Anti-hypochlorite and catalytic activity of commercially available moringa oleifera diet supplement

Article abstract of DOI:10.3390/molecules24183330

Aiming at the assessment of the pro-health, and especially anti-hypochlorite properties of Moringa oleifera species a representative, commercially available Moringa oleifera dietary supplement was used as a substrate for the preparation of aqueous Moringa extract. The anti-hypochlorite activity of the extract was assessed using the hypochlorite-specific coumarin-based fluorescence turn-off sensor, namely 7-diethylamino-coumarin-3-carboxylic acid (7-DCCA). This compound was synthesized via the Knoevenagel condensation of 4-diethylamino-2-hydroxybenzaldehyde with Meldrum’s acid and the Moringa extract was employed as a medium and catalyst. Moreover, the total phenolic content (TPC) as well as the reactive oxygen species (ROS)–scavenging ability of the aqueous Moringa extract were determined. The results obtained demonstrated the applicability of Moringa extract as an anti-hypochlorite agent. Additionally, the satisfactory yield of the 7-DCCA obtained suggests the usefulness of the extract as a catalyst and the reaction medium. The antioxidative potential of the extract was notably lower than that of the standard (TROLOX). Determination of TPC in 100 g of the dry weight (DW) of studied material revealed a high number of polyphones present.

Full text of DOI:10.3390/molecules24183330

molecules
Article
Anti-Hypochlorite and Catalytic Activity of
Commercially Available Moringa oleifera
Diet Supplement
1
,
2
3
, Alicja Matwijczuk 3
Karolina Starzak * , Bernadette Creaven , Arkadiusz Matwijczuk
and Dariusz Karcz 1
1
Department of Analytical Chemistry (C1), Faculty of Chemical Engineering and Technology, Cracow
University of Technology, Warszawska 24, 31-155 Cracow, Poland; dkarcz@chemia.pk.edu.pl
Centre of Applied Science for Health, Technological University Tallaght, Dublin 24, Ireland;
Bernie.Creaven@tudublin.ie
Department of Biophysics, University of Life Sciences in Lublin, Akademicka 13, 20-950 Lublin, Poland;
arkadiusz.matwijczuk@up.lublin.pl (A.M.); alicja.matwijczuk@up.lublin.pl (A.M.)
Correspondence: kstarzak@chemia.pk.edu.pl
2
3
*
ꢀ
ꢁꢂꢀꢃꢄꢅꢆꢇ
Academic Editors: Margarida Castell Escuer and Mariona Camps-Bossacoma
Received: 2 August 2019; Accepted: 11 September 2019; Published: 12 September 2019
ꢀ
ꢁꢂꢃꢄꢅꢆ
Abstract: Aiming at the assessment of the pro-health, and especially anti-hypochlorite properties of
Moringa oleifera species a representative, commercially available Moringa oleifera dietary supplement
was used as a substrate for the preparation of aqueous Moringa extract. The anti-hypochlorite
activity of the extract was assessed using the hypochlorite-specific coumarin-based fluorescence
turn-off sensor, namely 7-diethylamino-coumarin-3-carboxylic acid (7-DCCA). This compound was
synthesized via the Knoevenagel condensation of 4-diethylamino-2-hydroxybenzaldehyde with
Meldrum’s acid and the Moringa extract was employed as a medium and catalyst. Moreover, the total
phenolic content (TPC) as well as the reactive oxygen species (ROS)–scavenging ability of the aqueous
Moringa extract were determined. The results obtained demonstrated the applicability of Moringa
extract as an anti-hypochlorite agent. Additionally, the satisfactory yield of the 7-DCCA obtained
suggests the usefulness of the extract as a catalyst and the reaction medium. The antioxidative
potential of the extract was notably lower than that of the standard (TROLOX). Determination of TPC
in 100 g of the dry weight (DW) of studied material revealed a high number of polyphones present.
