Phytosterol, Lipid and Phenolic Composition, and Biological Activities of Guava Seed Oil

Abstract

Plant seeds have been found to contain bioactive compounds that have potential nutraceutical benefits. Guava seeds (Psidium guajava) are by-products in the beverage and juice industry; however, they can be utilized for a variety of commercial purposes. This study was designed to analyze the phytochemicals of the n-hexane extract of guava seed oil (GSO), to study its free-radical scavenging activity, and to monitor the changes in serum lipids and fatty acid profiles in rats that were fed GSO. The GSO was analyzed for phytochemicals using chromatographic methods. It was also tested for free-radical scavenging activity in hepatoma and neuroblastoma cells, and analyzed in terms of serum lipids and fatty acids. GSO was found to contain phenolic compounds (e.g., chlorogenic acid and its derivatives) and phytosterols (e.g., stimasterol, β-sitosterol and campesterol), and exerted radical-scavenging activity in cell cultures in a concentration-dependent manner. Long-term consumption of GSO did not increase cholesterol and triglyceride levels in rat serum, but it tended to decrease serum fatty acid levels in a concentration-dependent manner. This is the first study to report on the lipid, phytosterol and phenolic compositions, antioxidant activity, and the hepato- and neuro-protection of hydrogen peroxide-induced oxidative stress levels in the GSO extract.

Full text

molecules
Article
Phytosterol, Lipid and Phenolic Composition, and
Biological Activities of Guava Seed Oil
Adchara Prommaban 1, Niramon Utama-ang 2, Anan Chaikitwattana 3, Chairat Uthaipibull 4,
John B. Porter 5and Somdet Srichairatanakool 1, *
1Department of Biochemistry, Faculty of Medicine, Chiang Mai University, Chiang Mai 50200, Thailand;
waewaa@gmail.com
2Department of Product Development Technology, Faculty of Agro-Industry, Chiang Mai University,
Chiang Mai 50200, Thailand; niramon.u@cmu.ac.th
3
Department of Herb and Extract Business Development, Tipco Biotech Company, Prachuapkhirikhan 77000,
Thailand; anan@tipco.net
4
Protein-Ligand Engineering and Molecular Biology Laboratory, National Center for Genetic Engineering and
Biotechnology (BIOTEC), National Science and Technology Development Agency, Thailand Science Park,
Pathum Thani 12120, Thailand; chairat@biotec.or.th
5Department of Haematology, O’Gormond Building, Institute of Cancer Research, Huntley Street,
University College London, London WC1E 6BT, UK; j.porter@ucl.ac.uk
*Correspondence: somdet.s@cmu.ac.th; Tel.: +66-5393-5322; Fax: +66-5389-4031
Academic Editor: Panagiotis Zoumpoulakis
Received: 30 April 2020; Accepted: 20 May 2020; Published: 27 May 2020


Abstract:
Plant seeds have been found to contain bioactive compounds that have potential
nutraceutical benefits. Guava seeds (Psidium guajava) are by-products in the beverage and juice
industry; however, they can be utilized for a variety of commercial purposes. This study was
designed to analyze the phytochemicals of the n-hexane extract of guava seed oil (GSO), to study its
free-radical scavenging activity, and to monitor the changes in serum lipids and fatty acid profiles
in rats that were fed GSO. The GSO was analyzed for phytochemicals using chromatographic
methods. It was also tested for free-radical scavenging activity in hepatoma and neuroblastoma
cells, and analyzed in terms of serum lipids and fatty acids. GSO was found to contain phenolic
compounds (e.g., chlorogenic acid and its derivatives) and phytosterols (e.g., stimasterol,
β
-sitosterol
and campesterol), and exerted radical-scavenging activity in cell cultures in a concentration-dependent
manner. Long-term consumption of GSO did not increase cholesterol and triglyceride levels in rat
serum, but it tended to decrease serum fatty acid levels in a concentration-dependent manner. This is
the first study to report on the lipid, phytosterol and phenolic compositions, antioxidant activity,
and the hepato- and neuro-protection of hydrogen peroxide-induced oxidative stress levels in the
GSO extract.
Keywords:
Psidium guajava; seed; hexane extract; lipid; phenolic compounds;
phytosterols; antioxidant
1. Introduction
Guava (Psidium guajava L. Family Myrtaceae) is an important edible tropical fruit and a well-known
herbal plant that has been widely applied in folk and traditional medicine [
1
,
2
]. The leaves are known
to exhibit free-radical scavenging, inotropic, anti-glycemic, anti-hyperlipidemic, anti-hypertensive,
and anti-diarrheal activities [
3
–
8
]. The pulp and peel have been known to exert anti-neoplastic effects
on the induction of apoptosis and cell differentiation [
9
]. Guava seeds, a by-product of the beverage and
juice processing industry, are abundant in dietary fiber, proteins, fats, phenolics, flavonol glycosides,
Molecules 2020,25, 2474; doi:10.3390/molecules25112474 www.mdpi.com/journal/molecules

Molecules 2020,25, 2474 2 of 23
glutelins, tannins, saponin and amino acids [
10
–
14
]. In addition, guava seed oil (GSO) obtained from
red (P. cattleianum Sabin) and yellow (P. cattleianum var. lucidum Hort.) strawberry guava plants was
found to contain high amounts of fatty acids, of which linoleic acid (LA) was the most abundant [
15
].
Different methods/agents, involving heat, boiling, roasting, detergents and organic solvents,
have been used to obtain functional guava seed extracts. Additionally, GSO can be extracted by
using organic solvents such as acetone, petroleum ether, ethyl acetate and n-hexane. For example,
the sodium dodecyl sulfate extract of the seeds was shown to produce high yields of proteins that
were mostly glutelins [12]. Acetone extracts of the seeds were found to contain flavonoids, phenolics
and phenylethanoid glycosides [
16
]. The petroleum ether extract of GSO contained high amounts
of linoleic acid, while the n-hexane extract of GSO predominantly contained linoleic acid [
17
,
18
].
Likewise,
α
-tocopherol and
δ
-tocopherol were found to be present in GSO. Notably, the quantity and
nature of the tocopherols is of crucial importance regarding their oxidative stability [
17
]. Among
organic solvents, n-hexane was found to be the most efficient in fractionating a wide range of lipophilic
bioactive phytochemicals [19–21].
GSO possessed strong anti-oxidation and inhibitory activities against low-density lipoprotein
peroxidation and Gram-negative bacteria [
22
,
23
]. Recently, we have revealed that the edible hexane
extract of GSO is rich in linoleic acid, and contains some amounts of tocopherols, tocotrienols and
phenolic compounds [
24
]. It is highly likely that phytochemicals in GSO present nutraceutical effects
and benefits for health in humans. The aims of this study were to identify phenolic compounds,
phytosterols and lipids in the GSO using very sensitive chromatographic/mass spectrometric methods,
investigate the free-radical scavenging activity in hepatocytes and neuroblastoma cells, and evaluate
the serum lipid levels in rats that had been fed GSO.
2. Results
2.1. Identification of Lipids
Here, we present the high-performance liquid chromatography-electrospray
ionization-quadrupole-time of flight/mass spectrometry (HPLC-ESI-Q-TOF/MS) techniques,
that possess high efficiency, specificity and sensitivity for the detection and characterization of tentative
lipids in GSO. Though 13 peaks were resolved by chromatographic separation, only 6 peaks (No. 1–6)
were identified using the Analysis Software, and these represented the tentative compounds identified
as 4-hexyl-decanoic acid, sphinganine, 5S-hydroxyeicosatetraenoyl di-endoperoxide, didrovaltratum,
sphingofungin B, 13,14-dihydro-19(R)-hydroxyprostaglandin E1, eschscholtzxanthin, tetradecan-3-one
and xestoaminol C (Figure 1and Figure S1, Table 1).

Molecules 2020,25, 2474 3 of 23
Table 1. Qualitative analysis for lipids in guava seed oil using HPLC-ESI-QTOF/MS.
Peak TRTarget Mass Error Molecular
Formula
Exact Mass Observed Mass (m/z)Identification
No. (min) Score (ppm) (g/mol) [M +H]+[M +NH4]+[M +Na]+
10.576 96.14 3.86 C16H32 O2256.24 257.24 274.27 279.22 4-Hexyl-decanoic acid
96.14 3.54 C16H35 NO2273.27 274.27 291.30 296.25 Sphinganine
21.051 82.26 1.82 C20H34 O8402.23 403.23 420.26 425.21
5S-Hydroxyeicosatetraenoyl di-endoperoxide
89.23 −3.36 C22H32 O8424.21 425.21 442.24 447.20 Didrovaltratum
3 1.066 95.18 1.45 C20 H39NO6389.28 390.28 407.31 412.27 Sphingofungin B
4 1.094 96.85 2.19 C20 H36O6372.25 373.25 390.28 399.25 13,14-Dihydro-19(R)-hydroxyprostaglandin
E1
5 1.177 52.09 −1.18 C40 H54O2566.41 567.41 585.44 591.42 Eschscholtzxanthin
621.93 98.82 2.03 C14H28O 212.21 213.22 230.24 235.20 Tetradecan-3-one
98.82 1.88 C14H31 NO 229.24 230.24 247.27 252.22 Xestoaminol C
Abbreviations: m/z=mass to charge ratio, ppm =part per million, TR=retention time.

Molecules 2020,25, 2474 4 of 23
Molecules 2020, 25, x 4 of 24
Molecules 2020, 25, x; doi: www.mdpi.com/journal/molecules
Figure 1. HPLC-ESI-QTOF/MS profile of lipids in guava seed oil. Inserted figure shows a
magnification of area between 2 and 30 min.
2.2. Identification of Phytosterols
For the detection of polar constituents, trimethysilyl (TMS) obtained from
N-methyl-N-trimethylsilyltrifluoroacetamide (MSTFA) was able to derivatize certain
multiple-functional groups, including hydroxyl, amine, sulfate, and carboxyl groups. Hence, the
TMS derivatization of phytosterols and free fatty acids present in hexane extracts of GSO will
enhance the performance of gas chromatographic/mass spectrometric (GC/MS) analysis, and
provide characteristic ions in their electrospray ionization (ESI)-mass spectra. For the identification
of targeted compounds, the ESI-mass fragmentations of several types of analytes were preferentially
studied based on the mass spectra of the authentic standards (such as stimasterol, β-sitosterol,
sitostanal and campesterol) and the internal standard cholestane. Most of the TMS derivatives and
fatty acyl esters produced weak or intense molecular ions, abundant [M − 73]+ ions, and certain
characteristic ions in their ESI-mass spectra, thereby providing easy identification. The mass
fragmentation pathways of TMS-derivatized phytosterols and fatty acids were suggested and could
be tentatively identified. Total ion chromatograms (TIC), and the corresponding ESI-mass spectra of
typical TMS-derivatized fatty acids and phytosterols, for GSO are shown in Figures 2 and S2.
Figure 1.
HPLC-ESI-QTOF/MS profile of lipids in guava seed oil. Inserted figure shows a magnification
of area between 2 and 30 min.
2.2. Identification of Phytosterols
For the detection of polar constituents, trimethysilyl (TMS) obtained from
N-methyl-N-trimethylsilyltrifluoroacetamide (MSTFA) was able to derivatize certain
multiple-functional groups, including hydroxyl, amine, sulfate, and carboxyl groups. Hence,
the TMS derivatization of phytosterols and free fatty acids present in hexane extracts of GSO will
enhance the performance of gas chromatographic/mass spectrometric (GC/MS) analysis, and provide
characteristic ions in their electrospray ionization (ESI)-mass spectra. For the identification of targeted
compounds, the ESI-mass fragmentations of several types of analytes were preferentially studied
based on the mass spectra of the authentic standards (such as stimasterol,
β
-sitosterol, sitostanal and
campesterol) and the internal standard cholestane. Most of the TMS derivatives and fatty acyl esters
produced weak or intense molecular ions, abundant [M
−
73]
+
ions, and certain characteristic ions
in their ESI-mass spectra, thereby providing easy identification. The mass fragmentation pathways
of TMS-derivatized phytosterols and fatty acids were suggested and could be tentatively identified.
Total ion chromatograms (TIC), and the corresponding ESI-mass spectra of typical TMS-derivatized
fatty acids and phytosterols, for GSO are shown in Figure 2and Figure S2.

