International Journal of Food Science and Technology 2019 1

Original article
Free radical scavenging activities of phytochemical mixtures and
aqueous methanolic extracts recovered from processed coffee
leaves

Xiumin Chen,1,* David D. Kitts,2 Dayi Ji1 & Jian Ding1

1 Department of Food Science and Engineering, School of Food and Biological Engineering, Jiangsu University, # 301 Xuefu Road, Jingkou
District, Zhenjiang, Jiangsu Province 212013, China
2 Food, Nutrition, and Health, University of British Columbia, 2205 East Mall, Vancouver, BC, V6T 1Z4, Canada
(Received 29 September 2018; Accepted in revised form 12 December 2018)

Summary It is well known that the choice of extraction solvents greatly affects both phytochemical composition and
related bioactivity of plant extract. Herein, we show that coffee leaf extracts derived from 80% methanol
have no effect on basal nitric oxide production in Raw 264.7 cells, whereas, a different result was observed
with a previous hot water extract. Phytochemical mixtures formulated to contain equivalent amounts of
CQAs, mangiferin and rutin know to present in coffee leaves showed a relatively low (3.5–18.5%, TPC;
17.6–31.6%, ABTS; 7.9–13.6%, DPPH; and 8.5–29.7%, ORAC) contribution to antioxidant activities
when compared to the coffee leaf extracts. We conclude that the solvent polarity is particularly important
to the unique phytochemical mixture recovered, which in turn governs the bioactivity. This study also
demonstrated that other phytochemicals in coffee leaves that were not measured in this study are impor-
tant in assessing total antioxidant activity of this potential plant beverage.
Keywords Anti-inflammation, antioxidant, aqueous methanol extract, chlorogenic acids, coffee leaf, mangiferin.

Oxidative stress results in the over production of

Introduction
Traditional practices of using coffee leaves as tea have
been used for centuries in countries such as Ethiopia,
South Sudan and Indonesia (Ross, 2005). Coffee leaves
have also been used as ethnomedicines to ameliorate
disorders or diseases in the countries such as Haiti,
Uganda, Cuba, Nicaragua, Peru, et al (Ross, 2005;
Patay et al., 2016; Chen, 2018). Recently, researchers
who characterised the phytochemical components in
coffee leaves reported the presence of chlorogenic
acids, mangiferin, rutin, caffeine, trigonelline, carote-
noids, catechins, anthocyanin, quercitrin, kaempferol
and related glycosides (Ross, 2005; Campa et al., 2012;
Martins et al., 2014; Patay et al., 2016; Chen et al.,
2018). Although the underlying mechanisms for the
health benefits derived from coffee leaves remains to
be substantiated, it is plausible that the complex and
unique mixture of phytochemicals present in coffee
leaves determine the level of antioxidant and anti-
inflammatory activities recently reported in Chen et al.
(2018).
*Correspondent: E-mail: xmchen@ujs.edu.cn
reactive oxygen species (ROS) and reactive nitrogen
species (RNS) in biological systems, which contribute
to initiation of various diseases that include inflamma-
tion, diabetes, cardiovascular disease and cancer
(Moon and Shibamoto 2009). Plant phenolics derived
from foods are good resources of natural antioxidants.
A number of chemical based methods used to measure
antioxidant activity in various food systems including,
2,20 -azinobis (3-ethylbenzothiazoline-6-sulfonic acid;
ABTS), 2,2-diphenyl-1- picrylhydrazine (DPPH), oxy-
gen radical absorbance capacity (ORAC) and ferric
reducing/antioxidant power (FRAP) assays (Thaipong
et al., 2006). We have introduced a cell-based model
system using nitric oxide (NO) output from Caco-2
cells when challenged with an oxidative stress (Chen &
Kitts, 2008).
Due to the complexity of plant components that
contribute to the differences in polarity of phytochemi-
cals extracted using different solvents, efforts have
been directed to optimise the recovery of targeted phy-
tochemicals that are known to possess significant
bioactivities (Cheng et al., 2012). Ethanol, which is a
safe food grade solvent is commonly used for plant

