This document summarizes a study analyzing the flavonoid content of various citrus fruits and traditional Chinese medicinal foods from China. HPLC analysis found great diversity in flavonoid profiles. Huyou peel was highest in naringin (3.25%) and neohesperidin (2.76%). C. aurantium was also a good source of these compounds. Wenzhoumiju peel was richest in hesperidin (6.25%) and Zaoju highest in nobiletin (0.59%). LC-MS analysis first detected significant hesperidin (0.74%) in the root of Liangmianzhen, a Zanthoxylum species used as a spice. The study provides

1. Plant Foods for Human Nutrition 61: 57–65, 2006.
c 2006 Springer Science+Business Media, Inc. 57
DOI: 10.1007/s11130-006-0014-8
Citrus Flavonoids in Fruit and Traditional Chinese Medicinal Food Ingredients in China
YANHUA LU,1, ∗ CHONGWEI ZHANG,1 PETER BUCHELI2 & DONGZHI WEI1
1 State
Key Laboratory of Bioreactor Engineering, New World Institute of Biotechnology, East China University of Science and Technology, Mailbox 311,
Meilong Rd. No. 130, Shanghai 200237, China; 2 Nestl´ R&D Center Shanghai Ltd., Shanghai 201819, China (∗ author for correspondence; e-mail:
e
luyanhua@ecust.edu.cn£¬ dzhwei@ecust.edu.cn)
Published online: 1 July 2006
Abstract. Flavonoids-enriched tissues of citrus such as peel, immature as protectant against pathological hyperphosphorylation,
fruit and flower are consumed as culinary seasonings, tea ingredients environmental toxins, or side effects of chemotherapeutic
in China for centuries. This HPLC quantitative study on the five citrus
drugs [10].
flavonoids, naringin, hesperidin, neohesperidin, sinensetin and nobiletin
on a wide range of Chinese citrus fruits and several Traditional Chinese Recent research focuses on the bioactivities of poly-
Medicinal food ingredients in East China, revealed a great diversity in methoxylated flavones such as nobiletin and sinensetin.
flavonoid composition. Huyou peel (C. paradisi cv. Changshanhuyou) was Nobiletin is reported as a novel promising immunomod-
found to be the best naringin (3.25%) and neohesperidin (2.76%) source; ulatory and anti-inflammatory drug [11] and as an inhibitor
C. aurantium, a major ingredient of several citrus-related TCM, is also a to human mammary cancer cells [12]. Latest research also
suitable source of naringin and neohesperidin, and a good juice source for
flavanone glycosides; the peel of Wenzhoumiju (C. unshiu) is one of the demonstrates its anti-hepatitis C virus effect [13] and pro-
richest local species in hesperidin (up to 6.25%); Zaoju (C. subcompressa) tection against UVB-induced photoinflammation such as
has the highest content of nobiletin (0.59%), a polymethoxylated flavone. erythema (sunburn) and edema, photoaging and photocar-
LC-ES-MS analysis of Zanthoxylum genus for flavonoids revealed for the cinogenesis [14].
first time the presence of significant amounts (0.74%) of hesperidin in
Citrus flavonoid composition is the subject of well con-
the root of Liangmianzhen (Z. nitidum (Roxb.) DC), a relative of Sichuan
pepper, which is a spice widely used in China. ducted studies using HPLC in Japan [15, 16], the USA
[17, 18] and Spain [19]. Surprisingly, there are very few
Key words: Citrus, Flavonoid, Flavanone glycoside, Hesperidin, HPLC, studies in China about citrus flavonoids among locally
LC-MS, Naringin, Neohesperidin, Nobiletin, Polymethoxylated flavone, grown species and cultivars. Citrus species are cultivated in
Sinensetin, Traditional Chinese Medicine, TCM, Zanthoxylum
China for at least 1700 years and Chinese have believed
for centuries that food and drugs come from the same
source. Those ingredients are commonly used in Traditional
Introduction Chinese Medicine (TCM), and many of them are regulated
in China as foodstuff. For examples, sour orange flower
Intense interest in citrus flavonoids, mainly existing in the (Daidai flower—tea ingredient) and immature or mature
pericarp (albedo, membrane and the pith) of citrus fruits, whole citrus fruit (Fructus Aurantii Immaturus, Fructus
keeps for decades due to their versatile bioactivities, high Aurantii) are ingredients to treat flatulence and menopausal
consumption by consumers, and wide availability from in- symptoms [20]. The peel of mature citrus (Pericarpium
expensive raw materials in the citrus industry in form of Citri Reticulatae, pocket food and culinary seasoning) is
peel, juice and concentrate. used to reduce phlegm in the lung [21]. The peel of imma-
Citrus flavonoids are generally categorized into two ture citrus (Pericarpium Citri Reticulatae Viride) and dried
groups, flavanone glycosides (naringin, hesperidin, neo- fruit of Citrus medica L. var. Sarcodactylis (Noot.) Swingle
hesperidin, etc.) and polymethoxylated flavones (nobiletin, are prescribed for gut and digestion disorders [22, 23]. Ad-
sinensetin, tangeretin, etc.). Hesperidin is the best stud- ditionally, there are a few reports that indicate the existence
ied flavanone glycoside. It is known to improve vascu- of hesperidin in Zanthoxylum (Rutaceae) plants [24–27].
