PHYTOCHEMICAL INVESTIGATION OF FLAVONOIDS IN …

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109

Jasim et al. World Journal of Pharmacy and Pharmaceutical Sciences

PHYTOCHEMICAL INVESTIGATION OF FLAVONOIDS IN

DIFFERENT FRUIT PARTS OF IRAQI SOUR ORANGE

Ali Rahman Jasim*, Ahmed Abbas Husseinˡ and Abdul- Mutalib A. G. Nasser²

*Department of Pharmacognosy, College of Pharmacy, University of Baghdad, Baghdad,

Iraq.

ˡDepartment of Pharmaceutics, College of Pharmacy, University of Baghdad, Baghdad, Iraq.

²Department of Pharmacognosy, Baghdad College of Pharmacy, Baghdad, Iraq.

ABSTRACT

Citrus aurantium of the Rutaceae family is growing widely in Iraq.

Literature survey revealed that there was no phytochemical study

concerning Citrus aurantium fruits in Iraq. Flavonoids from citrus

genus have become of particular importance due to their broad

spectrum of biological activities, including anticarcinogenic, anti-

inflammatory and antiatherogenic properties; therefore a research on

Iraqi Citrus aurantium fruits will be of important value. This study is

concerned with the extraction, identification, isolation and purification

of some biologically important flavonoids in different fruit parts of

Iraqi Citrus aurantium including peels, pulps and juice. Extraction was

carried out by Soxhlet apparatus. The isolated compounds were identified by melting points

(M.P.) measurement, thin layer chromatography (TLC), high performance liquid

chromatography (HPLC), Fourier transforms infrared spectroscopy (FT-IR), Elemental

microanalysis (CHN) and1H nuclear magnetic resonance spectroscopy (

1H-NMR) analysis.

The most suitable extraction and identification methods were fully described in this study.

This study confirms the presence of Neohesperidin, Naringin, Tangeretin and Nobiletin

and the absence of Hesperidin and Narirutin in different fruit parts of Iraqi Citrus

aurantium. The separated flavonoids have important medicinal, therapeutic and biological

activities.

KEYWORDS: Citrus aurantium, Neohesperidin, Naringin, Tangeretin, Nobiletin.

WORLD JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCES

SJIF Impact Factor 6.041

Volume 5, Issue 5, 109-128 Research Article ISSN 2278 – 4357

*Corresponding Author

Dr. Ali Rahman Jasim

Department of

Pharmacognosy, College of

Pharmacy, University of

Baghdad, Baghdad, Iraq.

Article Received on

01 March 2016,

Revised on 22 March 2016,

Accepted on 13 April 2016

DOI: 10.20959/wjpps20165-6682


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INTRODUCTION

Citrus aurantium (sour orange, also known as bitter orange or seville orange), is a well-

known Citrus species in Iraq where it is found in most Iraqi house gardens. It is a small tree,

about four meters tall[1]

, and because of its bitter and sour taste, it is not widely used as an

edible fruit. The juice of the fruit is used in salads for sour taste instead of lemon juice, and

the peel is used in the production of jam.[2]

Most of Citrus aurantium uses are medicinal

rather than culinary. In Asian herbal medicine, the dried fruit is used in treatment of digestive

disorders and in Western herbal medicine the dried fruit peels are used to stimulate appetite

and gastric secretion.[3]

In traditional Chinese medicine Citrus aurantium has been used for

thousands of years and such use could be an indirect marker of safety and efficacy.[4]

There is

thus a history of benign human consumption of Citrus aurantium fruit. Citrus aurantium has

a complex chemical makeup; it contains flavonoids, including neohesperidin, naringin,

tangeretin and nobiletin. Citrus aurantium also contains alkaloids and the most important and

major alkaloid is synephrine. Furanocoumarins are also present.[3]

Citrus flavonoids are

generally categorized into two groups, flavanone glycosides and polymethoxyflavones

(figure 1).