doi:10.1111/ijfs.14099
© 2019 Institute of Food Science and Technology
2 Antioxidant activities of coffee leaves X. Chen et al.
polyphenol extraction, whereas methanol, a non-food
grade solvent, is more efficient in extracting polyphe-
nols with lower molecular weight; in comparison,
aqueous acetone which is more effective to extract
higher molecular weight flavanols (Thavamoney et al.,
2018). One good example of this is the use of
methanol as the solvent to extract total phenolics with
excellent recoveries reported from four vegetable by-
products that compared using hexane, chloroform and
ethyl acetate, respectively (Babbar et al., 2014).
Our previous study reported that hot water extracts
obtained from coffee leaves processed using different
tea processing methods, had different phytochemical
recoveries that greatly influenced specific measurements
of antioxidant and anti-inflammatory activities (Chen
et al., 2018). We reported in our previous study the fact
that hot water extraction of coffee leaves produced an
extract that has dual functions on NO production. We
also found that it was the water fraction which con-
tributed to the NO induction capacities, whereas, aque-
ous methanol fractions inhibited NO production (Chen
et al., 2019). Furthermore, the phytochemical mixtures
containing equivalent amount of 3-CQA, 5-CQA, 3,4-
diCQA, 3,5-diCQA, caffeine, mangiferin and rutin, as
present in corresponding coffee leaf extracts were not
found to be major contributors to the NO inhibitory
activities. However, the use of methanol as an extrac-
tion solvent for processed coffee leaves to recover
defined phytochemical profiles which react in chemical
based free radical (e.g. AAPH (2,20 -Azobis(2-amidino-
propane) dihydrochloride), DPPH, ABTS) and cell
based RNS (e.g. NO) scavenging activity needs to be
determined. The aims of the present study were to com-
pare the phytochemical composition and antioxidant
activities of both young and mature coffee leaves pro-
cessed by different tea processing methods when
extracted using 80% methanol. From these data we esti-
mate the contribution that phytochemical mixtures
have to elicit antioxidant activities of coffee leaf extracts
derived from different processing methods.
Materials and methods
Materials
Young (30–90 days) and mature (90–180 days) Coffea
arabica leaves were collected from the Finca La Aur-
ora coffee farm located in the Matagalpa area of
Nicaragua (13°030 35.5″N 85°540 19.0″W) and donated
by Wize Monkey Inc. (Vancouver, BC, Canada). Raw
264.7 (TIB-71) cell was purchased from ATCC
(Manassas, VA, USA). CGAs isoform standards
including 3-caffeoylquinic acid (3-CQA), 5-caffeoylqui-
nic acid (5-CQA), 3,4-dicaffeoylquinic acid (3,4-
diCQA), 3,5-dicaffeoylquinic acid (3,5-diCQA) were
purchased from Chengdu Must Bio-Technology Co.
International Journal of Food Science and Technology 2019
Ltd. (Chengdu, China). HPLC grade acetonitrile
(ACN) was purchased from Thermo Fisher Scientific
(Waltham, MA, USA). Fetal bovine serum (FBS) was
purchased from Invitrogen (Burlington, ON, Canada).
Other chemicals were analytical grade and were pur-
chased form Sigma-Aldrich (St. Louis, MO, USA).
Preparation and extraction of coffee leaves
Fresh coffee leaves were processed according to the
methods described in Chen et al. (2018) to produce
products that mimic different tea beverages, namely,
white-tea-process (WTP), Japanese-style-green-tea-pro-
cess (JGTP), Chinese-style-green-tea-process (JGTP),
oolong-tea-process (OTP) and black-tea-process (BTP)
coffee leaf teas. Young and mature coffee leaves were
labelled as Y and M, respectively. The dry coffee leaves
were ground into powder followed by extracted with
80% methanol at a sample to solvent ratio of 1 : 20 (W
: V) for three times with sonication (Elma S-30H ultra-
sonic cleanser, Tovatech, Germany) for 20 min at room
temperature. The samples were centrifuged at 5000 g
for 20 min and filtered through WhatmanÒ No. 4 filter
paper. The supernatant was combined and evaporated
under reduced pressure and then freeze-dried. All of the
extractions were performed at least three times and the
yield of the extract was calculated as: weight of extract/
weight of coffee leaf 9 100%.
HPLC analysis
Phytochemicals including, caffeine, 3-CQA, 5-CQA,
3,4-diCQA, 3,5-diCQA, mangiferin, isomangiferin and
rutin were analysed by the method described in Chen
et al. (2018). These eight phytochemicals were detected
simultaneously at one run. HPLC analysis were con-
ducted using Agilent 1100 LC system (Agilent, Santa
Clara, CA, USA).
Free radical scavenging capacities analysis
Total phenolic analysis
Total phenolic content (TPC) analysis was performed
based on the method described by Chen et al. (2018).
Briefly, coffee leaf extract, phytochemical mixtures
(PM) and gallic acid standard were mixed with 10-times
diluted Folin-Ciocalteu reagent for 1 min at room tem-
perature followed by incubated with Na2CO3
(75 mg mL1) for 30 min. The plate was read at
765 nm using a spectrometer and TPC was calculated as
mg gallic acid per g leaf. Triplicate measurements for
three independent extracts were performed in 96-well
plate.
ABTS analysis
The ABTS free radical scavenging capacity was mea-
sured based on the method described by Chen et al.
© 2019 Institute of Food Science and Technology
 Antioxidant activities of coffee leaves X. Chen et al. 3

