Journal of Food Measurement and Characterization (2024) 18:1302–1317

https://doi.org/10.1007/s11694-023-02272-1

ORIGINAL PAPER

A comprehensive characterization of polyphenol extracts from wasted

sour fruits by LC–MS/MS and evaluation of their antioxidant potentials
Waheed Ahmad1 · Akhtar Ali2 · Ali Mohsin1 · Xiaoguo Ji1 · Mahwash Aziz3 · Li Wang1 · Liming Zhao1,4

Received: 10 August 2023 / Accepted: 3 November 2023 / Published online: 6 December 2023
© The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature 2023

Abstract
Unripe grapes are pruned waste fruits of wine farming, while black lemons are dried products of agriculture waste lemons
from lime fields. These fruit wastes are a rich source of bioactive molecules, especially polyphenols that are beneficial metab-
olites for human health to reduce or inhibit the oxidative stress and can be utilized in foods or pharmaceuticals. Therefore,
this project aimed to study the wasted sour fruits for their polyphenols, antioxidant activities, and to support their symbiotic
utilization as therapeutic and pharmaceutical products. This research was completed with solvent extraction optimization of
polyphonic extracts, their in-vitro antioxidant activities using four different assays, and characterization of polyphenols by
LC-ESI-QTOF-MS/MS. Efficient extraction for phenolic compounds was obtained using 80% acidified methanol with 0.1%
formic acid. Results found that black lemons had a higher total phenolic content (TPC 10.11 ± 0.47 mg GAE/g), assimilated
to unripe grapes (8.75 ± 0.33 mg GAE/g). Moreover, black lemons were also found with higher antioxidant potential than
unripe grapes, possibly due to the presence of higher flavonoids, especially anthocyanins. 124 phenolic metabolites, includ-
ing 21 phenolic acids, 45 flavonoids, 8 isoflavonoids, 2 chalcones, 7 stilbenes, 15 lignans and 25 other compounds were
identified using mass spectrometry. In conclusion, these results supported the utilization of these products in combination
as therapeutical ingredients in the food, biomedicine, and pharmaceutical industries.

* Liming Zhao
[email protected]
1
State Key Laboratory of Bioreactor Engineering, East China
University of Science and Technology, Shanghai 200237,
China
2
School of Agriculture, Food and Ecosystem Sciences,
Faculty of Science, The University of Melbourne, Parkville,
VIC 3010, Australia
3
Department of Food Science and Technology, GCW
University, Faisalabad, Pakistan
4
Shanghai Collaborative Innovation Center
for Biomanufacturing Technology (SCICBT),
Shanghai 200237, China

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A comprehensive characterization of polyphenol extracts from wasted sour fruits by LC–MS/MS… 1303

Graphical abstract

Keywords Black lemons · Unripe grapes · Agricultural waste · Antioxidants · Phenolic acids

Introduction fields, plentiful in bioactive molecules, can be an essential

source of ingredients in high-value-added foods, cosmet-
Unripe grapes are the pruned agricultural waste fruits of the ics, and pharmaceutical industries. Unripe grapes have
wine industry, whereas black dry lemons are the processed anti-inflammatory, cardio-protective, anticancer, and anti-
form of waste fruits from the lime farming. Grapes ‘scientifi- diabetic properties, according to both in vitro and in-animal
cally known as Vitis vinifera L.’ are the enormous fruits on investigations [3]. The unique prospective uses of unripe
the earth, around 75 million tons/annum, mainly used in the grape extract in food and drinks are preservation, enrichment
industry that screens out a large amount of unripe grapefruits or direct usages in the human diet have recently been given
and thrown on the agricultural land. Immature grapes are renewed attention [4]. Meanwhile, lemon’s annual produc-
considered a newly discovered source of several potential tion is 25 million tons, which are mainly used as freshly
bioactive substances, including organic acids, phenolic com- extracted juice in food, cosmetics, and pharmaceuticals.
pounds, vitamins, and minerals that are highly valuable to Interestingly, in the harvesting season with high production,
the food, cosmetics, and biomedical fields [1]. Verjuice and its uses are low; at that time, fresh lemon is converted into
sour grape sauces are products derived from unripe grapes black lemon for whole year consumption. The black lemon
traditionally employed as seasoning or acidifying agents also referred as ‘loumi’, or ‘aswad’ is a lime boiled in salty
[2]. It is vital that immature grapes as waste in agriculture water and dried for preservation. The phenols in grapes,

13
1304 W. Ahmad et al.

including phenolic acids, flavones, flavanols, anthocyanins, of our ecosystem, which in turn influences the food we
stilbenes, and glutathione, serve an essential role in prohib- have access to. To reduce the impact of fruit wastes, it is
iting disorders related with oxidative stress, mainly due to important to screen out the compositional metabolites of
their antioxidant properties [5]. wasted fruits and converts them into valuable ingredients.
Grapes are rich in bioactive compounds like vitamins and Unripe grapes and black lemon (agricultural wastes) can
phytochemicals that act as potent antioxidants, scavenging be utilized in dietary food as taste and flavor enhancing
effect or formation inhibiting effect, thus protecting biomol- ingredients. Some phenolic compounds have already been
ecules from oxidation [5]. The concentration of phenols in discussed in previous reports [14–16]. Due to their intri-
grapes varies depending on factors like ripening stage, expo- cate nature, complex structure, and complex functional
sure to light, and availability of water and weather conditions activities, previous reports lack the comprehensive profil-
[6]. Although these fruits contain important antioxidants ing of these bioactive phenolics.
which have potential applications, received limited scientific It is hypothesized that unripe grapes and black lem-
attention and depend on various factors like variety, maturity ons, agricultural waste products, could be abundant in
level, processing, and season [7, 8]. A wide spectrum of phyto-metabolites and have strong antioxidant properties.
therapeutic benefits is offered by lemon and its components, Moreover, the growing interest of their availability in food
fruit liquid extract, oily part and skin. These are suitable for requested the identification and measurement of phenolic
commercial production as a therapeutic products since they metabolites to learn their symbiotic function in food qual-
exhibit anti-carcinogenic and cancer-chemo preventive char- ity and community health. Therefore, this study aimed to
acteristics [9]. It is primarily grown for alkaloids that also investigate unripe grapes and black lemons for their exten-
show antibacterial effects in crude extracts. Citrus fruit has sive evaluation of polyphenols and assessment of antioxi-
been shown in several studies to play a substantial role in dant properties. To complete the work, the phenolic com-
acting as an antimicrobial agent to prevent and inhibit either pounds from sour fruits were extracted and identified with
foodborne pathogens or rotting bacteria, thus protecting the liquid chromatography ESI-QTOF-MS/MS. Additionally,
body from the incidence of malignant tumors, especially in total metabolites; total phenolic contents (TPC), total flavo-
the stomach and colon [10]. Bioactive substances such as noid contents (TFC) and total tannin contents (TTC) were
phenolics, terpenoids, saponins, tannins, carotenoids, and quantified while antioxidant activities; 2,2-diphenyl-1-pic-
flavonoids are considered to be abundant in black lemons. ryl-hydrazyl-hydrate radical scavenging activity (DPPH),
Both agricultural waste products (black lemon and unripe hydroxyl radical scavenging assay (OH-RSA), 2,2-azino-
or immature grapes) are high in phytochemicals especially bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS), and
phenolics that are crucial for their development, reproduc- Ferrous ion chelating activity (FICA) were also analyzed.
tion, and defense against diseases and radiation effects. The This study supports the worthy potential and significance
biochemical characteristics of phytochemicals are significant of unripe grapes and black lemons as an essential supply
[11]. Oxidative stress is induced in the body by excessive of bioactive metabolites in many industries like cosmetics,
concentration of free radicals, but it can be mitigated by pharmaceuticals, nutraceuticals, and food sectors.
the natural antioxidants found in fruits like grapes and lem-
ons. Persistent oxidative stress has been linked to various
disorders including cancer, cardiovascular conditions, and
accelerated ageing. Natural antioxidants could therefore be Materials and methods
used to control the countless medical problems that result
from oxidized and other problems with metabolism [12]. Materials
These days, phenolic metabolites are receiving prominent
intentions for their broad spectrum in biomedical functions, For the extraction of samples and LC–MS performance,
including their capacity to chelate metals, alter signal trans- only analytical lab chemicals were employed. All analyti-
duction pathways, suppress cellular proliferation, and have cal related chemicals used for identification and evaluation
enzymatic activity [13]. were purchased from Sigma Aldrich (Darmstadt, Germany).
The excess amount of these agricultural wastes throw- Ethylene diamine tetra acetic acid (EDTA), Folin–Ciocal-
ing in the fields causes an increase in pollution and teu’s reagent, hydrated sodium acetate, vanillin, catechin,
environmental deterioration. In agriculture fields, these 2,2-diphenyl-1-picryl-hydrazyl-hydrate (DPPH), potassium
products become the habitat for disease-causing micro- ferrocyanide (III), 2,4,6 tripyridyl-s-triazine (TPTZ), 3-hyd-
organisms, insects, and pests. These organisms also robenzoic acid, 2,2-azino-bis 3-ethylbenzothiazoline-6-sul-
produce mycotoxins and other hazardous chemicals on fonic acid (ABTS), and quercetin were purchased from the
the decay of wastes that leach down into groundwater. Sigma Aldrich (Castle Hill, Australia) for the quantification
Increasing food waste poses negative effects on the health of polyphenols and antioxidant capability.