Keywords: Moringa oleifera; hypochlorite; scavenging; free radicals; antioxidative potential; inflammation;
coumarins; DPPH
1
. Introduction
Moringa (Moringa oleifera), commonly known as a ‘drumstick tree’ or ‘horseradish tree,’ is a tree
belonging to the species from the Moringa family (Moringaceae) and endemic to subtropical and
tropical regions of the world. It is often called the “tree of life” because of a high level of various
nutrients in its leaves, pods, and seeds [
an extremely valuable source of nutritional ingredients, especially in regions of the world threatened
by malnutrition [ ]. In developing countries, various parts of the Moringa tree are exploited with the
leaves being the most commonly used [ ]. The multitude of applications associated with Moringa
includes water and oil purification, biomass for biodiesel production, animal feeding, fencing, local
medicines, and the firewood [ ]. Moringa leaves are particularly rich in vitamins, antioxidants
1]. The ease with which it undergoes cultivation makes it
2
3
2,3
such as flavonoids, carotenoids, ascorbic acid, polyphenols, as well as tannins, saponins, proteins,
and minerals. Hence, they are commonly used in food preparations. Asian and African local
medicine uses them for treating various conditions such as malaria, parasitic diseases, typhoid fever,
Molecules 2019, 24, 3330; doi:10.3390/molecules24183330
www.mdpi.com/journal/molecules

Molecules 2019, 24, 3330
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swelling, skin problems, arthritis, hypertension, strengthening the immune system, eliciting lactation,
and many others [ ]. High level of zeatin in Moringa leaves makes the leaves’ extract a natural
plant growth enhancer [ ]. Other parts of the Moringa tree have been reported as lowering cholesterol
2–4
2
levels in humans, and showing diuretic, antispasmodic, antiulcer, antitumor, anticancer, antibacterial,
antifungal, and hepatoprotective activities [4].
In order to profit from beneficial properties of Moringa, markets around the world are full of
various medicinal, dietetic, and cosmetic preparations, available for people living in areas where
Moringa cultivation is impossible. Most of these diet supplements are capsules containing simple
powdered Moringa leaves. In our studies, we examined one of diet supplements from Cyprus available
on the market in central Europe in order to establish its antioxidant capacity and determine total
phenolic content in a commercially available product.
Our main goal was to check whether such products exhibit at least rudimentary activity attributed
to Moringa. Second, several research reports point at Moringa as exhibiting anti-inflammatory
−
and immunomodulatory effects [
2
], yet none of them focused on OCl (inflammation marker)
elimination by leaves extract. In addition, aiming at the examination of catalytic potential of the
Moringa extract a modified Knoevenagel condensation methodology was applied for the synthesis of
7-DCCA. Specifically, a green chemical approach for the synthesis of coumarin carboxylic acids [
5]
inspired us to use an aqueous Moringa extract as a medium and catalyst for the condensation of
4
-diethylamino-2-hydroxybenzaldehyde with Meldrum’s acid.
Taking into account, that most Moringa-based supplements available in Europe consist solely of
powdered Moringa leaves, the food supplement employed as a substrate in our studies was considered
representative. On the other hand, it is noteworthy that depending on the geographical origin or
cultivation conditions of the plant, the results obtained may exhibit slight differences [6–8].
2
. Results and Discussion
2
.1. Total Phenolic Content Determination
Polyphenols are considered to be responsible for the natural, antioxidant properties of plant
products. Hence, an investigation of their content in diet supplements seems necessary. Various
research on total phenolic content (TPC) in Moringa oleifera leaves show that their final content depends
on the variety of the plant, the season in which the plant material was harvested, and the location and,
therefore, climatic conditions at which it grew [6–8]. However, most of the available research data
were obtained with various organic solvents used for polyphenols extraction.
A modified Follin–Ciocalteu method, using gallic acid as a standard reference compound
and results expressed as gallic acid equivalents (GAE), was applied to determine TPC of Moringa
leaves extract. The amount of polyphenols determined in 100 g of the dry weight (DW) of studied
material was 4.04
±
0.11 g GAE. The value obtained is lower compared to that of methanolic extracts
]. Aiming at
9
.95–11.17 g/100 g DW [6] and similar to those of acetone extracts 2.87–3.19 g/100 g DW [
7
obtaining the relevant results in terms of aqueous conditions prevailing in the human body, all of our
experiments were performed using water as an extracting agent. This resulted in lower polyphenol
content determined most likely due to the poor aqueous solubility of various organic compounds,
present in Moringa leaves [
those reported for various seasonings, fruits, vegetables, or seeds clearly state that TPC in Moringa is
one of the highest of all plants tested [ 10 11]. Among more than 100 plant products tested only
2,9]. On the other hand, the comparison of the TPC value obtained with
7,8, ,
cloves, dried peppermint, star anise [11], and European elder [10] exhibited the TPC value higher
than that of Moringa. This implies that, regardless of the sparing aqueous solubility of polyphenols,
the high TPC content in Moringa leaves allows for transfer of these compounds into aqueous solution
at levels beneficial to human health. Moreover, even a diet supplement available commercially in the
area where Moringa species do not occur naturally contains a satisfactory amount of polyphenols.