(2017a). ABTS working solution (absorbance at
734 nm  0.7) was reacted with coffee leaf extract,
PM or Trolox standard followed by recording the
absorbance at 734 nm. The percentage inhibition was
calculated and the Trolox equivalent antioxidant
capacity (TEAC) is expressed as lmol Trolox per g
leaf.
activities of coffee leaf extracts were calculated as the
following (taking TPC assay as an example):
% contribution
TPC of PM
¼  100
TPC of corresponding coffee leaf extract

DPPH analysis Results and discussion

The DPPH free radical scavenging ability of coffee leaf
extract and PM were analysed according to the Phytochemical profiles of methanolic extracts of coffee
method described by Chen et al. (2017a). Coffee leaf leaves
extract, PM or Trolox standard was reacted with
The yield of coffee leaf extracts derived from aque-
DPPH and absorbance at 519 was read by a spectrom-
ous methanolic extraction and related phytochemical
eter. The TEAC was calculated same as above.
compositions are presented in Table 1. The HPLC
profile and the standard curves of the eight phyto-
ORAC analysis
chemicals is shown in Figures S1 and S2. The impact
The oxygen radical absorbance capacity (ORAC) value
of different processing methods on the yield of
of coffee leaf extract and PM was measured based on
methanolic extract was influenced by the age of cof-
the method described by Chen et al. (2017a). Coffee
fee leaves. For example, young leaves (e.g. OTP and
leaf extract, PM or Trolox standard was reacted with
BTP) that had undergone oxidation process had
fluorescein sodium salt and AAPH sequentially in 96-
greater extract yields than those recovered from
well black plate and the Fluorescence (kex = 485 nm;
mature leaves. However, mature leaves without prior
kem = 527 nm) was recorded for 1 h. The ORAC
oxidation tended to produce extracts that were
value was expressed as lmol Trolox per g leaf.
greater or equivalent to extracts recovered from
young leaves when processed in the same way. The
NO analysis
reasons that oxidation affects the extraction yield of
Raw 264.7 cell (TIB-71, ATCC) grown in Dulbecco’s
young vs. old leaves are not known. A plausible
Modified Eagle’s Medium (DMEM) supplement with
explanation, however, could be that there is a
10% fetal bovine serum, 100 U ml1 of penicillin and
decrease of polyphenol oxidase activities during the
100 lg ml1 of streptomycin were seeded at a density
development stages in the leaves which results in a
of 105 cells well1 in 96-well plate and cultured over-
varied transformation of constituents with unique
night followed by treated with coffee leaf extracts with
combined polarity (Mazzafera & Robinson, 2000).
or without interferon c (INF-c) and lipopolysaccharide
This would favour a relatively greater yield in young
(LPS). The NO scavenging capacity was evaluated by
leaves when extracted using methanol.
the ability of coffee leaf extract inhibiting NO produc-
Caffeine and 5-CQA are two of the most abundant
tion in INF-c and LPS-treated Raw 264.7 cells (Chen
phytochemicals present in coffee leaf extract. Com-
& Kitts, 2015). NO content in the cell culture super-
pared with the hot water extract, a greater efficiency of
natant was measured using Griess assay (Chen et al.,
caffeine, mangiferin and rutin recovery occurred using
2017b). The IC50 of NO inhibitory capacity was calcu-
80% methanol extraction. This finding is attributed to
lated using OriginPro software (OriginLab Corpora-
the lower polarity of methanol compared with water
tion, Northampton, MA, USA). Cell viability was
which favours the solubility of phenolic compounds in
accessed using 3-(4,5-dimethylthiazol -2-yl)-2,5-diphe-
aqueous methanol. Moreover, acoustic cavitation and
nyl tetrazolium bromide (MTT) assay and the cell via-
a mechanical effect, produced by ultrasound waves will
bilities in present study are all >90%.
also accelerate solvent penetration into plant tissue,
thus increasing the contact surface area between sam-
Contribution of PM to the antioxidant activities of coffee
ple and solvent and therefore enhancing extraction effi-
leaf extracts
ciency (Ghafoor et al., 2009). The higher temperature
The antioxidant activities of individual phytochemicals
used with boiling water extraction might also result in
including, 3-CQA, 4-CQA, 5-CQA, 3,4-diCQA, 3,5-
degradation of phenolic compounds. Similar to the
diCQA, 4,5-diCQA, caffeine, mangiferin and rutin were
results that observed with hot water extract, Japanese-
investigated. In addition, the antioxidant activities of
style-green-tea-process-young (JGTP-Y) and black-tea-
phytochemical mixtures that have equivalent content of
process-mature (BTP-M) coffee leaves contained the
3-CQA, 5-CQA, 3,4-diCQA, 3,5-diCQA, mangiferin
greatest and lowest measured phenolic compounds
and rutin as coffee leaf extracts were investigated and
among all ten types of coffee leaves, respectively.
percentage contribution of PM to the antioxidant