13
A comprehensive characterization of polyphenol extracts from wasted sour fruits by LC–MS/MS… 1305

Preparation and extraction of samples for phenolic was incubated at 27 °C for 5 min. At last, Sodium carbonate
metabolites (10% v/v, 25 µL) was added in the solution and incubated
again for 60 min at 27 °C. The sample’s absorbance was
Unripe grapes and black lemons were collected in May- taken at 760 nm. The amount of phenolic contents in total
2022 from a nearby market (Faisalabad city, Punjab, Paki- was measured against a standard curve of gallic acid (0–200
stan). Selected immature grapes were washed and dried µL/mL in methanol). Results were recorded using mg of
(Fig. 1B), while black lemon was collected as dry whole GAE (gallic acid equivalents) per gram of samples.
fruit (Fig. 1A). Both dried fruits were ground to convert
into a fine powder. Ground samples were kept at refrigera-
Flavonoids compounds
tion condition for other analysis. All the analysis was per-
formed at department of food science (GCW Faisalabad,
Flavonoid contents were analyzed as described by Sharifi
Pakistan). The extraction conditions for phenolic compounds
et al. [22] and Ali et al. [23] with some revisions. Sample
were optimized using methods given by Kiani et al. [17] and
extract (80 µL) was taken in 96-well plates and mixed with
Ali et al. [18] with slight modifications. Efficient extraction
­AlCl3 solution (80 µL) and sodium acetate aqueous solution
for phenolic compounds was obtained using 80% acidified
(120 µL, 50%). Then the solution was incubated at 27 °C for
methanol with 0.1% formic acid. Furthermore, samples were
2.5 h and absorbance was taken on the Thermo multiskan
extracted by mixing a 2 g sample with methanol (20 mL) and
GO microplate spectrophotometer at 440 nm. For the quan-
0.1% formic acid. Solutions were subjected to shaking in an
tification of flavonoid content, a standard curve (­ r2 = 0.999)
incubator shaking machine for 12 h at 10 °C and 150 rpm.
was constructed against 0–50 µg/mL of quercetin and results
After that solutions were subjected for centrifugation
were given in QE (mg of quercetin equivalents per gram)
(8000 rpm, 15 min). The buoyant was separated by syringe
unit.
filter (0.45 µL) and stored at − 20 °C for use of LC–MS/MS
and antioxidant activities performed in a week.
Tannin compounds
Quantification of polyphenols in black lemons
and unripe grapes Tannin compounds were analyzed as explained by Ali et al.
[24] after small changes. The prepared sample extract (25
Phenolic compounds µL) was mixed with 50 µL of vanillin (4%) in 96-well plate.
Then, 30 µL of ­H2SO4 (30%) was added and incubate for
The phenolic compound profile of samples was analyzed 15 min at 25 °C. After incubation, the absorbance of all
using the previously described method by Ali et al. [19], samples was recorded in triplicates at wavelength 500 nm.
Park [20] and Kiani et al. [21] with some modifications. A standard curve was prepared using catechin solution in
Sample extract (25 µL) was added in distilled water (200 µL) alcohol (0–1000 µg/mL) and noted as CE (mg catechin
and Folin Ciocalteu reagent (25% v/v, 25 µL) and solution equivalents per gram).

Fig. 1  Preparation of black

lemon powder (A) and unripe
grapes powder (B)

13
1306 W. Ahmad et al.

Measurement of antioxidant potential was developed to make standard with EDTA at concentration
0–50 g/mL for quantification, and the data was given as mg
ABTS radical scavenging assay of EDTA/g.

ABTS assay was analyzed as given by Severo et al. [25]. Phenolic compounds characterization using LC–MS/MS
Potassium persulfate solution (140 mM) and ABTS solution
(7 mM) were mixed and incubated for 16 h in the dark place The separation and identification of phenolic metabolites from
that generates ABTS + solution. Then ethanol was used to unripe grape and black lemon samples were carried out as
dilute the solution to record its absorbance at 734 nm to previously described by Ali et al. [27–29]. Both negative and
0.7 ± 0.02. ABTS + solution (290 µL) and sample extract positive modes were applied using Accurate-Mass QTOF Agi-
(10 µL) was added in a 96-well plate and incubated for six lent 6520. For the separation of phenolic chemicals, Synergi
min at 25 °C. The absorbance was recorded at 734 nm. By Hydro-RP with 4 µm particle size, 4.6 mm internal diameter,
constructing the standard curve against ascorbic acid con- 250 mm length, and 80 Å pore size was utilized, while keeping
centrations of 0–150 g/mL in water, the quantification was the flow rate constant at 800 µl/min. Mobile phase A contained
performed (expressed as mg AAE/g). formic acid (0.1%) in water and mobile phase B had formic
acid (0.1%) with acetonitrile. The gradient profile was as fol-
lows: Gradient elution program (phase B) lasting for 60 min
DPPH radical scavenging activity
at 800 µL/min flow rate was used. The elution was 10% at
0 min and increase to 20% at 10 min. It was 30% at 15 min
The DPPH assay for all samples was assessed in tripli-
and increase by 10% after each 5 min difference up to 100%
cate as explained by Fia et al. [14]. 25 µL sample extracts
at 50 min. Then gradient decreases as 75% at 55 min, 10%
were added with DPPH dye (275 µL, 0.1 M in methanol)
at 58 min and 5% at 60 min. Apart of 6 µL from the given
using 96-well plate method. The solution was incubated at
samples was applied. With the following parameters: 3500 V
25 °C for 30 min and recorded at wavelength of 517 nm
capillary voltage, 9 µL/min nitrogen gas flow rate, 500 V noz-
on a spectrophotometer. The radical scavenging capability
zle, temperature at 325 °C, atomizing at 45 psi and collision
was analyzed by preparing a standard curve of ascorbic acid
energies set as 10, 20, and 40 eV, complete scanning mode was
(0–50 µg/mL) in water. The results observed were recorded
accomplished in the region of 100–1300 amu.
as milligrams equivalents of ascorbic acid per gram of sam-
We extracted and identified phenolic compounds using
ple (mg AAE/g).
MassHunter workstation software (QTOF/6200 series
LC/MS Data Acquisition) with database library (PCDL)
Hydroxyl radical scavenging assay and PubChem [30]. Three treatments of each sample
were repeated and sixteen phenolic compounds were also
The Fenton-type reaction method was used to analyze the semi-quantified.
OH-RSA (Radical scavenging function of Hydroxyl group)
of extracted samples. In this method, 50 µL of anhydrous Statistical analysis
ferrous sulphate, F
­ eSO47H2O (6 mM) and hydrogen perox-
ide (50 µL, 6 mM, 30%) were added with 50 µL of sample Data recorded in the current study was given as mean val-
extract and incubated for 10 min at 25 °C. Then, addition of ues with standard deviation. Data was analyzed by ANOVA
50 µL 3-hydroxybenzoic acid (6 mM) was added to solution using SPSS version 25.0 (Inc., Chicago, USA) to observe
and wavelength of 510 nm was used to assess the absorb- the difference in means of all samples. Multiple rank tests
ance. Ascorbic acid concentrations between 0–300 g/mL by Tukey’s honestly significant differences (HSD) were also
were used to generate the standard curve and results were applied while keeping the significance level at p < 0.05.
expressed in mg of AAE/g. XLSTAT-2019.1.3 (Addinsoft Inc. USA) was employed to
investigate the relationship between antioxidant activity and
polyphenols.
Chelating activity of ­Fe2+ (FICA)

The chelating activity of Ferrous ions was measured by

Results and discussion
slightly modifying the methodology described by Patel [26]
and Ali et al. [24]. 85 µL of water, 50 µL of ferrous chloride
Quantification of total polyphenols of sour fruits
(2 mM) and 50 µL of ferrozine (5 mM) were added with 15
µL of extract and incubated at 25 °C for 10 min. A wavelength
Phenolic compounds have gained the great interest of sci-
of 562 nm was employed to measure the absorbance. A curve
entists due to their unique characteristics and beneficial