Flavanone Skeleton

Neohesperidin: R =Neohesperidose, R1=OH, R2 =CH3

Naringin: R =Neohesperidose, R1= R2 =H

Polymethoxyflavone Skeleton

Tangeretin: R= OCH3, R1=R2=H

Nobiletin: R= R1= OCH3, R2=H

Figure (1): Structures of some Citrus flavonoids.[5]

https://en.wikipedia.org/wiki/Synephrine#cite_note-64







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Flavonoids are a large group of various compounds.[6]

They are aromatic secondary plant

metabolites, derived from shikimate and phenylpropanoid pathways.[7]

Flavonoids are

important due to their pharmacological, physiological roles[8,9]

and their health benefits.[10]

They have a strong radical scavenging and antioxidant activity[11]

and are associated with

reduced risks for many chronic diseases[12]

; flavonoids can prevent some cardiovascular

disorders[13]

and certain kinds of cancers.[14]

They also have anti-inflammatory[15]

,

antimicrobial[16]

, antiviral[17]

, antiulcer[18]

and antiallergenic[19]

activities. Flavonoids can

inhibit platelet aggregation[20]

and have beneficial effects on capillary fragility.[21]

Epidemiological studies have shown an inverse association between the daily intake of some

flavonoids and the risk of certain chronic diseases and cancers, but further research is

required to assess the correlation between flavonoids consumption and human health

benefits.[22]

Recently, investigations of flavonoids in dietary sources have attracted an ever

growing interest due to their versatile well-established health benefits.[23]

There is no data on

the flavonoids composition of Iraqi Citrus aurantium, and to the best of our knowledge, there

has been no research into the flavonoid contents of Iraqi Citrus aurantium fruit peels, pulps

or juice. Therefore, the aim of this research is to study the bioactive flavonoid contents of

different fruit parts of Iraqi Citrus aurantium, so it can provides insight into how to explore

further the benefits of Citrus aurantium fruits for human health.

MATERIALS AND METHODS

Plant material: The ripe fruits of Iraqi Citrus aurantium were collected in January (2015)

from Baghdad in Iraq. The fruits were peeled and both peel (flavedo and albedo) and pulp

(juice sac and segment membrane) were air dried in the shade for two weeks (figure 2), then

the peels and pulps were pulverized by mechanical mills and weighed. The plant material

(Citrus aurantium fruit) was identified in the department of pharmacognosy /College of

pharmacy/ University of Baghdad and was authenticated by National Iraqi Herbarium.

Dried Peels after grinding Dried Pulps

Figure (2): Plant material



















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Preparation of juice sample: Although a large number of publications on the identification

of flavonoids in Citrus juices have already appeared over the past two decades[24]

, there is still

no available standardized procedure for sample extraction and preparation.[25]

The citrus fruit

juice was extracted by cutting the fruit in half and careful hand-squeezing to obtain the juice.

The juice was passed through a strainer to remove pulp and seeds. The freshly squeezed juice

was centrifuged for 10 min to separate the insoluble and the soluble fractions. Then the

supernatant was filtered.[26]

Extraction of peels and pulps: A one hundred gm. of dried powdered fruit peels and one

hundred gm. of dried powdered fruit pulps of Iraqi Citrus aurantium were defatted separately

with n-hexane for 24 hr. then filtered and allowed to dry at room temperature. The defatted

plant materials were packed separately in thimbles and placed in soxhlet extractors. Five

hundred ml of 80% methanol was used as a solvent and placed in a 1 liter round bottom flask

fitted with a soxhlet extractor. The extraction was continued for 12 hr. The extracts were

filtered and concentrated under reduced pressure to dryness using rotary evaporator at a

temperature not exceeding 40°C; the dry extracts were weighed and subjected to

identification, isolation and purification procedures.[27]

Identification of plant constituents by TLC: The methanolic extracts of fruit peels, pulps

and juice sample were examined by TLC, using readymade plates of silica gel GF254

(20×20cm) of 0.25mm thickness (MERCK).Detection was done by using UV light at 254 nm

and 366 nm. The flavonoids standards (Neohesperidin, Naringin, Hesperidin, Narirutin,

Tangeretin and Nobiletin) were purchased from Chengdu Biopurify Phytochemicals.