© 2019 Institute of Food Science and Technology International Journal of Food Science and Technology 2019
4 Antioxidant activities of coffee leaves X. Chen et al.

Table 1 Phytochemical content (mg g1 leaf) of coffee leaves extracted with methanol-water (80/20, v/v)

Sample Yield (%) Caffeine 3-CQA 5-CQA Mangiferin Isomangiferin Rutin 3,4-diCQA 3,5-diCQA

WTP-Y 26.9  0.8d

8.64  0.67b
ND 2.39  0.09h
2.88  0.24ef
0.44  0.03ef
2.64  0.12bc
0.12  0.01c
0.53  0.01f
WTP-M 34.7  0.8a 7.82  0.28bc 0.21  0.01b 11.18  0.65c 4.33  0.19c 0.58  0.02c 2.85  0.04b 0.10  0.01cd 0.71  0.03de
JGTP-Y 33.2  0.7ab 8.00  0.56bc 0.22  0.02b 21.15  0.08a 8.43  0.09a 1.47  0.02a 3.46  0.02a 0.28  0.02a 2.85  0.07a
JGTP-M 32.3  0.2b 8.11  0.33bc 0.30  0.02a 18.79  0.33b 6.31  0.20b 0.88  0.03b 2.49  0.03cd 0.09  0.01cd 1.75  0.03b
CGTP-Y 29.2  1.4c 10.18  0.67a 0.16  0.04c 6.25  0.56e 3.11  0.37de 0.48  0.03de 1.72  0.10ef 0.18  0.02b 1.64  0.13b
CGTP-M 34.0  1.2ab 7.67  0.34bc 0.18  0.00bc 9.87  0.43d 3.20  0.13de 0.46  0.02e 2.38  0.13d 0.10  0.00cd 1.13  0.06c
OTP-Y 26.7  0.1d 7.51  0.71bc ND 1.16  0.04i 0.64  0.03g 0.08  0.00g 0.73  0.08g 0.07  0.01de 0.13  0.01g
OTP-M 24.4  0.1e 10.92  0.48a 0.12  0.01d 3.44  0.17g 2.49  0.11f 0.37  0.01f 1.90  0.07e 0.10  0.00cd 0.63  0.01ef
BTP-Y 30.0  0.6c 11.33  0.35a ND 4.79  0.15f 3.43  0.10d 0.54  0.05cd 1.63  0.03f 0.11  0.00c 0.80  0.03d
BTP-M 24.3  0.7e 7.18  0.33c ND 0.91  0.03i 0.53  0.01g 0.06  0.00g 0.51  0.01h 0.05  0.00e 0.09  0.01g