Molecules 2020,25, 2474 5 of 23
Molecules 2020, 25, x 5 of 24
Figure 2. Total ion counts of phytosterols and lipids in guava seed oil analyzed using trimethylsillyl
(TMS) derivatization-gas chromatography/mass spectrometry (GC/MS).
Using the gas chromatography/mass spectrometry (GC/MS) scan mode, peak numbers 1–10
were identified by comparing the retention times (TR) of 13.25, 14.83, 14.89, 15.12, 17.70, 18.10, 20.41,
23.44, 23.70 and 24.37 min, respectively, and by comparing ESI-mass spectra against those of their
authentic standards. Minor components with less than 0.1% relative abundance in the TIC were not
considered of interest. As a result, they were identified as ethyl palmitate, ethyll linolenate, ethyl
linoleate, ethyl stearate, linoleic acid, linolenic acid, cholestane, β-sitosterol, stigmasterol and
campesterol, while sitostanal was not detected (Table 2). In stoichiometry, the amounts of
β-stimasterol, β-sitosterol and campesterol were found to be 297.61, 0.22 and 11.04 mg/100 g GSO,
respectively. This GC/MS method, combined with TMS derivatization, is a comprehensive chemical
method for the profiling analysis and quantitation of phytosterols in GSO. Taken together, all the
chromatographic analyses can provide relevant information on GSO by way of a direct comparison
of its chemical composition with the biological activities in the present study. Furthermore, such
information will be useful for predicting the biological and pharmacologic effects of GSO in
subsequent studies.
Table 2. Identification of phytosterols and lipids in guava seed oil using TMS derivatization-GC/MS.
Peak
No.
TR
(min) TIC
Exact
Mass
(g/mol)
Molecular
Formula
Observed Mass
(m/z)
Error
(%) Identification
1 13.25 2277755 284.5 C18H36O2 284 −0.18 Ethyl palmitate
2 14.83 2423828 308.5 C20H36O2 308 −0.16 Ethyll linolenate
3 14.89 5411461 310.5 C20H38O2 310 −0.16 Ethyl linoleate
4 15.12 359612 312.5 C20H40O2 312 −0.16 Ethyl stearate
5 17.77 3836542 352.6 C18H32O2 353 0.11 Linoleic acid TMS
6 18.10 17168 350.6 C18H30O2 352 0.4 Linolenic acid TMS
7 20.41 18788155 372.7 C27H48 372 −0.19 Cholestane
8 23.44 452445 486.9 C29H50O 484 −0.6 β-Sitosterol TMS
9 23.70 770310 485.8 C29H48O 485 −0.16 Stigmasterol TMS
10 24.37 12299199 472.9 C28H48O 472 −0.19 Campesterol TMS
11 25.99 324939 - - - - Unknown
Abbreviations: m/z = mass to charge ration, TIC = total ion count, TMS = trimethylsilyl, TR = retention time.
2.3. Liquid Chromatographic Analysis of Phenolic Compounds
In the chromatographic profile (Figure 3), 16 small peaks were detected in the range from 100 to
700 m/z. These peaks indicated that there are at least 16 possible phenolic compounds existing in the
GSO. Additional data on the high-performance liquid chromatography-single quadrupole
electrospray ionization/mass spectrometry (HPLC-ESI/MS) analysis, including retention times,
molecular ions and important fragment ions for tentative compounds, are presented in Figure S3a.
Figure 2.
Total ion counts of phytosterols and lipids in guava seed oil analyzed using trimethylsillyl
(TMS) derivatization-gas chromatography/mass spectrometry (GC/MS).
Using the gas chromatography/mass spectrometry (GC/MS) scan mode, peak numbers 1–10 were
identified by comparing the retention times (T
R
) of 13.25, 14.83, 14.89, 15.12, 17.70, 18.10, 20.41, 23.44,
23.70 and 24.37 min, respectively, and by comparing ESI-mass spectra against those of their authentic
standards. Minor components with less than 0.1% relative abundance in the TIC were not considered
of interest. As a result, they were identified as ethyl palmitate, ethyll linolenate, ethyl linoleate, ethyl
stearate, linoleic acid, linolenic acid, cholestane,
β
-sitosterol, stigmasterol and campesterol, while
sitostanal was not detected (Table 2). In stoichiometry, the amounts of β-stimasterol, β-sitosterol and
campesterol were found to be 297.61, 0.22 and 11.04 mg/100 g GSO, respectively. This GC/MS method,
combined with TMS derivatization, is a comprehensive chemical method for the profiling analysis and
quantitation of phytosterols in GSO. Taken together, all the chromatographic analyses can provide
relevant information on GSO by way of a direct comparison of its chemical composition with the
biological activities in the present study. Furthermore, such information will be useful for predicting
the biological and pharmacologic effects of GSO in subsequent studies.
Table 2. Identification of phytosterols and lipids in guava seed oil using TMS derivatization-GC/MS.
Peak
No.
TR
(min) TIC
Exact
Mass
(g/mol)
Molecular
Formula
Observed
Mass
(m/z)
Error
(%) Identification
1 13.25 2277755 284.5 C18H36O2284 −0.18 Ethyl palmitate
2 14.83 2423828 308.5 C20H36O2308 −0.16 Ethyll linolenate
3 14.89 5411461 310.5 C20H38O2310 −0.16 Ethyl linoleate
4 15.12 359612 312.5 C20H40 O2312 −0.16 Ethyl stearate
5 17.77 3836542 352.6 C18H32O2353 0.11 Linoleic acid TMS
6 18.10 17168 350.6 C18H30O2352 0.4 Linolenic acid TMS
7 20.41 18788155 372.7 C27H48 372 −0.19 Cholestane
8 23.44 452445 486.9 C29H50O 484 −0.6 β-Sitosterol TMS
9 23.70 770310 485.8 C29H48O 485 −0.16 Stigmasterol TMS
10 24.37 12299199 472.9 C28H48O 472 −0.19 Campesterol TMS
11 25.99 324939 - - - - Unknown
Abbreviations: m/z=mass to charge ration, TIC =total ion count, TMS =trimethylsilyl, TR=retention time.
2.3. Liquid Chromatographic Analysis of Phenolic Compounds
In the chromatographic profile (Figure 3), 16 small peaks were detected in the range from 100 to
700 m/z. These peaks indicated that there are at least 16 possible phenolic compounds existing in the
GSO. Additional data on the high-performance liquid chromatography-single quadrupole electrospray
ionization/mass spectrometry (HPLC-ESI/MS) analysis, including retention times, molecular ions and
important fragment ions for tentative compounds, are presented in Figure S3a. Authentic standards,
including gallic acid, catechin, tannic acid, rutin, isoquercetin, hydroquinine, eriodictyol and quercetin,
were analyzed and used as database, and are presented in Figure S3b. In comparison with the standards,

Molecules 2020,25, 2474 6 of 23
catechin, isoquercetin, eriodictyol and quercetin were detected in the GSO, while gallic acid, tannic
acid, rutin and hydroquinine would not be found.
In illustration, the fragmentations of phenolic compounds in the positive ion mode, eluted at
retention times of 9.47, 10.11, 12.64, 13.48, 14.02, 14.69, 16.11, 21.04, 29.53, 31.15, 32.32, 32.99, 34.47
and 35.09 min, were characterized as quinic acid, O-caffeoylquinic acid or chlorgenic acid, catechin,
apigenin-4-O-glycoside, ellagic acid-O-methoxyglucoside, dicaffeic acid, isoquercetin, O-caffeoylquinic
acid derivative, ellagic acid, eriodictyol, luteolin-7-O-rutinoside, quercetin, caffeoyl-glycosides or
cinnamoyl glycosides, and di-O-caffeoyquinic acid, respectively (Table 3). However, two other
compounds which were eluted at 17.22 and 25.84 min were not able to be identified. In term of
limitation, the database library was not available for identification of the targeted compounds, and it is
likely that the HPLC-ESI/MS analysis lacked sensitivity.
Molecules 2020, 25, x 6 of 24
Authentic standards, including gallic acid, catechin, tannic acid, rutin, isoquercetin, hydroquinine,
eriodictyol and quercetin, were analyzed and used as database, and are presented in Figure S3b. In
comparison with the standards, catechin, isoquercetin, eriodictyol and quercetin were detected in
the GSO, while gallic acid, tannic acid, rutin and hydroquinine would not be found.
In illustration, the fragmentations of phenolic compounds in the positive ion mode, eluted at
retention times of 9.47, 10.11, 12.64, 13.48, 14.02, 14.69, 16.11, 21.04, 29.53, 31.15, 32.32, 32.99, 34.47
and 35.09 min, were characterized as quinic acid, O-caffeoylquinic acid or chlorgenic acid, catechin,
apigenin-4-O-glycoside, ellagic acid-O-methoxyglucoside, dicaffeic acid, isoquercetin,
O-caffeoylquinic acid derivative, ellagic acid, eriodictyol, luteolin-7-O-rutinoside, quercetin,
caffeoyl-glycosides or cinnamoyl glycosides, and di-O-caffeoyquinic acid, respectively (Table 3).
However, two other compounds which were eluted at 17.22 and 25.84 min were not able to be
identified. In term of limitation, the database library was not available for identification of the
targeted compounds, and it is likely that the HPLC-ESI/MS analysis lacked sensitivity.
Figure 3. Total ion counts of HPLC-ESI/MS analysis for GSO. Peak identities are numbered in Table
3.
Figure 3.
Total ion counts of HPLC-ESI/MS analysis for GSO. Peak identities are numbered in Table 3.

Molecules 2020,25, 2474 7 of 23
Table 3. HPLC-ESI/MS identification of phenolic compounds for guava seed oil.
Peak
No.
TR
(min) TIC Exact Mass
(g/mol)
Molecular
Formula
Observed Mass
(m/z)Error (%) Possible Constituents/Compounds References
1 9.47 153000 192.1 C7H12O6[M −H]+194.1 1.03 Quinic acid [25]
2 10.11 174000 354.3 C16H18 O9[M −H]+354.9 0.17 O-Caffeoylquinic acid [25,26]
3 12.64 185000 290.3 C15H14 O6[M −H]+298.9 2.88 Catechin Authentic standard
4 13.48 152000 432.1 C21H20 O10 [M −H]+433.3 0.23 Apigenin-4-O-glycoside [27,28]
5 14.02 254000 496.2 C21H21 O14 [M −H]+497.2 0.2 Ellagic acid-O-methoxyglucoside [29,30]
6 14.69 254000 342.3 C18H14 O7[M −H]+342.9 0.15 Dicaffeic acid [26,28]
7 16.11 649000 464.1 C15H10 O7[M −H]+454.3 2.15 isoquercetin Authentic standard
8 17.22 318000 Unknown Unknown ND ND Unknown -
9 21.04 160000 452.1 Unknown [M −H]+453.2 0.27 O-Caffeoylquinic acid derivative [25]
10 25.84 129000 Unknown Unknown [M −H]+ND ND Unknown -
11 29.53 138000 302.2 C14 H6O8[M −H]+302.5 0.1 Ellagic acid [31,32]
12 31.15 306000 288.25 C15H12O6[M −H]+297.0 2.95 Eriodictyol Authentic standard
13 32.32 543000 594.5 C27H30 O15 [M −H]+595.5 0.17 Luteolin-7-O-rutinoside [24]
14 32.99 379000 302.2 C15H10 O7[M −K]+340.3 0.26 Quercetin Authentic standard
15 34.47 261000 382.0 C17H18 O10 [M −H]+383.3 0.34
Caffeoyl-glycosides or cinnamoyl glycosides
[26]
16 35.09 180000 516.45 C25H24O12 [M −H]+517.3 0.16 di-O-Caffeoyquinic acid [26]