Developing solvent systems[28-33]

A 100 ml volume of solvent system was placed in a glass tank (22.5gm×22cm×7cm) and

covered with a glass lid and allowed to stand for 45 minutes before use. A small amount of

each extract (1 mg dissolved in 1 ml solvent) was applied with standard samples (1mg/ml) to

TLC plates manually, using capillary tubes, after development, the plates were examined

under UV light at 254 and 366 nm. Different solvent systems were used for the detection of

flavonoids in different fruit parts

S1= n- Hexane: n-Butanol (85:15).

S2=Ethyl acetate: Formic acid: Glacial acetic acid: Water (100:11:11:27).

S3= n-Butanol: Water (50:50).

S4= Chloroform: Methanol: Water (23:12:2).







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Isolation of the active constituents

Isolation of flavonoids was done by preparative layer chromatography, using glass plates of

20cm x 20cm which were coated with slurry of silica gel GF 254 (2mm thickness) using

Jobling laboratory division TLC coater. The peels extract was applied as a concentrated

solution in a row of spots using capillary tube. Reference standards were applied at the right

side of the baseline. The application of the extract was repeated four times in each plate, one

should wait after each application until all the solvent is evaporated. The detection was done

by using UV light at a wave length of 254 nm (figure 3). The bands corresponding to the

standards were scrapped out and collected in a beaker mixed with a suitable solvent, stirred

and left a side for overnight, then filtered, and the filtrate was evaporated to dryness to give

precipitate. Recrystallization from methanol was done to get pure compounds. S1= n-

Hexane: n-Butanol (85:15) was used as a mobile phase for the isolation of Tangeretin and

Nobiletin.

Figure (3): Preparative layer Chromatography of Tangeretin and Nobiletin.

Qualitative and quantitative estimation of flavonoids by HPLC

High Performance Liquid Chromatography (HPLC) method was used for qualitative and

quantitative estimation of flavonoids. HPLC analysis was carried out using (Knauer /

Germany). Identifications were made by Comparism of retention times obtained at identical

chromatographic conditions of analyzed samples and authentic standards. The HPLC

conditions are listed in table (1).


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Table (1): HPLC conditions[29, 34, 35]

Sample Mobile phase Column Flow rate Detection

Naringin Acetonitrile:0.1%Acetic acid in

Water (30:70)

C18 150mm ×

4.6mm/5um

0.8 ml/ min UV. Detector at

ƛ 284 nm

Tangeretin Acetonitrile : Water (45:55) C18 150mm ×

4.6mm/5um

1ml / min UV. Detector at

ƛ 326 nm

Neohesperidin Acetonitrile:0.1%Acetic acid in

Water (30:70)

C18 150mm ×

4.6mm/5um

0.8 ml/ min UV. Detector at

ƛ 284 nm

Nobiletin Acetonitrile : Water (45:55) C18 150mm ×

4.6mm/5um

1ml / min UV. Detector at

ƛ 326 nm

Narirutin Acetonitrile:0.1%Phosphoric

acid in Water (30:70)

C18 150mm ×

4.6mm/5um

0.8ml/min UV. Detector at

ƛ 284nm

Hesperidin Methanol : Water (60:40) C18 5mm ×150mm 1.5 ml/ min UV. Detector at

ƛ 345 nm

RESULTS AND DISCUSSION

Four flavonoids were detected in Iraqi Citrus aurantium fruits; these compounds were

designated as C1, C2, C3 and C4 and were found to be Neohesperidin, Naringin,

Tangeretin and Nobiletin respectively. Although certain studies indicate the presence of

Hesperidin and/or Narirutin in Citrus aurantium grown in other countries[36]

; Hesperidin

and Narirutin were not detected in all studied fruit parts of Iraqi Citrus aurantium (peels,

pulps and juice) even when different concentrations of extracts were used in both TLC and

HPLC; however this could be attributed to many factors including heterogeneity and

complexity of soil like soil texture, structure and depth; moisture content and aeration which

create a big differences in the active constituents even in the same country[37]

; also the

genetic origin, harvesting time, and the part used of the plant.[4]