BTP-M, black-tea-process mature coffee leaves; BTP-Y, black-tea-process young coffee leaves; CGTP-M, Chinese-style-green-tea-process mature cof-
fee leaves; CGTP-Y, Chinese-style-green-tea-process young coffee leaves; JGTP-M, Japanese-style-green-tea-process mature coffee leaves; JGTP-Y,
Japanese-style-green-tea-process young coffee leaves; ND, not detectable; OTP-M, oolong-tea-process mature coffee leaves; OTP-Y, oolong-tea-pro-
cess young coffee leaves; WTP-M, white-tea-process mature coffee leaves; WTP-Y, white-tea-process young coffee leaves.
Data are shown as means  SD. Data in same column not sharing same letters are significantly different at P < 0.05.

compounds that have less active groups (Do et al.,

Antioxidant activities of coffee leaf extracts and
corresponding phytochemical mixtures
The chemical-based antioxidant assays are generally
based on hydrogen atom transfer (HAT) reaction (e.g.
ORAC assay) and electron transfer (ET) reaction (e.g.
Folin-Ciocalteu based TPC assay, DPPH and ABTS
assays). HAT reaction is used to quantitate the free
radical chain breaking reaction, whereas, ET reaction
measures the reducing capacity of antioxidants (Huang
et al., 2005). Except for the analysis methods, antioxi-
dant activity of plant extracts is also influenced by
physiochemical properties that include, solubility,
polarity and stability of potential bioactives in the
plant matrix (Nikousaleh & Prakash, 2016). In the pre-
sent study, the methanol extracts from JGTP-Y coffee
leaves possessed the greatest ABTS and DPPH free
radical scavenging activity and TPC and ORAC value
(P < 0.05), whereas, BTP-M methanol extract had the
lowest antioxidant activities (Table 2). Methanol
extracts of JGTP coffee leaves possessed greater ABTS
free radical scavenging activity than that obtained
from the hot water extract; however, in the case of
oxidised coffee leaves, including OTP and BTP leaves,
the findings were different. We conclude that the oxi-
dation of phytochemicals will influence the polarity of
products, thus affecting recovery in different solvents,
which in turn reflects the extent of bioactivity obtained
from the extract. The hydrogen bonds formed between
hydroxyl groups of phenolic compounds and organic
alcohol explain the greater TPC measured in the aque-
ous methanol JGTP-Y extracted samples, compared to
that of hot water extracts (Queimada et al., 2009). In
addition, increasing the water content in organic sol-
vent mixtures resulted in decreased TPC and antioxi-
dant activity due to the presence of phenolic
2014).
The use of methanol to extract OTP-M coffee leaves
produced lower NO inhibitory capacity compared to a
similar extraction using young leaves; the opposite was
observed for BTP leaves. This finding contrasted the
antioxidant activity results for these respective solvent
extracts and pointed to the fact that phytochemicals
measured in these extracts may not possess the same
degree of anti-inflammatory activity. Moreover, our
previous study also showed that NO inhibitory effects
of hot water gave different results compared to when
extracted with methanol; showing that OTP-Y and
BTP-M had greater IC50s compared with their mature
or young counterparts, respectively. Interestingly, hot
water BTP-M coffee leaf extracts were unique in affin-
ity to induce basal NO production in Raw 264.7 cells
(Chen et al., 2018), whereas, the aqueous methanol
extract showed no activity to induce NO. Further frac-
tionation of JGTP-Y and BTP-M hot water extracts,
produced significant NO induction activity (Chen
et al., 2019), thereby indicting that recovery of highly
hydrophilic compounds was responsible for this activ-
ity observed from boil water infusion.
Our previous study also found that the PM, includ-
ing 3-CQA, 5-CQA, 3,4-diCQA, 3,5-diCQA, mangi-
ferin, rutin and caffeine in coffee leaves were not the
major contributors to the NO inhibitory capacity
(Chen et al., 2019). In order to predict which phyto-
chemicals contribute to the antioxidant activity of cof-
fee leaf extracts, we measured the antioxidant activities
of individual phytochemicals (Table 3). Caffeine did
not show capacities to scavenge free radicals (data not
shown). In general, diCQAs (3,4-diCQA, 3,5-diCQA
and 4,5-diCQA) had greater antioxidant activities
compared with mono-CQAs (3-CQA, 4-CQA and 5-