Molecules 2020,25, 2474 8 of 23
2.4. Free-Radical Scavenging Activity
It was determined that GSO treatment showed an inhibitory effect on 1,1-diphenyl-2-picrylhydrazyl
radical (DPPH
•
) generation in a concentration-dependent manner (6.25–1000 mg/mL). In this
determination, the inhibition by GSO was almost complete at the concentration of 200 mg/mL
or 28.74
µ
g 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) equivalent (TE)/mL.
In comparison, Trolox and
α
-tocopherol were found to be even more effective in scavenging DPPH
•
than GSO, and in this determination the inhibition levels were dependent upon the concentrations of
20–60
µ
g/mL, and were complete above 60
µ
g/mL (Figure 4). In addition, GSO was measured at a half
maximal effective concentration (EC
50
) against Trolox, and the results revealed that GSO decreased the
initial DPPH•concentration by 50% at a concentration value of 139 g GSO/g [DPPH•].
Molecules 2020, 25, x 8 of 24
Molecules 2020, 25, x; doi: www.mdpi.com/journal/molecules
2.4. Free-Radical Scavenging Activity
It was determined that GSO treatment showed an inhibitory effect on
1,1-diphenyl-2-picrylhydrazyl radical (DPPH•) generation in a concentration-dependent manner
(6.25–1000 mg/mL). In this determination, the inhibition by GSO was almost complete at the
concentration of 200 mg/mL or 28.74 μg 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid
(Trolox) equivalent (TE)/mL. In comparison, Trolox and α-tocopherol were found to be even more
effective in scavenging DPPH• than GSO, and in this determination the inhibition levels were
dependent upon the concentrations of 20–60 μg/mL, and were complete above 60 μg/mL (Figure 4).
In addition, GSO was measured at a half maximal effective concentration (EC50) against Trolox, and
the results revealed that GSO decreased the initial DPPH• concentration by 50% at a concentration
value of 139 g GSO/g [DPPH•].
Treatment
Inhibi tion of DPPH
o
produc tion (%)
0
10
20
30
40
50
60
70
80
90
100
Trolox α-Tocopherol GSO
(μg/mL) (μg/mL) (μg TE/mL)
0
15.6
62.5
32.2
125
250
6.25
12.5
50
25
125
250
2.12
2.53
5.42
3.22
10.7
16.7
100
31.0
26.7
31.5
31.9
Figure 4. Inhibition of 1,1-diphenyl-2-picrylhydrazyl radical (DPPH•) generation by guava seed oil,
α-tocopherol and Trolox. Data obtained from two independent experiments performed in triplicate
are expressed as mean ± standard deviation (SD).
Furthermore, GSO and α-tocopherol were found to reduce reactive oxygen species (ROS) levels
in human hepatocellular carcinoma (HepG2) cells, in a concentration-dependent manner (p < 0.05 at
100 and 200 μg/mL), when compared with non-treated cells. In this determination, GSO was found
to be less effective at equal concentrations of α-tocopherol (Figure 5, left). Similarly, both GSO and
α-tocopherol dose-dependently suppressed the elevation of ROS levels in hydrogen peroxide
(H2O2)-induced human neuroblastoma (SH-SY5Y) cells, for which the degree of inhibition was
significant at 200 μg/mL of α-tocopherol (Figure 5, right). It is likely that antioxidant compounds,
including polar phenolic compounds (such as quinic acid and its derivatives, chlorogenic acid and
its derivatives, and hydrolysable tannins), phytosterols (such as stimasterol and campesterol) and
lipids (such as linoleic acid), exist as a consequence of GSO-protected oxidative stress in HepG2 and
SH-SY5Y cells, consequently preventing neurodegenerative diseases.
Figure 4.
Inhibition of 1,1-diphenyl-2-picrylhydrazyl radical (DPPH
•
) generation by guava seed oil,
α
-tocopherol and Trolox. Data obtained from two independent experiments performed in triplicate are
expressed as mean ±standard deviation (SD).
Furthermore, GSO and
α
-tocopherol were found to reduce reactive oxygen species (ROS) levels
in human hepatocellular carcinoma (HepG2) cells, in a concentration-dependent manner (p<0.05
at 100 and 200
µ
g/mL), when compared with non-treated cells. In this determination, GSO was
found to be less effective at equal concentrations of
α
-tocopherol (Figure 5, left). Similarly, both GSO
and
α
-tocopherol dose-dependently suppressed the elevation of ROS levels in hydrogen peroxide
(H
2
O
2
)-induced human neuroblastoma (SH-SY5Y) cells, for which the degree of inhibition was
significant at 200
µ
g/mL of
α
-tocopherol (Figure 5, right). It is likely that antioxidant compounds,
including polar phenolic compounds (such as quinic acid and its derivatives, chlorogenic acid and its
derivatives, and hydrolysable tannins), phytosterols (such as stimasterol and campesterol) and lipids
(such as linoleic acid), exist as a consequence of GSO-protected oxidative stress in HepG2 and SH-SY5Y
cells, consequently preventing neurodegenerative diseases.

Molecules 2020,25, 2474 9 of 23
Molecules 2020, 25, x 9 of 24
DMSO
ROS production (%)
0
20
40
60
80
100
120
140
GSO (μg/mL) α-Tocopherol (μg/mL)
6.25 12.5 25 50 100 200
HepG2 cells
6.25 12.5 25 50 100 200
*
*
6.25 12.5 25 50 100 200
GSO (μg/mL)
ROS production (%)
0
20
40
60
80
100
120
140
*
*
6.25 12.5 25 5 0 100 200
α-Tocopherol(μg/mL)
SH SY5Y cells
DMSO
Figure 5. Free-radical scavenging activity of reactive oxygen species in hydrogen peroxide-induced
human hepatocellular carcinoma (HepG2) and neuroblastoma (SH-SY5Y) cells by guava seed oil and
α-tocopherol. Data obtained from two independent experiments performed in triplicate are
expressed as mean ± SD values. * p < 0.05 when compared to non-treated cells. Abbreviations: GSO =
guava seed oil, ROS = reactive oxygen species.
2.5. Bioavailability of Serum Lipids in GSO-Fed Rats
As shown in Table 4, serum levels of total cholesterol did not change significantly in the rats
that had been fed DI (control), CO (reference oil) (30 g linoleic acid equivalent (LAE)/kg) and GSO (6
and 30 g LAE)/kg) for 90 d, irrespective of gender. The serum cholesterol levels were neither
influenced by the GSO and CO feeding nor GSO doses when compared with DI. Throughout the
study, serum triglyceride levels were found to be higher in male rats than in female rats (p > 0.05)
among all gender-based rat groups, while they were not different in mixed gender groups and
tended to decrease over the course of the study. For 90 d, serum triglyceride levels were decreased to
a greater degree in the CO (30 g LAE/kg) treatment (Δ 28.5 mg/dL) and the GSO (30 g LAE/kg)
treatment (Δ 16.5 mg/dL) than in the DI group (Δ 7.6 mg/dL) (P < 0.05), while serum triglyceride
levels were found to have increased in the GSO (6 g LAE/kg) treatment (Δ 24.3 mg/dL).
Here, we used a very sensitive high-performance liquid chromatography/fluorescence
detection (HPLC/FLD) method, together with alcoholic acid hydrolysis, for the quantitation of
derivatized fatty acids. Authentic fatty acids, including α-linolenic acid (ALA), arachidonic acid
(AA), palmitoleic acid (PLA), linoleic acid (LA), stearic acid (SA) and oleic acid (OA) (100 μM each),
were used to calibrate the column, on which they were positioned separately at TR of 19.90, 24.71,
26.72, 30.24, 46.46 and 49.66 min, respectively (Figure S4). Using the specific TR of the standard fatty
acids, serum concentrations of PLA, OA, SA, LA, ALA and AA were calculated, and are shown in
Table 5. The findings presented in Table 5 show very low or undetectable concentrations of serum
PLA and ALA in all rat groups. Feeding LA-rich CO and GSO (30 g LAE/kg) for 90 d did not increase
serum levels of LA, but did show a tendency to decrease them when compared to the control DI.
Importantly, there was a tendency of CO (30 g LAE/kg) and GSO (30 g LAE/kg) feeding to
significantly decrease the serum levels of OA, SA and AA in all rat groups (mixed genders), for
which the GSO was more efficient than the CO when compared with the DI control group. In
addition, GSO (30 g LAE/kg) was found to have lowered the serum SA level to a greater degree than
GSO (6 g LAE/kg) (p < 0.05). These results imply that GSO consumption potentially lowers the
plasma triglyceride levels, and can modulate the serum levels of fatty acids (e.g., stearic, oleic and
arachidonic acids), similarly to CO consumption at an equal concentration.
Figure 5.
Free-radical scavenging activity of reactive oxygen species in hydrogen peroxide-induced
human hepatocellular carcinoma (HepG2) and neuroblastoma (SH-SY5Y) cells by guava seed oil and
α
-tocopherol. Data obtained from two independent experiments performed in triplicate are expressed
as mean
±
SD values. * p<0.05 when compared to non-treated cells. Abbreviations: GSO =guava seed
oil, ROS =reactive oxygen species.
2.5. Bioavailability of Serum Lipids in GSO-Fed Rats
As shown in Table 4, serum levels of total cholesterol did not change significantly in the rats that
had been fed DI (control), CO (reference oil) (30 g linoleic acid equivalent (LAE)/kg) and GSO (6 and
30 g LAE)/kg
) for 90 d, irrespective of gender. The serum cholesterol levels were neither influenced
by the GSO and CO feeding nor GSO doses when compared with DI. Throughout the study, serum
triglyceride levels were found to be higher in male rats than in female rats (p>0.05) among all
gender-based rat groups, while they were not different in mixed gender groups and tended to decrease
over the course of the study. For 90 d, serum triglyceride levels were decreased to a greater degree in
the CO (30 g LAE/kg) treatment (
∆
28.5 mg/dL) and the GSO (30 g LAE/kg) treatment (
∆
16.5 mg/dL)
than in the DI group (
∆
7.6 mg/dL) (P<0.05), while serum triglyceride levels were found to have
increased in the GSO (6 g LAE/kg) treatment (∆24.3 mg/dL).
Here, we used a very sensitive high-performance liquid chromatography/fluorescence detection
(HPLC/FLD) method, together with alcoholic acid hydrolysis, for the quantitation of derivatized fatty
acids. Authentic fatty acids, including
α
-linolenic acid (ALA), arachidonic acid (AA), palmitoleic acid
(PLA), linoleic acid (LA), stearic acid (SA) and oleic acid (OA) (100
µ
M each), were used to calibrate the
column, on which they were positioned separately at T
R
of 19.90, 24.71, 26.72, 30.24, 46.46 and 49.66 min,
respectively (Figure S4). Using the specific T
R
of the standard fatty acids, serum concentrations of
PLA, OA, SA, LA, ALA and AA were calculated, and are shown in Table 5. The findings presented
in Table 5show very low or undetectable concentrations of serum PLA and ALA in all rat groups.
Feeding LA-rich CO and GSO (30 g LAE/kg) for 90 d did not increase serum levels of LA, but did show
a tendency to decrease them when compared to the control DI. Importantly, there was a tendency
of CO (30 g LAE/kg) and GSO (30 g LAE/kg) feeding to significantly decrease the serum levels of
OA, SA and AA in all rat groups (mixed genders), for which the GSO was more efficient than the
CO when compared with the DI control group. In addition, GSO (30 g LAE/kg) was found to have
lowered the serum SA level to a greater degree than GSO (6 g LAE/kg) (p<0.05). These results imply
that GSO consumption potentially lowers the plasma triglyceride levels, and can modulate the serum
levels of fatty acids (e.g., stearic, oleic and arachidonic acids), similarly to CO consumption at an
equal concentration.

Molecules 2020,25, 2474 10 of 23
Table 4.
Serum levels of total cholesterol and triglyceride from rats (5 male and 5 female each) treated with deionized water, corn oil (30 g LAE/kg) and guava seed oil
(6 g and 30 g LAE/kg) for 90 days. Data are expressed as individual and mean ±SD values. &p<0.05 when compared with the level at the baseline (day 0).
Serum Lipids Time DI CO (30 g LAE/kg) GSO (6 g LAE/kg) GSO (30 g LAE/kg)
5M 5F 5M,5F 5M 5F 5M,5F 5M 5F 5M,5F 5M 5F 5M,5F
Total cholesterol
(mg/dL)
Day 0 64.4 ±8.0 61.8 ±11.5 63.1 ±9.4 65.6 ±10.7 66.2 ±6.9 65.9 ±8.5 67.4 ±11.2 72.6 ±11.6 70.0 ±11.1 62.4 ±4.2 69.2 ±12.7 65.8 ±9.6
Day 90 66.8 ±13.5 51.8 ±8.5 59.3 ±13.3 62.2 ±4.8 65.4 ±6.1 63.8 ±5.5 72.8 ±16.4 62.2 ±4.8 67.5 ±12.7 74.0 ±6.5 62.6 ±10.1 68.3 ±10.0
Triglyceride
(mg/dL)
Day 0 87.0 ±24.2 33.2 ±8.2 60.1 ±33.1 100.4 ±24.6 44.8 ±4.3 72.6 ±33.7 70.0 ±17.4 41.8 ±10.0 55.9 ±20.0 89.4 ±34.2 60.8 ±9.9 75.1 ±28.1
Day 90 66.2 ±19.4 38.8 ±11.7 52.5 ±20.9 39.8 ±6.4 48.4 ±7.9 44.1 ±8.1 &118.6 ±45.1 39.8 ±6.4 79.2 ±51.4 77.4 ±20.2 39.8 ±9.8 58.6 ±24.8 &
Abbreviations: CO =corn oil, DI =deionized water, GSO =guava seed oil, LAE =linoleic acid equivalent, TC =total cholesterol, TG =triglyceride.
Table 5.
Levels of fatty acids of serum obtained from rats treated with deionized water, corn oil and guava seed oil for 90 days. Data are expressed as mean
±
SD
values. * p<0.05 when compared with deionized water; #p<0.05 when compared with the levels at a lower concentration.
Fatty Acid
Levels
DI CO (30 g LAE/kg) GSO (6 g LAE/kg) GSO (30 g LAE/kg)
5M 5F 5M,5F 5M 5F 5M,5F 5M 5F 5M,5F 5M 5F 5M,5F
Palmitoleic acid
(mg/dL)
0.72
±
0.29
ND
0.72
±
0.29
ND ND ND ND ND ND ND ND ND
Oleic acid
(mg/dL)
1.79
±
0.85 1.00
±
0.35 1.39
±
0.74 0.57
±
0.19 0.75
±
0.19
0.66 ±0.20 *
1.04
±
0.37 0.61
±
0.16
0.82 ±0.35 *
0.54
±
0.17 0.62
±
0.07
0.58 ±0.13 *
Stearic acid
(mg/dL)
4.39
±
1.38 2.55
±
0.77 3.47
±
1.44 1.57
±
0.65 1.84
±
0.37
1.71 ±0.52 *
2.74
±
0.43 1.38
±
0.25
2.06 ±0.79 *
1.37
±
0.22 1.33
±
0.15
1.35 ±0.18 *,#
Linoleic acid
(mg/dL)
1.70
±
0.66 0.90
±
0.31 1.30
±
0.64 0.97
±
0.41 1.41
±
0.37
1.19 ±0.43
1.05
±
0.35 0.59
±
0.15
0.82 ±0.35
0.82
±
0.24 0.78
±
0.11
0.80 ±0.18
α-Linolenic
acid (mg/dL) ND ND ND ND ND ND ND ND ND ND ND ND
Arachidonic
acid (mg/dL)
0.74
±
0.20 0.55
±
0.13 0.64
±
0.19 0.34
±
0.13 0.40
±
0.06
0.37 ±0.10 *
0.48
±
0.10 0.40
±
0.04
0.44 ±0.08 *
0.35
±
0.05 0.43
±
0.13
0.39 ±0.10 *
Abbreviations: CO =corn oil, DI =deionized water, F =female, GSO =guava seed oil, M =male, ND =not detectable.

