damascus
rose, biological conservation increased the antioxidant potential (DPPH)
and the content of polyphenolic compounds, including flavonoids. In the
case of Lavender and Damascus rose, these samples were characterized
by a higher pH, which could also have influenced these results (Table 2).
4. Discussion
Hydrosols, also known as hydrolates, are by-products of the hydro­
distillation of plants. They consist of the distillation water in which very
small amounts of essential oils remain dispersed. Hydrosols are widely
used in cosmetics, as ingredients of many products, e.g. creams, or on
their own as toners. Studies completed to date suggest hydrosols also
lend themselves to applications in the agri-food industry, to inhibit the
development of pathological microorganisms in food and to remove
biofilms constituting a threat to public health in food, pharmaceuticals
and beauty products [29]. These uses may help cut down on the use of
antibiotics and counteract the phenomenon of antimicrobial resistance
[30].
Hydrosols have also traditionally been used in diluted form in
refreshing drinks [18]. Their popularity can be attributed to their rich
chemical composition, as well as safety of use. They tend not to contain
many typical essential oils. They usually do not have a strong smell,
which could cause adverse reactions, such as headaches or contact
dermatitis. Generally, they can be applied directly to the skin or ingested
[18]. Despite their widespread use in many economic sectors, their
composition and properties have not been sufficiently explored. Scien­
tists tend to focus their attention on the essential oil composition in the
most popular aromatic plants, e.g. lavender or rose, and on their anti­
microbial properties. On the other hand, there are no studies comparing
the antioxidant properties and composition of hydrosols made from
different plant species, taking into account their origin, farming system,
part of plant or preservation method, while these factors may signifi­
cantly influence their properties.
In this paper, we examined seventeen different hydrosols from
fourteen plant species (Table 1). For the first time in scientific research,

Table 3
Antioxidant potential (DPPH, FRAP) and biochemical composition in plant hydrosol by classification, origin, method of preservatives and other factors. * p ≤ 0.05
between particular subgroup of hydrosol (Flower – a, Fruit - b, Leaves - c, Bulgaria - d, France - e, Non organic - f, Organic - g, Non preservatives - h, Chemical - i, Biological
- j).
FRAP DPPH TPC TFC pH

[µM Fe (II)/L] [%] [mg/L] [mg/L]

mean SD mean SD mean SD mean SD mean SD

Part of plant Flowera 2689.83c* 56.67 22.22b, c* 0.51 29.01b, c* 0.83 10.55b, c* 37.86 4.15 0.02
Fruitb 3929.48 238.97 8.44a* 0.49 14.43a* 0.71 5.86a* 20.18 3.38 0.01
Leavesc 3177.41a* 90.27 10.92a* 0.27 19.83a* 0.41 6.97a* 18.29 3.67 0.01
Origin Bulgariad 2632.97 123.34 7.40e* 0.33 13.66e* 0.89 8.99 29.85 3.52 0.01
Francee 3163.92 76.07 20.45d* 0.46 28.39d* 0.58 8.63 28.47 4.04 0.02
Farming system Non organicf 2792.27 138.82 7.25g* 0.36 14.42g* 0.76 8.62 30.07 3.66 0.02
Organicg 3125.30 63.33 21.72f* 0.46 29.31f* 0.62 8.79 28.22 4.02 0.02
Preservatives Nonh 2576.31i,j* 137.34 7.53i* 0.39 13.19i, j* 0.88 9.15i, j* 30.10 3.48i, j* 0.01
Chemicali 4933.37h, j* 112.33 7.51j* 0.20 17.68h, j* 0.34 2.54h, j* 24.91 3.58h, j* 0.02
Biologicalj 1975.42h, i* 18.08 33.61h, i* 0.65 39.55h, i* 0.78 12.99h, i* 30.12 4.37h, i* 0.02