International Journal of Food Science and Technology 2019 © 2019 Institute of Food Science and Technology
 Antioxidant activities of coffee leaves X. Chen et al. 5

Table 2 Total phenolic content and free radical scavenging capacities of coffee leaf extracted with methanol-water (80/20, v/v)

TPC (mg gallic ABTS (lmol DPPH (lmol ORAC (lmol Trolox
Sample acid per g leaf) Trolox per g leaf) Trolox per g leaf) per g leaf) NO (IC50) mg mL1

WTP-Y 40.1  0.5d 225  7d 382  28e 707  52d 1.34  0.10b
WTP-M 58.1  2.9c 361  21b 799  29ab 943  41bc 0.91  0.12c
JGTP-Y 75.4  0.9a 409  12a 820  27a 1115  65a 0.72  0.09c
JGTP-M 67.5  2.3b 367  9b 735  19b 978  24b 0.86  0.03c
CGTP-Y 45.1  2.9d 250  7d 604  4c 692  54d 0.82  0.08c
CGTP-M 56.7  3.1c 310  9c 654  12c 848  35c 0.83  0.03c
OTP-Y 15.1  0.6f 80  2f 193  8f 288  15f 0.88  0.10c
OTP-M 32.1  0.8e 132  1e 421  34de 420  19e 1.78  0.22a
BTP-Y 27.9  0.5e 149  9e 478  38d 460  4e 1.77  0.16a
BTP-M 12.4  1.2f 67  4f 181  19f 236  9f 1.46  0.11ab

BTP-M, black-tea-process mature coffee leaves; BTP-Y, black-tea-process young coffee leaves; CGTP-M, Chinese-style-green-tea-process mature cof-
fee leaves; CGTP-Y, Chinese-style-green-tea-process young coffee leaves; JGTP-M, Japanese-style-green-tea-process mature coffee leaves; JGTP-Y,
Japanese-style-green-tea-process young coffee leaves; OTP-M, oolong-tea-process mature coffee leaves; OTP-Y, oolong-tea-process young coffee
leaves; WTP-M, white-tea-process mature coffee leaves; WTP-Y, white-tea-process young coffee leaves.
Data are shown as means  SD. Data in same column not sharing same letters are significantly different at P < 0.05.

Table 3 Antioxidant activities of individual phytochemical standards†

DPPH (lmol ORAC (lmol

TPC (mg gallic acid g1) ABTS (lmol Trolox g1) Trolox g1) Trolox g1)

3-CQA 445  29c 2825  71bc 1943  131de 11 004  409bc

4-CQA 448  32c 2762  154bc 2130  153cd 10 714  478bc
5-CQA 454  33c 2692  103c 1960  155cd 9286  308c
3,4-diCQA 513  13bc 3087  85b 2501  173ab 15 597  1275a
3,5-diCQA 558  1ab 3203  213b 2698  140a 16 703  543a
4,5-diCQA 594  31a 3845  145a 2605  109ab 16 912  781a
Mangiferin 499  16bc 3942  318a 2562  59ab 12 032  651b
Rutin 450  17c 1891  213d 2309  27bc 11 201  579bc

†
Caffeine did not show detectable antioxidant activities.
Data in same column not sharing same letters are significantly different at P < 0.05.