lar integrity and to decrease capillary permeability [1], Sichuan pepper (Zanthoxylum bungeanum Maxim.) and
and is used as supplementation for patients of blood ves- green Sichuan pepper (Zanthoxylum schinifolium Sieb. Et
sel fragility and permeability complaints [2]. Pharmaco- Zucc.) are both TCM that are also used as culinary spices,
logical properties, ranging from anti-inflammatory and and Liangmianzhen (Zanthoxylum nitidum (Roxb.) DC) is
analgesic effects [3], inhibitory effects on oral carcino- used in toothpaste in China as an anti-inflammatory ingre-
genesis [4, 5], to a clinically demonstrated estrogen-like dient.
effect against menopausal symptoms [6], are also reported. We report the composition of citrus flavonoids in differ-
Furthermore, together with naringin, it appears to possess ent species and cultivars of citrus fruits grown in three of
cholesterol-lowering [7] and potential anti-cancer effects the largest citrus producing provinces (Zhejiang, Fujian and
[8]. On the other hand, naringin is reported for antiulcer Jiangxi) in China, and in several TCM ingredients regulated
effect on gastric lesions induced by ethanol in rats [9] and in China as foodstuff (except Liangmianzhen).

2. 58
Materials and Methods Sample Preparation
Chemicals Fruit juices were made from fresh fruit by hand squeezing
through a double-layer cloth and a 30 min ultrasonic treat-
Standard compounds of hesperidin, naringin, nobiletin were ment at 40◦ C before HPLC determination [28]. Citrus peel
gifts from Tianjin Jianfeng Natural Products Ltd. (P. R. (flavedo and albedo) and fruit pulp (juice sac and segment
China); neohesperidin was purchased from Sigma-Aldrich membrane) after juice squeezing were oven-dried at 55◦ C
Chemie GmbH (Germany) and sinensetin from Chromadex until constant weight was reached. Dried peel and fruit pulp
Inc. (USA). were milled (IKA Works A11 Basic Mill, Germany) into
powder (particle size 0.355 mm, 90% mesh passed). All
TCM ingredients were milled into powder and oven-dried
Fruit Samples at 55◦ C until constant weight was reached. Approximately
0.5 g of powder of each dried sample was treated for 60 min
Most of the fresh citrus fruit samples were collected from at 40◦ C in an ultrasonic bath with 20 mL of methanol, and
the Citrus Research Institute, Zhejiang Academy of Sci- after paper filtration (Xin Xing quantitative filter paper,
ence, Huangyan, Zhejiang. Exceptions were Tonggan (Cit- medium rate, type 102, Hangzhou Fuyang Special Paper
rus tankan Hayata.) and Yongchunlugan (Citrus reticulata Industry Co., Ltd, China) adjusted to 25 mL in a volumetric
Blanco.) from Yongchun (Fujian), peel of Lemon (Citrus flask.
limon L. Burm.f) from Sichuan, peel of Huyou (Citrus par- All prepared sample solutions including standard so-
adisi cv. Changshanhuyou) from Changshan (Zhejiang), lutions were passed through a 0.45 µm membrane fil-
Wenzhoumiju (Citrus unshiu Marc.), Foshou (Citrus med- ter (regenerated cellulose membrane, Uniflo 13/0.45/RC,
ica L. var. Sarcodactylis (Noot.) Swingle) and Jingan (For- Schleicher & Schuell, Germany) prior to the injection into
tunella crassifolia Swingle) from Jinhua (Zhejiang) and the HPLC system.