Molecules 2020,25, 2474 11 of 23
3. Discussion
Natural products derived from several parts, of plants including leaves, roots, bark, rhizome,
stock, pulp and seeds, have provided unparalleled sources of chemical diversity that possess bioactive
molecules of valuable interest. Polyunsaturated fatty acids (PUFA) are rich in tree-born seed oils,
and have been claimed to be rich in lipophilic antioxidants that are highly susceptible to oxidation,
possibly leading to the generation of rancid oil and secondary lipid peroxides. So far, hyphenated
analytical methods, such as GC/MS and HPLC/MS, have been applied for the efficient detection and
characterization of targeted molecules. Likewise, GC/MS is a sensitive method for the comprehensive
characterization of volatile small molecules such as fatty acids. Predominantly, HPLC-ESI/MS and high
performance liquid chromatography-electrospray ionization/mass spectrometry/mass spectrometry
(HPLC-ESI/MS/MS)) are powerful analytical techniques, with high sensitivity and accuracy, that can
be used to determine the compound profile of plant materials and natural products [
33
]. Moreover,
HPLC-ESI-Q-TOF/MS provides higher resolution, faster speeds and less solvent consumption, leading
to a rapid and sensitive characterization of certain unexpected natural products [34,35].
Our recent findings have shown that the hexane extract of GSO was abundant with linoleic
acid (most abundant at 69.95% of total fatty acids), followed by oleic acid, palmitic acid, stearic
acid, arachidic acid and
α
-linolenic acid. Consistently, GSO (Psidium gaujava L.) extraction with
n-hexane solvent revealed a high content of linoleic acid (60.03% of total fatty acids) as the main
fatty acid component [
18
]. In comparison, petroleum ether extract of GSO gave a higher yield of
linoleic acid (78.4% of total fatty acids) [
17
], suggesting that both the method employed and the
solvent administered can affect the different percentage yields of linoleic acid. In studies on lipids,
reverse-phase HPLC-ESI/MS is a robust and popular technique that is commonly used. In the
literature, xestoaminol C obtained from a Fiji sponge Xestospongia sp. was reported to be an extremely
active agent against parasites and microbes [
36
]. Not surprisingly, eschscholtzxanthin, which is the
predominant pigment carotenoid in poppy petals, was found in the hexane extracts of GSO [
37
].
In this study, we have detected the presence of certain possible lipids, including 4-hexyl-decanoic
acid, sphinganine, 5S-hydroxyeicosatetraenoyl di-endoperoxide, didrovaltratum, sphingofungin B,
13,14-dihydro-19(R)-hydroxyprostaglandin E1, eschscholtzxanthin, tetradecan-3-one and xestoaminol
C, in GSO. Surprisingly, none of these compounds have ever been reported to be isolated in the hexane
extracts of GSO, but some compounds have been described. For instance, 4-hexyl-decanoic acid
(isopalmitic acid) is a natural major branch-chain saturated fatty acid that is present in the leaves of Abies
pindrow, and probably produced as a lipophilic adsorbent during hexane extraction [
38
,
39
]. Interestingly,
sphingoganin and sphingofungin, existing in natural seed oils, and xestoaminol C found in New Zealand
Brown Alga Xiphophora chondrophylla, showed strong anti-candidiasis and anti-tubercular activities [
40
–
42
]. Notably, Eschscholtzxanthin is one of the red pigment carotenoids that is synthesized in the leaves
of several plants as a response to photoinhibitory conditions during winter acclimation and displays
antioxidant activity [
43
]. Consistently, we have demonstrated the anti-leukemic and anti-plasmodium
activities of GSO [
24
]. Recently, we reported that the hexane extract of GSO was abundant with
α
-tocopherol (23.0 mg/kg) and
β
-tocotrienol (70.5 mg/kg) [
17
,
44
]. With regard to potential health benefits,
phytosterols could play an important role in facilitating certain biological activities such as free-radical
scavenging and plasma lipid-modulating effects. Here, we have analyzed GSO and found the presence
of certain phytosterols, such as
β
-sitosterol (297.61 mg/100 g), stigmasterol (0.22 mg/100 g), campesterol
(11.04 mg/100 g), and other neutral lipids. In comparison, hexane extracts of Panax quinquefolium ginseng
and Cajanus cajan seed oils contained phytosterols such as squalene, oxidosqualene, campesterol,
stigmasterol, clerosterol,
β
-sitosterol,
β
-amyrin,
δ
(5)-avenasterol,
δ
[5,24(25)]-stigmasterol, lupeol,
δ
(7)-sitosterol,
δ
(7)-avenasterol, 24-methylenecycloartanol and citrostadienol [
45
,
46
]. Additionally,
hexane extracts of Alyssum homolocarpum seed oil were abundant with
β
-sitosterol (3.3 mg/g) and
campesterol (0.86 mg/g), which readily transverse the blood-brain barrier [
47
]. Moreover, a variety of
phytosterols, and their contents in hexane-extractable seed oils derived from Cajanus cajan, nutmeg,
white mustard, anise, coriander and caraway, have been demonstrated [46,48].

Molecules 2020,25, 2474 12 of 23
We have revealed that the hexane extracts of GSO contained a total phenolic content of
45.57 ±0.97 µg
gallic acid equivalent (GAE)/g of GSO [
43
]. In this research, the HPLC-ESI single
quadrupole/MS combined with correlation analysis of measured versus predicted mass spectra
was used to afford the rapid characterization of small organic molecules, particularly the phenolic
compounds present in GSO. Possible compounds included quinic acid, chlorogenic acid, catechin,
apigenin-4-O-glycoside, ellagic acid-O-methoxyglucoside, dicaffeic acid, isoquercetin, chlorogenic
acid derivative, ellagic acid, eriodictyol, luteolin-7-O-rutinoside, quercetin, caffeoyl-glycosides or
cinnamoyl-glycosides and di-O-caffeoyquinic acid. In the case presented here, some compounds
were identified after being compared with corresponding compounds that had been previously
identified in natural products. Nonetheless, ESI single quadrupole/MS has traditionally presented
certain limitations, such as unavailable authentic compounds as references, an incomplete database
library, less ability to observe compounds of low polarity, and low sensitivity and mass resolution for
detection when compared with HPLC-ESI/MS/MS or HPLC-ESI-QTOF/MS. In addition, the raw mass
spectrometry data showed that many fragments were difficult to isolate for interpretation. Evidently,
caffeoylquinic acid (chlorogenic acid) isomers, quinic acid derivatives, di-O-caffeoylquinic acid and
caffeoylquinic acid derivatives were found in different proportions in the extracts obtained from
Prunus domestica,Salicornia gaudichaudiana, Galphimia glauca, Gymnaster koraiensis, Artemisia princeps
Pampanini and Foeniculum vulgare Mill, depending on organic solvent extractions [
49
–
55
]. Additionally,
apigenin, luteolin and their glycosides are acknowledged as the predominant phenolic compounds
in the hexane extracts of plant seeds, and these exert strong antioxidant or free-radical scavenging
abilities, or altogether effects [
46
,
56
–
58
]. It is likely that the chlorogenic acid, caffeic acid derivatives,
apigenin and its glycosides, exhibit antioxidant, free-radical scavenging and anti-inflammatory effects,
suggesting that GSO would be significantly beneficial for living cells and human health.
In fact, PUFA, tocopherols, tocotrienols, phytosterols and phenols are the most important natural
antioxidants that are present in vegetable and seed oils. Hence, the hexane extracts of seed oils that
possess anti-oxidative compounds can also exert free-radical scavenging properties. Among available
antioxidant assays, the DPPH
•
scavenging assay is a simple colorimetric method that can be applied
for the study in both hydrophobic (e.g., hexane extract) and hydrophilic environments. In this study,
we found that GSO was able to decrease levels of DPPH
•
in aqueous environments, and H
2
O
2
-induced
oxidative stress in HepG2 and SH-SY5Y cells in a concentration-dependent manner, even though
it was less efficient than the reference antioxidants Trolox (water-soluble vitamin E analogue) and
α
-tocopherol, respectively. Obviously, GSO presented EC
50
of the DPPH
•
scavenging activity at a dose
of 139 g oil/g [DPPH
•
] or 19.97
µ
g TE/g [DPPH
•
], which then exerted antioxidant activity. Similarly,
the researchers have shown that GSO reduced the initial DPPH
•
concentration by 50% (EC
50
) at a
dose of 12.9 g oil/g [DPPH
•
] [
17
], which is potentially more powerful than our experiments with GSO.
However, our GSO experiments demonstrated a lower value of EC
50
than other oils extracted from
walnuts, peanuts, almonds, hazelnuts and pistachio nuts [
59
]. Moreover, we have proposed that the
hepato- and neuro-protective effects acknowledged in GSO would be mediated by active phytosterols
and phenolic compounds, via scavenging radicals that are generated during the production of oxidative
stress. Notably, antioxidant activity may occur as a result of the presence of hydrophobic compounds
(e.g., carotenoids, tocopherols, tocotrienols and linoleic acid per se) [
60
–
62
], along with the polar
phenolics [
63
] that are present in certain plant seed oils including GSO, as was observed in this study.
Zhang and Liu have demonstrated that the extracts derived from millets (Setaria italic and Panicum
miliaceum) contain chlorogenic acid, caffeic acid, xanthophylls and zeaxanthin which could scavenge
peroxyl radicals in 2,2-azobis-amidinopropane-induced HepG2 cells [
64
,
65
]. Likewise, the chlorogenic
acid and di-O-caffeoylquinic acid present in plant extracts showed protective effects against oxidative
stress-induced hepatic (HepG2), neuroblastoma (SH SY5Y), and nepheochromocytoma (P-12) cell
damage [
53
,
54
,
66
,
67
]. Moreover,
β
-sitosterol and its glucoside obtained from the Gastrocotyle hispida
extract exhibited DPPH
•
-scavenging activity in a concentration-dependent manner [
68
]. Pretreatment
with phytosterols, including
β
-sitosterol, daucosterol and pectolinarin obtained from Cirsium setidens

