Industrial Crops & Products 136 (2019) 93–101

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Industrial Crops & Products

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

Advantages of contemporary extraction techniques for the extraction of T

bioactive constituents from black elderberry (Sambucus nigra L.) flowers
⁎
Vujanović Milenaa, , Majkić Tatjanab, Zengin Gökhanc, Beara Ivanab, Cvetanović Aleksandraa,
⁎
Mahomoodally Fawzi Mohammadd, Radojković Marijaa,
a
Faculty of Technology, University of Novi Sad, Bulevar cara Lazara 1, 21000, Novi Sad, Serbia
b
Faculty of Sciences, University of Novi Sad, Trg Dositeja Obradovića 3, 21000, Novi Sad, Serbia
c
Science Faculty, Selcuk University, Konya, Turkey
d
Faculty of Science, University of Mauritius, 230 Réduit, Mauritius

A R T I C LE I N FO A B S T R A C T

Keywords: Since ancient times, elderberry (Sambucus nigra L.) has been used in traditional medicine, but also as a sup-
Sambucus nigra plement in various beverages and dishes. Although elderberry is a widespread species in the Balkan Peninsula, it
Elderflower is not sufficiently exploited and it is barely chemically and biologically characterized. The present study was
Extraction focused on the phytochemical composition and biological effects of S. nigra flowers’ extracts, obtained by dif-
Phenolic profile
ferent techniques (microwave-assisted extraction (MAE), ultrasound-assisted extraction (UAE) and maceration
Biological activity
(MAC)) in the presence of different solvents (50% ethanol (EtOH) or water (H2O)). The phytochemical profiles of
the obtained extracts were determined by LC–MS/MS and spectrophotometric techniques: rutin and 5-O-caf-
feoylquinic acid were found to be the major compounds in all extracts.
To evaluate its biological potential, antioxidant, neuroprotective (acetyl – and butyrylcholinesterase inhibi-
tion (AChE and BChE)), antityrosinase and antilipase abilities were tested. MAE with 50% EtOH exhibited the
strongest antioxidant activity, which was in good correlation with the high content of phenolics. The best AChE
inhibitory effect was observed by MAC with H2O extract. However, the strongest tyrosinase inhibitors were 50%
EtOH extracts. The presented results prove the high biological and industrial potential of S. nigra flowers and
open new directions towards novel product design.

1. Introduction quality extracts (Vazquez-Roig and Picó, 2015). Consequently, different

techniques have arisen, but the special place in scientific research in the
Nowadays, in many branches of industry which produce and use process of isolating bioactive compounds from plants is occupied by
plant extracts, special attention is directed to the development of ex- microwave-assisted (MAE) and ultrasonic-assisted (UAE) extractions.
traction techniques which are economically sustainable, but also en- These techniques enhance the extraction efficiency by unique phe-
vironmentally-friendly (Chemat et al., 2012; Cunha and Fernandes, nomena, which are occurring during the processes – in UAE it is cavi-
2018). The commonly used extraction techniques tend to be replaced tation, while in MAE it is an interaction between microwaves and polar
due to the recognized drawbacks, such as high consumption of solvents molecules in the media. Both UAE and MAE are suitable for the ex-
and plant material, usage of organic solvents, high energy consumption, traction of plant bioactive molecules such as polyphenols, providing
etc. The new approaches should offer technologies which are able to extracts with a higher concentration of active compounds and enhanced
reduce the use of organic solvents, minimize energy consumption, en- biological activity. Different studies have already demonstrated the
able faster processing of samples and cost-effective production of high- advantages of these techniques over conventional ones (Belwal et al.,

Abbreviations: 50%EtOH extract, 50% ethanol extract; ABTS, 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid); AChE, acetylcholinesterase; BChE, butyr-
ylcholinesterase; CE, Catechin equivalent; CUPRAC, cupric reducing antioxidant capacity; EDTA, ethylenediaminetetraacetic acid equivalents; FRAP, ferric reducing
antioxidant power; GAE, Gallic acid equivalent; GALE, Galantamine equivalents; GRAS, Generally Recognized As Safe; H2Oextract, water extract; KAE, Kojic acid
equivalents; LC–MS/MS, liquid chromatography-mass spectrometry; MAC, maceration; MAE, microwave assisted extraction; OE, orlistat equivalents; PCA, principal
component analysis; TE, trolox equivalents; UAE, ultrasound assisted extraction; VIP, variable importance prediction; WHO, World Health Organization
⁎
Corresponding authors.
E-mail addresses: milenavujanovic@uns.ac.rs (V. Milena), ramarija@uns.ac.rs (R. Marija).

https://doi.org/10.1016/j.indcrop.2019.04.058
Received 15 March 2019; Received in revised form 23 April 2019; Accepted 27 April 2019
Available online 08 May 2019
0926-6690/ © 2019 Elsevier B.V. All rights reserved.
V. Milena, et al. Industrial Crops & Products 136 (2019) 93–101