Our results are in agreement

with those obtained from studies on Citrus aurantium grown in the United States of America

which also confirmed the absence of Hesperidin and Narirutin in Citrus aurantium.[38]

Neohesperidin and Naringin levels in Iraqi Citrus aurantium fruits are higher than those in

Citrus aurantium grown in China, United States of America and Japan.[39, 40]

The highest

concentrations of flavonoids were found in the peels (table 4). Polymethoxyflavones

(Tangeretin and Nobiletin) occur less frequently in citrus juices due to their lipophilic

nature (lack of sugar moieties) and hence their low water solubility. The presence of traces of

Polymethoxyflavones (Tangeretin and Nobiletin) in hand-squeezed juices of Iraqi Citrus

aurantium may probably be due to the contamination of juices with the peel constituents

during sample preparation.[41]

C3 and C4 (Polymethoxyflavones) were isolated by preparative

layer chromatography and were identified by M.P. measurement, TLC, HPLC, FT-IR

spectroscopy, CHN analysis and 1H-NMR analysis; C1 and C2 (flavanone glycosides) were






115


identified by TLC and HPLC. The results obtained from HPLC analysis regarding the

qualitative identification of Citrus aurantium fruit constituents were identical with those

obtained from TLC.

Selection of the best developing solvent systems in TLC

S3= n-Butanol: Water (50:50)[33]

was found to be the best developing solvent system for the

separation of Neohesperidin and Naringin and S1= n- Hexane: n-Butanol (85:15) was

found to be the best developing solvent system for the separation of Tangeretin and

Nobiletin.

Identification of the separated compounds (Flavonoids)

Measuring melting points

The isolated compounds were identified from their sharp melting points using electro–

thermal melting point apparatus (Stuart / UK), Compound 3 (C3) showed a melting point of

(154-157°C) compared to Tangeretin standard melting point (155-156°C) and Compound 4

(C4) showed a melting point of (137-138°C) compared to Nobiletin standard melting point

(137-138°C).

TLC

TLC confirms the presence of two flavanone glycosides (Neohesperidin (C1) and Naringin

(C2)) and two polymethoxylated flavones (Tangeretin (C3) and Nobiletin (C4)) and the

absence of Hesperidin and Narirutin in all studied fruit parts of Iraqi Citrus aurantium

(peels, pulps and juice), using different developing solvent systems (figure 4), The flavonoids

appeared as a single spots having the same color and Rf values as that of reference standards

(table 2).


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.

Figure (4): TLC of samples obtained from different fruit parts of Iraqi Citrus aurantium

H: Hesperidin Standard.

NR: Narirutin Standard.

NS:Neohesperidin Standard.

NG: Naringin Standard.

F1a: Methanolic extract of Citrus aurantium fruit peels.

FP1a: Methanolic extract of Citrus aurantium fruit Pulps.

Ja: Citrus aurantium juice sample.

Table (2): Rf Values of Standards and corresponding compounds in extracts obtained

from different fruit parts of Iraqi Citrus aurantium using different developing solvent

systems.

Solvent System S1 S2 S3 S4

Rf Value of Neohesperidin Standard 0.533 0.646 0.891

Rf Value of C1 in Peels 0.530 0.646 0.891

Rf Value of C1 in Pulps 0.530 0.646 0.891

Rf Value of C1 in Juice 0.532 0.640 0.880

Rf Value of Naringin Standard 0.577 0.70 0.955




Rf Value of Tangeretin Standard 0.30 0.913 0.733




Rf Value of Nobiletin Standard 0.157 0.88 0.678





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HPLC

HPLC confirms the presence of Neohesperidin, Naringin, Tangeretin and Nobiletin and

the absence of Hesperidin and Narirutin in Iraqi Citrus aurantium fruits (peels, pulps and

juice) as shown in table (3) and Figures (from 5a to 5n). The percentages of the compounds

in peels and pulps were given in table (4).

Table (3): Retention times (in minutes) of standards, isolated compounds and the

corresponding detected compounds in different extracts of different fruit parts (peels,

pulps and juice) of Iraqi Citrus aurantium.