4
K. Jakubczyk et al.
the hydrosols were grouped according to the country of origin (France,
Bulgaria), farming system (organic/conventional), part of plant
(flowers, leaves, fruits), and preservation method: with the use of
chemical factors (Potassium sorbate, Sodium benzoate, Citric acid),
biological factors (radish root ferment) or without preservatives.
There are few studies dedicated to the antioxidant properties of hy­
drosols, but their findings are consistent with those obtained in this
study, showing a moderate antioxidant capacity. An analysis of hydro­
sols obtained from coconut and arecanut showed they could effectively
inhibit DPPH (25.98–81.83%) and ABTS (4.75–63.57%), showing
strong antioxidant and reducing power (FRAP). The hydrosol made from
coconut flower had the highest total phenolic content, while arecanut
flower hydrosol had the best scavenging effect on DPPH [31]. Similar
findings are also reported in our study in terms of reducing power
(FRAP) for the majority of studied parameters; in other cases, high TFC
or TPC levels were associated with a higher antioxidant capacity, but
with reducing potential this relationship was not observed. These dif­
ferences may point to some other mechanisms at play or presence of
certain masked bioactive compounds [31]. The main mechanism may
also involve a cross-reaction of radicals emerging from reactions be­
tween highly reactive peroxyl radicals and antioxidants [32]. In addi­
tion, both methods have certain limitations and different reactivities of
standard antioxidants were found in both tests, with glutathione
showing low reactivity in the FRAP assay [33,34]. Moreover, Calendula
arvensis L. hydrosol showed very strong antioxidant activity (IC50
25.1 mg/L observed in the DPPH test), comparable with that of the
synthetic antioxidant BHT (17.3 mg/L). These properties are probably
associated with its chemical profile, particularly the relatively high
percentage of oxygenated sesquiterpenes [35]. In a study on hydrosol
extracts obtained by liquid–liquid extraction (LLE) of Daucus muricatus
L., high phenolic contents were observed and all extracts showed
reductive effects, which increased with an increase in concentration
(FRAP). All of the hydrosols were able to reduce the stable,
purple-colored radical DPPH into yellow-colored DPPH-H, reaching
50% of reduction with an IC50: 3.64 μg/mL (flowers), 5.15 μg/mL
(roots), 5.83 μg/mL (aerial parts), 7.19 μg/mL (stems) and 7.50 μg/mL
(leaves). All the tested samples showed stronger antioxidant activity
than BHT [36]. A study on rose hydrosols demonstrated antioxidant
potential to be concentration-dependent. At concentrations of 0.5% and
1% R. alba L. hydrosol showed 6% and 13% inhibition, respectively,
whereas the values obtained for R. damascena Mill. hydrosol demon­
strated very low inhibitory potential. At concentrations of 10%, the
inhibitory effect of R. damascena Mill. hydrosol was about half of that of
R. alba L; however, at concentrations of 15%, the values reached 22%
and 20%, respectively [37].
Findings from the present study show that antioxidant potential is
not only determined by the plant species, but it is also significantly
influenced by plant origin, use of organic farming systems, application
of different preservation methods (notably natural) as well as part of the
plant used. Significantly higher results were obtained in the group of
hydrosols preserved using natural methods. Peptides obtained from
radish root extend the shelf life of the hydrosol, at the same time
providing exceptional moisturizing and stabilizing effects, and
enhancing antioxidant properties. Plant hydrosols are commonly
referred to as floral waters, due to the fact that flowers are most often
used to obtain them. Our study confirms that floral hydrosols had the
highest antioxidant potential expressed as percentage of DPPH radical
inhibition, as well as total content of polyphenols and flavonoids, i.e.
compounds responsible for antioxidant properties. The results obtained
with flower 2689.83 µM Fe(II)/L, 22.22% antioxidant activity, TPC
29.01 mg/L, TFC 10.55 mg/L, pH - 4.15 (Table 3). Scientific literature
confirms that phenolic compounds may promote antioxidant activity,
showing direct antioxidant effects due to the presence of hydroxyl
groups, which may act as donors of H2 [4,36]. DPPH is a stable free
radical that can accept an electron or hydrogen radical to become a
stable diamagnetic molecule. DPPH radical reacts with suitable reducing
5
Biomedicine & Pharmacotherapy 142 (2021) 112033
agent producing new bond, thus changing the color of the solution.
Flavonoids has been reported to be the most significant determinant
ROS scavenging because it donates hydrogen and an electron to hy­
droxyl, hydroperoxyl, and peroxynitrite radicals, stabilizing them and
giving rise to a relatively stable flavonoids radical.
The findings obtained in our study are consistent with those of other
authors, who confirm that antioxidant activity is associated with a high