2017; Rao and Rathod, 2017). were the same as in the case of UAE, while the power of microwave
At the same time, there is an increasing interest, by both science and irradiation was 360 W.
industry, for the wild growing plant species, due to their simple culti- As for the maceration (MAC), the plant sample (5 g) was extracted
vation, high availability, and abundance with secondary metabolites. using 50% EtOH or H2O (150 mL) as a solvent. The extraction condi-
The Balkan Peninsula is rich in a large number of wild growing plants, tions were set according to the procedure previously described in detail
and one of the most abundant is black elderberry (Sambucus nigra L.), by Cvetanović et al. (2019).
which belongs to the Adoxaceae family. Its common names are elder, All the obtained extracts were stored in a dark glass bottle at −20 °C
elderberry, black elder, European elder, European elderberry and to prevent oxidative damage.
European black elderberry (ITIS, 2016). All parts of this plant (flower,
bark, leaf, and fruits) are the rich sources of dietary phytochemicals, 2.3. Determination of total phenolics (TPC) and flavonoids (TFC) contents
such as carbohydrates, lipids, terpenoids, flavonoids, phenolic acids,
alkaloids, etc. (Agalar (2019)). Elderflowers have been used in tradi- TPC and TFC were measured according to the well-known spec-
tional medicine for treatment of inflammation, skin disorders, diuretic, trophotometrically procedures Singleton and Rossi (1965) and
colds, fever and other respiratory disturbances (Blumenthal et al., 2000; Radojković et al. (2016), respectively. The obtained results for were
Weiss and Fintelmann, 2000; EMA, 2008). What is more, scientific calculated and expressed as mg GAE/g extract in case of TPC, and mg
evidence implied that elderberry flowers can be used both in the pre- CE/g extract in case of TFC.
vention and therapy of various diseases, due to the expressed anti-in-
flammatory (Olejnik et al., 2015), antioxidant (Dawidowicz et al., 2.4. LC–MS/MS analysis of phenolic compounds
2006) and antimicrobial (Arjoon et al., 2012) activity. Apart from tra-
ditional medicine, the elderberry flower is often used in the traditional LC–MS/MS method published by Orčić et al. (2014) was used for the
diet, usually for the preparation of beverages, such as tea or juice. determination of phenolic compounds and quinic acid in the obtained
What is astonishing is the fact that although the products of elder- elderflower extracts. For all identified compounds, different analytical
berry flowers are very popular in everyday, traditional use, there is not standards were used, while the quantification of the present compounds
a single commercial product on the market. On the other hand, the rich was carried out by MS/MS technique. The samples and standards were
chemical composition and presence of a large number of bioactive in- analyzed using Agilent Technologies 1200 Series high-performance li-
gredients, as well as a multitude of flowers on the plant tree, clearly quid chromatography coupled with Agilent Technologies 6410 A Triple
indicate the potential of elderflower use in various industries that are Quad tandem mass spectrometer with an electrospray ion source and
related to pharmaceutical, cosmetic or nutritional functional products. controlled by Agilent Technologies MassHunter Workstation soft-
For this purpose, obtaining highly potent bioactive extracts is the first ware—Data Acquisition (ver. B.03.01). For all the compounds, the peak
and the most important step. Therefore, the goal of this study was to areas were determined using Agilent MassHunter Workstation soft-
investigate different extraction techniques in order to obtain highly ware—Qualitative Analysis (ver. B.03.01). The calibration curves were
bioactive S. nigra flower extracts. In the frame of this paper, elderberry plotted and the concentrations of the samples calculated using the
extracts were prepared by one conventional - maceration (MAC) and OriginLabs Origin Pro (ver. 8.0) software.
two modern (UAE and MAE) techniques in the presence of different
solvents (50% EtOH or H2O) and compared in terms of their chemical 2.5. Evaluation of biological activity
composition and bioactivity. To the best of our knowledge, the ability
of the obtained extracts to inhibit acetylcholinesterase, butyr- 2.5.1. Antioxidant activity
ylcholinesterase, tyrosinase, and lipase was explored for the first time in Antioxidant activity of the obtained extracts was assessed using
this study. several different in vitro assays: ABTS•+, FRAP, CUPRAC, phospho-
molybdenum and metal chelating assays, according to the procedures
2. Material and methods described elsewhere (Beara et al., 2017; Zengin et al., 2018). In case of
the first fourth assays obtained results were expressed as trolox
2.1. Plant materials equivalents (mg TE/g E), while in the case of fifth assay EDTA
equivalents were used (mg EDTA/g E).
Sambucus nigra L. plant material was collected in June 2017 in
Pljevlja (Montenegro). The specimen voucher (S. nigra L., No. 2-1512) 2.5.2. Enzyme inhibitory activity
was prepared and identified by Milica Rat, Ph.D., and deposited at the Previously described enzymatic methods were used for the de-
Herbarium of the Department of Biology and Ecology (BUNS termination of AChE, BChE, tyrosinase, and lipase inhibitory ability
Herbarium), University of Novi Sad, Faculty of Sciences, The Republic (Mollica et al., 2017; Zengin et al., 2017; Mollica et al., 2018, respec-
of Serbia. tively). The inhibitory effects were expressed as standard inhibitor
After drying the plant material was grounded using a laboratory mill equivalents galantamine (mg GALE/g E) for AChE and BChE; kojic acid
and was stored according to the procedure described by Vujanović (mg KAE/g E) for tyrosinase; orlistat (mg OE/g E) for lipase).
et al., 2019. The average diameter of the crushed plant material was
determined by the sifting analysis. The value of the average particle 2.6. Statistical analysis
diameter was 0.23 ± 0.04 mm.
Statistical analysis used in this study was done according to the
2.2. Sample preparation previously described methodology (Zengin et al., 2019). All statistical
analyses were performed using the STATISTICA (StatSoft, Dell) for
Ultrasonic water (Branson, USA) bath was used for ultrasound-as- Windows version 13.2.
sisted extraction (UAE). The extracts were prepared by mixing 5 g of
plant material with 150 mL of solvents (H2O or 50% EtOH). After 3. Results and discussion
30 min of sonification at 40 kHz, the mixture was filtrated and obtained
extracts were stored in the freeze (Veličković et al., 2017). 3.1. Total bioactive constituents
Microwave-assisted extraction (MAE) was carried out in modified
domestic equipment previously described in Đurović et al. (2017). For The flower of S. nigra is a very attractive and insufficiently exploited
the extraction process, solvents-to-sample ratio and extraction time source of highly bioactive secondary metabolites, such as polyphenols.