Compound

Retention

time of the

standard

compound

Retention time

of the isolated

compound

Retention

time of the

compound in

peels extract

Retention

time of the

compound in

pulp extract

Retention

time of the

compound in

juice sample

Nobiletin 6.86 6.86 6.88 6.88 6.88

Tangeretin 10.35 10.35 10.35 10.353 10.35

Neohesperidin 11.12 _ 11.122 11.122 11.12

Naringin 7.95 _ 7.955 7.95 7.95

Hesperidin 4.00 Not detected Not detected Not detected Not detected

Narirutin 9.85 Not detected Not detected Not detected Not detected

Table (4): The percentages of flavonoids in the peels and pulps of Iraqi Citrus aurantium

fruit.

Compound

Percentage of the

Compound in the

peels

Percentage of the

Compound in the

pulps

Nobiletin 0.9 0.35

Tangeretin 0.43 0.15

Neohesperidin 1.15 0.4

Naringin 1.3 0.46

Hesperidin Not detected Not detected

Narirutin Not detected Not detected

Figure (5a): HPLC chromatogram of Tangeretin standard using acetonitrile: water

(45:55) as a mobile phase.


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Figure (5b): HPLC chromatogram of compound 3 (C3) using acetonitrile: water (45:55)

as a mobile phase.

Figure (5c): HPLC chromatogram of Nobiletin standard using acetonitrile: water


Figure (5d): HPLC chromatogram of Compound 4 (C4) using acetonitrile: water



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Figure (5e): HPLC chromatogram of methanolic extract of Citrus aurantium fruit peels

using acetonitrile: water (45:55) as a mobile phase.

Figure (5f ): HPLC chromatogram of methanolic extract of Citrus aurantium fruit pulps

using acetonitrile: water (45:55) as a mobile phase.

Figure (5g): HPLC chromatogram of Citrus aurantium fruit juice using acetonitrile:

water (45:55) as a mobile phase.


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Figure (5h): HPLC chromatogram of Neohesperidin standard using acetonitrile:

0.1%Acetic acid in Water (30:70) as a mobile phase.

Figure (5i): HPLC chromatogram of Compound 1 (C1) using acetonitrile: 0.1%Acetic

acid in Water (30:70) as a mobile phase.

Figure (5j): HPLC chromatogram of Naringin standard using acetonitrile: 0.1%Acetic



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Figure (5k): HPLC chromatogram of Compound 2 (C2) using acetonitrile: 0.1%Acetic


Figure (5l): HPLC chromatogram of methanolic extract of Citrus aurantium fruit peels

using acetonitrile: 0.1%Acetic acid in Water (30:70) as a mobile phase.

Figure (5m): HPLC chromatogram of methanolic extract of Citrus aurantium fruit

pulps using Acetonitrile: 0.1%Acetic acid in Water (30:70) as a mobile phase.


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Figure (5n): HPLC chromatogram of Citrus aurantium fruit juice

using Acetonitrile: 0.1%Acetic acid in Water (30:70) as a mobile phase.

CHN analysis: Elemental microanalysis was performed (using EuroEA Elemental

analyzer/Italy) for the isolated compounds to confirm their chemical structure and purity

(table 5).

Table (5): Elemental Microanalysis of the isolated compounds

FT. IR

The identification of the isolated compounds was further confirmed by FT-IR spectroscopy

using Shimadzu FT-IR-8400S Infrared Spectrometer, as in figure 6a and table (5a) for

Compound 3 (C3) and figure 6b and table (5b) for Compound 4 (C4).[42]

Figure (6a): FT-IR spectrum of Compound 3 (C3).

Isolated Compound C% H% N%

Found Calculated Found Calculated Found Calculated

C3 (Tangeretin) 66.162 64.51 6.048 5.81 _ _

C4 (Nobiletin) 63.873 62.68 5.714 5.51 _ _


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Table (5a): Characteristic FT-IR Absorption Bands (in cm-1

) of Compound 3 (C3).

Figure (6b): FT-IR spectrum of Compound 4 (C4).

Table (5b): Characteristic FT-IR Absorption Bands (in cm

-1) of Compound 4 (C4).