phenolic content. High TFC or TPC levels were associated with a higher
antioxidant capacity, but with reducing potential this relationship was
not observed (Wild lavender - 39.87% - antioxidante activity - TPC -
41.32; Rosemary - 33.53% - TPC - 40.24; Roman chamomile - 36.60% -
TPC - 44.23; Roman chamomile - 36.60). The radical reduction DPPH
test uses preformed radicals and determines the decrease in absorbance,
while the FRAP test measures the formed ferrous ions through increased
absorbance. Therefore, it is recommended to use analyzes based on
various mechanisms. In the studies of Chaves et al. the DPPH method
showed higher results compared to the FRAP method, these tests also
showed different sensitivity [38].
Shen et al. demonstrated that total phenolic contents of hydrosols
from different parts of arecanut and coconut ranged from 0.17 to
3.88 ug GAE/mL [31]. Total phenolic contents amounted to 5.2 mg
GAE/L for rose hydrosol [39], 54.5 and 49.0 mg GAE/g for aqueous
extracts of arecanut of root and adventitious root, respectively [40], and
406.43 ug GAE/mL for areca flower extracts [41]. Hydrosols made from
the flowers of C. aurantium were found to contain total phenolics at
0.803 ± 0.1 mg GAE/g of extract, and total flavonoids at
0.324 ± 0.09 mg QE/g of extract [42]. Hydrosols of Rosa alba L. and
Rosa damascena Mill. were also examined for their total phenolic content
(TPC). It was determined that the hydrosol of Rosa alba L. contained
72.72 μg GAE/mL, which was more than twice the amount in Rosa
damascena – 32.52 μg GAE/mL [37].
Hydrosols are neutral to slightly acidic, with a pH value ranging from
3 to 7. The normal facial skin pH lies between 4.7 and 5.6. Hence, the pH
level of hydrosols should fall in the same range, because a higher or
lower pH might have a negative effect and cause skin problems. In our
study, the pH of plant hydrosols was in the range of 3.31–5.42,
depending on plant species and preservation method. The hydrosols
with radish root ferment were characterized by the optimal pH (4.37),
which, together with their high antioxidant potential, supports their use
in everyday skin care. Thanks to their optimal pH value, these hydrosols
can restore the skin’s acidity, e.g. after using alkaline cleansing prod­
ucts. This is very important in skin care, whether healthy or affected by
pathological conditions, for instance helping maintain a healthy mi­
crobial environment on the skin surface. Our findings in this regard are
consistent with those of other authors [17,37,43]. In a study by Labadie
et al., pH values were diverse and ranged from 4 to 7, depending on
plant species and conditions of filtration and storage [17].
Hydrosols available in the European cosmetics market originate
mainly from France and Bulgaria. Our study confirmed that products
from France have a higher content of bioactive compounds and signif­
icantly stronger antioxidant properties. A similar correlation was noted
in hydrosols made from organically-grown material. It can therefore be
concluded that antioxidant capacity of hydrosols is not determined
solely by plant species, and consequently by chemical composition, but
also by other factors taken into account in this study.
5. Conclusion
The high antioxidant properties of hydrosols can be employed in
cosmetology, to fight against skin ageing or natural prevention and
treatment of skin diseases, as well as in the food industry. Due to the
high antioxidant potential of hydrosols, they are capable of inhibiting
oxidation processes, thus potentially extending the shelf life of foods or
cosmetic products. Antioxidant potential as such is not determined
solely by plant species, but also depends on factors like part of the plant,
its origin and preservation method. Therefore, for the best antioxidant
K. Jakubczyk et al.
capacity, one should choose hydrosols of organic origin, made from
flowers, originating from France and preserved using natural methods.
Nonetheless, further research on this subject is needed.
Funding
The project is financed under the program of the Minister of Science
and Higher Education under the name “REGIONALNA INICJATYWA
DOSKONAŁOŚCI” in 2019–2022 (Project No. 002/RID/2018/19).
CRediT authorship contribution statement
Karolina Jakubczyk: Conceptualization, Funding acquisition,
Investigation, Methodology, Project administration, Supervision,
Writing – original draft. Aleksandra Tuchowska: Writing – original
draft. Katarzyna Janda-Milczarek: Methodology, Resources, Supervi­
sion, Writing – review & editing.
Declaration of Competing Interest
The authors declare that they have no conflict of interest. We declare
that this manuscript is original, has not been published before and is not
currently being considered for publications elsewhere. The authors
declare they have no actual or potential competing financial interests.
This article does not contain any studies with human or animal subjects.
Acknowledgement
Not applicable.
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