Molecules 2020,25, 2474 13 of 23
and Aster scaber extracts, considerably decreased ROS levels in H
2
O
2
-induced neuroblastoma (SK-N-SH)
cells [69].
Although GSO have exhibited important nutraceutical properties, such as high contents of linoleic
acid, tocopherols and tocotrienols, anti-oxidation and wound healing effects, long-term consumption
of the GSO may lead to dyslipidemia and increased levels of atherogenic index. Evidently, GSO
(
30 g LAE/kg
) and CO (6 g LAE/kg) treatments were found to lower the serum levels of triglycerides
significantly, when compared to treatments without GSO. Similarly, the GSO and CO treatments could
significantly decrease the serum levels of fatty acids, including oleic, stearic and arachidonic acids,
when compared to treatments without GSO. In these treatments, the GSO (30 g LAE/kg) seemed to
display a level of efficiency that was equal to the CO at an equal concentration but was greater than
that of the GSO (6 g LAE/kg). From previous instances, long-term consumption of LA-rich corn oil
and ALA-rich perilla oil revealed a trend of reducing serum triglyceride and cholesterol levels in
rodents, when compared with the DI control. However, the consumption of flaxseed oil and CO
did not change the serum cholesterol and triglyceride levels in humans. In the study, serum ALA
was undetectable in all rat groups, while serum LA levels were lower in CO and GSO groups than
in the DI group. A previous study has demonstrated that grape seed oil and corn oil, that were
rich in linoleic acid or omega-6 fatty acids, tended to decrease serum cholesterol levels in rats [
70
].
Similarly, linoleic acid-rich niger seed oil was found to display a hypolipidemic effect through the
facilitation of lipid transportation and metabolism, possibly lowering the risk of cardiovascular disease
development [
71
]. Consistently, a combination diet of proteins, perilla seed oil, linoleic acid and
α
-linolenic acid (per oral) was found to decrease plasma triglyceride and cholesterol levels in rats [
72
].
Importantly, the supplementation of caffeoylquinic acid (or chlorogenic acid), which is rich in the
extracts obtained from chicory (Cichorium intybus) seeds and GSO, decreased levels of the triglyceride
and atherogenic index, but increased levels of antioxidant capacity in the serum of rats that were fed
with a high fructose/glucose diet [
73
]. Conversely, feeding rats with a berry seed oil-supplemented
diet did not significantly influence the plasma levels of triglycerides, while the total cholesterol values,
which included high-density and low-density lipoprotein fractions, were not changed [
74
]. The results
suggest that GSO rich in
α
-linoleic acid, phytosterols and chlorogenic acid could be utilized as a source
of functional lipids, in the synthesis of essentially longer n-6 fatty acids, and the protection of oxidative
stress-induced hepatocytes and neuronal cells.
4. Materials and Methods
4.1. Chemicals and Reagents
Arachidonic acid (AA), boron trifluoride (BF
3
), 4-bromomethyl-7-methoxycoumarin (Br-MMC),
18-crown-6, 2
0
,7
0
-dichlorodihydrofluorescein diacetate (DCFH-DA), 1,1-diphenyl-2-picrylhydrazyl
(DPPH), Folin-Ciocalteu reagent, formic acid, hydrogen peroxide, linoleic acid (LA),
α
-linolenic acid
(ALA), oleic acid (OA), palmitic acid (PA), palmitoleic acid (PLA), phosphoric acid, stearic acid (SA),
α
-tocopherol, and 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) were purchased
from Sigma-Aldrich Company (St. Louis, MO, USA). Acetonitrile, acetic acid, ethanol, ethyl acetate,
n-hexane, heptadecanoic acid, n-heptane, isopropanol, methanol, n-pentane (the highest pure HPLC
grade), N-methyl-N-trimethylsilyltrifluoroacetamide (MSTFA, LiChropur
™
, purity
≥
98.5%) and
deionized water (DI) (Milli-DI
®
, Resistivity at 25
◦
C>1 M
Ω·
cm) were purchased from Merck Chemical
Company (Merck KGaA, Darmstadt, Germany). Dulbecco’s modified Eagle medium (DMEM), Ham’s
F12 Nutrient, fetal bovine serum (FBS), penicillin-streptomycin (10,000 U/L) and trypsin-ethylene
diamine tetra acetic acid (trypsin-EDTA) were purchased from Gibco Technologies (Thermo Fisher
Scientific, Waltham, MA, United States). Corn oil (CO) (density 0.91–0.93 g/mL, compositions of 2–3 g
SA, 11–13 mg PA, 25–31 g OA, 59 g LA, 1 g ALA, 0.3 g trans-fats and 14.3 mg
α
-tocopherol in 100 g of
oil) was purchased from a supermarket that was located in the city of Chiang Mai.

Molecules 2020,25, 2474 14 of 23
4.2. Preparation of GSO Extract
Dried guava seeds (P. guajava var Pan See Tong) (200 g) were ground and extracted with n-hexane
(1000 mL) in a Soxhlet apparatus (boiling point in the range of 60–80
◦
C) for 8 h. The extract was then
filtered through a Buchner funnel with Whatman’s No. 1 filter paper and concentrated at 60
◦
C using
a rotatory evaporator [
75
]. The GSO obtained was decolorized with activated charcoal and stored
in a plastic container in the dark at 4
◦
C for further study. From the chromatographic analyses, we
found that the GSO contained linoleic acid, palmitic acid and oleic acid (69.95%, 6.14% and 10.47%
of total fatty acids, respectively), with a n3/n6 ratio of 1:224;
α
-tocopherol (23.0 mg/kg);
β
-tocopherol
(1.5 mg/kg);
γ
-tocopherol (1.4 mg/kg);
β
-tocotrienol (70.5 mg/kg);
δ
-tocotrienol (17.4 mg/kg) and
γ-tocotrienol (4.0 mg/kg) [24].
4.3. High-Performance Liquid Chromatography-Electrospray Ionization-Quadrupole Time-of-Flight/Mass
Spectrometry for Lipids
GSO was analyzed for its polar phenolic compounds at the Central Laboratory, Faculty of
Agriculture, Chiang Mai University, Chiang Mai, Thailand, using the HPLC-ESI-QTOF/MS method
that was established by Gu and colleagues with slight modifications [
35
]. The HPLC instrument
was equipped with an ESI-QTOF/MS machine (Agilent Acquisition SW version 6200 series TOF/6545
series LC/Q-TOF), QTOF Firmware Version 25.698 (Agilent Technologies, Santa Clara, CA, USA).
Mobile phase A (acetonitrile) and mobile phase B (0.1% formic acid) were degassed at 25
◦
C for
15 min. The GSO (20 mg) was constituted in 1.0 mL of the A:B mixture (1:1, v/v), filtered using a
syringe filer (polyvinylidene fluoride type, 0.45
µ
m pore size, Millipore, MA, USA) and put into HPLC
vials. The flow rate was set to be 0.35 mL/min, the injection volume was measured at 10
µ
L for each
sample, and the running time was 60 min. Chromatographic separation was carried out on a column
(InfinityLab Poroshell 120 EC-C18 type, 2.1 mm
×
100 mm, 2.7
µ
m, Agilent Technologies, Santa Clara,
CA, USA) that was regulated thermally at 40
◦
C. The ESI-MS/MS spectra were recorded using the
Agilent Q-TOF mass spectrometer (Agilent Technologies, Santa Clara, CA, USA). In the MS system,
nitrogen gas nebulization was set at 45 pounds per inch
2
with a flow rate of 5 L/min at 300
◦
C, and the
sheath gas was set at 11 L/min at 250
◦
C. In addition, the capillary and nozzle voltage values were
set at 3.5 kV and 500 V, respectively. A complete mass scan was conducted as a mass to charge ratio
(m/z) ranging from 200 to 3200. All the operations, acquisition and analysis of the data were monitored
using Agilent LC-Q-TOF-MS MassHunter Acquisition Software Version B.04.00 (Agilent Technologies,
Santa Clara, CA, USA) “Find by Be” algorithm to generate a list of accurate mass matches-compounds.
Peak identification was performed in positive modes using the library database, and the identification
scores were further selected for characterization and m/zverification.
4.4. Trimethylsilylation Derivatization-Gas Chromatography/Mass Spectrometry of Phytosterols
4.4.1. Derivatization
The trimethylsilyl (TMS) derivatization-GC/MS of phytosterols for GSO was performed at the
Central Laboratory (North Branch), Department of Land Development, Ministry of Agriculture
and Cooperation, Chiang Mai, Thailand using the method established by Lee et al. with slight
modifications [
76
]. Briefly, GSO (100
µ
L) was evaporated to dryness under the nitrogen stream.
The dried residue was re-dissolved in 1% pyridine in ethyl acetate (40
µ
L) and MSTFA (100
µ
L),
and derivatized at 80
◦
C for 30 min. After cooling, the resulting solution was diluted with 0.4 mL of
ethyl acetate and 60
µ
L of cholestane (internal standard). A 4-
µ
L aliquot of the resulting solution was
directly injected into the GC/MS system. All of the derivatives were analyzed using GC-MS scan mode.
4.4.2. Gas Chromatography/Mass Spectrometry
GC-MS analysis was performed in full scan mode on the GC system (Agilent Technologies Model
6890N, Deutschland, GmbH, Waldbronn, Germany) that was directly coupled to the MS detector

Molecules 2020,25, 2474 15 of 23
(Agilent Technologies Model 5973 inert, Palo Alto, CA, USA). Chromatographic separation was
performed using a capillary column (DB-5MS, a dimension of 30 m
×
0.25 mm, 0.25
µ
m film thickness,
an Agilent J&W Scientific (Folsom, CA, USA). Ultra-high purity helium was used as the carrier gas at
a flow rate of 1.5 mL/min, with an inlet temperature 270
◦
C and an auxiliary temperature of 280
◦
C,
for a running time of 35 min. The sample solution was injected in split mode (split ratio 10:1) at
280
◦
C. The electrospray ionization (ESI) energy was set at 70 eV. For the single quadrupole MS
system, the temperatures of the ion source and the interface were set at 150
◦
C and 230
◦
C, respectively.
The mass scan ranged from 40 to 500 m/z. The selected ion monitoring (SIM) mode was set at 272,
382, 394 and 486 m/zfor TMS-derivatized sterols, while the scanned mode was set in a range of 40 to
500 m/zfor TMS-derivatized unknown values. The oven temperature was programmed as follows:
80
◦
C (held for 3 min), ramped to 110
◦
C at 10
◦
C/min (held for 5 min), increased to 190
◦
C (held for
3 min), ramped to 220
◦
C at 10
◦
C/min (held for 4 min), and increased to 280
◦
C at 15
◦
C/min (held
for 13 min). In the phytosterol groups,
β
-stigmasterol, sitosterol, sitostanol and campesterol were
identified using authentic reference standards. In addition, the mass fragments of the analytes were
compared with the data of known compounds using a comprehensive mass-spectral library (Wiley
version 7.0, www.wiley.com) for identification of the targeted molecules. In terms of method validation,
the limit of detection (LOD) was 0.5 mg/kg (ppm), the limit of quantitation (LOQ) was 1.20 mg/kg
(ppm), and the recovery value was 70–110%.
4.5. High-Performance Liquid Chromatography-Electrospray Ionization/Mass Spectrometry of
Phenolic Compounds
Qualitative analysis of phenolic compounds was carried out at the Central Laboratory (North
Branch), Department of Land Development, Ministry of Agriculture and Cooperation, Chiang Mai
using the HPLC/MS method established by Cuyckens and colleagues with slight modifications [
77
].
The HPLC system (Agilent Technologies 1100 Series, Deutschland GmbH, Waldbronn, Germany)
consisted of a quaternary pump (G1311A), an online vacuum degasser (G1322A), an autosampler
(G1313A), a thermostated column compartment (G1316A) and a photodiode array (PDA) detector
(G1315A). The outlet of the PDA was coupled directly to the atmospheric pressure ESI interface of
the mass spectrometer (MS) detector (Agilent Technologies 1100 LC/MSD SL, Palo Alto, CA, USA)
through a flow splitter (1:1). In further analysis, GSO (20 mg) was constituted in 1.0 mL of the mixture
of solvent A (acetonitrile) and solvent B (10 mM formate buffer pH 4.0) (1:1, v/v) and filtered through
a syringe filter (polytetrafluoroethylene membrane, 25 mm diameter, 0.45-
µ
m pore size, Corning
®
)
before being used, and was then injected (20
µ
L) into the system. Chromatographic separation was
carried out on a column (LiChroCART RP-18e, 150 mm
×
4.6 mm, 5
µ
m particle size; Purospher STAR,
Merck, Darmstadt, Germany) operated at 40
◦
C. The mobile phases A and B were run at a flow rate
of 1.0 mL/min under the gradient program of 100% B (0% A) for an initial period of 5 min, 0–20%
A from 5 to 10 min, 20% A from 10 to 20 min, 20–40% A from 20 to 60 min, 40% A for 3 min, and
followed by an initial 100% B for 5 min. PDA detection was set at 270 nm. MS analysis was done in
positive ESI mode, and spectra were acquired within the mass to charge ratio (m/z) ranging from 100 to
700. For the single quadrupole MS system, the ESI energy was set at 70 eV, while the temperatures
of the ion source and the interface were set at 150
◦
C and 230
◦
C, respectively. Nitrogen was used as
the nebulizing, drying and collision gas. The capillary temperature was set to 320
◦
C, the nebulizer
pressure was set to 60 psi, and the drying gas flow rate was set to 13 L/min. Capillary voltages were
set to 3500 V (positive) and 150 V (negative). The oven temperature was programmed as follows:
80
◦
C (held for 3 min), ramped to 110
◦
C at 10
◦
C/min (held for 5 min), increased to 190
◦
C (held for
3 min), ramped to 220
◦
C at 10
◦
C/min (held for 4 min), and increased to 280
◦
C at 15
◦
C/min (held for
13 min). Accurate mass measurements were performed by the auto mass calibration method using an
external mass calibration solution (ESI-L Low Concentration Tuning Mix; Agilent calibration solution
B). Herein, the LOD, LOQ and recovery value were found to be 0.5 mg/kg, 1.20 mg/kg and 70–110%,
respectively. The chromatographic and mass spectrometric analyses and prediction of the chemical