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V. Milena, et al. Industrial Crops & Products 136 (2019) 93–101

Only a few publications mention the qualitative and quantitative con- compounds from elderflower, using both 50% EtOH and H2O (417.6
tent of polyphenols in elderflowers (Christensen et al. (2010); Mikulic- and 322.6 mg GAE/g E). UAE yielded a slightly lower amount of phe-
Petkovsek et al., 2015; Thi Ho et al., 2016). One of the challenges in this nolics than MAE (362.5 for 50% EtOH extract and 279.1 mg GAE/g E
study was the fact that due to the high variety of secondary metabolites, for H2O extract). Although MAC is the traditional extraction technique,
there is no single solvent or optimal extraction technique for their ex- in the process of isolating the biologically active compounds from el-
traction in general, and also, there is no specific recommendation for derflower, it achieved very good results. Same as in MAE and UAE, 50%
their extraction from elderflower either. Hence, this paper focused on EtOH has been proved to be a more efficient solvent than H2O. Re-
investigating and defining the most efficient extraction technique re- garding the content of total flavonoids in the investigated elderflower
garding the capacity and use of ˮgreen solventsˮ (ethanol and water) for extracts, the results followed the same trend. It turned out that 50%
the isolation of polyphenols from elderflower. The emphasis is placed EtOH is a better solvent than H2O, while modern extraction techniques
on the use of non-toxic solvents, such as ethanol and water, which are have shown greater efficiency than the traditional ones. The highest
characterized as GRAS (Generally Recognized As Safe). Alcohols (or content of flavonoids was in extracts obtained by MAE, UAE, and MAC,
mixtures of alcohol and water) were used to extract polyphenols from respectively.
plant material, as these compounds are often more soluble in solvents The content of the total polyphenolic compounds obtained by the
less polar than water. Ethanol has some advantages: low toxicity to Folin–Ciocalteu method does not provide a complete qualitative and
humans, and antiseptic properties, which reduce microbiological con- quantitative description of the polyphenolic compounds contained in
tamination of the extracts (Lemos et al., 2017). For these reasons, 50% the extracts, due to the possible presence of interfering compounds
EtOH was used for the extraction, as well as the other solvent – H2O, (sugars, aromatic amines, organic acids, etc.) affecting the measure-
chosen because traditional elderflower beverages are prepared with tap ment results. Therefore, a detailed analysis of the chemical composition
water (Mikulic-Petkovsek et al., 2015). using LC–MS/MS technique was done in order to detect certain differ-
The total content of phenolic and flavonoid compounds (TPC and ences among the extracts.
TFC) in H2O and 50% EtOH extracts were determined using spectro-
photometric assays. The obtained results are presented in Fig. 1. MAE
was the most suitable technique for the isolation of total phenolic

Fig. 1. Total phenolics and flavonoid content in elderflower extracts.

Different letters indicate differences in the tested extracts (p < 0.05).