Gongju (Citrus kinokuni hort. ex. Tanaka) from Nanfeng
(Jiangxi). All these samples were collected in November
2004.
HPLC Chromatography
Traditional Chinese Medicine (TCM) Samples Quantitative analysis was performed on Agilent HPLC
system 1100 series (degasser G1322A, quaternary pump
Most TCM samples were purchased from Shanghai Huayu G1311A, autosampler G1313A, column heater G1316A
TCM Ltd. The rest were collected locally: Fructus Au- and diode array detector G1315B). Samples were sep-
rantii Immaturus (Citrus aurantium L.) from Zhejiang, arated on a Kromasil column (100-5C18-250A, Ø
green Fructus Aurantii (Poncirus trifoliata (L.) Rafin) from 4.6 mm × 250 mm, 5 µm, C18 reverse phase) at 35◦ C, flow
the Fujian Institute of Drug Control, Foshou (Citrus med- rate 1 mL/min, UV wavelengths: 284 and 332 nm. The gra-
ica L. var. Sarcodactylis (Noot.) Swingle) from Fujian dient elution program was as follows: initial 15-min run of
Jiaochen Fuoshou Production Base, green Sichuan pepper 40% methanol (v/v), followed by a 5-min linear gradient
(Zanthoxylum schinifolium Sieb. Et Zucc.) from Chongqing to 100% methanol and hold for 5 min, linear gradient to
Jiangjin Government, and Fructus Aurantii and Fructus Au- 40% methanol in 1 min and hold for 30 min. The injection
rantii Immaturus (Citrus aurantium Linn.) from Jiangxi volume is 10 µL. Peak purity check and identification were
Xingan Chinese Medicine Production Administration carried out by 190–400 nm UV scan through diode array
Bureau. detector (DAD) on software Agilent ChemStation Plus rev.
A.10.02.
Preparation of Standards
LC-ESI-MS identification
Naringin, hesperidin, neohesperidin, sinensetin and no-
biletin standard compounds were dissolved with methanol LC-MS identification of hesperidin occurring in TCM of the
in an ultrasonic bath for 30 min at 40◦ C. The solutions Zanthoxylum genus was performed under the same HPLC
were adjusted in 50 mL volumetric flasks to obtain a conditions mentioned above, and coupled with a Finnigan
2160 mg/L-naringin, 1428 mg/L-hesperidin, 1408 mg/L- LCQ Deca XP MAX mass spectrometer (San Jose, CA).
neohesperidin, 80 mg/L-sinensetin and 118 mg/L-nobiletin 5 µL of each sample and 3 µL of mixed standard solu-
stock solution. 0.1, 1, 3, 5, 7 and 9 mL of the stock solution tion (hesperidin 0.0178 g + neohesperidin 0.0100 g/25 mL
were daily diluted with methanol and adjusted respectively methanol) were injected and separated under the same
to 10 mL in volumetric flasks for making calibration curves. linear gradient program mentioned above at 0.6 mL/min

3. 59
Figure 1. HPLC profiles of (i) a standard mix of flavonoids and their online UV spectrum (190–400 nm); (ii) Zaoju (C. subcompressa Tanaka) peel
extract; (iii) Fructus Aurantii Immaturus (C. aurantium L., Shanghai Huayu TCM Ltd.) extract at 284 and 332 nm, NG: naringin; HP: hesperidin; NP:
neohesperidin; SN: sinensetin; NB: nobiletin.
flow rate. The 284 nm profile was recorded. MS analy- Quantitative Analysis
sis was performed with an electrospray ionization interface
(ESI) in positive ion mode. The ionization conditions were A complete summary of the results on flavonoids in fresh
optimized as follows: source voltage 5.00 kV, sheath gas fruit and TCM ingredients of 27 citrus and 3 non-citrus
flow rate 40 psi, capillary voltage 15 V, capillary tempera- cultivars from Zhejiang, Fujian and Jiangxi Province in
ture 275◦ C and scan range 200–1500 m/z. China is presented in Table 1 and 2. In Table 1, naringin
is not detected in peel and fruit pulp of nearly all the fresh
fruits except 2.11 ± 0.07% and 0.79 ± 0.06% in peel and
Results and Discussion fruit pulp of Goutoucheng (C. aurantium Linn.) respec-
tively, and 3.25 ± 0.37% in peel of Huyou (C. paradisi cv.