Molecules 2020,25, 2474 16 of 23
formula, including the exact mass calculation, were performed by Mass Hunter software version
B.04.00 build 4.0.479.0 (Agilent Technology, Santa Clara, CA, USA). Available authentic phenolics
(1 mg/mL each) such as gallic acid, catechin, tannic acid, rutin, isoquercetin, hydroquinine, eriodictyol
and quercetin were also analyzed and used as database. In addition, MS data were searched for in
published literature repositories.
4.6. Determination of Free-Radical Scavenging Activity
Antioxidant activity of GSO was assayed using the DPPH
•
-scavenging method [
78
,
79
]. Briefly,
GSO (0–1000 mg/mL ethyl acetate) or Trolox (0–250
µ
g/mL ethanol) was mixed in equal volumes with
0.2 mM DPPH
•
solution and incubated at 25
◦
C in the dark for 30 min. The OD of the colored product
was measured at 515 nm against the reagent blank. The percentage of free-radical scavenging activity
was calculated by applying the following formula:
% DPPH•scavenging =[1 −(ODsample −ODblank sample)/ODcontrol]×100 (1)
The values of GSO and Trolox decreased the initial DPPH
•
concentration by 50% (EC
50
) and were
calculated by graphically plotting the percentage of the remaining DPPH•concentrations [17].
In addition, cellular ROS scavenging activity was determined in human hepatocellular
carcinoma (HepG2) and neuroblastoma (SH-SY5Y) cells by using the fluorescent dichlorofluorescein
(DCF)-fluorometric method [
80
]. In principle, DCFH-DA substrate simply diffuses into the cells
and is hydrolyzed by cellular esterase to produce 2
0
,7
0
-dichlorofluorescein (reduced), which will be
subsequently oxidized by existing ROS to a green fluorescence DCF product. Fluorescence intensity
(FI) is directly proportional to the amount of ROS in the cells.
In assay, HepG2 cells were cultured in DMEM supplemented with 10% FBS, penicillin G (100 U/mL)
and streptomycin (100
µ
g/mL) at 37
◦
C in a humidified atmospheric 5% CO
2
incubator. The cells
(10
4
cells/well) were incubated with GSO or
α
-tocopherol at concentrations of 0–200
µ
g/mL, which
were previously diluted with DMSO (control) for 24 h at 37
◦
C. Afterwards, the treated cells were
incubated with a 10
µ
M DCFH-DA solution at 37
◦
C for 30 min and washed twice with PBS. After
being challenged with 100
µ
M hydrogen peroxide (H
2
O
2
), kinetic FI was measured at wavelengths of
λex
485 nm and
λem
530 nm using a flow cytometer (Guava
®
easyCyte, Merck, Darmstadt, Germany).
SH-SY5Y cell line was purchased from the American Type Culture Collection (ATCC
®
CRL2266
™
,
Manassa, VA, USA). Cells were cultured in DMEM and Ham’s F12 Nutrient Mixture (ratio 1:1,
by volume) supplemented with 20% (v/v) FBS, 100 U/mL penicillin and 100
µ
g/mL streptomycin, and
maintained at 37
◦
C in a 5% CO
2
incubator [
81
]. Similarly, SH-SY5Y cells (1
×
10
4
cells/well and viability
>80%) were seeded in 96-well plates for 24 h, then treated with GSO or
α
-tocopherol (12.5–200
µ
g/mL)
or DMSO for 24 h at 37
◦
C. Afterwards, the treated cells were incubated with 10
µ
M DCFH-DA at
37
◦
C for 30 min, washed twice with PBS solution and challenged with 100
µ
M H
2
O
2
. The FI values
were then measured at wavelengths of
λex
485 nm and
λem
530 nm with 1-h kinetic mode using a flow
cytometer (Guava®easyCyte, Merck, Darmstadt, Germany).
4.7. Analysis Serum Lipids in Guava Seed Oil-Fed Rats
The protocol for the study involving animals was approved by the Ethical Committee for Animal
Studies of the Medical Faculty, Chiang Mai University (Protocol Number 43/2558). Wistar rats
were randomly divided into 4 groups (5 male and 5 female rats in each group), and fed with a
nutritionally-balanced commercial diet (No. C.P. 082, Perfect Companion Group Co. Ltd., Bangkok,
Thailand) comprised of crude protein 24%, fat 4.5%, fiber 5%, minerals (Ca 1.0%, Na 0.20%, K 1.17%, Mg
0.23%, Mn 171 ppm, Cu 22 ppm, Zn 100 ppm, Fe 180 ppm, Se 0.1 ppm) and vitamins (A 20,000 IU/kg, D
4000 IU/kg, E 100 mg/kg, B
1
5 mg/kg, B
2
20 mg/kg, B
6
20 mg/kg, B
12
0.036 mg/kg, niacin 100 mg/kg, folic
acid 6 mg/kg, biotin 0.4 mg/kg, pantothenic acid 60 mg/kg). The rats were orally administered with DI,
CO [30 g linoleic acid equivalent (LAE)/kg], and GSO (6 g LAE and 30 g LAE/kg) for 90 d. Fasting

Molecules 2020,25, 2474 17 of 23
blood was collected on days 0 and 90, and serum was separated for the analysis of total cholesterol and
triglyceride levels using a Randox
®
automated analyzer (Randox Laboratories Ltd., County Antrim,
United Kingdom) according to the manufacturer’s instructions.
In addition, serum fatty acid levels were quantified using the high-performance liquid
chromatography/fluorescence detection (HPLC/FLD) method [
82
]. With regard to the assay, serum
(100
µ
L) was firstly spiked with an internal standard of 5 mM heptadecanoic acid (20
µ
L), and then
incubated with Dole’s reagent (isopropanol: n-heptane: 2 M phosphoric acid =40:10:1 by volume)
(500
µ
L) at room temperature for 5–10 min. Secondly, the mixture was incubated with n-heptane (200
µ
L)
and water (300
µ
L), and centrifuged at 1000
×
gfor 5 min. Thirdly, the upper-layer heptane extract (200
µ
L) was aspirated, evaporated using nitrogen gas, and incubated with a derivatizing reagent (200
µ
L)
made of 10 mg Br-MMC, 26.5 mg 18-crown-6 and 100 mg potassium carbonate in 10 mL of acetonitrile
at 60
◦
C for 15 min, in order to produce a fluorogenic methyl-7-methoxycoumarin fatty acid (MMC-FA)
derivative. The derivative solution was passed through a 0.45
µ
m nylon-membrane filter and analyzed
using the HPLC/FLD system. The conditions included a column (C18 type,
4.6 mm ×250 mm
, 5
µ
m
particle size, Agilent Technologies, Santa Clara, California, United States), a mobile-phase solvent
(acetonitrile:DI =85:15, v/v), a flow rate of 1.5 mL/min, fluorescence detection (
λex
325 nm,
λem
398 nm)
and a data recorder/integrator using Millenium 32 HPLC Software (version 3.2, Waters Corporation,
Milford, MA, USA, 2000). Serum fatty acid values were identified by comparison with the specific
T
R
of the authentic fatty acids including
α
-linolenic, arachidonic, palmitoleic, stearic and oleic acids.
Concentrations of serum fatty acids were determined from the standard curves constructed from
different concentrations.
4.8. Statistical Analysis
Data were analyzed using the SPSS program (IBM SPSS Statistics V 22.0, (IBM Coporation, Ormonk,
NY, USA, 2013 shared license by Chiang Mai University) and are presented as mean
±
standard
deviation (SD) or mean
±
standard error of mean (SEM) values. Statistical significance was determined
using one-way analysis of variance (post-hoc =Tukey-HSD). p<0.05 was considered significant.
5. Conclusions
In conclusion, our studies show that guava seed oil (Psidium guajava) contains phenolic compounds
and phytosterols, which are free-radical scavenging and have protective effects against cell damage in
hepatic- and neuro-cell lines. Chronic oral administration to rats at high doses was associated with
significant lowering of plasma triglycerides, without alteration in serum cholesterol, similar to that
seen with corn oil administration at the same dose. The potential clinical relevance of the findings
merits further investigation.
Supplementary Materials:
The following are available online. Figure S1: HPLC-ESI-QTOF/MS for lipids of
GSO. Figure S2: GC/MS for phytosterols of GSO. Figure S3: (
a
) LC-ESI/MS for phenolics of GSO; (
b
) LM/MS for
authentic phenolics. Figure S4: HPLC FLD of authentic FA (100 uM) E2.
Author Contributions:
A.P. designed and conducted experiments, analyzed data, and wrote a draft manuscript;
N.U. designed and advised on experiments of food and natural products. C.U. designed experiments and offered
comments; A.C. supplied GSO samples and information; J.B.P. supervised the study of free-radical scavenging
effects of GSO in HepG2 cells and proofread the revised manuscript; S.S. conceived of and designed the study &
experiments, and contributed to the discussion and the proofreading of the manuscript. All authors have read
and approved of the final draft of the manuscript.
Funding:
This work was funded by the Research and Researchers for Industries (RRI) PhD. Program, Thailand
Science Research and Innovation (formerly Thailand Research Fund) (PHD56I0036); British Council-Newton Fund
(PhD. Placement) through Miss Adchara Prommaban; and the Faculty of Medicine Endowment Fund, Chiang Mai
University through Associate Professor Somdet Srichairatanakool, PhD (015/2560).
Acknowledgments:
We gratefully acknowledge the Research and Researchers for Industries (RRI) Program
through Adchara Prommaban (PHD56I0036) of Thailand Science Research and Innovation (formerly Thailand
Research Fund), Newton Fund (Placement of Adchara Prommaban) through Thailand British Council and Thailand
Research Fund, the Faculty of Medicine Endowment Fund, Chiang Mai University through Associate Professor

Molecules 2020,25, 2474 18 of 23
Somdet Srichairatanakool, for providing funding, as well as Tipco BioTech Company, Prachuapkhirikhan, Thailand
for supplying the guava seed oil. We thank Russell K. Hollis at the Language Center, Chiang Mai University and
John B. Porter for the proofreading of the manuscript.
Conflicts of Interest: The authors declare no conflict of interest.
Abbreviations
AA arachidonic acid
ALA α-linolenic acid
BF3boron trifluoride
Br-MMC 4-bromomethyl-7-methoxycoumarin
CO corn oil
DCF dichlorofluorescein
DCFH-DA 20,70-dichlorodihydrofluorescein diacetate
DI deionized water
DMEM Dulbecco’s modified Eagle medium
DPPH 1,1-diphenyl-2-picrylhydrazyl
EC50 a half effective concentration
ESI electrospray ionization
F female
FBS fetal bovine serum
FI fluorescent intensity
GAE gallic acid equivalent
GC/MS gas chromatography/mass spectrometry
GSO guava seed oil
HPLC-ESI/MS high-performance liquid chromatography-electrospray ionization/mass spectrometry
HPLC-ESI-QTOF/MS high-performance liquid chromatography-electrospray ionization-quadrupole time of
flight/mass spectrometry
HepG2 human hepatocellular carcinoma cells
HPLC/FLD high-performance liquid chromatography/fluorescence detection
H2O2hydrogen peroxide
LA linoleic acid
LAE linoleic acid equivalent
LOD limit of detection
LOQ limit of quantitation
M male
MMC-FA methyl-7-methoxycoumarin fatty acid
MSTFA N-methyl-N-trimethylsilyltrifluoroacetamide
m/zmass to charge ratio
ND not determined
OA oleic acid
OD optical density
P. Psidium
P-12 nepheochromocytoma cell
PA palmitic acid
PDA photodiode array
PLA palmitoleic acid
Ppm part per million
PUFA polyunsaturated fatty acid
ROS reactive oxygen species
SA stearic acid