Molecules 2019, 24, 3330
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2
.2. Antioxidative Assay
The physiological conditions inside the human body are diverse and span from strongly acidic
elements in the initial parts of the digestive system (pH 2–3), through weak acidic conditions occurring
inside the white blood cells (pH 5.0–5.5) [12 13], to slightly alkaline pH of body fluids (pH 7.4).
,
Taking into account the fact that the dietary supplements are usually administered orally, and their
active ingredients are transported with blood and physiological fluids, they must have the ability
to withstand the changing pH conditions. All further analyses were performed at three various pH
conditions, which include 3, 5, and 7.4.
Determination of antioxidative potential was performed with a standard spectrophotometric test
•
using DPPH radicals solution. The rate at which the solutions of analytes tested reduce the constant
concentration of radicals determines the antioxidant capacity of the compounds tested. The results
•
obtained are compared to the standard that is the ethanolic TROLOX solution. The DPPH radical
reduction method was utilized due to the fact that, in the wavelength range in which the measurements
are performed, Moringa extracts do not exhibit any absorption bands.
•
Figure 1 shows the percentage of DPPH reduced at various pH values in the presence of
increasing concentrations of Moringa extract. The slope indicates the antioxidant capacity in an inverse
•
relationship between the percentage of DPPH inhibited and concentration of the Moringa extract.
In more detail, the lower the extract concentration required to reduce a fixed number of radicals,
the higher the antioxidant capacity of the extract tested. Similar graphs for ascorbic acid and TROLOX
are included in Supplementary files (Figures S1 and S2, respectively).
+
Previous studies proved that changes in H concentration lead to changes in the mechanism of
DPPH scavenging by polyphenols. Increase in the pH value (in acidic range) leads to a decrease of the
reaction rate between extract components and DPPH radicals [14,15]. Thus, higher DPPH inhibition
observed at a pH of 5 than at a pH of 3 (Figure 1) results from lower antioxidant activity of the Moringa
extract observed in less acidic media.
•
Figure 1. DPPH reduction percentage of aqueous Moringa extracts at various pH values after 30
◦
minutes of reaction at 25 C.
Based on the results obtained, the IC50 value determining the analyte concentration required to
reduce fixed, initial radical concentration by 50%, was calculated (Figure 2).

Molecules 2019, 24, 3330
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Figure 2. IC50 values of Moringa extracts, ascorbic acid (AA), and TROLOX at various pH values.
The IC50 values for Moringa leaves extracts at each pH tested were significantly higher, compared
to those of pure ascorbic acid (AA) and TROLOX, which indicates their much lower antioxidant activity.
The relatively low values may also be a consequence of water being used as an extracting solvent for
raw, plant material. It is well-known that the antioxidant properties of plant products originate mainly
from polyphenols and flavonoids, which are poorly (if at all) soluble in water. The existing studies
show that organic solvents used for the extraction increase the final yield of polyphenols extracted,
which increases the antioxidant capacity of extracts tested [16]. Investigations into Moringa extracts
obtained using various solvents state that the extraction with moderate polarity solvents, such as
butanol or ethanol gives the best results in terms of antioxidant properties of the final extract [17].
However, the use of such solvents to prepare a product for use in a health shop or as a food supplement
would be limited. While typically pharmaceutical medication is taken in milligrams, food supplements
and health food capsules tend to be taken in grams. Therefore, the relative comparison of antioxidant
activity is difficult to make. It is also very likely that, in our studies, the compounds with the ability
•
to reduce DPPH were not fully extracted from plant material, and, hence, the aqueous Moringa
•
extracts obtained demonstrated the relatively poor DPPH -scavenging ability. On the other hand, it is
worth emphasizing that the reason for using aqueous extract in our research was mimicking in vitro
physiological conditions, which prevail in humans, especially in those body parts that interact with
food supplements. In this context, the previously reported IC50 value for aqueous Moringa extract
(
0.517 mg/mL) [17] correlates well with those obtained from our studies (0.472 mg/mL, 0.351 mg/mL,
and 0.673 mg/mL at the pH of 3, 5, and 7.4, respectively).
Another, although highly unlikely, reason for low antioxidant activity of the Moringa extract tested
is the quality of the substrate. Based on the description of the product provided by the manufacturer
on the packaging, one can assume that the substrate consists solely of powdered Moringa oleifera leaves
and no additional supplementary substances are added. Information on where the plant material
comes from, at what stage of vegetative development the plant has been harvested [2,6,18], and how it
is stored, can have a significant impact on the final results as well. The IC50 value of aqueous extract
similar to that of Reference [17] implies that the substrate used in our studies does not contain any
additional substances or the additives present (if any) did not affect the IC50 value. This hypothesis,
however, remains unverified, due to the lack of information regarding the nature of potential additives
present in the food supplement tested. Nevertheless, the commercially available diet supplement
tested can undoubtedly serve as a source of natural antioxidants.