13
A comprehensive characterization of polyphenol extracts from wasted sour fruits by LC–MS/MS… 1307

potential as anti-inflammatory, antioxidant, anti-diabetic, the same rows. All data is presented as mean (n = 3) with
anti-microbial and anti-carcinogenic properties. The phe- standard deviation ( ±).
nolic content and antioxidant assays of unripe grapes and Flavonoids are the prevalent group of phenolic metabo-
black lemons are presented in Table 1. lites in herbs, therapeutical plants and fruits that attracted for
The Folin Ciocalteu method was used for the deter- their health benefits. It was the first time that bioactive was
mination of total phenolic contents (TPC). The analy- studied in unripe grapes and black lemon together as sour
sis revealed that the phenolic contents observed in black fruit powder used as traditional spices in many countries.
lemon (10.11 ± 0.47 mg GAE/g) were higher (p < 0.05) as Total phenolic and flavonoids are significantly different in
compared to the phenolic contents found in unripe grapes these forms of sour fruits.
(8.75 ± 0.33 mg GAE/g). Previous studies by de Camargo
[31] have also reported similar results in which grapes were Antioxidant activities of sour fruit samples
analyzed for their antioxidant potential with two distinct
methods, based on chemical reactions, using ethanol and Antioxidants are considered vital ingredients for several
water and results revealed total phenolic contents observed products that perform functions like radical oxygen scaven-
in grapes with water extraction was least (76.11 ± 3.31 mg gers, reducing agents, metal chelators, and ­H+ ion donors.
CE/g) and the maximum was observed with ethanol/water To analyze the antioxidant abilities of unripe grapes and
extraction (119.7 ± 5.92 mg CE/g). The TPC results might be black lemon extracts, various assays were performed includ-
differentiate due to different maturity levels of fruit used in ing DPPH assay, ABTS assay, ferrous ion chelating activity
this study and later studies for the measurement of phenolic (FICA) and OH-RSA. Antioxidant potential results for both
contents. Total Flavonoid content analysis demonstrated that samples (black lemon and unripe grapes) are demonstrated
black lemon showed a considerably (p < 0.05) high amount in Table 1.
of flavonoid contents (6.26 ± 0.65 mg QE/g) in comparison The scavenging free radical ability of samples in the bio-
to unripe grape samples (4.94 ± 0.31 mg QE/g). Another logical process is usually estimated by using the inexpensive
study revealed similar results in which citrus fruit was ana- assay known as DPPH, which is determined on the capacity
lyzed for its flavonoid contents and results reported that the of electron and hydrogen ion contribution. When combined
maximum amount of TFC was observed in lemon pulp indi- with sample extracts, DPPH, a free radical with a stable
cating 1.46 ± 0.05 mg RE/g FW [32]. Moreover, black lemon nitrogen molecule, loses some of its bluish-purple colors [3].
also contains significantly (p < 0.05) higher levels of total DPPH radical scavenging activity exhibited that black lemon
tannin contents indicated as 2.11 ± 0.26 mg CE/g in contrast (21.42 ± 0.73 mg AAE/g) showed a significantly (p < 0.05)
to unripe grapes that observed as 1.37 ± 0.18 mg CE/g. Tan- higher level of antioxidant potential than unripe grapes
nin contents observed by another red grapes study revealed (14.52 ± 0.63 mg AAE/g). Previous studies by Ivanišová,
the total tannin contents was observed maximum in the Kántor, and Kačániová [34] on the antioxidant activity and
extract of pomace which was 79.3 mg CE/g [33]. However, total polyphenol content of several grape varieties revealed
the phenolic compound profile of black lemon was observed similar radical scavenging activities and the maximum was
significantly higher making it a suitable natural antioxidant demonstrated by the Dornfelder variety as 0.73 ± 0.03 mg
or a functional ingredient in food production. TEAC/g of fresh fruit. Another study by Eddine et al. [35]
Total tannin contents (TTC), Total flavonoids contents on the biochemical, antioxidant and microbiological prop-
(TFC), Total phenolic contents (TPC), Cathechin equiva- erties of grapes revealed similar results with grape extract
lence (CE), Quercetin equivalence (QE), and Gallic acid (119.670 mg GAE/L) and was higher with grape juice
equivalence (GAE). All values are significantly different in (182.170 mg GAE/L).
ABTS assay results indicated that unripe grapes
(34.71 ± 3.08 mg AAE/g) showed substantially (p < 0.05)
lower activities than black lemon (57.32 ± 2.68 mg AAE/g).
Table 1  Quantification of total polyphenols and antioxidant analysis Furthermore, a chelating assay of ferrous ion was carried out
Variables Black lemon Unripe grapes as an indication of the antioxidant capabilities of the selected
samples and results indicated significantly (p < 0.05) higher
TPC (mg GAE/g) 10.11 ± 0.47 8.75 ± 0.33
potential of black lemon (4.21 ± 0.39 mg EDTA/g) in con-
TFC (mg QE/g) 6.26 ± 0.65 4.94 ± 0.31
trast to unripe grapes (3.47 ± 0.22 mg EDTA/g). Previous
TTC (mg CE/g) 2.52 ± 0.70 1.28 ± 0.40
studies have also revealed [36] the same results. In addi-
DPPH (mg AAE/g) 21.42 ± 0.73 14.52 ± 0.63
tion, the scavenging activity of OH radical was performed to
ABTS (mg AAE/g) 57.32 ± 2.68 34.71 ± 3.08
estimate the antioxidant profile which demonstrated similar
FICA (mg EDTA/g) 4.21 ± 0.39 3.47 ± 0.22
results showing significantly higher activities (p < 0.05) in
OH-RSA (mg AAE/g) 28.87 ± 1.39 17.16 ± 0.93
black lemon (28.87 ± 1.39 mg AAE/g) while unripe grapes

13
1308 W. Ahmad et al.

(17.16 ± 0.93 mg AAE/g) showed the least amount of anti- represent the fraction ion at m/z 163. The fraction ions of
oxidant potential. compound 12 (observed only in unripe grapes) identified
as Caffeic acid (m/z 179.0350) was observed at m/z 135.
Phenolic compounds characterization using LC–MS Compound 13 identified in both samples as Cinnamic acid
(m/z 147.0451) revealed the fraction ions m/z 103. Other
Lemons and grapes have recently gained scientists' interest compounds were also observed like 1,2,2'-Triferuloylgen-
as they have been identified to contain a high amount of bio- tiobiose (m/z 871.2655) and Verbascoside A (m/z 669.239).
active metabolites. These bioactive metabolites especially Similar results were also analyzed by Ali, Cottrell, and Dun-
phenolic compounds have shown potential health effects in shea [16] who used different Australian fruits for identifica-
various studies [35, 37]. An untargeted screening and clas- tion of anthocyanins and non-anthocyanins compounds by
sification of phenolic metabolites from the targeted sam- using LC/MS–MS QTOF technique. Their results identify
ples of unripe grapes and black lemon were achieved using 10 different hydroxycinnamic acids and some of which were
the advanced technique of LC-ESI-QTOF-MS/MS. It is a similar to the compounds observed in the current study.
modern analytical method that enables us to identify all the There were two Hydroxyphenylacetic acid com-
bioactive components of plant-based foods. PCDA (Personal pounds number 20 and 21, identified as 2-Hydroxy-2-pheny-
Compound Database Library) was used for the qualitative lacetic acid (m/z 153.0546) and 3, 4-Dihydroxyphenylacetic
characterization of the compounds through Agilent Mass acid (m/z 169.0496) with their fraction ions at m/z 109 and
Hunter software (version B.06.00). The results observed m/z 169, respectively, detected in both targeted products.
regarding the characterization of phenolic compounds in
unripe grapes and black lemons are provided in Table 2. Flavonoids
LC/MS analysis identified a total of one hundred twenty-four
(124) phenolic compounds that included 21 phenolic acids, Biological functions make flavonoids the prevalent group
45 flavonoids, 8 isoflavonoids, 7 stilbenes, 2 chalcones, 15 of plant source secondary metabolites and are used in
lignans and 25 other polyphenols. pharmacological, medical, and cosmetic applications [38].
Results of this work found forty five flavonoids in target
Phenolic acids samples. Flavonoids were observed higher in number than
all the other phenolic compounds from the targeted samples.
Phenolic acids are secondary metabolites in plants com- Observed categories of flavonoids included 4 Flavanols, 4
monly known for their promising health benefits. These Flavanones, 7 Flavones, 11 Flavonols, 8 Isoflavonoids and
compounds have many biological functions including anti- others.
tumor, cardiovascular protective, anti-inflammation capacity,
antimicrobial and anti-aging functions [16]. and that’s why Anthocyanins
phenolic acids are widely used in many health industries.
Twenty one phenolic acids were found in the unripe grapes A significant amount of anthocyanins were detected via
and black lemon samples that include nine Hydroxybenzoic LC–MS identification. Anthocyanins are water soluble,
acids, ten Hydroxycinnamic acids and two hydroxyphe- colored compounds consisting of hydroxyls position at
nylacetic acids. Elimination of Hexosyl moiety and CO2 third, fifth and seventh position in ring B [39] and have very
from their base generate fragmented pattern in these com- high antioxidant potential along with certain anti-cancer,
pounds. Compound number 1 & 2 benzoic acid derivatives anti-tumor, and anti-carcinogenic properties [40]. The first
were identified as Protocatechuic acid 4-O-glucoside (m/z compound identified was compound number 22 (Table 2)
315.0721) and Gallic acid 3-O-gallate (m/z 323.0398) and as Delphinidin 3, 5-O-diglucoside (m/z 628.1645) and its
they exhibited their fraction ions at m/z 153, m/z 169, and m/z fraction ions were observed at m/z 303. Malvidin 3-O-(6''-
125. Similar compounds were also observed in another study acetylgalactoside) was another anthocyanin observed
[21] where eight hydroxybenzoic acids and fifteen hydroxy- (compound 24), which revealed its fraction ions at m/z 331.
cinnamic acids were identified using LC–MS technique on Pelargonidin 3-O-(6''-malonyl-glucoside) and Cyanidin
several kinds of Australian herbs. Other hydroxybenzoic 3-O-sambubioside 5-O-glucoside were identified as com-
acids observed were Gallic acid (m/z 169.0142), Ellagic acid pounds 26 and 28, and their fraction ions were recorded
(m/z 3000.9990), and benzoic acid (m/z 121.0295). at m/z 520 and m/z 744, respectively, Cyanidin 3-O-(6''-
Ten hydroxycinnamic acids were also identified in the caffeoylglucoside), Cyanidin 3-O-rutinoside and Cyanidin
sour fruit samples. Compounds number 10 and 11 only 3,5-O-diglucoside screened as compounds 31, 34 and 37,
observed in black lemon that identified as Cinnamoyl respectively, showed that their ions begin at m/z 303, and
glucose (m/z 311.1125) represent its fraction ions at m/z m/z 287. Compound number 36, Pelargonidin 3-O-gluco-
311 and p-Coumaric acid 4-O-glucoside (m/z 325.0929) sylrutinoside (m/z 742.2291) catalyzed at m/z 271. Cyanidin