CQA) and mono-CQA isomers had similar antioxidant
activities, indicating that the position of esterification
did not affect the antioxidant activities of mono-CQA.
These results are similar to Xu et al. (2012). The order
of the free radical scavenging capacity of mangiferin
and rutin varied depending on the antioxidant assay
methods used. For example, mangiferin exhibited the
greatest ABTS radical scavenging capacity, whereas,
when analysed by the ORAC assay, it displayed equiv-
alent antioxidant activity compared with mono-CQAs
and rutin. Rutin had similar TPC and ORAC value
compared with mono-CQA, however, ABTS scaveng-
ing capacity was much lower compared to other phy-
tochemicals. Although, each molecule of mangiferin
and rutin both has four aromatic hydroxyl groups, the
orientation of hydroxyl groups may produce different
antioxidant activity. Mangiferin is a xanthone and the
catechol moiety of mangiferin enables it chelate iron
and showing free radical scavenging capacity (Khare
& Shanker, 2016). Rutin is a flavonoid that contains a
30 ,40 -ortho-dihydroxy (catechol) group in the B ring
and 2,3-conjugation double bond, which contributes to
its antioxidant activity (Seyoum et al., 2006).
The antioxidant activities and the percentage contri-
bution of PM to the antioxidant activities of coffee
leaf extracts were present in Table 4. The total amount
of 3-CQA, 5-CQA, 3,4-diCQA, 3,5-diCQA, mangiferin
and rutin only accounted for a range of 0.2–3.8% of
the weight of coffee leaves. The contribution of PM to
the antioxidant activities measured by TPC, ABTS,
DPPH and ORAC assays was 3.5–18.5%, 17.6–31.6%,
7.9–13.6% and 8.5–29.7%, respectively. This result
indicates that these PM are not major contributors to
the antioxidant activities observed herein. We therefore
suggested that other factors including phytochemicals
that were not measured in the extracts in present study
involved in the observed antioxidant activities. On the
other hand, the unit antioxidant activity of PM was
relatively high, considering the low concentration of
PM in the coffee leaf extract.

© 2019 Institute of Food Science and Technology International Journal of Food Science and Technology 2019
6 Antioxidant activities of coffee leaves X. Chen et al.

Table 4 Antioxidant activities of phytochemical mixtures and the contribution (%) to antioxidant activities of coffee leaf extract

TPC ABTS DPPH ORAC

PMC‡
† 1 § 1 1
PM (lmol Trolox g ) % (lmol Trolox g ) % (lmol Trolox g ) % (lmol Trolox g1) % g per 100 g

WTP-Y 3.0  0.6f 7.5 45.4  4.8c 20.2 45.1  2.1e 11.8 112.1  8.3ef 15.9 0.9
WTP-M 7.6  0.1b 13.0 63.4  7.9b 17.6 73.8  5.6c 9.2 213.1  17.6c 22.6 2.0
JGTP-Y 14.0  1.2a 18.5 107.6  7.2a 26.3 111.6  4.7a 13.6 330.8  20.9a 29.7 3.8
JGTP-M 11.5  0.6b 17.0 106.3  12.5a 29.0 89.4  1.4b 12.2 278.0  23.8b 28.4 3.1
CGTP-Y 5.8  0.4de 12.8 51.8  2.2bc 20.7 51.5  2.3d 8.5 120.9  4.3e 17.5 1.4
CGTP-M 6.2  0.6cd 10.9 57.1  2.6bc 18.4 55.4  3.0d 8.5 157.5  8.5d 18.6 1.7
OTP-Y 0.5  0.4g 3.5 16.6  4.7d 20.8 15.3  5.9f 7.9 24.4  0.3g 8.5 0.3
OTP-M 3.6  0.1f 11.1 41.7  2.5c 31.6 46.3  3.3de 11.0 81.2  2.4f 19.3 0.9
BTP-Y 4.5  0.1ef 16.1 43.0  2.0c 28.9 48.5  0.4de 10.1 92.3  65.7ef 20.1 1.1
BTP-M 0.5  0.2g 3.9 13.9  3.6d 20.7 16.6  2.6f 9.2 23.1  1.0g 9.8 0.2

†
PM: phytochemical mixtures that have equivalent amount of 3-CQA, 5-CQA, 3,4-diCQA, 3,5-diCQA, mangiferin and rutin as corresponding coffee leaves.
‡
PMC: phytochemical mixture content (g per 100 g) in different coffee leaves. The content equals to the sum of 3-CQA, 5-CQA, 3,4-diCQA, 3,5-
diCQA, mangiferin, isomangiferin and rutin from Table 1.
§
%: Percentage contribution of phytochemical mixtures to the antioxidant activities of coffee leaves.
Data in same column not sharing same letters are significantly different at P < 0.05.

Figure 1 Principal component analysis. (a)

Score plot of coffee leaves according to pro-
cessing method and age of leaves; (b) Load-
ing plot of variables (caffeine, 3-CQA, 5-
CQA, 3,4-di-CQA, 3,5-di-CQA, mangiferin,
isomangiferin, rutin, sum [total phytochemi-
cals detected by HPLC]), TPC, ABTS and
DPPH radical scavenging activity, ORAC,
IC50 of NO scavenging activities.