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V. Milena, et al. Industrial Crops & Products 136 (2019) 93–101

Table 1
Polyphenolic profile of the obtained extracts (μg/mg extract).
Extraction technique MAE UAE MAC MAE UAE MAC
Solvent

Compound 50% EtOH H2O

p-Hydroxybenzoic acid 0.007 ± 0.000 c 0.009 ± 0.001 c 0.010 ± 0.001 c 0.015 ± 0.001c 0.071 ± 0.004b 0.187 ± 0.011a
Protocatechuic acid 0.059 ± 0.005d 0.053 ± 0.004d 0.064 ± 0.005d 0.080 ± 0.006c 0.129 ± 0.010b 0.220 ± 0.018a
Gentisic acid 0.008 ± 0.001d 0.007 ± 0.000e 0.010 ± 0.001 b 0.011 ± 0.001a 0.008 ± 0.001c 0.006 ± 0.000f
p-Coumaric acid 0.035 ± 0.002c 0.028 ± 0.003c 0.033 ± 0.003c 0.042 ± 0.004c 0.518 ± 0.036a 0.406 ± 0.002b
Vanillic acid < LoD < LoD < LoD < LoD 0.097 ± 0.010a 0.091 ± 0.009a
Gallic acid 0.030 ± 0.003b 0.008 ± 0.001d 0.005 ± 0.000d 0.021 ± 0.002c 0.010 ± 0.001d 0.089 ± 0.008a
Esculetin 0.007 ± 0.000b 0.005 ± 0.001c 0.008 ± 0.000a 0.005 ± 0.000d 0.003 ± 0.000e 0.008 ± 0.000a
Caffeic acid 0.129 ± 0.008c 0.079 ± 0.002d 0.156 ± 0.013c 0.188 ± 0.026b 0.153 ± 0.019c 0.272 ± 0.022a
Quinic acid 4.180 ± 0.674d,e 3.752 ± 0.122e 5.615 ± 0.561c 4.728 ± 0.404d 7.214 ± 0.721b 8.395 ± 0.840a
Ferulic acid 0.009 ± 0.001e 0.010 ± 0.001d 0.013 ± 0.001b 0.012 ± 0.001c 0.016 ± 0.002a < LoD
Naringenin 4.418 ± 0.447a 2.066 ± 0.145c 2.651 ± 0.199b 1.694 ± 0.019c 0.252 ± 0.014d 0.351 ± 0.025d
Catechin 0.103 ± 0.010a 0.051 ± 0.005b 0.038 ± 0.004c 0.030 ± 0.003c < LoD < LoD
Epicatechin 0.118 ± 0.012a` 0.069 ± 0.007b 0.034 ± 0.003c 0.019 ± 0.002d < LoD < LoD
Quercetin 0.076 ± 0.008b,c,d 0.067 ± 0.007c,d 0.124 ± 0.012b 0.035 ± 0.004d 0.103 ± 0.010b,c 0.560 ± 0.056a
Isorhamnetin 0.062 ± 0.004c 0.033 ± 0.002d,e 0.058 ± 0.003c,d 0.007 ± 0.000e 0.141 ± 0.008b 0.432 ± 0.026a
5-O-Caffeoylquinic acid 56.49 ± 2.825 a 55.23 ± 2.762a 54.60 ± 2.730 a 44.97 ± 2.248b 2.836 ± 0.142d 16.19 ± 0.810c
Kaempferol 3-O-glucoside 1.349 ± 0.171a 1.463 ± 0.108a 1.400 ± 0.189a 1.292 ± 0.047a 0.487 ± 0.043b 0.284 ± 0.020c
Quercetin -3-O-hexoside 4.328 ± 0.260a 4.333 ± 0.260a 4.293 ± 0.258a 4.071 ± 0.244a 0.496 ± 0.030c 1.440 ± 0.086b
Rutin 76.77 ± 2.303c 91.39 ± 2.742a 81.06 ± 2.432b 61.64 ± 1.849d 1.612 ± 0.048e 2.188 ± 0.066e
Morin 0.025 ± 0.000c 0.026 ± 0.000c 0.033 ± 0.0.00b < LoD < LoD 0.107 ± 0.001a
Ellagic acid 0.256 ± 0.005c 0.289 ± 0.006a 0.276 ± 0.005b 0.215 ± 0.004d 0.064 ± 0.001f 0.093 ± 0.002e
Ursolic acid 28.33 ± 0.000a 23.80 ± 0.000b 21.05 ± 0.000c 1.754 ± 0.000d < LoD < LoD
Kaempferol 0.035 ± 0.002c 0.024 ± 0.002d 0.039 ± 0.002b 0.005 ± 0.000e 0.040 ± 0.003b 0.072 ± 0.001a

*
± 3SD. Means within each row with different letters (a–e) differ significantly (p ≤ 0.05).
*
< LoD limit of detection.
Analyzed, but not detected: Apiin, Apigenin, Apigenin 7-O-glucoside, Baicalin, Baicalein, Chrysoeriol, Daidzein, Genistein, Epigallocatechin gallate, Luteolin,
Luteolin 7-O-glucoside, Matairesinol, Myricetin, Sinapic acid, Syringic acid, Secoisolariciresinol, Vitexin.