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2
.3. Determination of Anti-Hypochlorite Potential
2
.3.1. Synthesis of Coumarin-Derived Hypochlorite-Sensitive Fluorescent Probe 7-DCCA
The anti-hypochlorite potential of aqueous Moringa extract was determined based on the
fluorescence quenching of the coumarin-derived hypochlorite-sensitive probe 7-DCCA. This compound
was synthesized based on numerous reports, which documented the use of Meldrum’s acid as a substrate
for the synthesis of coumarin 3-carboxylic acid derivatives [19
to various crop-derived products such as juices, extracts, or even waste waters employed as both
media and catalysts in the Knoevenagel condensation-like reactions of Meldrum’s acid [ 22]. It is
–21]. A number of these papers refer
5,
particularly worth emphasizing that the majority of these reactions were carried out at eco-friendly
conditions such as room temperature, aqueous media, and only a slightly acidic pH. These requirements
were fulfilled by our aqueous Moringa extract and, hence, we attempted a green synthesis of the
target compound using the extract as medium and the catalyst in the synthesis of the 7-DCCA probe
(
Figure 3). The reaction yield was referenced to that of the carried out one in 10% aqueous acetic acid
and compared with the previously reported data.
Figure 3. Aqueous Moringa extract-mediated synthesis of the fluorescent probe 7-DCCA.
The 7-DCCA product was obtained with a moderate yield of 42%, and was lower than that
obtained from the reaction carried out in dilute acetic acid, which resulted in a 56% yield. Additionally,
it remained in a stark contrast with excellent yields reported elsewhere [5,22]. A majority of unreacted
salicylaldehyde substrate was recovered from the reaction mixture at the recrystallization step,
while no unreacted Meldrum’s acid was found. This suggests that the Moringa extract may consists
of components with the ability to react with Meldrum’s acid, which lowers the yield of 7-DCCA,
even though verification of this hypothesis would require an additional set of experiments. Nevertheless,
the room temperature and slight acidic pH of the aqueous Moringa extract were considered sufficient
for the isolation of a high purity 7-DCCA product with a satisfactory yield.
2
.3.2. Anti-Hypochlorite Assay
Hypochlorite is formed in human neutrophils upon inflammation. Due to its high reactivity,
it may damage host cells. Hence, it is important to monitor its presence and remove its excess from cells
and body fluids. It is especially important in the case of people suffering from chronic inflammation.
Hypochlorous acid (HOCl) belongs to the category of bactericidal and highly reactive chlorine species
(
RCS). Endogenously, HOCl is formed as a final product on the course of enzymatic reactions occurring
in the activated neutrophils phagosomes and is one of the major compounds involved in the activity
of the innate immune system [23]. However, its overproduction, e.g., in people diagnosed with
chronic inflammation, may lead to numerous and irreversible damage to host cells and tissues. In the
acidic interior of the lysosomal vesicles, the HOCl balance moves toward the molecular chlorine
Cl [13]. However, formed promptly and in large quantities, hypochlorous acid can also migrate
2
through the membranes of the phagolysosomal vesicles, and then through the neutrophil membranes
to body fluids [24]. The release of residual HOCl and other reactive oxygen species (ROS) from active
neutrophils also occurs at the time of their apoptosis after phagocytosis of the pathogen. The pH of body
fluids (pH 7.4) shifts the balance of the aqueous HOCl solution toward the formation of hypochlorite
−
ions OCl [13], while also having strong oxidizing and chlorinating properties. The presence of HOCl

Molecules 2019, 24, 3330
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and its derivatives in intercellular spaces may lead to the local irritation of epithelial tissue cells,
damage of proteins, nucleotides, DNA, RNA, fatty acids, or cholesterol [25 28]. The harmful effect
–
of hypochlorite on the body is manifested in people suffering from chronic inflammation, e.g., joints,
where the immune system is stimulated to produce antibacterial compounds, and, thus, maintains
a local concentration of hypochlorite and other ROS on an elevated level, which contributes to further
stimulation of the immune system and further increases production of bactericides. It contributes
to increasing interest in recent years in the search for compounds capable of detecting an elevated
concentration of hypochlorite in tissues and cells in vivo, as well as in the search for compounds capable
of scavenging it.