NMR analysis: The NMR spectra were taken by dissolving the sample in dimethyl

sulphoxide (DMSO) – d6 and run on NMR Spectrometer. All chemical shifts reported are in

reference to tetra methyl silane (TMS) at 0 part per million (ppm).

Functional group Group frequency wave

number (cm-1

)

Assignment

C-H

C-H

C=O

C=C

C-H

C-O

C-H and C=C

3010.98

2945.40,2839.31

1647.26

1608.69, 1587.47

1512.24

1464.02,1361.79

1265.35

829.42, 798.56

Aromatic C-H stretching

C-H stretching (aliphatic)

C=O stretching (conjugated)

Aromatic C=C stretching

C-H bending (aliphatic)

Ether C-O stretching

Out of plane C-H and C=C bending

of benzene ring.

Functional group Group frequency wave

number (cm-1

)

Assignment

C-H

C-H

C=O

C=C

C-H

C-O

C-H and C=C

3012.91

2947.33, 2841.24

1647.26

1606.67,1587.47

1564.32

1462.09, 1361.79

1263.42

827.49, 798.56

Aromatic C-H stretching

C-H stretching (aliphatic)

C=O stretching (conjugated)

Aromatic C=C stretching

C-H bending (aliphatic)

Ether C-O stretching

Out of plane C-H and C=C bending

of benzene ring.


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The NMR measurement was carried out in Al-Albayt University, Al-Mafraq, Jordan; using

Euro-vectorEA 3000A NMR spectrometer apparatus / Italy.

The NMR spectrum of Compound 3, was characterized and when compared with the

literature, showed good agreement with NMR data of Tangeretin.[43]

All the aromatic

protons are observed, and the five methoxyl groups were also prominent figure (7a). 1HNMR

(300 MHz, DMSOd6) δ[ppm]: 8.02(2H,d,H2',H6'); 7.16 (2H,d,H3',H5'); 6.77 (1H,s,H3);

4.02 (3H,s,OCH3); 3.97-3.78 (12H,m, 4xOCH3).

Tangeretin, R1=R3= H, R2=R4 =R5 =R6 =R7 = OCH3

Figure (7a):

1H-NMR spectrum of Compound 3 (C3).

In the NMR spectrum of Compound 4, the aromatic protons of ring (B) were displayed as a

doublet for each H6', H2' and H5' at δ =7.65, 7.55 and 7.19 ppm respectively as in figure

(7b), also, Characteristic peak demonstrated as a singlet at δ= 6.88 ppm due to H3 at ring C.

In addition the six methoxyl groups were assigned at δ=3.98-3.79 ppm as a multiplet. All the

NMR data were in good agreement with those reported in the literature for Nobiletin.[44]

1HNMR (300 MHz, DMSOd6) δ [ppm]: 7.65 (1H,d,Ar-H6'); 7.55 (1H,d,Ar-H2'); 7.19

(1H,d,Ar-H5'); 6.88 (1H,s,H3); 4.13(3H,s,OCH3); 4.03;(3H,s,OCH3) ;3.98-3.79

(12H,m,4xOCH3).


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Nobiletin, R3= H, R1= R2=R4 =R5 =R6 =R7 = OCH3

Figure (7b): 1H-NMR spectrum of Compound 4 (C4).

CONCLUSION

Citrus aurantium is easily available and is effective in treatment of different diseases like

cardiovascular diseases, cancer, circulatory diseases and digestive disorders. So it is

important to give more research attention for this plant, such research will increase its

therapeutic applications. Citrus aurantium fruit byproducts could be interesting not only due

to their important fiber content but also due to their higher levels of flavonoids than that

present in juice, these flavonoids have important medicinal activities particularly cholesterol

lowering and more recently their use in fighting cancer.

The results revealed that Iraqi Citrus aurantium is an unchallenged source of naringin and

neohesperidin, and a good juice source for these flavanone glycosides; Iraqi Citrus aurantium

fruits contain mainly glycosylated flavanones particularly naringin and neohesperidin which

were found to be the major flavonoids. This study provides insight into how to explore

further the benefits of Iraqi Citrus aurantium fruits for human health.

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