Food Chemistry: X 15 (2022) 100368

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Food Chemistry: X
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The flavonoid profiles in the pulp of different pomelo (Citrus grandis L.

Osbeck) and grapefruit (Citrus paradisi Mcfad) cultivars and their in
vitro bioactivity
Mei Deng , Lihong Dong , Xuchao Jia , Fei Huang , Jianwei Chi , Zafarullah Muhammad , Qin Ma ,
Dong Zhao , Mingwei Zhang *, Ruifen Zhang *
Sericultural & Agri-Food Research Institute Guangdong Academy of Agricultural Sciences/Key Laboratory of Functional Foods, Ministry of Agriculture/Guangdong Key
Laboratory of Agricultural Products Processing, Guangzhou 510610, PR China

A R T I C L E I N F O A B S T R A C T

Keywords: Previous results indicated that the flavonoid profiles might have varietal differences in pomelo, but detailed
Pomelo information is unknown. We previously isolated 4 new flavonoids, cigranoside C, D, E, F, in Citrus grandis
Grapefruit Shatianyu pulp. However, their distribution in different pomelo cultivars remains to be explored. Therefore, the
Flavonoid profiles
flavonoid profiles and in vitro bioactivity of the pulp from 5 pomelo and 1 grapefruit cultivars commonly
UHPLC-ESI MS/MS
consumed in China were investigated. Fourteen flavonoids were identified, cigranoside C, D, E were detected in
Antioxidant activity
In vitro enzyme inhibition these pomelo and grapefruit. Naringin and cigranoside C were the major flavonoids in grapefruit, Guanximiyu-
W, Guanximiyu-R and Liangpingyu, while melitidin and rhoifolin was the predominant flavonoid in Shatianyu
and Yuhuanyu, respectively. Pomelo and grapefruit showed strong antioxidant activity, and were potent in­
hibitors of pancreatic lipase with IC50 values of 11.4–72.6 mg fruit/mL except Shatianyu. Thus, pomelo and
grapefruit are natural antioxidants and possess anti-obesity potential.

1. Introduction dihydrochalcone and neohesperidin, in the pulp of pomelo cultivars

from Thailand and found that naringin was their main flavonoid. Xi et al.
Flavonoids are the most common group of polyphenols in fruits and (2014) revealed that naringin was the predominant flavonoid in pomelo,
can contribute to reducing the risk of many chronic diseases, such as while naringin and neohesperidin were the predominant flavonoids in
metabolic syndrome, type 2 diabetes mellitus and cardiovascular dis­ grapefruits. In our previous study, we isolated 4 new flavonoids
eases, possibly due to their antioxidant activity and digestive enzyme (cigranoside C, D, E, F), together with 2 firstly reported flavonoids
inhibitory activity (Liu, 2003; Sun, Warren and Gidley, 2019; Wu et al., (neoeriocitrin and bergamjuicin) in pomelo. Besides, the main flavonoid
2017; Zhu et al., 2014). Citrus fruits, greatly popular in the word, are we isolated from Shatianyu pulp was melitidin (Deng et al., 2021). These
abundant in flavonoids, especially flavanones in aglycone or glycoside different results from our lab and others indicated that the flavonoid
forms (Khan & Dangles, 2014; Lu et al., 2020). Pomelo (Citrus grandis profiles in pomelo pulp might have varietal difference. Furthermore, the
(L.) Osbeck) is a kind of Citrus fruits and cultivated widely in southern distribution of the 6 newly isolated flavonoids from Shatianyu in
China. C. grandis cvs. Guanximiyu, Shatianyu, Liangpingyu and Yuhua­ different pomelo cultivars remains to be explored. Therefore, it is
nyu are representative pomelo cultivars in China (Zhang et al., 2011). necessary to further analyze the compositions and contents of flavonoids
Grapefruit (C. paradise Mcfad), a hybrid of sweet orange (C. sinensis) and among different pomelo cultivars.
sweet pomelo (C. maxima Burm) mainly distributed in South Africa and Antioxidant activity and digestive enzymes (pancreatic lipase,
the European Union, is also highly appreciated by Chinese consumers. α-amylase and α-glucosidase) inhibitory effects are the important effects
Earlier studies have preliminarily revealed the flavonoid profiles of of flavonoids, which account for their many health benefits. The bio­
the pulp of pomelo and grapefruit. Mäkynen et al. (2013) detected 6 activities of flavonoids are closely related to their molecular structure
flavonoids, including naringin, naringenin, hesperidin, hesperetin, (Liu et al., 2017; Salahuddin et al., 2020; Su et al., 2014). Previous