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A comprehensive characterization of polyphenol extracts from wasted sour fruits by LC–MS/MS… 1309

Table 2  Characterization of polyphenol compounds from sour fruits by LC–MS/MS-QTOF

No Name Formula Retention time ESI ± Theoretical Observed Mass error Product ions Sample
(m/z) (m/z) (ppm)

Phenolic Acids
Hydroxybenzoic
acids
1 Protocatechuic acid C13H16O9 4.037 ** [M − ­H]− 315.0721 315.0746 7.9 153 U
4-O-glucoside
2 Gallic acid C7H6O5 7.388 ** [M − ­H]− 169.0142 169.0144 0.6 125 B
3 Ellagic acid C14H6O8 7.414 ** [M − ­H]− 300.9990 300.9990 0.0 284, 257 B
4 3-O-Methylgallic C8H8O5 11.584 ** [M − ­H]− 183.0299 183.0299 0.0 169 B
acid
5 Paeoniflorin C23H28O11 13.732 [M + ­H]+ 481.1705 481.1744 8.1 481 B
6 Protocatechuic acid C7H6O4 15.860 ** [M − ­H]− 153.0193 153.0195 1.3 109 U
7 Gallic acid C14H10O9 15.993 ** [M − ­H]− 323.0398 323.0381 − 5.5 169, 125 B
3-monogallate
8 p-Hydroxybenzoic C7H6O3 16.818 ** [M − ­H]− 137.0234 137.0243 − 4.3 93 U, B
acid
9 Benzoic acid C7H6O2 21.713 ** [M − ­H]− 121.0295 121.0294 − 0.8 103, 93 U
Hydroxycinnamic
acids
10 Cinnamoyl glucose C15H18O7 9.676 ** [M − ­H]− 311.1125 311.1114 − 3.5 311 B
11 p-Coumaric acid C15H18O8 16.629 ** [M − ­H]− 325.0929 325.0936 2.2 163 B
4-O-glucoside
12 Caffeic acid C9H8O4 17.639 ** [M − ­H]− 179.0350 179.0351 0.6 135 U
13 Cinnamic acid C9H8O2 18.021 ** [M − ­H]− 147.0451 147.0441 − 6.8 103 U, B
14 Dihydroferulic acid C10H12O4 18.986 [M + ­H]+ 197.0809 197.0818 4.57 197 B
15 1,2,2'-Triferuloyl- C42H46O20 22.826 [M + ­H]+ 871.2655 871.2631 − 2.75 871 B
gentiobiose
16 Verbascoside A C31H40O16 28.316 [M + ­H]+ 669.2389 669.2399 1.49 669 B
17 Rosmarinic acid C18H16O8 35.023 ** [M − ­H]− 361.0918 361.0906 − 3.32 197, 179, B
18 p-Coumaroyl C11H10O5 52.416 [M + ­H]+ 223.0601 223.0615 6.28 223 B
glycolic acid
19 p-Coumaric acid C9H8O3 54.433 ** [M − ­H]− 163.0400 163.0411 6.7 119 B, U
Hydroxypheny-
lacetic acids
20 2-Hydroxy-2-phe- C8H8O3 15.035 ** [M + ­H]+ 153.0546 153.0553 4.57 109 U, B
nylacetic acid
21 3,4-Dihydroxyphe- C8H8O4 18.218 [M + ­H]+ 169.0496 169.0505 5.32 169 U, B
nylacetic acid
Flavonoids
Anthocyanins
22 Delphinidin C27H31O17 4.061 [M + ­H]+ 628.1634 628.1645 1.75 303 U
3,5-O-digluco-
side
23 Peonidin 3-O-(6''- C24H25O12 4.649 [M + ­H]+ 506.1419 506.1429 1.98 506 B
acetyl-galacto-
side)
24 Malvidin 3-O-(6''- C25H27O13 4.707 [M + ­H]+ 536.1525 536.1569 8.21 331 B
acetyl-galacto-
side)
25 Peonidin C16H13O6 4.737 [M + ­H]+ 302.0785 302.0775 − 3.31 301 B
26 Pelargonidin C24H23O13 4.737 [M + ­H]+ 520.1212 520.1240 5.38 520 B
3-O-(6''-malonyl-
glucoside)

13
1310 W. Ahmad et al.

Table 2  (continued)
No Name Formula Retention time ESI ± Theoretical Observed Mass error Product ions Sample
(m/z) (m/z) (ppm)

27 Cyanidin 3-O-sam- C32H39O20 4.926 [M + ­H]+ 744.2108 744.2090 − 2.42 744 B

bubioside
5-O-glucoside
28 Isopeonidin C21H21O10 6.102 [M + ­H]+ 434.1208 434.1234 5.99 434 B
3-O-arabinoside
29 Peonidin 3-O-ruti- C28H33O15 6.147 [M + ­H]+ 610.1893 610.1895 0.33 610 B
noside
30 Cyanidin 3-O-(6''- C30H27O13 6.174 [M + ­H]+ 596.1525 596.1518 − 1.17 287 U, B
p-coumaroyl-
glucoside)
31 Cyanidin 3-O-(6''- C30H27O14 10.608 [M + ­H]+ 612.1474 612.1487 2.12 303 U, B
caffeoyl-gluco-
side)
32 Peonidin 3-O-(6''- C31H29O13 12.273 [M + ­H]+ 610.1681 610.1663 − 2.95 610 B
p-coumaroyl-
glucoside)
33 Peonidin C34H43O21 13.090 [M + ­H]+ 788.2370 788.2397 3.43 788 B
3-O-digluco-
side-5-O-gluco-
side
34 Cyanidin 3-O-ruti- C27H31O15 16.818 [M + ­H]+ 596.1736 596.1733 − 0.50 287 B
noside
35 Peonidin C28H33O16 18.040 [M + ­H]+ 626.1842 626.1839 − 0.48 626 B
3-O-sophoroside
36 Pelargonidin C33H41O19 18.642 [M + ­H]+ 742.2315 742.2291 − 3.23 271 B
3-O-glucosyl-
rutinoside
37 Cyanidin C27H31O16 20.732 [M + ­H]+ 612.1685 612.1634 − 8.33 287 U
3,5-O-digluco-
side
38 Cyanidin C21H21O11 21.552 [M + ­H]+ 450.1157 450.1138 − 4.22 287 U
3-O-galactoside
39 Delphinidin C21H21O12 21.651 [M + ­H]+ 466.1106 466.1087 − 4.08 303 U
3-O-galactoside
40 Malvidin 3-O-(6''- C32H31O14 24.225 [M + ­H]+ 640.1787 640.1808 3.28 640 B
p-coumaroyl-
glucoside)
41 Cyanidin 3-O-glu- C33H41O20 53.612 [M + ­H]+ 758.2264 758.2257 − 0.92 287 U, B
cosyl-rutinoside
Flavanols
42 Epigallocatechin C34H36O22 18.781 [M + ­H]+ 797.1771 797.1764 − 0.88 797 U
3-O-gallate-7-O-
glucoside-4''-O-
glucuronide
43 Procyanidin trimer C45H38O18 20.808 [M + ­H]+ 867.2131 867.2162 3.57 867 U
C1
44 Procyanidin dimer C30H26O12 21.056 ** [M − ­H]− 579.1497 579.1529 5.53 579 U
B1
45 (-)-Epigallocat- C21H22O13 36.003 [M + ­H]+ 483.1133 483.1126 − 1.45 483 B
echin 7-O-glucu-
ronide
Flavanones
46 Didymin C28H34O14 13.090 [M + ­H]+ 595.2022 595.2032 1.68 577, 287 B
47 6-Geranylnarin- C25H28O5 15.226 [M − ­H]− 407.1864 407.1890 6.39 287, 243, 159, B
genin 119

13
A comprehensive characterization of polyphenol extracts from wasted sour fruits by LC–MS/MS… 1311

Table 2  (continued)
No Name Formula Retention time ESI ± Theoretical Observed Mass error Product ions Sample
(m/z) (m/z) (ppm)