Various research reports have identified the ability of coumarin derivatives to penetrate cell
membranes [29–32]. 7-DCCA (Figure 3) is well-known for its fluorescent properties [30,33] and, thus,
can be applied as a potential in vivo sensor for biological applications. Our recent studies [34] have
shown that the fluorescence intensity of 7-DCCA is quenched linearly with increasing concentration of
hypochlorite, which allows it to be potentially used for quantitate determination of hypochlorite at each
−
pH tested. Therefore, to our best knowledge, we have studied, for the first time, the hypochlorite (OCl )
ions-scavenging ability of Moringa leaves’ extract using a coumarin-derived probe, 7-(diethylamino)
coumarin-3-carboxylic acid (7-DCCA).
TheresultspresentedinthisstudydemonstratetheapplicabilityofMoringaextractasananti-hypochlorite
agent and utilize the 7-DCCA probe as a marker of its presence. Various pH conditions occurring in various
parts of the human body differentiate the hypochlorous acid forms present in vivo
experiments were carried out again in three buffers, which are pH’s 3, 5, and 7.4. Figure 4 shows fluorescence
intensity changes in 150 M (0.04 mg/mL) of 7-DCCA solution (dotted lines) upon an increase in the
concentration of NaOCl at three pH values.
[13]. Therefore, all the
µ
1
1
5000
2000
Moringa pH 3
Moringa pH 5
Moringa pH 7.4
probe pH 3
probe pH 5
probe pH 7.4
9
6
3
000
000
000
0
0
0.02
0.04
0.06
0.08
NaOCl [mM]
Figure 4. Comparison of changes in fluorescence intensity of 7-DCCA (0.04 mg/mL): under the influence
of NaOCl (dotted lines), and, after 15 min of Moringa extract (0.5 mg/mL), incubation with NaOCl
(
permanent lines). The measurements were carried out at
λ
Ex
289 nm,
λ
Em
460 nm, and at a temperature
◦
of 25 C.
In each case, a linear decrease in signal intensity was observed, which corresponds well with our
previous data. This experiment served as a blank in the determination of anti-hypochlorite activity of
the Moringa extract [34]. In the case of the Moringa extract, the samples were tested in an identical
manner except that the 7-DCCA probe was added after 15 minutes of extract incubation with NaOCl
(Figure 4). In blank samples, the reaction between NaOCl and the probe occurs immediately and
remains unchanged once finished (as evidenced in our previous studies) [34]. Therefore, in the case of

Molecules 2019, 24, 3330
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blank samples, the 15 minute delay was not necessary, while, in the case of Moringa, the 15 minute
delay was necessary in order to allow the active compounds from the extract to react with reactive
chlorine species.
A direct comparison of results obtained from varied pH samples is difficult due to the fact that the
7
-DCCA probe itself shows significant differences in its fluorescence intensity at various environmental
conditions. Apparently, the presence of Moringa extract introduces a series of additional intermolecular
interactions, which result in varied fluorescence quantum yields of the probe and, thus, differences
in the initials fluorescence intensities of 7-DCCA observed [35].
It is clear that the aqueous Moringa extract notably slows down the rate of fluorescence quenching
of 7-DCCA at each pH tested after just 15 minutes of the reaction. The slope is given in Figure 4.
It reflects the most significant difference in the fluorescence intensity drop, which happens at a pH of 7.4.
Therefore, the lowest activity of the extract is at this pH. The slope obtained from measurements carried
out at pH 3 points at the highest anti-hypochlorite activity observed in these conditions. This trend
may result from a pH-dependent variety of RCS [13], which, in turn, may result in differences in the
nature of interactions occurring between RCS and the extract components.
Molecular chlorine, which dominates at a low pH, may react relatively easily with polyphenol-rich
extract via an electrophilic substitution mechanism. The chlorination of polyphenols present in the
Moringa extract utilizes the Cl species, which manifests in a persistent fluorescence of the 7-DCCA
2
−
probe. Compared to Cl , the OCl species, which dominate at a neutral and basic pH, demonstrate
2
−
lower reactivity toward phenols. Although the OCl is well-known for its strong oxidizing character,
its concentration seems insufficient for successful oxidation of the polyphenol-rich extract. On the
−
other hand, the OCl can still chlorinate the 7-DCCA probe [34], which results in quenching of its
fluorescence emission. Therefore, the overall anti-hypochlorite activity of the aqueous Moringa extract
is high at a low pH, while at neutral and basic conditions, it is relatively lower. These results may vary
depending on the solvent used for the extraction from the Moringa powder. Moreover, these finding
confirm that the 7-DCCA probe gives a reliable response in a wide pH range.