* Corresponding authors.
E-mail addresses: [email protected] (M. Zhang), [email protected] (R. Zhang).

https://doi.org/10.1016/j.fochx.2022.100368
Received 9 February 2022; Received in revised form 23 May 2022; Accepted 12 June 2022
Available online 16 June 2022
2590-1575/© 2022 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-
nc-nd/4.0/).
M. Deng et al. Food Chemistry: X 15 (2022) 100368

studies showed that the flavonoid extracts of citrus peels (Huang et al., 2.5. Measurement of total flavonoid contents (TFC)
2020) or the digesta of citrus fruits (Sun, Tao, Huang, Ye and Sun, 2019)
exhibited CAA activity and pancreatic lipase inhibitory activity. The The TFC was measured according to the sodium borohydride/
differences in antioxidant and enzyme inhibitory activity of flavonoids chloranil-based (SBC) assay (He et al., 2008) and the results were pre­
from different pomelo pulp are still unknown. sented as mg catechin equivalents (CE)/100 g FW of the pulp sample.
In order to clarify the varietal differences of pomelo pulp in flavonoid
profiles and bioactivity, 5 representative pomelo cultivars together with 2.6. Analysis of flavonoid compositions
a grapefruit cultivar commonly consumed in China were analyzed in the
present study to determine their compositions and contents of flavo­ The separation of phenolic extracts of different pomelo and grape­
noids in the pulp; and to compare their differences in antioxidant ac­ fruit cultivars was conducted on a Thermo Scientific Dionex UltiMate
tivity and inhibitory activity to α-amylase, α-glucosidase and pancreatic 3000 UHPLC (Tempe, Arizona, USA) with a Waters HSS C18 column
lipase. (1.8 μm, 2.1 × 100 mm, MA, USA). Acetonitrile (solvent A) and 0.4%
aqueous acetic acid (v/v, solvent B) were used as the mobile phase. The
2. Materials and methods elution of flavonoid compounds using the following conditions: 0–10
min, 5–8% A; 10–20 min, 8–12% A, 20–22 min, 12–14% A, 22–52 min,
2.1. Materials 14% A. Other analysis conditions were as follows: injection volume, 2
μL; flow rate, 1 mL/min; column temperature, 30 ◦ C; detection wave­
Shatianyu was purchased from Meizhou county, Guangdong prov­ length, 280 nm.
ince, China, in December 2018. Liangpingyu was obtained from ESI-MS analysis was performed on a Thermo Scientific TSQ Endura
Liangping county, Chongqing province, China, in November 2018. Triple Quad LC/MS/MS (Suwanee, GA, USA) equipped with an ion trap
Guanximiyu with red (Guangximiyu-R) and white (Guangximiyu-W) mass spectrometer and a diode array detector. The negative mode was
pulp were purchased from Pinghe county, Fujian province, China, in chosen to conduct electrospray ionization using the following condi­
October 2018. Yuhuanyu was obtained from Yuhuan county, Zhejiang tions: spray needle voltage, 4000 V; capillary temperature, 350 ◦ C; dry
province, China, in November 2018. Grapefruits were collected from the gas, 10 L/min; collision energy, 10–30 V; mass spectra, m/z 100–1000.
local supermarket in Guangzhou in October 2018.
2.7. Analysis of antioxidant activity