48 Hesperidin C28H34O15 22.590 [M + ­H]+ 611.1971 611.1974 0.49 303 U, B

49 Hesperetin C22H22O12 23.133 [M + ­H]+ 479.1184 479.1199 3.13 301 U
3'-O-glucuronide
Flavones
50 Nobiletin C21H22O8 3.726 ** [M − ­H]− 401.1242 401.1225 − 4.24 401 U, B
51 Apigenin 6-C-glu- C21H20O10 4.175 ** [M − ­H]− 431.0983 431.0990 1.62 269 U, B
coside
52 Apigenin 7-O-glu- C21H24O9 6.652 [M − ­H]− 419.1347 419.1327 − 4.77 417 U
coside
53 Apigenin 6,8-di-C- C27H30O15 16.818 [M + ­H]+ 595.1658 595.1691 5.54 595 B
glucoside
54 6-Hydroxyluteolin C21H20O11 21.552 [M + ­H]+ 449.1079 449.1109 6.68 303, 285 U
7-O-rhamnoside
55 Diosmin C28H32O15 22.666 ** [M − ­H]− 609.1814 609.1868 8.86 301 B, U
Flavonols
56 Kaempferol C33H40O21 4.530 [M + ­H]+ 773.2135 773.2150 1.94 773 B
3,7,4'-O-triglu-
coside
57 Isorhamnetin C22H20O13 4.608 [M + ­H]+ 493.0977 493.0979 0.41 493 B
3-O-glucuronide
58 Kaempferol C33H40O20 16.034 [M + ­H]+ 757.2186 757.2204 2.38 757 B
3-O-glucosyl-
rhamnosyl-galac-
toside
59 Kaempferol C21H19O11 16.662 [M + ­H]+ 448.1000 448.1041 9.15 448 B
7-O-glucoside
60 Myricetin C15H10O8 16.934 [M + ­H]+ 319.0449 319.0451 0.63 319 U
61 Isorhamnetin C28H32O16 18.040 [M + ­H]+ 625.1763 625.1801 6.08 625 B
3-O-glucoside
7-O-rhamnoside
62 Kaempferol C33H40O19 18.642 [M + ­H]+ 741.2237 741.2236 − 0.13 741 B
3-O-(2''-rhamno-
syl-galactoside)
7-O-rhamnoside
63 Dihydroquercetin C15H12O7 22.220 [M + ­H]+ 305.0656 305.0668 3.93 305 U
64 Kaempferol C21H18O12 23.133 [M + ­H]+ 463.0871 463.0899 6.05 463 U
3-O-glucuronide
65 Dihydromyricetin C21H22O12 24.926 [M + ­H]+ 467.1184 467.1174 − 2.14 467 B, U
3-O-rhamnoside
66 Quercetin C15H10O7 28.760 [M + ­H]+ 303.0499 303.0525 8.58 285, 169 U
Isoflavonoids
67 6''-O-Acetylglycitin C24H24O11 4.055 [M + ­H]+ 489.1392 489.1380 − 2.45 489 B
68 6''-O-Acetylgen- C23H22O11 4.737 [M + ­H]+ 475.1235 475.1246 2.32 475 B
istin
69 4',7-Dihydroxyiso- C15H14O3 12.000 [M − ­H]− 241.0870 241.0877 2.90 241 B
flavan
70 Violanone C17H16O6 26.037 [M + ­H]+ 317.1020 317.1034 4.41 317 U
71 Dihydroformon- C16H14O4 30.090 [M + ­H]+ 271.0965 271.0969 1.48 271 U, B
onetin
72 3'-O-Methylequol C16H16O4 33.387 [M + ­H]+ 273.1122 273.1124 0.73 273 B
73 2-Dehydro-O-des- C15H12O4 46.877 [M + ­H]+ 257.0809 257.0822 5.06 257 B
methylangolensin
74 Dihydrobiocha- C16H14O5 49.864 [M + ­H]+ 287.0914 287.0920 2.09 269, 203, 201, B
nin A 175

13
1312 W. Ahmad et al.

Table 2  (continued)
No Name Formula Retention time ESI ± Theoretical Observed Mass error Product ions Sample
(m/z) (m/z) (ppm)

Chalcones
75 Phloridzin C21H24O10 7.307 ** [M − ­H]− 435.1297 435.1294 − 0.69 435 B, U
76 Phloretin 2'-O-glu- C21H22O11 22.220 [M + ­H]+ 451.1235 451.1256 4.66 275 U
curonide
Stilbenes
77 4'-Hydroxy-3,4,5- C17H18O4 4.150 [M + ­H]+ 287.1278 287.1272 − 2.09 287 U
trimethoxystil-
bene
78 Piceatannol C20H22O9 6.120 ** [M − ­H]− 405.1191 405.1186 − 1.23 405 U, B
3-O-glucoside
79 Dihydroresveratrol C14H14O3 11.128 ** [M − ­H]− 229.0870 229.0872 0.87 229, 81 B
80 3'-Hydroxy- C17H18O5 15.553 ** [M − ­H]− 301.1081 301.1090 2.99 301 B
3,4,5,4'-
tetramethoxystil-
bene
81 Resveratrol C14H12O3 21.552 [M + ­H]+ 229.0859 229.0876 7.42 211, 167, 127 U
82 3,4,5,4'-Tetrameth- C26H44N4O2S 21.609 [M − ­H]− 475.3112 475.3136 5.05 475 B
oxystilbene
83 Piceatannol C14H12O4 54.463 [M + ­H]+ 245.0809 245.0823 5.71 245 B
Lignans
84 7-Hydroxy- C20H22O7 4.649 [M + ­H]+ 375.1439 375.1437 − 0.53 375 B
matairesinol
85 Schisantherin A C30H32O9 5.663 [M − ­H]− 535.1973 535.1946 − 5.04 535 B
86 Lariciresinol C20H24O6 6.147 ** [M − ­H]− 359.1500 359.1515 4.18 329, 192, 178, B, U
175, 160
87 2-Hydroxyenterol- C18H18O5 13.283 ** [M − ­H]− 313.1081 313.1083 0.64 255 U, B
actone
88 2-Hydroxyentero- C18H22O5 13.698 [M − ­H]− 317.1394 317.1378 − 5.05 299, 287, 269, U, B
diol 257
90 Schisanhenol C23H30O6 14.084 [M − ­H]− 401.1969 401.1975 1.50 401 B
91 Secoisolaricires- C20H26O6 15.100 [M − ­H]− 361.1656 361.1635 − 5.81 346, 315, 223, U
inol 165
92 Arctigenin C21H24O6 15.373 ** [M − ­H]− 371.1500 371.1486 − 3.77 356, 312, 295 U, B
93 1-Acetoxypinores- C22H24O8 17.769 [M − ­H]− 415.1398 415.1407 2.17 357 B
inol
94 Matairesinol C20H22O6 20.903 ** [M − ­H]− 357.1343 357.1352 2.52 357 U
95 Deoxyschisandrin C24H32O6 22.641 [M − ­H]− 415.2126 415.2148 5.30 402, 361, 347, B
301
96 Lariciresinol- C30H36O10 23.921 [M + ­H]+ 557.2381 557.2411 5.38 557 B
sesquilignan
97 Schisandrin C C22H24O6 33.649 ** [M + ­H]+ 385.1646 385.1664 4.67 385 B
98 Todolactol A C20H24O7 35.800 [M + ­H]+ 377.1595 377.1614 5.04 377 B
99 Tigloylgomicin H C28H36O8 41.043 [M + ­H]+ 501.2483 501.2503 3.99 501 B
Other Polyphe-
nols
100 Mellein C10H10O3 17.370 ** [M − ­H]− 177.0557 177.0555 − 1.13 133 U, B
101 Esculetin C9H6O4 47.592 [M + ­H]+ 179.0339 179.0347 4.47 179 B
102 Urolithin C C13H8O5 49.008 [M + ­H]+ 245.0445 245.0440 − 2.04 245 B
103 Urolithin A C13H8O4 50.738 [M + ­H]+ 229.0496 229.0504 3.49 229 B
104 Coumarin C9H6O2 50.738 [M + ­H]+ 147.0441 147.0443 1.36 103, 91 U, B
105 4-Hydroxycou- C9H6O3 52.252 [M + ­H]+ 163.0390 163.0398 4.91 163 B, U
marin

13
A comprehensive characterization of polyphenol extracts from wasted sour fruits by LC–MS/MS… 1313

Table 2  (continued)
No Name Formula Retention time ESI ± Theoretical Observed Mass error Product ions Sample
(m/z) (m/z) (ppm)

106 Scopoletin C10H8O4 54.433 [M + ­H]+ 193.0496 193.0505 4.66 193 B, U

107 Rosmanol C20H26O5 20.652 ** [M − ­H]− 345.1707 345.1728 6.08 301 U, B
108 Carnosic acid C20H28O4 41.346 [M + ­H]+ 333.2061 333.2060 − 0.30 333 B, U
109 Carvacrol C10H14O 45.040 [M + ­H]+ 151.1118 151.1125 4.63 151 B
110 Carnosol C20H26O4 53.472 [M + ­H]+ 331.1904 331.1880 − 7.25 331 B, U
111 3,4-DHPEA-EDA C17H20O6 4.297 [M − ­H]− 319.1187 319.1191 1.25 275, 195 B
112 p-HPEA-EDA C17H20O5 6.646 [M − ­H]− 303.1238 303.1230 − 2.64 303 B
113 p-HPEA-AC C10H12O3 12.551 [M − ­H]− 179.0713 179.0702 − 6.14 137, 119 U, B
114 Phlorin C12H16O8 4.536 [M − ­H]− 287.0772 287.0781 3.14 287 U
115 Pyrogallol C6H6O3 9.783 [M + ­H]+ 127.0390 127.0395 3.94 127 B
116 Salvianolic acid D C11H10O6 11.933 [M + H] + 239.0550 239.0563 5.44 239 B
117 Quinic Acid C7H12O6 13.178 [M − ­H]− 191.0561 191.0572 5.76 171, 127, 85 U
118 Catechol C6H6O2 14.968 [M + ­H]+ 111.0441 111.0449 7.20 111 U, B
119 p-Anisaldehyde C8H8O2 18.986 [M + ­H]+ 137.0597 137.0600 2.19 122, 109 U, B
120 Arbutin C12H16O7 28.287 [M + ­H]+ 273.0969 273.0980 4.03 273 B
121 [6]-Gingerol C17H32O4 38.383 [M − ­H]− 299.2228 299.2222 − 2.01 299 U, B
122 Psoralen C11H6O3 48.711 [M + ­H]+ 187.0390 187.0396 3.21 187 B
123 Xanthohumol C21H22O5 49.864 [M + ­H]+ 355.1540 355.1544 1.13 355 B
124 Juglone C10H6O3 50.702 [M + ­H]+ 175.0390 175.0395 2.86 175 B