3.2. Polyphenolic profile guidance. The dominant flavonoid was rutin and its highest con-
centration was detected in 50% EtOH and H2O MAE extracts, (91.39
LC–MS/MS analysis of the selected 47 phenolics resulted in quan- and 61.64 μg/mg, respectively). The presence of rutin in all extracts is
titative determination of 23 examined compounds (Table 1), while of great importance because of its extraordinary biological activity.
corresponding chromatograms are shown in Fig. 2. Different studies have demonstrated that rutin has considerable biolo-
Presented research has led to great results. It has been established gical properties such as anti – Alzheimer, sedative, anticonvulsant and
that phenolic acids were dominant in H2O extracts, while flavonoids antidiabetic activity (Ganeshpurkar and Saluja, 2017).
were leading in 50% EtOH extracts. Quinic acid, a precursor in the If this investigation is compared with the research carried out by
synthesis of phenolic compounds, is present in considerable con- Mikulic-Petkovsek et al. (2015), it can be noticed that in their research
centrations, predominantly in the MAC H2O extract and MAE 50% of the extracts the dominant compounds were 5-O-caffeoylquinic acid
EtOH extracts. The 5-O-caffeoylquinic acid is the main phenolic acid in and rutin, too, but in higher concentrations. These differences can be
all extracts, particularly high in the 50% EtOH and H2O extracts ob- explained by the diverse origin of the material, different conditions
tained by MAE extraction (56.49 and 44.97 μg/mg E, respectively). 5-O- under which the extraction process was conducted, as well as the use of
caffeoylquinic acid is formed by the isomerization of chlorogenic acid distinct solvents for isolation of the biologically active compounds. The
and identified in fruits, leaves and other vegetal tissues (Dawidowicz variation in the number of present compounds in the current and the
and Typek, 2010). As for the process of isomerization, it can take place comparable study can be explained by stress factors such as tempera-
during the extraction of the plant material. Some studies have shown ture, moisture and soil nutrients, which might be responsible for the
that chlorogenic acid has antioxidant, antiviral (Jassim and Naji, 2003), dissimilarity in polyphenols content in the same plant species (de Matos
antibacterial (Rodriguez de Sotillo et al., 1998) and antifungal (Bowles Nunes et al., 2014; Nantongo et al., 2018). To the best of our knowl-
and Miller, 1994) activity and does not exhibit a toxic effect. Due to its edge, there are only a few studies about the phenolic profile of S. nigra
antioxidative activity, it can contribute to the prevention of type 2 flower, and this is the only one in which the phenolic profile of Mon-
diabetes and cardiovascular diseases (Paynter et al., 2006). The pre- tenegro species is characterized. High content of rutin could be po-
sence of ursolic acid in the examined extracts is very important because tentially used for distinguishing Montenegrian S. nigra from the species
scientific studies were carried out in vitro and in vivo, are declared this located in some northern European regions, but the lack of studies on
acid as a chemoprotective agent (Iqbal et al., 2018). Interestingly, ur- the phenolic profile of this species limits the possibility of comparing
solic acid is present only in 50% EtOH extracts, where the values ranged the S. nigra bioactive compounds from different regions.
from 21.05 to 28.33 μg/mg, and in low concentration in MAE H2O Based on the performed analysis, it has been shown that some
extract, which was not expected since ursolic acid is a triterpenic acid, phenolic compounds are not present in the extracts obtained using
soluble in organic solvents and insoluble in water. This can be ex- modern extraction techniques, while they are detected in the extracts
plained by the power of microwave radiation. Increased radiation obtained by MAC. This result can be explained by the assumption that
power causes local warming in plant cells, which leads to rapid buildup there was a degradation of thermolabile compounds during the ex-
destruction of plant cells and facilitated diffusion of intracellular con- traction process carried out using MAE and UAE, due to the influence of
tents, due to which ursolic acid is also identified in the MAE H2O ex- temperature, while the extracts obtained by MAC were not exposed to
tract (Chan et al., 2011). In order to analyze the chemoprotective effect the temperature. The obtained results of the phytochemical screening
of elderflower extracts, some future studies can be based aimed at this can be of great value in the evaluation of the genus Sambucus members’

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V. Milena, et al. Industrial Crops & Products 136 (2019) 93–101

Fig. 2. LC–MS/MS chromatograms of S. nigra extracts.

a) MAE 50% EtOH extract, b) UAE 50% EtOH extract, c) MAC 50% EtOH extract, d) MAE H2O extract, e) UAE H2O extract, f) MAC H2O.
1: quinic acid, 2: gallic acid, 3: protocatechuic acid, 4: 5-O-caffeoylquinic acid, 5: catechin, 6: gentisic acid, 7: p-hydroxybenzoic acid, 8: epicatechin, 9: esculetin, 10:
caffeic acid, 11: p-coumaric acid, 12: ferulic acid, 13: rutin, 14: quercetin-3-O-hexosid, 15: ellagic acid,16: kaempferol 3-O-glucoside, 17: morine, 18: naringenin, 19:
quercetin, 20: kaempferol, 21: isorhamnetin, 22: ursolic acid, 23: vanillic acid.