2.2. Chemicals and reagents The oxygen radical absorbance capacity (ORAC) was measured ac­
cording to the method of Huang et al. (2002) and the results were shown
Cigranoside A, B, C, D, E, F, bergamjuicin, neoeriocitrin, melitidin, as μmol Trolox equivalents (TE)/100 g FW of the pulp sample. The
rhoifolin, and naringin were prepared in our laboratory (Deng et al., cellular antioxidant activity (CAA) was determined using the method
2021). Hesperidin, neohesperidin, narirutin, isoquercitrin, (+)-catechin, reported by Wolfe and Liu (2007) and the results were shown as μmol
quercetin, gallic acid, Folin–Ciocalteu reagent, DCFH-DA, Trolox, quercetin equivalents (QE)/100 g FW of the pulp sample.
AAPH, fluorescein sodium, 4-methylumbelliferyl oleate (4-MUO),
α-glucosidase, α-amylase and pancreatic lipase, and 4-nitrophenyl-α-D- 2.8. In vitro enzymes inhibition assays
glucopyranoside (pNPG) were obtained from Sigma Aldrich Co. (St.
Louis, MO, USA). HBSS, new bovine calf serum and DMEM (H) medium 2.8.1. α-Amylase inhibition
were obtained from Gibco Life Technologies (Grand Island, NY, USA). The α-amylase inhibitory activity was measured according to the
HepG2 human liver cancer cells were purchased from the ATCC method reported by Salahuddin et al. (2020). A 96-well microplate was
(Rockville, MD, USA). Acetonitrile and glacial acetic acid in HPLC-grade seeded with diluted samples (40 μL), α-amylase (0.25 U/mL, 40 μL) and
were purchased from Thermo Fisher Scientific (Suwanee, GA, USA). PBS (20 μL), respectively, and incubated at 37 ◦ C for 3 min. Then, sol­
uble starch solution (1 mg/mL, 20 μL) was added, and the microplate
was incubated at 37 ◦ C for another 4 min. After the enzyme reaction was
2.3. Extraction of phenolics stopped by adding HCL (1 mol/L, 20 μL), 60 μL of iodine reagent con­
taining 5 mmol/L potassium iodine and 5 mmol/L iodine was added to
Phenolics were extracted following the method of Zhang et al (2013). the microplate. Then, the absorbance was taken at 650 nm. The
The pulp of grapefruit and pomelo obtained by peeling were homoge­ α-amylase inhibition (%) was determined as follows: [1-(A2-A1)/(A4-
nized using a blender (WBL2521H, Midea Group Co., Ltd., Foshan, A3)]*100%, where A1 is the absorbance of the samples in the above
Guangdong, China). Subsequently, each pulp sample (100 g) with 80% measurement; A2 is the absorbance of the measurement in which the
aqueous acetone (1:2, w/v) were further homogenized at 5000 rpm for enzyme was replaced by PBS; A3 is the absorbance of the measurement
5 min in an ice bath using an STSRH-300 homogenizer (Shanghai Sotin in which the samples were replaced by PBS; A4 is the absorbance of the
Intelligent Equipment Co., Ltd., Shanghai, China). The homogenates measurement in which both the samples and enzyme were replaced by
were centrifuged at 4000g for 10 min at 4 ◦ C (TG16, Shanghai Lu PBS.
Xiangyi Centrifuge Instrument Co. Ltd., China). Then, the residue was
repeated the above extraction steps and the pooled supernatants were 2.8.2. α-Glucosidase inhibition
condensed to dry at 45 ◦ C using a rotary evaporator (N-1300V, Tokyo The α-glucosidase inhibitory activity was determined following the
Rika Machinery Co., Ltd.). Finally, the condensed phenolics were dis­ method of Lin et al. (2015). A 96-well microplate was seeded with 20 µl
solved with 25 mL distilled water and stored at − 20 ◦ C for further of samples, 40 μL of PBS and 10 μL of α-glucosidase (0.2 U/mL),
analysis. respectively, and incubated at 37 ◦ C for 15 min. Then, 20 μL of 5 mmol/L
pNPG was added, and the microplate was incubated at 37 ◦ C for another
6 min. After the enzyme reaction was stopped by adding 100 μL of 200
2.4. Measurement of total phenolic contents (TPC) mmol/L Na2CO3, the absorbance was taken at 405 nm. The α-glucosi­
dase inhibition (%) was determined as follows: [A1-(A2-A3)]/A1*100%,
The Folin-Ciocalteu colorimetric method (Dewanto et al., 2002) was where A2 is the absorbance of the samples in the above measurement；
used to determine the TPC and the results were presented as mg gallic A1 is the absorbance of the measurement in which the samples were
acid equivalents (GAE)/100 g fresh weight (FW) of the pulp sample. replaced by PBS；A3 is the absorbance of the measurement in which the