For better clarity, the results obtained are given as IC50 [mM] values (Table 1). The higher the IC50
value, the higher the hypochlorite-scavenging activity.
Table 1. IC50 value of NaOCl concentration required to quench fluorescence of 150 µM of 7-DCCA
by 50% with or without the presence of compounds with potential anti-hypochlorite activity. Results
◦
obtained after 15 minutes of analytes incubation with an increasing concentration of NaOCl at 25 C.
IC50 [mM]
pH 3
pH 5
pH 7.4
Moringa (
7
ascorbic acid
0.41
±
±
±
0.02
0.18 ± 0.01
0.07 ± 0.00
1.11 ± 0.07
0.12 ± 0.01
0.06 ± 0.00
1.22 ± 0.13
aq)
-DCCA 1
0.07
1.81
1
0.00
0.09
2
2
blank, reference.
The percentage of 7-DCCA fluorescence quenched is directly proportional to the hypochlorite
−
concentration. Therefore, the higher the concentration of ClO required to reduce emission intensity of
the probe is, the higher the anti-hypochlorite potential of the compound tested is.
The blank samples consisted of only a mixture of NaOCl and the 7-DCCA probe and, regardless of
the significant differences in the fluorescence intensity of the probe at various pHs (Figure 4), the amount
of NaOCl required to quench 7-DCCA fluorescence by 50% in blank samples was almost identical and
oscillated at around 0.07 mM (for 0.15 mM of the 7-DCCA probe in each sample). This corresponds
well with our previous studies [34], where equimolar probe: hypochlorite ratio was assumed.
In the case of the Moringa extract-containing samples, the IC50 value decreased with an increase in pH,
which indicates higher anti-hypochlorite activity at lower pH values. Physiological conditions inside of
neutrophil phagocytes in which HOC1 is formed in vivo are characteristic of a pH of 5.0–5.5 [12,13].

Molecules 2019, 24, 3330
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It is expected that compounds, which are present in the Moringa extract and particularly those
responsible for hypochlorite scavenging, would also demonstrate similar behavior in a living organism.
Such a hypothesis is valid based on an assumption that the anti-hypochlorite components of the
Moringa extract permeate through the neutrophil cell membranes. Verification of this hypothesis,
however, would require a more in-depth research aiming primarily at the identification of those
−
molecules, which react with OCl , and possess the ability to permeate biological membranes [36].
Additionally, promising results were obtained from pH 7.4 samples, where the IC50 values were
slightly lower compared to those with a pH of 5. The pH of 7.4 reflects the conditions prevailing
in body fluids, which are relative easily accessible for any type of active substance from the digestive
system. In this context, it is particularly noteworthy that, at a pH of 7.4, only 0.5 mg/mL of aqueous
−
Moringa extract is capable of removing half of the total OCl amount present in the sample (0.06 mM
−
−
OCl required to quench fluorescence in the samples without extract and 0.12 mM OCl in the sample
with extract) (Table 1) after only 15 minutes of incubation.
The results obtained are even more promising once it was noted that all measurements were
◦
carried out at 25 C, which shows the ability of Moringa extract to act at room temperature. It is,
therefore, expected that a similar action may take place at a physiological temperature of the human
body as well. Increased concentration of extract in the samples together with an extended reaction
time with NaOCl in vitro would likely result in further improvement in anti-hypochlorite activity.
On the other hand, recent studies state that only 5% to 10% of total phenolic intake can be absorbed
from a small intestine [36], which means that effects resulting from the increased extract concentration
in vitro may differ from those occurring in vivo. Therefore, additional bioavailability studies should be
performed in order to determine the maximum concentration of extract necessary for the most effective
absorption of its active compounds from the digestive system to the bloodstream.
The anti-hypochlorite activity results obtained from Moringa extract were referenced to those
performed with well-known anti-hypochlorite agent ascorbic acid (AA) (see supplementary file
Figure S3). The IC50 values for 0.005 mg/mL of AA were nearly four to 10 fold higher compared to
those of 0.5 mg/mL of Moringa extract (Table 1). However, it is worth noting that AA is highly soluble
in water, while Moringa extract is rich in various, active compounds with limited aqueous solubility.
Application of organic solvents for extraction significantly improve the final results for the extract,
but, again, our goal was estimating the physiological conditions in the experiments performed in vitro
.