**Compounds were screened in both modes [M + ­H]+/[M − ­H]−. Samples were presented with abbreviations; Black lemon (B), Unripe grapes
(U)

3-O-galactoside (m/z 450.1138), Delphinidin 3-O-galacto- Hesperidin (m/z 611.1974) was observed in both samples,
side (m/z 466.1087), and Cyanidin 3-O-glucosylrutinoside whereas Hesperetin 3-O-glucuronide (m/z 479.1199) was
(758.2257) were characterized as compounds 38, 39 and 41. observed only in unripe grapes, from their fraction ions
The fraction ions of the observed compounds were formed on m/z 303 and m/z 301 apart. Same analyses were also
at m/z 287 and m/z 303, respectively. discussed by another study [42] in which they used same
technique to characterize Australian olive’s phenolic and
Flavanols their antioxidant capabilities.

Procyanidin trimer C1 (m/z 867.213) was first observed

flavanols as compound 43, in positive mode and produced Flavones
fraction ions at m/z 867 only identified in unripe grapes. In
addition, two other compounds were also only identified in A preliminary identification of compound number 50 (Nobi-
unripe grapes are Epigallocatechin 3-O-gallate-7-O-gluco- letin) and 51 (Apigenin 6-C-glucoside) was observed in both
side-4''-O-glucuronide and Procyanidin dimer B1 forming samples at m/z 401.1225 and m/z 431.0990, respectively.
fraction ions at m/z 797 for compound 42 and m/z 579 for The resulting ions m/z 237, m/z 188, m/z 145, m/z 59 and
compound 44. Similar (-)-Epigallocatechin 7-O-glucuronide m/z 269 in the MS2 experiment were created by the reduc-
compounds were only observed in black lemon and previous tion of carbon dioxide molecule (44 Da), C ­ 3O2, and water
results revealed the presence of similar compounds in lemon molecule (86 Da), respectively. Apigenin 6,8-di-C-glucoside
and mint samples [17, 41] generated ions at m/z 577, and m/z 383, by the reduction of
­CO2, ring A from flavonoids, and the ring B from flavo-
Flavanones noids in MS/MS. Apigenin 6,8-C-arabinosideC-glucoside
(m/z 565.1605) and 6-Hydroxyluteolin 7-O-rhamnoside
The compound 47, 6-Geranylnaringenin (m/z 407.1890) (449.1109) were identified as producing their fraction ions
was identified in black lemon only in negative mode at m/z 271, and m/z 303, m/z 285, respectively. Compound
revealed its fraction ions on m/z 287, m/z 243, m/z 159 number 55, Diosmin was observed in each sample however,
and m/z 119. Didymin (m/z 595.2032), as compound 46, 6-Hydroxyluteolin as compound 54, was only identified in
revealed its fraction ions on m/z 577 and m/z 287. unripe grapes.

13
1314 W. Ahmad et al.

Flavonols lignans from the selected variety of samples. This is the

first study reporting on the identification and characteriza-
Compounds number 56 and 57 in Table 2 were observed tion of lignin bioactive molecules from black lemon sam-
as Kaempferol 3,7,4'-O-triglucoside and Isorhamnetin ples. 7-Hydroxymatairesinol (m/z 375.1437) and Schisan-
3-O-glucuronide forming fraction ions at m/z 773 and m/z therin A (m/z 535.1946) were identified in black lemon
493, respectively. Kaempferol 3-O-glucosyl-rhamnosyl- sample only. Negative mode observed lignin compounds
galactoside (m/z 757.2204) and Kaempferol 7-O-glucoside were Lariciresinol with m/z 359.1515, 2-Hydroxyentero-
(m/z 448.1041) were recognized positively in black lemon diol with m/z 317.1378, Schisanhenol with m/z 401.1975,
sample only. Whereas, Myricetin (m/z 319.0451) and Secoisolariciresinol with m/z 361.1635, 1-Acetoxypinores-
Kaempferol 3-O-glucuronide (m/z 463.0899) were identi- inol with m/z 415.1407, and Deoxyschisandrin with
fied as compounds number 60 and 64 that were found in 415.2148. In addition, another compound was observed
unripe grapes only. Schisandrin C in black lemon with [M + ­H ] + only and
formed its fraction ions on m/z 385. Another study [46]
Isoflavonoids conducted on phytochemicals with cancer-fighting proper-
ties revealed some compounds observed from the lignan
Seven Isoflavonoids were observed in black lemon sam- profile of unripe grapes were similar to the compounds
ples 3'-O-Methylequol and Dihydrobiochanin A as com- identified in the present research.
pounds 72 and 74, respectively, in positive modes at m/z
273.1124 and m/z 287.0920. 6''-O-Acetylglycitin (m/z
489.1380) and 6''-O-Acetylgenistin (m/z 475.1246) were Other polyphenols
only observed in black lemon producing fraction ions at m/z
489 and 475, respectively. Compound 71 was recognized Coumarins and their derivatives
as Dihydroformononetin producing ions at m/z 271 in both
samples. An Isoflavonoid was also identified from samples Seven coumarins derivatives were identified from both
of unripe grapes termed as Violanone (m/z 317.1034) form- samples, two of which were Esculetin and Coumarin at
ing its ions on m/z 317. [M + ­H]+ mode only with fraction ions m/z 179, m/z 103
and m/z 91, where Mellein was observed in positive as well
Stilbenes negative mode at m/z 133. Black lemon was observed to
have Urolithin C and Urolithin A fragmenting at m/z 245
This study observed seven stilbenes from samples of black and m/z 229, respectively. Other hydroxycoumarins found
lemon and grapes. Compound 78, Piceatannol 3-O-gluco- to be present in both targeted products were 4-Hydroxy-
side (m/z 405.1186) was analyzed positively as well nega- coumarin (m/z 163.0398) and Scopoletin (m/z 193.0505)
tively and found to be present in both samples. Numerous observed in positive modes as compound 105 and 106,
studies have been conducted on piceatannol, compound respectively.
as stilbene having two phenol rings found in red wine and
ripened grapes and has considerable biomedical benefits
including anti-tumor, anti-inflammation, antioxidant and Terpenes and tyrosols
anti-atherosclerotic effects [43, 44]. On the other side,
Dihydroresveratrol and 3'-Hydroxy-3,4,5,4-tetramethoxy Four phenolic terpenes were characterized from both unripe
stilbene were observed in black lemon only. They were grapes and black lemon samples out of which Rosmanol
observed to produce their fraction ions on m/z 229, m/z 81 was identified that formed fraction ions at m/z 301. Other
and m/z 301, respectively. Other stilbenes observed were compounds were all identified in [M + ­H]+ given as Car-
4'-Hydroxy-3,4,5-trimethoxystilbene and resveratrol only in nosic acid (Compound 108), carvacrol (compound 109) and
unripe grapes and formed their fraction ions at m/z 287, m/z Carnosol (compound 110) observed to be fragmenting at
211, m/z 167, and m/z 127. m/z 333, m/z 151 and m/z 331, respectively. Carvacrol (m/z
151.1125) was the only phenolic terpene identified in black
Lignans lemon as compound 109.
Three tyrosols were identified as 3,4-DHPEA-EDA and
LC–MS-characterized bioactive substances, including lig- p-HPEA-EDA, only in black lemon samples fragmenting at
nans, exhibit exceptional antioxidant and anti-carcinogenic m/z 275, m/z 195, and m/z 303, respectively. p-HPEA-AC
activities. Human ovarian, breast, and prostate cancers (compound number 113) was the only tyrosol identified in
are protected or prevented from spreading by consuming both the samples of unripe grapes and black lemon at m/z
plant lignans [45]. LC–MS helped to identify 15 different 179.0702 with fraction ions at m/z 137 and m/z 119.