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M. Deng et al. Food Chemistry: X 15 (2022) 100368

enzyme and pNPG were replaced by PBS. cultivars showed higher TPC than pineapple, banana, peach, lemon,
pear and orange (70.6–94.3 mg GAE/100 g FW) (Wolfe et al., 2008).
2.8.3. Pancreatic lipase inhibition Therefore, pomelo, especially cultivars like Liangpingyu and Shatianyu,
The pancreatic lipase inhibitory activity was measured according to are excellent dietary sources of phenolics.
the method of Zhu et al. (2014). A buffer containing 13 mmol/L Tris, 1.3
mmol/L CaCl2 and 150 mmol/L NaCl was used to prepare 4-MUO and 3.2. Total flavonoid contents
lipase. Briefly, 25 μL of samples and 50 μL of 4-MUO (0.1 mmol/L) were
added to a 96-well microplate, respectively. Then, the lipase (50 U/mL, The TFC of the grapefruit and pomelo cultivars are presented in
25 μL) was added to initiate the enzyme reaction. After the 96-well Fig. 1. The TFC ranged from 13.4 (Guanximiyu-R) to 193.3 (Liangpin­
microplate was incubated at 25 ◦ C for 30 min, the enzyme reaction gyu) mg CE/100 g FW with the CV of 90.1%, indicating significant ge­
was stopped by adding sodium citrate (0.1 mol/L, pH 4.2, 100 μL). notype differences in TFC among pomelo and grapefruit cultivars.
Finally, the fluorescence intensity was measured (excitation, 355 nm; Liangpingyu had the highest TFC, followed by grapefruit, Shatianyu and
emission, 460 nm). The pancreatic lipase inhibition (%) was calculated Yuhuanyu (p < 0.05). Guanximiyu-W and Guanximiyu-R had the lowest
as follows: [1-(A2-A1)/(A4-A3)]*100%, where A2 is the fluorescence of TFC (p > 0.05). In previous studies, the TFC of the pulp from different
the samples in the above measurement; A1 is the fluorescence of the litchi, apple and strawberry varied from 39.4 to 129.8 (averaging 67 mg
measurement in which the enzyme was replaced by buffer; A3 is the CE/100 g FW), 35.7–46.8, and 46.2–70.5 mg CE/100 g FW, respectively
absorbance of the measurement in which both the samples and enzyme (Meyers et al., 2003; Wolfe et al., 2003; Zhang et al., 2013). The average
were replaced by buffer; A4 is the absorbance of the measurement in content of the TFC in grapefruit and pomelo was 64.3 mg CE/100 g FW,
which the samples were replaced by buffer. which was higher than most of the fruits mentioned above.