HPLC Analysis Validation Changshanhuyou). Neohesperidin in fruit pulp and juice
is only detected in four cultivars of fresh fruit such as
A set of typical calibration curves were linear (R = 0.9998– 0.3 ± 0.02% in fruit pulp and 208.3 ± 21.5 mg/L in juice
0.9999) in the range of 14–1300 mg/L (hesperidin, in Goutoucheng (C. aurantium Linn.), 20.2 ± 3.7 mg/L,
neohesperidin), 21–1900 mg/L (naringin), 0.8–70 mg/L 77.4 ± 1.9 mg/L and 20.4 ± 0.3 mg/L in juice of Ham-
(sinensetin) and 1.2–100 mg/L (nobiletin). Peel of Huyou lin (C. sinensis Osbeck), Liubenchen (C. sinensis × C.
(Citrus paradisi cv. Changshanhuyou) was chosen for a re- reticulata) and Jingan (Fortunella crassifolia Swingle)
covery test. Approximately the same quantities of the five respectively. Sinensetin and nobiletin almost do not
standard samples to sample contents were added into sam- occur in fruit pulp and juice except 1.1 ± 0.2 mg/L and
ple powder and extracted together. On average, 112, 103, 9.0 ± 1.6 mg/L in juice of Penggan (C. reticulata Blanco.)
95, 90 and 106% of naringin, hesperidin, neohesperidin, respectively.
sinensetin and nobiletin, respectively, were recovered in Among the cultivars examined, the fresh fruit peel
triplicate experiments. A repeatability test was done with of three cultivars of C. unshiu, are the richest in hes-
six replicates and the RSD (relative standard deviation) for peridin (5.86–6.25%). Gongju (C. kinokuni) in Jiangxi and
the five flavonoids ranged from 3.0 to 4.1%. Peak purity Goutoucheng (C. aurantium) in Zhejiang contain high con-
was checked through DAD UV spectrum scanning to help centrations of hesperidin in juice (302 mg and 184 mg/L,
verifying separation conditions and identifying flavonoid respectively). The peel of Chinese huyou (C. paradisi cv.
peaks in addition to comparing with standards peaks and Changshanhuyou) is the highest in both naringin (3.25%)
their retention times (Figure 1). and neohesperidin (2.76%). Zaoju (C. subcompressa), a

4. 60
Table 1. Citrus flavonoid content in fresh citrus
Naringin Hesperidin Neohesperidin Sinensetin Nobiletin
Scientific name Local name Juice (mg/L) Peela (%) Fruit pulp (%) Juice (mg/L) Peel (%) Peel (%) Peel (%)
C. aurantium Linn Goutoucheng 522.4 (27.9) ND 0.20 (0.02) 184.2 (120.2) 1.04 (0.03) 0.01 (0.00) 0.08 (0.00)
C. erythrosa Hort Zhuhong ND 3.81 (0.06) 2.93 (0.72) 41.3 (1.0) ND 0.01 (0.00) 0.43 (0.04)
C. kinokuni Hort Gongju ND 4.23 (0.21) 2.31 (0.01) 302.4 (13.8) ND 0.05 (0.00) 0.45 (0.01)
C. limon (L.) Burm.f Lemon 1.59 (0.45) ND ND ND