13
A comprehensive characterization of polyphenol extracts from wasted sour fruits by LC–MS/MS… 1315

Table 3  Quantification/semi- No Compounds Formula RT Unripe grapes Back lemon

quantification of targeted
phenolic compounds 1 Gallic acid C7H6O5 7.388 13.95 ± 9.34 393.53 ± 32.78
2 Protocatechuic acid C7H6O4 15.860 308.56 ± 10.05 533.56 ± 20.05
3 Caffeic acid C9H8O4 17.639 76.32 ± 7.06 390.96 ± 46.79
4 Cinnamic acid C9H8O2 18.021 82.18 ± 5.35 380.73 ± 19.90
5 Dihydroferulic acid C10H12O4 18.986 1.56 ± 0.67 10.39 ± 0.75
6 Dihydromyricetin 3-O-rhamnoside C21H22O12 24.926 NQ 84.30 ± 1.42
7 Rosmarinic Acid C18H16O8 35.023 70.95 ± 1.67 73.69 ± 1.09
8 Ellagic acid C14H6O8 7.414 NQ 78.54 ± 0.60
9 p-coumaric acid C9H8O3 54.433 307.46 ± 1.20 296.48 ± 0.59
10 Kaempferol 7-O-glucoside C21H19O11 16.662 19.42 ± 1.27 31.16 ± 1.79
11 Quercetin C15H10O7 28.760 228.95 ± 13.75 NQ
12 Myricetin C15H10O8 16.934 72.40 ± 0.80 NQ
13 Diosmin C28H32O15 22.666 115.60 ± 4.19 590.73 ± 40.88
14 Pyrogallol C6H6O3 9.783 233.12 ± 13.91 393.53 ± 29.69
15 Scopoletin C10H8O4 54.433 169.20 ± 18.87 139.74 ± 2.89
16 Coumarin C9H6O2 50.738 335.10 ± 23.06 524.80 ± 53.28

RT retention time, NQ not quantified

Other phenolic compounds Calculation of selected phenolic compounds

In addition to the mentioned categories of phenolic com- Sixteen compounds were measured in sour fruit samples
pounds some additional polyphenols were also found. (unripe grapes and black lemon) that are presented in
Compound number 114 only observed in unripe grapes was Table 3. Most prevalent class are flavonoids in these fruits.
identified as Phlorin (­ C12H16O8) with m/z 287.0781, and its Black lemon was observed to have higher num-
fraction ions were observed on m/z 287. Miscellaneous poly- ber of flavonoids, and highest amount of diosmin was
phenols observed from both targeted samples included Cat- calculated (590.73 ± 40.88 µg/g). Dihydromyricetin
echol (m/z 111.0449), p-Anisaldehyde (m/z 137.0600), and 3-O-rhamnoside (84.30 ± 1.42 µg/g), and ellagic acid
[6]-Gingerol (m/z 299.2222). Compounds 120 and 124 were (78.54 ± 0.60 µg/g) were only quantified in black lemon.
identified as Arbutin and Juglone with their fraction ions at Coumarin (335.10 ± 23.06 µg/g) is highest phenol quanti-
m/z 273 and m/z 175 in given order, both observed only in fied in unripe grapes. Protocatechuic acid was calculated
black lemon sample. Another polyphenol was characterized in unripe grapes (308.56 ± 10.05 µg/g) and black lemon
with negative mode from unripe grapes and known as Quinic (533.56 ± 20.05 µg/g). Quercetin (228.95 ± 13.75 µg/g) and
Acid (m/z 191.0572). Myricetin (72.40 ± 0.80 µg/g) are two compounds that are
Using LC–MS/MS-QTOF, we were able to evaluate and only present in unripe grapes. The concentration of caf-
identify 124 phenolic metabolites with their fraction ions feic acid was much higher (390.96 ± 46.79 µg/g) in black
in sour fruit samples. Both of these fruit’s phenolic con- lemon than in unripe grapes (76.32 ± 7.06 µg/g). Gallic acid
stituents have not yet been the subject of a single study. The (13.95 ± 9.34 µg/g) and p-coumaric acid (307.46 ± 1.20 µg/g)
assessment of bioactive components in these particular fruits were calculated in unripe grapes but lower concentration
forms can provide a new field of study towards revealing of p-coumaric acid (296.48 ± 0.59 µg/g) also quantified in
their significant health advantages and new applications. The black lemon. The lowest found phenol was Dihydroferulic
use of this sophisticated analytical method has a significant acid quantified in unripe grapes (1.56 ± 0.67 µg/g) and black
promise for the discovery of novel bioactive chemicals. Low lemon (10.39 ± 0.75 µg/g). Overall, the results showed that
collision energies is the drawback of not having ability to all the quantified compounds in black lemon were higher
find the natural ring of phenols LC–MS/MS analysis that than in unripe grapes. The total amount of polyphenols
underwent alteration. depends on the maturity of fruits.

13
1316 W. Ahmad et al.

Conclusion 5. C. Dani, D. Bonatto, M. Salvador, M.D. Pereira, J.A.P. Henr-

iques, E. Eleutherio, Antioxidant protection of resveratrol and
catechin in Saccharomyces cerevisiae. J. Agric. Food Chem. 56,
In conclusion, a comprehensive profiling of polyphenols 4268–4272 (2008)
in unripe grapes and black lemons was investigated by 6. D.O. Adams, Phenolics and ripening in grape berries, Am. J.
LC–ESI–QTOF-MS/MS, including the finding of some Enol. Viticult. (2006)
7. F. Tinello, A. Lante, Evaluation of antibrowning and antioxidant
phenolic compounds not previously reported in these sour activities in unripe grapes recovered during bunch thinning.
fruits. The obtained results show these agricultural waste Aust. J. Grape Wine Res. 23, 33–41 (2017)
products to be abundant in polyphenols as well as confirm- 8. Ş Kıvrak, İ Kıvrak, E. Karababa, Nutritional value, phenolic,
ing the important contribution of anthocyanins to its total vitamin and mineral contents of a fermented beverage from
grapes of’Merlot’and’Cabernet Sauvignon’. MITTEILUNGEN
polyphenols. Further, results summarize the importance of KLOSTERNEUBURG 66, 255–265 (2016)
performing comprehensive identification and characteriza- 9. S. Chaisawadi, W. Methawiriyasilp, D. Thongbute, T. Chayawat-
tions of phenolic compounds in commonly ignored forms tana, clean production of commercial freeze-dried lime powder
of traditional products. This study identified 124 bioactive for medicinal herb and nutritional health benefits, in, Interna-
tional Society for Horticultural Science (ISHS), Leuven, Bel-
compounds including 21 phenolic acids, 41 flavonoids, 8 gium, pp. 29–35 (2005)
isoflavonoids, 2 chalcones, 7 stilbenes, 15 lignans and 25 10. N. Singh, N.S. Yarla, N.J. Siddiqi, M. de Lourdes Pereira,
other compounds in sour fruit samples. The complemen- B. Sharma, Features, pharmacological chemistry, molecular
tary profile of phenolic compounds in both samples makes mechanism and health benefits of lemon, medicinal chemistry
(Shariqah (United Arab Emirates)), 17, 187–202 (2021)
it interesting to utilize especially as dietary supplements 11. A.-M. Chiorcea-Paquim, T.A. Enache, E. De Souza Gil, A.M.
that enhance sensory and therapeutic properties. This study Oliveira-Brett, Natural phenolic antioxidants electrochemistry:
confirmed that black lemon and unripe grapes have a high towards a new food science methodology. Compr. Rev. Food
level of antioxidant functions and can be utilized in nutra- Sci. Food Saf. 19, 1680–1726 (2020)
12. Ş Kıvrak, T. Göktürk, İ Kıvrak, Determination of phenolic com-
ceuticals food, pharmaceuticals and cosmetics for different position of Tilia Tomentosa flowers using UPLC-ESI-MS/MS.
health effects. Indeed, their inclusion in a single product can Int. J. Second. Metab. 4, 249–256 (2017)
also be applied, combining their complementary phenolic 13. D. Del Rio, A. Rodriguez-Mateos, J.P. Spencer, M. Togno-
composition, which could refer to synergies between the lini, G. Borges, A. Crozier, Dietary (poly)phenolics in human
health: structures, bioavailability, and evidence of protective
health’s effects studied for both sour fruits. effects against chronic diseases. Antioxid. Redox Signal. 18,
1818–1892 (2013)
Acknowledgements The authors thank all the team and funding
14. G. Fia, C. Gori, G. Bucalossi, F. Borghini, B. Zanoni, A natu-
sources (The National Key Research and Development Program of
rally occurring antioxidant complex from unripe grapes: the
China (2022YFC2105100) and Shanghai Collaborative Innovation
case of Sangiovese (v. Vitis vinifera). Antioxidants, 7, 27 (2018)
Center for Biomanufacturing Technology) for their support in this
15. Ş Kıvrak, İ Kıvrak, E. Karababa, Analytical evaluation of phe-
research work.
nolic compounds and minerals of Opuntia robusta J.C. Wendl
and Opuntia ficus-barbarica A. Berger. Int. J. Food Prop. 21,
Declaration
229–241 (2018)
16. B. Singh, J.P. Singh, A. Kaur, N. Singh, Phenolic composition,
Conflict of interest The authors declare no any conflict of interest. antioxidant potential and health benefits of citrus peel. Food
Res. Int. 132, 109114 (2020)
17. H.S. Kiani, W. Ahmad, S. Nawaz, M.A. Farah, A. Ali, Opti-
mized extraction of polyphenols from unconventional edible
plants: LC-MS/MS profiling of polyphenols, biological func-
References tions, molecular docking, and pharmacokinetics study. Mol-
ecules 28, 6703 (2023)
1. C. Beres, G.N.S. Costa, I. Cabezudo, N.K. da Silva-James, A.S.C. 18. A. Ali, J.J. Cottrell, F.R. Dunshea, Identification and charac-
Teles, A.P.G. Cruz, C. Mellinger-Silva, R.V. Tonon, L.M.C. terization of anthocyanins and non-anthocyanin phenolics from
Cabral, S.P. Freitas, Towards integral utilization of grape pomace Australian native fruits and their antioxidant, antidiabetic, and
from winemaking process: a review. Waste Manage. 68, 581–594 anti-Alzheimer potential. Food Res. Int. 162, 111951 (2022)
(2017) 19. A. Ali, J.J. Cottrell, F.R. Dunshea, Antioxidant, alpha-glucosi-
2. F. Cosme, T. Pinto, A. Vilela, Phenolic compounds and antioxi- dase inhibition activities, in silico molecular docking and phar-
dant activity in grape juices: a chemical and sensory view. Bever- macokinetics study of phenolic compounds from native Austral-
ages 4, 22 (2018) ian fruits and spices. Antioxidants 12, 254 (2023)
3. A. Teixeira, N. Baenas, R. Dominguez-Perles, A. Barros, E. Rosa, 20. J.S. Park, J.-W. Woo, G.-H. Choi, D.S. Choi, M.Y. Jung, Chloro-
D.A. Moreno, C. Garcia-Viguera, Natural bioactive compounds genic acid profiles and antioxidant potentials of 17 sweet potato
from winery by-products as health promoters: a review. Int. J. varieties cultivated in Korea: impact of extraction condition and
Mol. Sci. 15, 15638–15678 (2014) classification by hierarchical clustering analysis (2015)
4. Â.C. Salvador, M.M.Q. Simões, A.M.S. Silva, S.A.O. Santos, 21. H.S. Kiani, B. Ali, M.K. Al-Sadoon, H.S. Al-Otaibi, A. Ali,
S.M. Rocha, A.J.D. Silvestre, Vine waste valorisation: integrated LC-MS/MS and GC-MS identification of metabolites from the
approach for the prospection of bioactive lipophilic phytochemi- selected herbs and spices, their antioxidant, anti-diabetic poten-
cals. Int. J. Mol. Sci. 20, 4239 (2019) tial, and chemometric analysis. Processes 11, 2721 (2023)