2.9. Statistical analysis

3.3. Flavonoid compositions and contents

All measurements were conducted in triplicates and the results were

The compositions and contents of flavonoids in grapefruit and
presented as the mean ± SD. Statistical analysis were conducted by one-
pomelo varieties were analyzed by UHPLC-ESI MS/MS. Fourteen fla­
way ANOVA of SPSS 19.0 software, and p < 0.05 indicated statistical
vonoids were detected in the determined grapefruit and pomelo. The
significance. Pearson correlation was used to analyze the correlation
MS, MS-MS data and retention time of the 14 flavonoid standards are
between variables.
presented in Table 1. The flavonoid compositions and contents are
presented in Table 2. All the 14 flavonoids were detected in grapefruit,
3. Results and discussion but narirutin and neohesperidin were not detected or below the detec­
tion limit in the 5 pomelo cultivars. In Guanximiyu-W and Guanximiyu-
3.1. Total phenolic contents R, naringin was the most abundant flavonoid, followed by rhoifolin and
cigranoside C, and the 3 compounds accounted for 91% and 86% of the
The TPC of the grapefruit and pomelo cultivars are presented in detected flavonoids in Guanximiyu-W and Guanximiy-R, respectively.
Fig. 1. The TPC ranged from 91.8 (Guanximiyu-R) to 170.9 (Liangpin­ Melitidin was the largest amount of flavonoid in Shatianyu, followed by
gyu) mg GAE/100 g FW with the coefficient of variation (CV) of 24.9% bergamjuicin, naringin, cigranoside B and cigranoside A, and the 5
in the determined pomelo. Liangpingyu had the highest TPC, followed compounds possessed 97% of the detected flavonoids in Shatainyu.
by Shatianyu, grapefruit and Yuhuanyu (p < 0.05). Guanximiyu-W and Nevertheless, Rhoifolin was the most abundant flavonoid in Yuhuanyu,
Guanximiyu-R had the lowest TPC (p > 0.05). The pomelo and grape­ followed by naringin, and the 2 compounds took up 84% of the detected
fruit cultivars in the present study showed higher TPC than pomelo flavonoids in Yuhuanyu. Naringin was the most abundant flavonoid in
cultivars in Thailand (51.9–94.9 mg GAE/100 g FW) (Mäkynen et al., Liangpingyu, followed by cigranoside C, melitidin, neoeriocitrin and
2013). This is partly due to different methods used for phenolics rhoifolin, and the 5 compounds accounted for 98% of the detected fla­
extraction, together with the different cultivars tested. The TPC of vonoids in Liangpingyu. Naringin was the largest amount of flavonoid in
Liangpingyu was higher than those of cherry (Wolfe et al., 2008) and the grapefruit, followed by narirutin, cigranoside C, neohesperidin, hes­
majority of litchi cultivars (Zhang et al., 2013) (101.5–170.5 mg GAE/ peridin and melitidin, and the 6 compounds possessed 98% of the
100 g FW). Furthermore, the determined pomelo and grapefruit detected flavonoids in grapefruit.
As firstly separated compounds from Shatianyu with new structures
in our previous work (Deng et al., 2021), cigranoside C, D and E had
never been reported before in pomelo and grapefruit. Their contents

Table 1
The retention time, MS and MS-MS data of flavonoid standards observed by
UHPLC-ESI-MS/MS analysis.
Compound Retention time (min) [M-H]− (m/z) MS2 ions (m/z)

Cigranoside C 10.97 595 567, 259, 577

Isoquercitrin 19.71 463 300, 271
Neoeriocitrin 21.53 595 459, 151, 576
Narirutin 24.58 579 271, 151, 295, 313
Naringin 26.47 579 459, 271, 235
Cigranoside E 27.65 883 619, 577, 659
Bergamjuicin 27.96 885 579, 621
Rhoifolin 28.13 577 269, 413
Hesperidin 28.53 609 301, 325, 242
Neohesperidin 30.48 609 301, 286, 343, 242
Cigranoside A 32.38 723 677, 659, 580, 621
Cigranoside B 34.87 723 677, 659, 580, 451
Melitidin 36.76 723 677, 579, 621, 661
Fig. 1. TPC and TFC of different pomelo and grapefruit cultivars. Bars with no
Cigranoside D 37.28 721 268, 577, 619, 659
letters in common are significantly different (p < 0.05).