The available literature consists of countless reports on a variety of biological assays performed on
Moringa oleifera extracts. On the other hand, the reports on anti-hypochlorite activity of Moringa species are
scarce. Thus, the results presented in this study may potentially open a new chapter in researching this plant.
3
. Materials and Methods
3
.1. Materials
All chemicals used were of a reagent grade or higher. All solvents were of 99% purity or higher
(HPLC grade). DPPH (α,α-diphenyl-β-picrylhydrazyl), Folin-Ciocalteu reagent, TROLOX (6-hydroxy-2,5,7,8-
tetramethylchroman-2-carboxylic acid), gallic acid, 4-diethylamino-2-hydroxybenzaldehyde, and Meldrum’s
acid were purchased from Sigma Aldrich (St. Louis, MO, USA). Sodium hypochlorite was purchased from
´
ChemPur (Piekary Sla˛skie, Poland). Methanol and ethanol, ascorbic acid, and sodium carbonate were
purchased from Avantor (Gliwice, Poland). Moringa oleifera powdered leaves were purchased as commercially
available in the European diet supplement Moringa slim (Azurelab Holdings Ltd., Nicosia, Cyprus).
3
.2. Instrumentation
Steady-state fluorescence measurements and UV-Vis spectrophotometric measurements were
performed on a Tecan Infinite 200 microplate reader (Tecan Austria GmbH, Grödig/Salzburg, Austria).
The NMR spectrum of the coumarin-derived probe was acquired on a Bruker Avance III spectrometer
− −1
500 MHz), using DMSO d6 as a solvent. The infrared spectra were recorded in the region of 4000 cm
(

Molecules 2019, 24, 3330
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to 400 cm 1 on a Shimadzu IR Spirit Fourier-Transform Infrared spectrophotometer equipped with
−
an ATR adapter. HPLC-ESI-MS analyses were performed on an LCMS-8030 mass spectrometer
(Shimadzu, Kyoto, Japan), according to the method described previously [34]. All HPLC-MS analyses
were performed in a positive ion mode.
3
.3. Methods
3
.3.1. Extract Preparation
The human body is primarily an aquatic environment. Therefore, in order to imitate the
physiological conditions during all experiments performed, all Moringa extracts were prepared in water
without any organic solvents. Additionally, 100 mg of powdered leaves were suspended in 10 mL
of demineralized water. The mixture was then vigorously shaken and kept for extraction in room
temperature for 15 min, and then centrifuged at 5000 rpm for 10 min. The supernatant was collected and
◦
stored at −18 C for total phenolic content determination, antioxidant, and anti-hypochlorite assays.
3
.3.2. Total Phenolic Content Determination
Total phenolic content (TPC) determination of aqueous Moringa extract was performed using
a modified Folin-Ciocalteu assay. A volume of 0.5 mL of Moringa extract (5 mg/mL) was mixed
◦
with 0.5 mL of Folin-Ciocalteu reagent (FCR). After 3 min at 25 C, 1.5 mL of sodium carbonate
solution (saturated) was added and the sample was filled with water up to 50 mL. The absorbance was
◦
measured at λmax 735 nm after 30 min of incubation of samples in a water bath at 40 C. The results
were presented in grams of gallic acid equivalents (GAE) per 100 g of the sample dry weight.
3
.3.3. Antioxidant Assay
To the following wells of transparent, 96-well plate, increasing concentrations of Moringa solution
was applied. Therefore, its final concentration in 200
from 0 to 0.5 mg/mL. Samples of the reference compound, which were ascorbic acid (AA) and TROLOX
TROL), were prepared the same way and their final concentration in 200 L of the total volume of each
µL of the total volume of each sample ranged
(
µ
sample ranged from 0 to 0.009 mg/mL and 0 to 0.015 mg/mL, respectively. Just before the measurement,
•
all wells were supplemented with 1 mM ethanolic solution of DPPH radicals so that their final
concentration in each well was 200
µM. The plate was shaken on an internal shaker of the reader for
1
0 s to ensure thorough mixing of reagents in all wells. The spectrophotometric measurements started
◦
λ 500–550 nm with a 1 nm wavelength step at 25 C for 30 min. The results obtained
in the range of
were the average of five exposures of each sample with a beam of light. All experiments were repeated
three times.
3
.3.4. Synthesis of 7-Diethylamino-Coumarin-3-Carboxylic Acid
The suspension of 1g Moringa powder in 100 mL of demineralized water was subjected to
ultrasound irradiation at room temperature for 10 min and the solid was then filtered off. The filtrate
was used as a medium and catalyst for the synthesis of 7-diethylamino-coumarin-3-carboxylic acid.