medicinal properties and their further exploitation as a source of 3.3. Biological activity
bioactive compounds.
In order to get an overall conclusion whether the determined phe- 3.3.1. Antioxidant activity
nolic could be used to emphasize some difference between the extracts, In this study, several antioxidant tests were used, because the an-
a principal component analysis (PCA) was performed (Fig. 3). The first tioxidant properties of plant extracts can be manifested through various
two components (F1 and F2), accounted for 86.46% of the explained mechanisms. Antioxidant activity S. nigra extracts was examined using
variance if we take into consideration the content of the determined radical scavenging (ABTS•+), reducing power (FRAP and CUPRAC),
phenolic acids and flavonoids. 50% EtOH extracts are located in the metal chelating and phosphomolybdenum assays. The results are de-
same part of the diagram (the lower right side), mainly according to the picted in Table 2.
distinguished content of quercetin 3-O-hexoside, rutin, and kaempferol- The best radical scavenger activity was achieved by 50% EtOH and
3-O-glucoside and chlorogenic acid, which could be potentially used as H2O extracts obtained by MAE, whose values range from 212.7 to
the differentiation factors. This grouping implicates a separation based 209.1 mg TE/g E, respectively. Based on statistical analysis, it seems
on the solvent used for the extraction. The distant position of H2O ex- that there is no difference in the activity of these two extracts. The
tracts is due to a high content of p-coumaric acid in H2O UAE extracts, lowest radical scavenger activity was demonstrated by the H2O ex-
while H2O MAC stands out as the best solvent and extraction technique tracts, which were obtained by MAC and UAE (173.8 and 167.0 mg TE/
for the most of the phenolic acid. g E, respectively). The best radical scavenger activity of the 50% EtOH
extract of MAE can be related to the high concentration of total and

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V. Milena, et al. Industrial Crops & Products 136 (2019) 93–101

Fig. 3. Principal component analysis of phe-

nolic acids and flavonoids content in S. nigra
flower extracts (a) Distribution of variables on
loadings plot; (b) distribution of elderflower
samples on scores plot. Abbreviations: (a); (b).
Ex1 – 50% EtOH UAE extract; Ey1 – 50% EtOH
MAE extract; Ez1 – 50% EtOH MAC extract;
Vx1 – water UAE extract; Vy1 – water MAE
extract; Vz1 – water MAC extract.

individual phenolic compounds. ameliorated, which in the past century could not be relieved.
The reducing power activity of the extracts was tested by FRAP and Nevertheless, the modern era has its challenges, too. Nowadays, people
CUPRAC assays (Table 2). The 50% EtOH MAE extract, which had the are exposed to various factors that influence the development of
highest total phenolic and flavonoid content, showed the strongest re- modern-day illnesses. According to the World Health Organization
ducing power activity (260.9 for FRAP and 383.8 mg TE/g E for CU- (WHO), some of today's most common diseases are neurodegenerative,
PRAC), followed by H2O extracts MAE (256.2 for CUPRAC and cardiovascular diseases and metabolic disorders. The scientific com-
194.8 mg TE/g E for FRAP). 50% EtOH extract UAE (239.3 for FRAP munity is intensively searching for the discovery of natural remedies in
and 355.0 mg TE/g E for CUPRAC) had higher activity than 50% EtOH order to help the population recover from the most common diseases
extract MAC, which is the same trend as in the radical scavenger ac- and to improve their health. In this respect, different enzymes may
tivity. prove vital for managing various diseases including Alzheimer’s dis-
The H2O MAE extract and 50% EtOH MAC extract exhibited the best ease, obesity, and diabetes (Srivastava et al., 2017). For this reason, S.
antioxidant effect in the phosphomolybdenum assay (1.64 and nigra flower extracts were analyzed as potential natural enzyme in-
1.74 mmol TE/g E, respectively). Similarly, in the metal chelating test, hibitors of (AChE), (BChE), tyrosinase and lipase. The obtained results
the H2O MAE extract has achieved the best effect, while in the case of are summarized in Table 4, showing us the great potential of this el-
the 50% EtOH extract, UAE was dominated (52.42 and 61.57 mg derberry species in this respect. In addition, the results are unique, as
EDTA/g E). this type of research has never been done before.
On the basis of the obtained antioxidant potential, it can be con- We have researched the effect of the elderberry flower on the fol-
cluded that the best extraction technique is the MAE extraction, and the lowing enzymes, whose excessive activity is the cause of many modern
most suitable solvent 50% EtOH, since the extracts obtained by this day diseases.
technique showed very strong antioxidant activity. Inhibition of cholinesterases, the key enzymes in the breakdown of
The result of the regression analysis (Table 3 and Fig. 4) indicates acetylcholine, is considered as one of the management strategies
that all antioxidant activities are positively correlated with the content against several neurological disorders. The inhibition of cholinesterases
of total phenols and flavonoids, as well as the content of rutin and 5-O- results from an increase in the concentration of acetylcholine in the
caffeoylquinic acid, as the dominant compounds. Bearing in mind that brain, which subsequently causes an increase in communication be-
rutin and 5-O-caffeoylquinic acid have exceptional biological potential tween the brain nerve cells (Pope and Brimijoin, 2018). In the present
and are present in high concentrations in the investigated extracts, the study, all the extracts showed AChE inhibitory effect and the greatest
strong antioxidant activity of the extracts can be explained by the inhibitory effect was obtained by H2O and 50% EtOH extracts obtained
presence of these compounds in their composition, which, in a sy- by MAC (3.79 and 3.73 mg GALE/g E, respectively). The best inhibition
nergistic effect with other phenolic components, contributes to a of BChE is achieved by H2O and 50% EtOH extracts obtained by UAE
stronger antioxidant activity (Pereira et al., 2018; Forino et al., 2015). (1.55 and 0.50 mg GALE/g E, respectively).
Another enzyme worth for this investigation was tyrosinase.
3.3.2. Enzyme inhibitor activity Tyrosinase is the main enzyme of melanin synthesis. If the activity of
With the advancement of medicine, numerous diseases have been this enzyme is not controlled during the synthesis of melanin pigment,