Molecules 2020,25, 2474 19 of 23
SD standard deviation
SEM standard error of mean
SH-SY5Y and SK-N-SH human neuroblastoma cells
TC total cholesterol
TE Trolox equivalent
TG triglyceride
TIC total ion chromatogram
TMS trimethylsilyl
Trolox 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid
trypsin-EDTA trypsin-ethylene diamine tetraacetic acid
TRretention time
References
1.
Gutierrez, R.M.; Mitchell, S.; Solis, R.V. Psidium guajava: A review of its traditional uses, phytochemistry and
pharmacology. J. Ethnopharmacol. 2008,117, 1–27. [CrossRef]
2.
Morais-Braga, M.F.B.; Carneiro, J.N.P.; Machado, A.J.T.; dos Santos, A.T.L.; Sales, D.L.; Lima, L.F.;
Figueredo, F.G.; Coutinho, H.D.M. Psidium guajava L., from ethnobiology to scientific evaluation: Elucidating
bioactivity against pathogenic microorganisms. J. Ethnopharmacol.
2016
,194, 1140–1152. [CrossRef] [PubMed]
3.
Conde Garcia, E.A.; Nascimento, V.T.; Santiago Santos, A.B. Inotropic effects of extracts of Psidium guajava L.
(guava) leaves on the guinea pig atrium. Braz. J. Med. Biol. Res. 2003,36, 661–668. [CrossRef] [PubMed]
4.
Ojewole, J.A.O. Hypoglycemic and hypotensive effects of Psidium guajava Linn. (Myrtaceae) leaf aqueous
extract. Methods Find. Exp. Clin. Pharm. 2006,27, 689–695. [CrossRef] [PubMed]
5.
Ojewole, J.A.O.; Awe, E.O.; Chiwororo, W.D.H. Antidiarrhoeal activity of Psidium guajava Linn. (Myrtaceae)
leaf aqueous extract in rodents. J. Smooth Muscle Res. 2008,44, 195–207. [CrossRef] [PubMed]
6.
Deguchi, Y.; Miyazaki, K. Anti-hyperglycemic and anti-hyperlipidemic effects of guava leaf extract.
Nutr. Metab. 2010,7, 1–10. [CrossRef] [PubMed]
7.
Adesida, A.; Farombi, E.O. Free radical scavenging activities of guava extract
in vitro
.Afr. J. Med. Med. Sci.
2012,41, 81–90.
8.
Sen, S.S.; Sukumaran, V.; Giri, S.S.; Park, S.C. Flavonoid fraction of guava leaf extract attenuates
lipopolysaccharide-induced inflammatory response via blocking of NF-kappaB signalling pathway in
Labeo rohita macrophages. Fish. Shellfish Immunol. 2015,47, 85–92. [CrossRef]
9.
Bontempo, P.; Doto, A.; Miceli, M.; Mita, L.; Benedetti, R.; Nebbioso, A.; Veglione, M.; Rigano, D.; Cioffi, M.;
Sica, V.; et al. Psidium guajava L. anti-neoplastic effects: Induction of apoptosis and cell differentiation.
Cell Prolif. 2012,45, 22–31. [CrossRef]
10.
Nair, S.; Nagar, R.; Gupta, R. Antioxidant phenolics and flavonoids in common Indian foods. J. Assoc.
Physicians India 1998,46, 708–710.
11.
Opute, F.I. The component fatty acids of Psidium guajava seed fats. J. Sci. Food Agric.
1978
,29, 737–738.
[CrossRef]
12.
Bernardino-Nicanor, A.; Anon, M.C.; Scilingo, A.A.; Davila-Ortiz, G. Functional properties of guava seed
glutelins. J. Agric. Food Chem. 2005,53, 3613–3617. [CrossRef]
13.
Chang, Y.P.; Tan, M.P.; Lok, W.L.; Pakianathan, S.; Supramaniam, Y. Making use of guava seed (Psidium
guajava L): The effects of pre-treatments on its chemical composition. Plant. Foods Hum. Nutr.
2014
,69, 43–49.
[CrossRef]
14.
Michael, H.N.; Salib, J.Y.; Ishak, M.S. Acylated flavonol glycoside from Psidium gauijava L. seeds. Die. Pharm.
2002,57, 859–860.
15.
Biegelmeyer, R.; Andrade, J.M.M.; Aboy, A.L.; Apel, M.A.; Dresch, R.R.; Marin, R.; Raseira, M.d.C.B.;
Henriques, A.T. Comparative analysis of the chemical composition and antioxidant activity of red (Psidium
cattleianum) and yellow (Psidium cattleianum var. lucidum) strawberry guava fruit. J. Food Sci.
2011
,76,
C991–C996. [CrossRef] [PubMed]
16.
Salib, J.Y.; Michael, H.N. Cytotoxic phenylethanol glycosides from Psidium guaijava seeds. Phytochem
2004
,
65, 2091–2093. [CrossRef] [PubMed]

Molecules 2020,25, 2474 20 of 23
17.
Malacrida, C.R.; Jorge, N. Fatty acids and some antioxidant compounds of Psidium guaijava seed oil.
Acta Aliment. 2013,42, 371–378. [CrossRef]
18.
Arain, A.; Sherazi, S.T.H.; Mahesar, S.A.; Sirajuddin. Spectroscopic and chromatographic evaluation of
solvent extracted guava seed oil. Int. J. Food Prop. 2017,20, S556–S563. [CrossRef]
19.
Garg, S.; Talwar, G.P.; Upadhyay, S.N. Comparison of extraction procedures on the immunocontraceptive
activity of neem seed extracts. J. Ethnopharmacol. 1994,44, 87–92. [CrossRef]
20.
Adeleke, M.A.; Popoola, S.A.; Agbaje, W.B.; Adewale, B.; Adeoye, M.D.; Jimoh, W.A. Larvicidal efficacy of
seed oils of Pterocarpus santalinoides and tropical Manihot species against Aedes aegypti and effects on aquatic
fauna. Tanzan J. Health Res. 2009,11, 250–252. [CrossRef]
21.
Hossain, S.J.; Islam, M.R.; Pervin, T.; Iftekharuzzaman, M.; Hamdi, O.A.A.; Mubassara, S.; Saifuzzaman, M.;
Shilpi, J.A. Antibacterial, anti-diarrhoeal, analgesic, cytotoxic activities, and GC-MS profiling of Sonneratia
apetala (Buch.-Ham.) Seed. Prev. Nutr. Food Sci. 2017,22, 157–165.
22.
Huang, H.Y.; Chang, C.K.; Tso, T.K.; Huang, J.J.; Chang, W.W.; Tsai, Y.C. Antioxidant activities of various
fruits and vegetables produced in Taiwan. Int. J. Food Sci. Nutr. 2004,55, 423–429. [CrossRef]
23.
Pelegrini, P.B.; Murad, A.M.; Silva, L.P.; dos Santos, R.C.P.; Costa, F.T.; Tagliari, P.D.; Bloch Jr, C.; Noronha, E.F.;
Miller, R.N.G.; Franco, O.L. Identification of a novel storage glycine-rich peptide from guava (Psidium guajava)
seeds with activity against Gram-negative bacteria. Peptides 2008,29, 1271–1279. [CrossRef]
24.
Prommaban, A.; Utama-Ang, N.; Chaikitwattana, A.; Uthaipibull, C.; Srichairatanakool, S. Linoleic acid-rich
guava seed oil: Safety and bioactivity. Phytother. Res. 2019,33, 2749–2764. [CrossRef]
25.
Chen, H.J.; Inbaraj, B.S.; Chen, B.H. Determination of phenolic acids and flavonoids in Taraxacum formosanum
Kitam by liquid chromatography-tandem mass spectrometry coupled with a post-column derivatization
technique. Int. J. Mol. Sci. 2012,13, 260–285. [CrossRef]
26.
Yu, F.; Qian, H.; Zhang, J.; Sun, J.; Ma, Z. Simultaneous quantification of eight organic acid components
in Artemisia capillaris Thunb (Yinchen) extract using high-performance liquid chromatography coupled
with diode array detection and high-resolution mass spectrometry. J. Food Drug Anal.
2018
,26, 788–795.
[CrossRef]
27.
Baeza, G.; Sarria, B.; Bravo, L.; Mateos, R. Exhaustive qualitative LC-DAD-MS
n
analysis of arabica green
coffee beans: Cinnamoyl-glycosides and cinnamoylshikimic acids as new polyphenols in green coffee.
J. Agric. Food Chem. 2016,64, 9663–9674. [CrossRef] [PubMed]
28.
Plazonic, A.; Bucar, F.; Males, Z.; Mornar, A.; Nigovic, B.; Kujundzic, N. Identification and quantification
of flavonoids and phenolic acids in burr parsley (Caucalis platycarpos L.), using high-performance liquid
chromatography with diode array detection and electrospray ionization mass spectrometry. Molecules
2009
,
14, 2466–2490. [CrossRef] [PubMed]
29.
Ma, J.Y.; Zhou, X.; Fu, J.; Hu, T.; Or, P.M.; Feng, R.; He, C.Y.; Chen, W.J.; Zhang, X.; Chen, Y.; et al. Metabolite
profiling analysis of FR429, an ellagitannin purified from Polygonum capitatum, in rat and human liver
microsomes, cytosol and rat primary hepatocytes
in vitro
.Chem. Biol. Interact.
2014
,220, 33–40. [CrossRef]
[PubMed]
30.
Ma, J.Y.; Zhou, X.; Fu, J.; He, C.Y.; Feng, R.; Huang, M.; Shou, J.W.; Zhao, Z.X.; Li, X.Y.; Zhang, L.; et al.
In vivo
metabolite profiling of a purified ellagitannin isolated from polygonum capitatum in rats. Molecules
2016,21, 1110. [CrossRef] [PubMed]
31.
Simirgiotis, M.J.; Benites, J.; Areche, C.; Sepulveda, B. Antioxidant capacities and analysis of phenolic
compounds in three endemic Nolana species by HPLC-PDA-ESI-MS. Molecules
2015
,20, 11490–11507.
[CrossRef] [PubMed]
32.
Xia, J.X.; Zhao, B.B.; Zan, J.F.; Wang, P.; Chen, L.L. Simultaneous determination of phenolic acids and
flavonoids in Artemisiae Argyi Folium by HPLC-MS/MS and discovery of antioxidant ingredients based on
relevance analysis. J. Pharm. Biomed. Anal. 2019,175, 112734. [CrossRef]
33.
Hossain, M.B.; Rai, D.K.; Brunton, N.P.; Martin-Diana, A.B.; Barry-Ryan, C. Characterization of phenolic
composition in Lamiaceae spices by LC-ESI-MS/MS. J. Agric. Food Chem.
2010
,58, 10576–10581. [CrossRef]
[PubMed]
34.
Tang, J.; Dunshea, F.R.; Suleria, H.A.R. LC-ESI-QTOF/MS characterization of phenolic compounds from
medicinal plants (hops and juniper berries) and their antioxidant activity. Foods
2020
,9, 7. [CrossRef]
[PubMed]