C. paradisi cv. Huyou 0.13 (0.01) 2.76 (0.28) ND 0.03 (0.00)
Changshanhuyou
C. reticulata Blanco Penggan ND 3.60 (0.00) 1.31 (0.13) 64.4 (8.0) ND 0.04 (0.00) 0.42 (0.01)
C. reticulata Blanco Yongchunlugan 25.7 (0.5) 3.58 (0.64) 1.50 (0.08) 117.3 (12.8) ND 0.05 (0.00) 0.48 (0.01)
C. sinensis Osbeck Hamlin 57.3 (10) 4.48 (0.10) 2.31 (0.40) 72.1 (3.5) ND 0.05 (0.00) 0.10 (0.00)
C. sinensis × C. reticulata Liubenchen 197.9 (5.8) 2.59 (0.02) 1.55 (0.20) 68.6 (1.4) ND 0.03 (0.00) 0.04 (0.00)
C. subcompressa Zaoju ND 3.50 (0.01) 2.50 (0.31) 33.0 (3.5) ND 0.04 (0.00) 0.59 (0.00)
C. succosa Hort Bendizao ND 4.90 (0.21) 2.45 (0.51) 50.7 (0.4) ND 0.02 (0.00) 0.12 (0.01)
C. tankan Hayata Tonggan ND 4.35 (0.22) 1.31 (0.03) 95.2 (1.2) ND 0.02 (0.00) 0.14 (0.00)
C. tardiferax Hort ManJu ND 3.10 (0.03) 1.90 (0.02) 35.6 (30.2) ND 0.04 (0.00) 0.30 (0.00)
C. unshiu Marc Wenzhoumiju Jinghua ND 5.86 (0.19) 83.8 (0.9) ND ND 0.01 (0.00)
C. unshiu Marc Wenzhoumiju mid-late ND 6.25 (0.10) 1.45 (0.01) 75.4 (5.3) ND 0.01 (0.00) 0.02 (0.00)
season
C. unshiu var. praecox Gongchuan early season ND 6.18 (0.06) 1.70 (0.04) 70.8 (4.3) ND 0.01 (0.00) 0.02 (0.00)
Fortunella crassifolia Jingan 56.5 (0.8) ND 0.11 (0.00) 47.4 (0.5) ND ND ND
Swingle
Note. Content% (dry wt.) for dried samples; Content mg/L for juice; Peel = flavedo + albedo; Fruit pulp = juice sac + segment membrane.
a Standard deviation is shown in parentheses (N = 3).

5. 61
Table 2. Citrus Flavonoid content (%) in TCM ingredients
Scientific name Local name, origin Tissuea Naringin Hesperidin Neohesperidin Sinensetin Nobiletin
C. aurantium L Fructus Aurantii Whole fruit 6.81 (0.27) 0.10 (0.00) 6.49 (0.22) 0.01 (0.00) 0.07 (0.00)
Immaturus, Zhejiang
C. aurantium L Fructus Aurantii, Xingan, Whole fruit 3.50 (0.10) 0.49 (0.021) 6.10 (0.10) 0.01 (0.00) ND
Jiangxi
C. aurantium L Fructus Aurantii Whole fruit 10.78 (0.05) 0.72 (0.01) 6.89 (0.08) 0.01 (0.00) 0.11 (0.00)
Immaturus, Jiangxi
C. aurantium L Fructus Aurantii, Hunan Whole fruit 0.42 (0.02) 2.76 (0.08) 0.28 (0.01) 0.01 (0.00) 0.01 (0.00)
C. aurantium L Daidai, Zhejiang Flower 3.46 (0.07) 0.49 (0.01) 6.89 (0.08) ND ND
C. aurantium L Fructus Aurantii Whole fruit 4.68 (0.74) 2.12 (0.19) 6.10 (0.70) 0.01 (0.00) 0.06 (0.01)
Immaturus, Xingan,
Jiangxi
Poncirus trifoliata (L.) Green Fructus Aurantii Whole fruit 1.06 (0.00) 0.28 (0.00) 0.68 (0.04) 0.18 (0.02) 0.02 (0.00)
Rafin Immaturus, Fujian
a Note. Standard deviation is shown in subscript number (N = 3); ND = not detected.