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M. Deng et al. Food Chemistry: X 15 (2022) 100368

Table 2
Flavonoid compositions of pomelo and grapefruit cultivars (μg/100 g FW). Values with no letters in common in each row are significantly different (p < 0.05). Tr: trace;
nd: not detected.
Compounds Guanximiyu-W Guanximiyu-R Shatianyu Yuhuanyu Liangpingyu Grapefruit

Cigranoside A 55.1 ± 1.7 cd 49.7 ± 4.2 cd 1145 ± 85 a 17.2 ± 0.9 d 172 ± 2 b 99.9 ± 3.1 c
Cigranoside B 93.1 ± 2.4 d 84.8 ± 3.3 d 1773 ± 40 a 19.4 ± 1 e 321 ± 12 b 168 ± 5 c
Cigranoside C 800 ± 36 c 544 ± 21 d 188 ± 11 f 404 ± 7 e 3375 ± 113 a 3165 ± 132 b
Cigranoside D 83.8 ± 1.6 c 75.7 ± 3.2 cd 380 ± 12 a 101 ± 6 b 69.3 ± 3.9 d 6.25 ± 0.11 e
Cigranoside E 106 ± 2 b 98.8 ± 4.7 c 203 ± 5 a 81.9 ± 1.9 d 22.0 ± 0.8 e 10.6 ± 0.9 f
Bergamjuicin 191 ± 3 bc 227 ± 6 b 5148 ± 91 a 10.2 ± 0.6 e 69.8 ± 3.8 d 142 ± 2 c
Neoeriocitrin 185 ± 3 d 82.9 ± 1.6 f 104 ± 6 e 481 ± 9 b 1473 ± 15 a 380 ± 10 c
Melitidin 478 ± 19 c 330 ± 17c 23338 ± 874 a 194 ± 7 c 1745 ± 100 b 1320 ± 17 b
Rhoifolin 4023 ± 46 b 1768 ± 37 c 164 ± 6 f 4135 ± 91 a 1025 ± 37 d 569 ± 29 e
Naringin 6875 ± 72 c 3613 ± 111 d 2448 ± 100 d 3245 ± 129 d 30100 ± 885 b 40430 ± 770 a
Hesperidin 1.7 ± 0.2 b 1.1 ± 0.1 b 1.35 ± 0.05 b 0.38 ± 0.01 b 2.8 ± 0.2 b 1670 ± 43 a
Neohesperidin Tr Tr Tr Tr Tr 2356 ± 107
Narirutin nd nd nd nd nd 21973 ± 664
Isoquercitrin 15.9 ± 0.4 d 12.1 ± 1 de 9.88 ± 0.83 e 109 ± 7 a 23.6 ± 1.2 c 35.5 ± 2.1 b
Sum 12907 ± 82 d 6887 ± 191 f 34902 ± 1175 c 8798 ± 105 e 38398 ± 1672 b 72325 ± 1554 a