International Journal of Food Science and Technology 2019 © 2019 Institute of Food Science and Technology

Correlation between phytochemicals and free radical scav-
enging activities
PCA analysis and correlation coefficients were per-
formed to further interpret the complexity of results
presented in Fig. 1 and Table S1, respectively.
Phenolic compounds except for 3,4-diCQA were cor-
related with TPC, ABTS and DPPH scavenging
activity and ORAC value, however, only TPC and
ORAC values were correlated with the NO inhibi-
tory activity (Table S1). This result indicated that
phenolic compounds other than what were measured
herein contribute to the NO scavenging patterns
derived from these coffee leaf extracts. Our finding
that the phytochemical mixtures which contained
equivalent amount of measured phytochemicals also
did not inhibit NO production strongly points to
other phytochemicals, not yet identified in coffee
leaves that contribute to NO scavenging capacity
(Chen et al., 2019).
Principle component analysis also showed clearly
that two components contributed to 75.8% and
11.6% of the total variance, respectively. Score plot
graph (Fig. 1a) showed five separate groups. Except
for caffeine and 3-CQA, the methanol extract derived
from JGTP-Y coffee leaf had the highest other mea-
sured phytochemicals, and showed high antioxidant
activities. In contrast, the BTP-M and OTP-Y coffee
leaves had the lowest phenolic compound content
and this corresponded to low antioxidant activities.
BTP-Y and OTP-M possessed the lowest NO inhibi-
tory activities. Mature (JGTP-M, CGTP-M and
WTP-M) and young (WTP-Y and CGTP-Y) leaves
that were processed by green or white tea processing
methods, had similar characteristics, respectively. Our
finding indicates that under non-oxidation conditions,
the leaf age influences the properties of coffee extract
more dramatically than the processing method used
to generate the coffee leaves for beverage. However,
when coffee leaves were treated with an oxidation
process, both age of leaf and processing method vari-
ables produced a significant interaction on antioxi-
dant activities. This finding was different from the
results obtained using the hot water extract. Observ-
ing the loading plot (Fig. 1b) and the correlation of
coefficient (Table S1) also confirmed that the caffeine
content was not correlated with the other bioactive
variables, but the presence of phenolic compounds
was correlated with antioxidant activity.
Conclusions
Methanol extracts of JGTP-Y coffee leaves possessed
the highest recovery of phenolic compounds, which in
turn corresponded to higher antioxidant activities. The
extraction solvent selected to recover the coffee leaf
extracts influenced NO scavenging, or induction
© 2019 Institute of Food Science and Technology
Antioxidant activities of coffee leaves X. Chen et al. 7
capacity dramatically. Aqueous methanol solvent
extraction had no effect on basal NO production, but
did show activity to inhibit NO activity. The impact of
the solvent on recovery yield and antioxidant activities
of coffee leaf extracts was also affected by the oxida-
tion process. We recommend that different solvents
should be used to extract coffee leaves when assessing
specific bioactivity; more so, depending on how the
leaves were initially processed and the age of the leaf.
Similar to the impact of phytochemical mixtures on
NO inhibitory, we also found that the phytochemical
mixtures prepared to define coffee leaves were not a
major contributor to the antioxidant activities reported
herein. Further research is needed to elucidate what
specific phytocompounds, or phytochemical mixtures
from coffee leaves, contribute to the antioxidant activi-
ties reported in this study.
Acknowledgments
This study was supported by Jiangsu University senior
talent start up fund (No. 4111360002) to XC and
NSERC-Engage (499039) and MITACS accelerate
(IT08181) grants to DDK. We thank Arnaud Petitval-
let and Max Rivest from Wize Monkey Inc. (Vancou-
ver, BC, Canada) for the kind donation of coffee
leaves and a contribution to the MITACS accelerate
grant, as well as the Finca La Aurora coffee farm in
Nicaragua for providing samples of coffee leaves.
Conflict of interest
The authors have no conflicts of interest to declare.
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Supporting Information
Additional Supporting Information may be found in
the online version of this article:
Table S1. Correlation coefficient (r) of different
variables.
Figure S1. HPLC profile of coffee leaf extract.
Figure S2. Standard curves and regression equations
of different phytochemicals.
© 2019 Institute of Food Science and Technology