typical cultivar in Huangyan (Zhejiang) has the highest collected in Spain and France are reported to contain
content of polymethoxylated flavones, especially nobiletin 12.35% of flavonoids, with neohesperidin (5.44%) and
(0.59%) (Figure 1). Sour orange (C. aurantium) in TCM naringin (1.93%) being the most important ones [30]. In
form, though not a commercial fruit in China due to its addition, the present results agree with a Chinese study
sourness, is a good alternative to grapefruit (C. paradisi) that reported a level of 2.8–7.7% for naringin, and 0.5–
for naringin and neohesperidin source. Consistent with a 0.8% for hesperidin [31]. Green Fructus Aurantii Immatu-
Chinese report [29], its naringin concentration in juice rus (Poncirus trifoliata (L.) Rafin.) is still locally used as
(522 mg/L) is the highest. Moreover, it is an important substitute to C. aurantium in Fujian though the naringin
ingredient of TCM such as Zhiqiao (Fructus Aurantii), content is much lower than the C. aurantium samples. Jin-
Zhishi (Fructus Aurantii Immaturus) (Figure 1), and even gan (Fortunella crassifolia Swingle), the pickled whole
the flower of sour orange (Daidai flower), which are all fruit (including peel) used as candy, is mostly not de-
in the list of TCM ingredients regulated as foodstuff in tected for the five citrus flavonoids in the peel and pulp but
China (Table 2). For example, the flowers of C. aurantium positive for flavanone glycosides in juice. The tea
Table 3. Hesperidin data comparison of the three richest cultivars of Citrus fruits grown in the USA, China, Japan and Spain
Scientific name Local name Tissue Hesperidin (%) Data source
C. limon Santa Teresa #1, CRC #3894 Albedo 0.66 USDAa [17]
C. reticulata × Poncirus Unknown, CRC #2619 Albedo 0.51
trifoliata
C. limon Santa Teresa #1, CRC #3894 Flavedo 0.51
C. sinensis Fisher, CRC #3645 Albedo 0.32
C. unshiu Marc Wenzhoumiju mid-late season Peel 6.25 Chinese fruit
C. unshiu var. praecox Gongchuan early season Peel 6.18
C. unshiu Marc Wenzhoumiju Peel 5.86
C. aurantium L Fructus Aurantii (Hunan) Whole fruit 2.76 TCM
C. aurantium L Fructus Aurantii Immaturus (Xingan) Whole fruit 2.12
C. aurantium L Fructus Aurantii Immaturus (Jiangxi) Whole fruit 0.72
C. depressa Hayatab Unknown Whole fruit 27.50 Spanishc [19]
C. aurantium L.b Bouquet de Fleurs Whole fruit 2.95
C. aurantium L Cajel Whole fruit 2.13
C. hanayu Hanayu Edible part 2.00 Japanesed [15, 16]
C. reshn Cleopatra Edible part 1.91
C. unshiu Unshu Edible part 1.60
a Samples were separated into flavedo and albedo, frozen at − 20◦ C and ground into small pieces.
b Whole immature fruit.
c Samples were dried (50◦ C in ventilated oven), ground into fine powder.
d Samples were separated into peel and edible part (consisting of juice sac and segment epidermis), freeze-dried and ground into powder.

6. 62
Table 4. Data comparison of the three richest cultivars of Citrus juice in flavanone glycosides (naringin, hesperidin, neohesperidin)
reported from the USA and China
Flavanone
Scientific name Local name Content (mg/L) Origin glycoside
C. aurantium L Bouquet de Fleurs sour orange 362 Floridaa [18] Naringin
C. aurantium L Seville 244
C. aurantium L African 243
C. aurantium L Goutoucheng 522.4 China
C. sinensis × C. reticulata Liubenchen 197.9
C. sinensis Osbeck Hamlin orange 57.3
C. sinensis Ruby Blood sweet orange 254 Floridaa [18] Hesperidin
C. sinensis Late Navel sweet orange 232
C. sinensis Pera sweet orange 208
C. kinokuni Gongju 302 China
C. reticulata Blanco Yongchunlugan, mandarin 117.3
C. tankan Hayata Tonggan 95.2
C. paradisi K-Early tangelo 720 Floridaa [18] Neohesperdin
C. aurantium L Bouquet de Fleurs sour orange 209
C. aurantium L Seville 147
C. aurantium L Goutoucheng 208.3 China
C. sinensis × C. reticulata Liubenchen 77.4
Fortunella crassifolia Swingle Jingan 20.4
a Sample preparation: freshly extracted juice samples were passed through a double layer of cheesecloth.