13
A comprehensive characterization of polyphenol extracts from wasted sour fruits by LC–MS/MS… 1317

22. J. Sharifi-Rad, S. Song, A. Ali, V. Subbiah, Y. Taheri, H.A.R. 34. E. Ivanišová, A. Kántor, M. Kačániová, Antioxidant activity and
Suleria, LC-ESI-QTOF-MS/MS characterization of phenolic com- total polyphenol content of different varieties of grape from the
pounds from Pyracantha coccinea M.Roem. and their antioxidant small Carpathians wine region of Slovakia (2019)
capacity. Cell. Mol. Biol. 67, 201–211 (2021) 35. N. Salah Eddine, S. Tlais, A. Alkhatib, R. Hamdan, Effect of four
23. A. Ali, H. Wu, E.N. Ponnampalam, J.J. Cottrell, F.R. Dunshea, grape varieties on the physicochemical and sensory properties of
H.A.R. Suleria, Comprehensive profiling of most widely used unripe grape verjuice. Int. J. Food Sci. 2020, 6457982 (2020)
spices for their phenolic compounds through LC-ESI-QTOF- 36. A. Rauf, G. Uddin, J. Ali, Phytochemical analysis and radical
MS(2) and their antioxidant potential. Antioxidants 10(5), 721 scavenging profile of juices of Citrus sinensis, Citrus anrantifolia,
(2021) and Citrus limonum. Organ. Med. Chem. Lett. 4, 5 (2014)
24. A. Ali, Y.M. Bashmil, J.J. Cottrell, H.A.R. Suleria, F.R. Dunshea, 37. H. Salih Mahdi, A. Parveen, Biosynthesis, characterization
LC-MS/MS-QTOF screening and identification of phenolic com- and antibacterial activity of gold nanoparticles (Au-NPs) using
pounds from australian grown herbs and their antioxidant poten- black lemon extract, Materials Today: Proceedings, 18 (2019)
tial. Antioxidants 10(11), 1770 (2021) 5164–5169
25. J. Severo, A. Tiecher, F.C. Chaves, J.A. Silva, C.V. Rombaldi, 38. A.N. Panche, A.D. Diwan, S.R. Chandra, Flavonoids: an overview.
Gene transcript accumulation associated with physiological and J. Nutr. Sci. 5, e47 (2016)
chemical changes during developmental stages of strawberry cv. 39. S.A. Aherne, N.M. O’Brien, Dietary flavonols: chemistry, food
Camarosa. Food Chem. 126, 995–1000 (2011) content, and metabolism. Nutrition 18, 75–81 (2002)
26. R.M. Patel, Ferrous ion chelating activity (FICA)-a compara- 40. G. Garrido-Bañuelos, A. Buica, W. du Toit, Relationship between
tive antioxidant activity evaluation of extracts of eleven naturally anthocyanins, proanthocyanidins, and cell wall polysaccharides
growing plants of Gujarat, India. Int. J. Sci. Res. 2, 426–428 in grapes and red wines. A current state-of-art review. Crit. Rev.
(2012) Food Sci. Nutr. 62, 7743–7759 (2022)
27. A. Ali, S.M. Kiloni, P.R. Cáceres-Vélez, P.R. Jusuf, J.J. Cottrell, 41. S. Kumar, A. Singh, B. Kumar, Identification and characterization
F.R. Dunshea, Phytochemicals, antioxidant activities, and toxico- of phenolics and terpenoids from ethanolic extracts of Phyllanthus
logical screening of native australian fruits using Zebrafish embry- species by HPLC-ESI-QTOF-MS/MS. J. Pharm. Anal. 7, 214–222
onic model. Foods 11, 4038 (2022) (2017)
28. A. Ali, J.J. Cottrell, F.R. Dunshea, Characterization, antioxidant 42. A. Ali, J.J. Cottrell, F.R. Dunshea, Antioxidant, alpha-glucosidase
potential, and pharmacokinetics properties of phenolic com- inhibition activities, comprehensive metabolite fingerprinting of
pounds from native australian herbs and fruits. Plants 12(5), 993 Australian black and green olives and their antioxidant and phar-
(2023) macokinetics properties. Separations 10, 354 (2023)
29. A. Ali, H.F. Zahid, J.J. Cottrell, F.R. Dunshea, A comparative 43. H. Piotrowska, M. Kucinska, M. Murias, Biological activity of
study for nutritional and phytochemical profiling of Coffea ara- piceatannol: leaving the shadow of resveratrol. Mutat. Res. 750,
bica (C. arabica) from different origins and their antioxidant 60–82 (2012)
potential and molecular docking. Molecules 27, 5126 (2022) 44. Y.-L. Tang, S.-W. Chan, A review of the pharmacological effects
30. S. Kim, J. Chen, T. Cheng, A. Gindulyte, J. He, S. He, Q. Li, B. of piceatannol on cardiovascular diseases. Phytother. Res. 28,
Shoemaker, P. Thiessen, B. Yu, L. Zaslavsky, J. Zhang, E. Bol- 1581–1588 (2014)
ton, PubChem 2023 update. Nucleic Acids Res. 51(D1), D1373– 45. Q. Cui, R. Du, M. Liu, L. Rong, Lignans and their derivatives
D1380 (2023). https://​doi.​org/​10.​1093/​nar/​gkac9​56 from plants as antivirals. Molecules 25 (2020)
31. A.C. de Camargo, A.C.T. Biasoto, A.R. Schwember, D. Granato, 46. T.J. Shree, S. Poompavai, S.M. Begum, V. Gowrisree, S. Hemala-
G.B. Rasera, M. Franchin, P.L. Rosalen, S.M. Alencar, F. Shahidi, tha, E. Sieni, R. Sundararajan, Cancer-fighting phytochemicals:
Should we ban total phenolics and antioxidant screening methods? another look. J. Nanomed. Biother. Discov. 9, 162 (2019)
The link between antioxidant potential and activation of NF-κB
using phenolic compounds from grape by-products. Food Chem. Publisher's Note Springer Nature remains neutral with regard to
290, 229–238 (2019) jurisdictional claims in published maps and institutional affiliations.
32. X. Dong, Y. Hu, Y. Li, Z. Zhou, The maturity degree, phenolic
compounds and antioxidant activity of Eureka lemon [Citrus Springer Nature or its licensor (e.g. a society or other partner) holds
limon (L.) Burm f.]: a negative correlation between total phe- exclusive rights to this article under a publishing agreement with the
nolic content, antioxidant capacity and soluble solid content. Sci. author(s) or other rightsholder(s); author self-archiving of the accepted
Hortic. 243, 281–289 (2019) manuscript version of this article is solely governed by the terms of
33. Y. Zhao, K. Tran, M. Brennan, C. Brennan, Kinetics of ultrasonic such publishing agreement and applicable law.
extraction of polyphenols, anthocyanins and tannins from five dif-
ferent New Zealand grape pomaces. Int. J. Food Sci. Technol. 56,
2687–2695 (2021)

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