Table 2
Antioxidant properties of elderflower extracts.
Extraction technique Solvent ABTS FRAP CUPRAC Phosphomolybdenum Metal chelating mg (EDTAE/g E)c
(mg TE/ g E)a (mg TE/g E)a (mg TE/g E)a (mmol TE/g E)b

MAE 50% EtOH 212.74 ± 0.78a 260.92 ± 7.90a 383.78 ± 5.08a 1.68 ± 0.10a 53.84 ± 0.53b
UAE 206.94 ± 4.92a 239.35 ± 7.95b 355.01 ± 5.14b 1.60 ± 0.04a 61.57 ± 1.33a
MAC 189.04 ± 2.18b 208.91 ± 5.36c 325.43 ± 9.69c 1.74 ± 0.07a 52.13 ± 2.63b
MAE H2O 209.06 ± 1.34a 194.82 ± 0.95d 256.23 ± 3.47d 1.64 ± 0.06a 52.42 ± 0.84b
UAE 173.79 ± 4.25c 136.58 ± 1.62f 208.52 ± 3.91f 1.18 ± 0.03c 32.35 ± 1.64c
MAC 167.03 ± 2.44c 179.51 ± 2.60e 230.48 ± 5.43e 1.20 ± 0.04b 28.60 ± 0.07c

a
mg trolox equivalent per g of extract.
b
mmol trolox equivalent per g of extract.
c
mg EDTA equivalent per g of extract.
d
± 3SD. Means followed by different letters (a–f) differ significantly at p < 0.05.

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V. Milena, et al. Industrial Crops & Products 136 (2019) 93–101

Table 3
Results of regression analysis between rutin and 5-O-caffeoylquinic acid content and investigated biological activities.
Correlation factor R2

Antioxidant assays Enzyme inhibitor assays

ABTS FRAP CUPRAC PM MC AChE BChE Tyrosinase Lipase

Rutin 0.740 0.804 0.825 0.794 0.870 0.665 0.211 0.888 0.285
5-O-caffeoyquinic acid 0.830 0.890 0.900 0.870 0.922 0.767 0.287 0.920 0.346

PM – Phosphomolybdenum assay.
MC – Metal chelating assay.

there is an increased melanin synthesis. Decreasing tyrosinase activity extracts to various cosmetic or food products that positively affect our
has been targeted for the betterment or prevention of the conditions health.
related to the hyperpigmentation of the skin, such as melasma and age To provide further insights on the tested extracts, we performed a
spots (Ando et al., 2007). Amazingly, all of the analyzed extracts multivariate statistical analysis. The results are given in Figs. 4 and 5.
showed very strong inhibitory activity against enzyme tyrosinase. 50% Apparently, we observed a strong correlation between the total content
EtOH extracts obtained by conventional and modern extraction tech- of bioactive compounds and antioxidant properties. However, enzyme
niques have shown the best inhibitory activity against tyrosinase but inhibitory properties (except for tyrosinase) were not correlated with
without statistically significant difference (257.1 for MAC; 255.5 for these compounds. As a result, ıt can be saıd that the observed enzyme
MAE; 255.4 mg KAE /g E for UAE). Also, the H2O extracts have shown inhibitory properties can be explained with the presence of other
the very good inhibitory activity of tyrosinase. The values obtained are compounds such as alkaloids and terpenoids or the interactions (an-
quite similar, giving small priority to MAC. tagonistic or synergetic) of phytochemicals.
Lastly, the analyzed enzyme was lipase, which is a digestive en- On the other hand, the regression analysis between the ability of
zyme, essential for the proper digestion of food. It hydrolyzes fat to tyrosinase inhibition and the content of the dominant phenolic com-
triglycerides and fatty acids, which helps the digestion process. At this pounds shows that 5-O-caffeoylquinic acid greatly contributes to the
point, lipase inhibition can be considered as one of the effective examined activity. Perhaps this acid could be used as a tyrosinase in-
methods for controlling obesity. By inhibiting lipase, the fat is not ab- hibitor, while the flowers may be the starting raw material for isolating
sorbed but is actually excreted from the body (Jawed et al., 2018). 5-O-caffeoylquinic acid. In the principal component analysis (PCA), two
Curiously enough, the flower extracts of S. nigra showed very good principal components were obtained summarizing 90.2% of the total
lipase inhibitory activity. In relation to other enzymes, where the better variance and we observed four clusters in the heat map analysis from
activity is achieved with 50% EtOH extracts, in the case of lipase, the PCA (Fig. 5). However, the 6 samples were clearly discriminated into
stronger inhibitory activity is achieved by H2O water extracts. The H2O two groups in Factorial plan 1–2 of sPLS-DA and the similar classified in
UAE extract proved to be the best, followed by 50% EtOH MAE extract. the heat map analysis from sPLS-DA. Consequently, the classification
This is the first scientific study in which the inhibitory activity of the conducted using sPLS-DA was better compared with PCA (Fig. 5). To
enzyme of the plant type S. nigra was studied which gives this paper that end, the bioactivities accountable wıth a sıgnıfıcant difference
special significance. Therefore, there was no possibility of any com- between the importance of the tested samples (VIP score) were iden-
parison with the existing literature data. The results of this investiga- tified. VIP (variable importance in the prediction) values of FRAP,
tion showed the great potential of the examined plant species and are CUPRAC, and tyrosinase assays were larger than 1, indicating that these
opening the possibility for further research, in order to apply these bioactivities were the main contributors to the variabilities of the tested