4
-diethylamino-2-hydroxybenzaldehyde (1.0 g, 5 mmol) and Meldrum’s acid (0.75 g, 5 mmol) was
dissolved in 30 mL of aqueous Moringa extract and stirred at room temperature for 24 h. The orange
solid formed was filtered off, dried, and recrystallized from methanol, which yielded dark-orange
crystals of 7-diethylamino-coumarin-3-carboxylic acid: Yield 0.56 g (42%), C H NO (261.28 g/mol),
1
4
15
4
+
1
m/z [M+H] 262.00, H-NMR (DMSO):
δ
= 12.48 ppm (s, 1H, H3 (-COOH)), 8.58 (s, 1H, H4), 7.64 (d,
1
4
H, H5, J = 9.04 Hz), 6.80 (dd, 1H, H6, J = 9.04 Hz, J = 2.30 Hz), 6.57 (d, 1H, H8 J = 2.30 Hz), 3.49 (q,
1
2
H, (-CH -)), 1.14 (t, 6H, (-CH )).
2
3

Molecules 2019, 24, 3330
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3
.3.5. Anti-Hypochlorite Assay
To the fallowing wells of black, 96-well plate, 20 µL of 25 mM acetate (pH 3 and 5) or phosphate
pH 7.4) buffers and NaOCl were added. The final concertation of hypochlorite in the total volume
200 L) of samples ranged from 0 to 0.84 mM. Next, 10 L of Moringa extract solution was applied
L of 1 mM probe solution was added. The plate was shaken for 10 s
on the reader shaker and then the fluorescence measurement was started. All samples were excited by
(
(
µ
µ
and, after 15 min of a reaction, 30
µ
light at a wavelength of
λ
289 nm and the fluorescence spectra at λ 320–700 nm were recorded at
Exc
◦
25 C. The results obtained were the average of five exposures of each sample with a beam of light.
All experiments were repeated three times. Analysis for the reference substance ascorbic acid 10 µL of
0
.1 mg/mL solution in each well were made analogously.
4
. Conclusions
TotalphenoliccontentdeterminedintheMoringaoleiferasampletestedwas4.04 ± 0.11 g GAE/100 g DW
.
The antioxidative potential determined for the aqueous extract is notably lower than that of the
standard (TROLOX), which may result from the low aqueous solubility of compounds responsible
for radical scavenging.
The research carried out for the first time to determine the ability of Moringa leaves extract to
scavenge hypochlorous ions in vitro gave satisfactory results. At the pH value adequate to that of the
−
body fluid environment (7.4), the extract was able to reduce the OCl ions concentration present in the
◦
solution by 50% after 15 min of the reaction carried out at 25 C. Considering the preliminary stage of
research carried out, this is undoubtedly a satisfactory result. Understanding the mode of action of the
extract, however, requires a more elaborate approach.
The research reported in this study aimed at examining a commercially available Moringa oleifera
dietary supplement for its ability to provide an effect similar to that attributed to fresh plant
extracts. The results obtained allow the assumption that the benefits for human health properties
of Moringa oleifera species can also be enjoyed by people living in areas where this plant does not
occur endemically.
Supplementary Materials: The following are available online. Figure S1: DPPH reduction percentage of ascorbic
◦
acid at various pH values after 30 minutes of reaction at 25 C. Figure S2: DPPH reduction percentage of TROLOX
◦
at various pH values after 30 min of reaction at 25 C. Figure S3: Changes in fluorescence intensity of 7-DCCA
(
0.04 mg/mL) after 15 min of ascorbic acid (0.005 mg/mL) incubation with NaOCl. The measurements were carried
◦
out at λEx 289 nm, λEm 460 nm, and at a temperature of 25 C.
Author Contributions: Conceptualization, methodology, investigation, visualization, resources, writing—original
draft preparation, writing—review and editing, K.S. and D.K. Resources, writing—review and editing, B.C.
Resources, A.M. (Arkadiusz Matwijczuk) and A.M. (Alicja Matwijczuk).
Funding: This research received no external funding.
Conflicts of Interest: The authors declare no conflict of interest.
Abbreviations
7
AA
-DCCA
7-diethylamino-coumarin-3-carboxylic acid
ascorbic acid
DW
FCR
GAE
dry weight
Folin–Ciocalteu reagent
gallic acid equivalents
hypochlorite ion
−
OCl
RCS
ROS
TPC
TROL
reactive chlorine species
reactive oxygen species
total phenolic content
TROLOX

Molecules 2019, 24, 3330
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©
2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).