Molecules 2020,25, 2474 21 of 23
35.
Gu, C.; Howell, K.; Dunshea, F.R.; Suleria, H.A.R. LC-ESI-QTOF/MS characterisation of phenolic acids and
flavonoids in polyphenol-rich fruits and vegetables and their potential antioxidant activities. Antioxidants
2019,8, 405. [CrossRef] [PubMed]
36.
Jimenez, C.; Crews, P. Novel marine sponge amino acids, 10. Xestoaminols from Xestospongia sp. J. Nat. Prod.
1990,53, 978–982. [CrossRef] [PubMed]
37.
Phillip, D.; Hobe, S.; Paulsen, H.; Molnar, P.; Hashimoto, H.; Young, A.J. The binding of Xanthophylls to the
bulk light-harvesting complex of photosystem II of higher plants. A specific requirement for carotenoids
with a 3-hydroxy-beta-end group. J. Biol. Chem. 2002,277, 25160–25169. [CrossRef]
38.
Burdi, D.K.; Samejo, M.Q.; Bhanger, M.I.; Khan, K.M. Fatty acid composition of Abies pindrow (west himalayan
fir). Pak. J. Pharm. Sci. 2007,20, 15–19.
39.
Mohamed, M.H.; Wilson, L.D.; Headley, J.V. Tunable polymeric sorbent materials for fractionation of model
naphthenates. J. Phys. Chem. B 2013,117, 3659–3666. [CrossRef]
40.
Nenoff, P.; Haustein, U.F.
In vitro
activity of phytosphingosines against Malassezia furfur and Candida albicans.
Acta Derm. Venereol. 2002,82, 170–173. [CrossRef]
41.
Oh, M.J.; Cho, Y.H.; Cha, S.Y.; Lee, E.O.; Kim, J.W.; Kim, S.K.; Park, C.S. Novel phytoceramides containing
fatty acids of diverse chain lengths are better than a single C18-ceramide N-stearoyl phytosphingosine to
improve the physiological properties of human stratum corneum. Clin. Cosmet. Investig. Derm.
2017
,10,
363–371. [CrossRef] [PubMed]
42.
Dasyam, N.; Munkacsi, A.B.; Fadzilah, N.H.; Senanayake, D.S.; O’Toole, R.F.; Keyzers, R.A. Identification
and bioactivity of 3-epi-xestoaminol C isolated from the New Zealand brown alga Xiphophora chondrophylla.
J. Nat. Prod. 2014,77, 1519–1523. [CrossRef]
43.
Hormaetxe, K.; Hernandez, A.; Becerril, J.M.; Garcia-Plazaola, J.I. Role of red carotenoids in photoprotection
during winter acclimation in Buxus sempervirens leaves. Plant. Biol. 2004,6, 325–332. [CrossRef] [PubMed]
44.
Da Silva, A.C.; Jorge, N. Bioactive compounds of oils extracted from fruits seeds obtained from agroindustrial
waste. Eur. J. Lipid Sci. Tech. 2016,118, 1600024. [CrossRef]
45.
Beveridge, T.H.; Li, T.S.; Drover, J.C. Phytosterol content in American ginseng seed oil. J. Agric. Food Chem.
2002,50, 744–750. [CrossRef] [PubMed]
46.
Hassan, E.M.; Matloub, A.A.; Aboutabl, M.E.; Ibrahim, N.A.; Mohamed, S.M. Assessment of
anti-inflammatory, antinociceptive, immunomodulatory, and antioxidant activities of Cajanus cajan L.
seeds cultivated in Egypt and its phytochemical composition. Pharm. Biol. 2015,54, 1380–1391. [CrossRef]
47.
Hamedi, A.; Ghanbari, A.; Razavipour, R.; Saeidi, V.; Zarshenas, M.M.; Sohrabpour, M.; Azari, H. Alyssum
homolocarpum seeds: Phytochemical analysis and effects of the seed oil on neural stem cell proliferation and
differentiation. J. Nat. Med. 2015,69, 387–396. [CrossRef]
48.
Kozlowska, M.; Gruczynska, E.; Scibisz, I.; Rudzinska, M. Fatty acids and sterols composition, and antioxidant
activity of oils extracted from plant seeds. Food Chem. 2016,213, 450–456. [CrossRef]
49.
Kalleli, F.; Bettaieb Rebey, I.; Wannes, W.A.; Boughalleb, F.; Hammami, M.; Saidani Tounsi, M.; M’Hamdi, M.
Chemical composition and antioxidant potential of essential oil and methanol extract from Tunisian and
French fennel (Foeniculum vulgare Mill.) seeds. J. Food Biochem. 2019,43, e12935. [CrossRef]
50.
Kayano, S.; Yamada, N.F.; Suzuki, T.; Ikami, T.; Shioaki, K.; Kikuzaki, H.; Mitani, T.; Nakatani, N. Quantitative
evaluation of antioxidant components in prunes (Prunus domestica L.). J. Agric. Food Chem.
2003
,51, 1480–1485.
[CrossRef]
51.
Costa, F.; Jerz, G.; Figueiredo Fde, S.; Winterhalter, P.; Leitao, G.G. Solvent system selectivities in countercurrent
chromatography using Salicornia gaudichaudiana metabolites as practical example with off-line electrospray
mass-spectrometry injection profiling. J. Chromatogr. A 2015,1385, 20–27. [CrossRef] [PubMed]
52.
Gonzalez-Cortazar, M.; Herrera-Ruiz, M.; Zamilpa, A.; Jimenez-Ferrer, E.; Marquina, S.; Alvarez, L.;
Tortoriello, J. Anti-inflammatory activity and chemical profile of Galphimia glauca.Planta Med.
2013
,80, 90–96.
[CrossRef] [PubMed]
53.
Jho, E.H.; Kang, K.; Oidovsambuu, S.; Lee, E.H.; Jung, S.H.; Shin, I.S.; Nho, C.W. Gymnaster koraiensis and its
major components, 3,5-di-O-caffeoylquinic acid and gymnasterkoreayne B, reduce oxidative damage induced
by tert-butyl hydroperoxide or acetaminophen in HepG2 cells. BMB Rep.
2013
,46, 513–518. [CrossRef]
[PubMed]

Molecules 2020,25, 2474 22 of 23
54.
Lee, S.G.; Lee, H.; Nam, T.G.; Eom, S.H.; Heo, H.J.; Lee, C.Y.; Kim, D.O. Neuroprotective effect of caffeoylquinic
acids from Artemisia princeps Pampanini against oxidative stress-induced toxicity in PC-12 cells. J. Food Sci.
2011,76, C250–C256. [CrossRef] [PubMed]
55.
Kayano, S.; Kikuzaki, H.; Fukutsuka, N.; Mitani, T.; Nakatani, N. Antioxidant activity of prune (Prunus
domestica L.) constituents and a new synergist. J. Agric. Food Chem. 2002,50, 3708–3712. [CrossRef]
56.
Menichini, F.; Losi, L.; Bonesi, M.; Pugliese, A.; Loizzo, M.R.; Tundis, R. Chemical profiling and
in vitro
biological effects of Cardiospermum halicacabum L. (Sapindaceae) aerial parts and seeds for applications in
neurodegenerative disorders. J. Enzym. Inhib. Med. Chem. 2013,29, 677–685. [CrossRef]
57.
Kenny, O.; Smyth, T.J.; Hewage, C.M.; Brunton, N.P. Antioxidant properties and quantitative UPLC-MS
analysis of phenolic compounds from extracts of fenugreek (Trigonella foenum-graecum) seeds and bitter
melon (Momordica charantia) fruit. Food Chem. 2013,141, 4295–4302. [CrossRef]
58.
Ablat, A.; Halabi, M.F.; Mohamad, J.; Hasnan, M.H.; Hazni, H.; Teh, S.H.; Shilpi, J.A.; Mohamed, Z.; Awang, K.
Antidiabetic effects of Brucea javanica seeds in type 2 diabetic rats. BMC Complement. Altern. Med.
2017
,17,
94–107. [CrossRef]
59.
Arranz, S.; Cert, R.; P
é
rez-Jim
é
nez, J.; Cert, A.; Saura-Calixto, F. Comparison between free radical scavenging
capacity and oxidative stability of nut oils. Food Chem. 2008,110, 985–990. [CrossRef]
60.
Gorinstein, S.; Martin-Belloso, O.; Katrich, E.; Lojek, A.; Ciz, M.; Gligelmo-Miguel, N.; Haruenkit, R.;
Park, Y.S.; Jung, S.T.; Trakhtenberg, S. Comparison of the contents of the main biochemical compounds and
the antioxidant activity of some Spanish olive oils as determined by four different radical scavenging tests.
J. Nutr. Biochem. 2003,14, 154–159. [CrossRef]
61.
Assiri, A.M.; Hassanien, M.F. Bioactive lipids, radical scavenging potential, and antimicrobial properties of
cold pressed clove (Syzygium aromaticum) oil. J. Med. Food 2013,16, 1046–1056. [CrossRef] [PubMed]
62.
Ali, Q.; Anwar, F.; Ashraf, M.; Saari, N.; Perveen, R. Ameliorating effects of exogenously applied proline on
seed composition, seed oil quality and oil antioxidant activity of maize (Zea mays L.) under drought stress.
Int. J. Mol. Sci. 2013,14, 818–835. [CrossRef]
63.
Alu’datt, M.H.; Rababah, T.; Alhamad, M.N.; Al-Mahasneh, M.A.; Almajwal, A.; Gammoh, S.; Ereifej, K.;
Johargy, A.; Alli, I. A review of phenolic compounds in oil-bearing plants: Distribution, identification and
occurrence of phenolic compounds. Food Chem. 2017,218, 99–106. [CrossRef] [PubMed]
64.
Zhang, L.Z.; Liu, R.H. Phenolic and carotenoid profiles and antiproliferative activity of foxtail millet.
Food Chem. 2014,174, 495–501. [CrossRef]
65.
Zhang, L.; Liu, R.; Niu, W. Phytochemical and antiproliferative activity of proso millet. PLoS ONE
2014
,
9, e104058. [CrossRef] [PubMed]
66.
Kim, S.S.; Park, R.Y.; Jeon, H.J.; Kwon, Y.S.; Chun, W. Neuroprotective effects of 3,5-dicaffeoylquinic acid
on hydrogen peroxide-induced cell death in SH-SY5Y cells. Phytother. Res.
2005
,19, 243–245. [CrossRef]
[PubMed]
67.
Gonzalez-Burgos, E.; Liaudanskas, M.; Viskelis, J.; Zvikas, V.; Janulis, V.; Gomez-Serranillos, M.P. Antioxidant
activity, neuroprotective properties and bioactive constituents analysis of varying polarity extracts from
Eucalyptus globulus leaves. J. Food Drug Anal. 2018,26, 1293–1302. [CrossRef] [PubMed]
68.
Shahat, A.A.; Hidayathulla, S.; Khan, A.A.; Alanazi, A.M.; Al Meanazel, O.T.; Alqahtani, A.S.; Alsaid, M.S.;
Hussein, A.A. Phytochemical profiling, antioxidant and anticancer activities of Gastrocotyle hispida growing
in Saudi Arabia.Acta Trop. 2019,191, 243–247. [CrossRef]
69.
Chung, M.J.; Lee, S.; Park, Y.I.; Lee, J.; Kwon, K.H. Neuroprotective effects of phytosterols and flavonoids
from Cirsium setidens and Aster scaber in human brain neuroblastoma SK-N-SH cells. Life Sci.
2016
,148,
173–182. [CrossRef]
70.
Asadi, F.; Shahriari, A.; Chahardah-Cheric, M. Effect of long-term optional ingestion of canola oil, grape seed
oil, corn oil and yogurt butter on serum, muscle and liver cholesterol status in rats. Food Chem. Toxicol.
2010
,
48, 2454–2457. [CrossRef]
71.
Mekonnen, Z.; Gebreselema, A.; Abere, Y. Effect of locally manufactured niger seed oil on lipid profile
compared to imported palm and sunflower oils on rat models. J. Lipids 2018,2018, 8. [CrossRef] [PubMed]
72.
Sakono, M.; Yoshida, K.; Yahiro, M. Combined effects of dietary protein and fat on lipid metabolism in rats.
J. Nutr. Sci. Vitam. 1993,39, 335–343. [CrossRef]

Molecules 2020,25, 2474 23 of 23
73.
Jurgonski, A.; Juskiewicz, J.; Zdunczyk, Z.; Krol, B. Caffeoylquinic acid-rich extract from chicory seeds
improves glycemia, atherogenic index, and antioxidant status in rats. Nutrition
2011
,28, 300–306. [CrossRef]
[PubMed]
74.
Pieszka, M.; Tombarkiewicz, B.; Roman, A.; Migdal, W.; Niedziolka, J. Effect of bioactive substances found in
rapeseed, raspberry and strawberry seed oils on blood lipid profile and selected parameters of oxidative
status in rats. Environ. Toxicol. Pharm. 2013,36, 1055–1062. [CrossRef] [PubMed]
75.
Zahir, A.A.; Rahuman, A.A.; Kamaraj, C.; Bagavan, A.; Elango, G.; Sangaran, A.; Kumar, B.S. Laboratory
determination of efficacy of indigenous plant extracts for parasites control. Parasitol. Res.
2009
,105, 453–461.
[CrossRef] [PubMed]
76.
Lee, J.Y.; Park, N.H.; Lee, W.; Kim, E.H.; Jin, Y.H.; Seo, E.K.; Hong, J. Comprehensive chemical profiling
of Pinellia species tuber and processed Pinellia tuber by gas chromatography-mass spectrometry and liquid
chromatography-atmospheric pressure chemical ionization-tandem mass spectrometry. J. Chromatogr. A
2016,1471, 164–177. [CrossRef]
77.
Cuyckens, F.; Claeys, M. Optimization of a liquid chromatography method based on simultaneous electrospray
ionization mass spectrometric and ultraviolet photodiode array detection for analysis of flavonoid glycosides.
Rapid Commun. Mass Spectrom. 2002,16, 2341–2348. [CrossRef]
78. Kano, M.; Takayanagi, T.; Harada, K.; Makino, K.; Ishikawa, F. Antioxidative activity of anthocyanins from
purple sweet potato, Ipomoera batatas cultivar Ayamurasaki. Biosci. Biotechnol. Biochem.
2005
,69, 979–988.
[CrossRef]
79.
Pangjit, K.; Udomsuk, L.; Upanan, S.; Pongjanta, A.; Chansiw, N.; Srichairatanakool, S. Iron-chelating and
anti-hemolytic properties of ethanolic extract of lotus (Nelumbonucifera gaertn) leaves. J. Med. Assoc. Thai.
2016,99, S58–S66.
80.
Agarwala, S.; Rao B., N.; Mudholkar, K.; Bhuwania, R.; Satish Rao, B.S. Mangiferin, a dietary xanthone
protects against mercury-induced toxicity in HepG2 cells. Env. Toxicol. 2012,27, 117–127. [CrossRef]
81.
Shen, L.; Zhao, H.Y.; Du, J.; Wang, F. Anti-tumor activities of four chelating agents against human
neuroblastoma cells. In Vivo 2005,19, 233–236. [PubMed]
82.
Puttmann, M.; Krug, H.; von Ochsenstein, E.; Kattermann, R. Fast HPLC determination of serum free fatty
acids in the picomole range. Clin. Chem. 1993,39, 825–832. [CrossRef] [PubMed]
Sample Availability: Samples are available from the authors.
©
2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).