ingredient Foshou (C. medica) is overall not detected for 3–7 mm). The high levels in the peel of Huyou (C. paradisi
the five flavonoids. cv. Changshanhuyou, 3.25%), a relative to grapefruit,
Comparing the presented results with the data published confirmed this observation. Opposite to the occurrence
(a) by the United States Department of Agriculture of naringin, hesperidin content varies widely among
(USDA) about 114 cultivars grown in California [17], (b) different citrus species such as C. limon and C. sinensis
by a Japanese group on 66 Citrus species and near-citrus in California, C. unshiu in Zhejiang, China, C. hanayu
relatives [15, 16], and (c) by a Spanish group on immature and C. reshni in Japan, and C. depressa and C. aurantium
and mature fruits of six citrus species [19], the results in Spain (Table 3). High levels of flavanones in TCM
revealed that C. paradisi is an unchallenged naringin ingredients and immature fruits in Spain mainly result from
source for cultivars grown in the USA (Star Ruby, 1.13% fruit collection at an early stage of maturation when the
in albedo), Japan (Marsh grapefruit, 1.46% in edible part) flavanones are being synthesized [19]. That also explains
and Spain (37.8% in whole immature fruit of a size of why, for example, their naringin and neohesperidin content
mAU 12.101 100 12.02 NP
200 284nm
HP HP 13.93
150 13.822 Positive Ion TIC
50
Relative Abundance
100 Hesperidin and
NP neohesperidin
50
0
0 01 5 10 15
0 5 10 15
mAU 100 12.08
284nm 1# Positive Ion TIC
600
400
1# 50
Zanthoxylum
nitidum (Roxb.) DC.
12.040
200
5
00 2 4 6
0
0 5 10 20 25
5 10
Time (min) Time (min)
Figure 2. LC and TIC (Total Ion Chromatograph) profiles of Liangmianzhen (Zanthoxylum nitidum (Roxb.) DC.)
compared to hesperidin and neohesperidin standard solution. 1# is supposed to be the hesperidin peak.

7. 63
Figure 3. MS online spectrum of Liangmianzhen (Peak 1#) compared to hesperidin and neohesperidin standard solution (m/z: 200–1500).
is higher than in mature Citrus samples in US and Japan Fujian which is the no.1 citrus fruit exported from China,
[15–17]. Content of flavanone glycosides in Citrus juice has as well a reasonably high hesperidin content in juice
was compared also with 52 citrus cultivars from Florida, (117 mg/L).
USA [18] (Table 4). Because a 5% (v/v) addition of C. au-
rantium is permitted in frozen concentrated orange juice by LC-ESI-MS Identification of Hesperidin in Liangmianzhen
FDA [32], C. aurantium and C. sinensis are valuable juice (Zanthoxylum nitidum (Roxb.) DC.)
source for flavanone glycosides in the USA as indicated
by their high levels found in the study from Florida [18]. As hesperidin was found to be present in significant
Yongchunlugan (C. reticulata Blanco), the mandarin from amounts in Liangmianzhen, the sample was further ana-

8. 64
lyzed by LC-ESI-MS. Because of the identical molecular References
weight of hesperidin and neohesperidin, a mixed solution
of these two standards was injected into the LC-MS as ref- 1. Pizzorno J, Murray M (1999) Textbook of Natural Medicine, (2nd
erence, and peak 1# was found to be consistent with that edn.). New York: Churchill Livingstone, p 1393.
of hesperidin (Figure 2). Further MS spectrum comparison 2. Garg A, Garg S, Zaneveldand LJD, Singla AK (2001) Chemistry and
Pharmacology of the Citrus Bioflavonoid Hesperidin. Phytother Res
(Figure 3) led to the conclusion that peak 1# was identical 15: 655–669.
with that of hesperidin. The content of hesperidin in the root 3. Galati EM, Monforte MT, Kirjavainen S, Forertieri AM, Tripodo
of Liangmianzhen was determined as being 0.74 ± 0.01%. MM (1994) Biological effects of hesperidin, a Citrus flavonoid.
This result agreed with an earlier report [25] that hesperidin Part 1. Anti-inflammatory and analgesic activity. Farmaco 49: 709–
712.
crystals were observed in a morphological and histolog-
4. Tanaka T, Makita H, Ohnishi M, Hirose Y (1994) Chemoprevention
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