Fig. 4. Statistical Evaluation (A: Factorial plan 1–2 of the PCA results obtained from biologicals activities of six extracts and eigenvalue; B: Relationship between total
bioactive compounds and biological activities; C: Clustering of extracts in according to biological activities based on Heatmap from PCA).

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Table 4
Enzyme inhibition activity of elderflower extracts.
Extraction technique Solvent AChE inhibition BChE inhibition Tyrosinase inhibition Lipase inhibition
mg GALE/ g Ea mg GALE/ g Ea mg KAE/g Eb mg OE/g Ec

MAE 50% EtOH 3.32 ± 0.29b 0.40 ± 0.15d 255.54 ± 0.96a 16.25 ± 0.62c
UAE 3.39 ± 0.07b 0.50 ± 0.11c 255.43 ± 3.15a 11.80 ± 0.42e
MAC 3.73 ± 0.09a 0.49 ± 0.18c 257.11 ± 1.56a 10.05 ± 1.02f
MAE H2O 1.41 ± 0.09d 0.50 ± 0.08c 93.08 ± 0.85c 18.78 ± 1.89b
UAE 2.03 ± 0.26c 1.55 ± 0.01a 89.61 ± 2.11c 41.04 ± 1.19a
MAC 3.79 ± 0.05a 1.06 ± 0.22b 100.44 ± 0.86b 14.28 ± 0.32d

Different letters indicate differences in tested extracts (p < 0.05).

a
mg galantamine equivalent per g of extract.
b
mg kojic acid equivalent per g of extract.
c
mg orlistat equivalent per g of extract.
d
± 3SD.

Fig. 5. Statistical Evaluation (A: sPLS-DA results obtained from biologicals activities of the tested extracts; variables explaining le largest part of variance between the
two species (vip score) and variables weight on the first component in two species; B: Clustering of extracts in according to biological activities based on Heatmap
from sPLS-DA).

extracts. The results from the present findings will contribute towards es-
tablishing baseline data on the biological properties of S. nigra. These
results can be the starting point in further research in order to create a
4. Conclusion product that could successfully deal with/react to/affect different types
of disease, following further in vivo and clinical assessments. It is an-
In the present study, the influence of conventional (MAC) and ticipated that this study will open up new opportunities for future bio-
modern extraction techniques (MAE and UAE) on the process of iso- prospects of this invaluable medical plant.
lating biologically active substances from elderflowers were in-
vestigated. MAE proved to be the most efficient extraction technique Acknowledgments
while 50% EtOH was a better solvent than H2O. Although MAE has
been favored, the other applied techniques can also be of importance, The authors of this study are grateful to Dr. Milica Rat, University of
since the amounts of the isolated compounds in all the cases were Novi Sad Faculty of Sciences, Department of Biology and Ecology,
considerable. All of the extracts were very rich in polyphenols, parti- Republic of Serbia, for the support in terms of confirmation and de-
cularly rutin, 5-O-caffeoylquinic acid and ursolic acid, which are, at position of Sambucus nigra L. at the BUNS Herbarium.
least partially, responsible for exerted biological activities. The extracts This research study was supported by the Ministry of Education,
obtained by MAE and 50% EtOH solvent had the highest content of Science and Technological Development of the Republic of Serbia
bioactive compounds and exhibited very strong antioxidant activity in (Grant No. 172058).
all applied assays, except for the phosphomolybdenum assay.
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