ranged from 188 (Shatianyu) to 3375 (Liangpingyu), 6.25 (grapefruit) to 3.4. Antioxidant activity
380 (Shatianyu), 10.6 (grapefruit) to 203 (Shatianyu) μg/100 g FW,
respectively. The contents of cigranoside C ranked second or third place The antioxidant activity of the pomelo and grapefruit cultivars
in Liangpingyu, Guanximiyu-W, Guanximiyu-R and grapefruit among all evaluated by ORAC and CAA are presented in Fig. 2. The ORAC values of
the detected flavonoids. Although cigranoside F was also a new flavo­ these pomelo and grapefruit ranged from 678.9 to 1590.2 μmol TE/100
noid isolated from Shatianyu, it was not detected in the pomelo and g FW with a CV of 31.1%, indicating significant genotype differences in
grapefruit cultivars. These might be attributed to the fact that cigrano­ the ORAC activity among pomelo and grapefruit cultivars. Liangpingyu
side F was below the detection limit in Shatainyu or did not exist in other showed the highest ORAC activity (p < 0.05), followed by Shatianyu and
pomelo cultivars. Cigranoside A, cigranoside B, bergamjuicin and neo­ grapefruit. The ORAC values of the latter two were higher than that of
eriocitrin were previously found in traditional Chinese medicine, such as Yuhuanyu (p < 0.05). Guanximiyu-W and Guanximiyu-R presented the
the pericarp of C. grandis huajuhong (cigranoside A and B) (Ma et al., lowest ORAC activity (p > 0.05) among the determined cultivars. Highly
2018), the juice of C. bergamia bergamot (bergamjuicin) (Formisano significant correlations were observed between the ORAC activity and
et al., 2019) and the rhizomes of Drynaria fortunei (Kunze et Mett.) J. Sm phenolic contents of the tested pomelo and grapefruits, and the correl­
(neoeriocitrin) (Yang et al., 2015). However, the 4 flavonoids had not ative coefficient r was as high as 0.93 (p < 0.01). Although the ORAC
been detected in pomelo pulp before, and their contents ranged from activities of pomelo and grapefruit are lower than that of some citrus
17.2 (Yuhuanyu) to 1145 (Shatainyu), 19.4 (Yuhuanyu) to 1773 (Sha­ fruits, such as orange and lemon (2887 and 1848 μmol TE/100 g FW,
tainyu), 10.2 (Yuhaunyu) to 5148 (Shatianyu) and 82.9 (Guanximiyu-R) respectively) (Wolfe et al., 2008), they are comparable to those of pear,
to 1473 (Liangpingyu) μg/100 g FW, respectively. Melitidin and rhoi­ nectarine, watermelon, avocado, kiwifruit, mango, pineapple and ba­
folin were previously detected in the juice of pomelo and grapefruit, but nana (565–1759 μmol TE/100 g FW) (Wolfe et al., 2008).
their contents were calculated based on naringin equivalent (Zhang The CAA values of these pomelo and grapefruits ranged from 12.6 to
et al., 2011). In the present study, melitidin and rhoifolin, with the 48.4 μmol QE/100 g FW with a CV of 56.7%, indicating that the CAA
contents ranging from 194 (Yuhuanyu) to 23,338 (Shatainyu) and 164 activity showed higher genotype differences than the ORAC activity
(Shatainyu) to 4135 (Yuhuanyu) μg/100 g FW, respectively, were among pomelo and grapefruit. Liangpingyu presented the highest CAA
determined by absolute quantification. Naringin was the predominant activity followed by Shatianyu and Yuhuanyu (p < 0.05). In comparison,
flavonoid in citrus fruits including sour orange, grapefruit and pomelo the CAA value of the former was approximate twice that of the latter
(Gattuso et al., 2007). Its contents ranged from 2448 (Shatianyu) to two. The CAA activity of grapefruit was lower than those of other
40,430 (grapefruit) μg/100 g FW among the tested pomelo and grape­
fruit. This was comparable to the pomelo cultivars in Thailand (25–39
mg/100 g FW) (Chaiwong & Theppakorn, 2010). Hesperidin, neo­
hesperidin and narirutin were mainly reported in grapefruit, sweet or­
anges, lemons, and limes (Gattuso et al., 2007; Khan & Dangles, 2014;
Tocmo et al., 2020). These 3 flavonoids were mainly detected in
grapefruit in the present study, with the contents of 1670, 2356 and
21,973 μg/100 g FW, respectively. These was comparable to the levels in
grapefruit reported previously (Igual et al., 2013).
TFC of the tested pomelo and grapefruit determined by calculating
the monomer flavonoid contents was quite different from those
measured by an SBC assay. Using the former method, grapefruit showed
the highest TFC among these pomelo and grapefruit, almost twice that of
Shatianyu and Liangpingyu. However, using the SBC assay, Liangpingyu
showed the highest TFC, which was 3.8 and 2.5 times that of Shatianyu
and grapefruit, respectively. The reason for the inconsistence between
the two results might be that there were other undetected flavonoids
among pomelo and grapefruit cultivars besides the 14 flavonoids
detected in the present study, which could be inferred from the liquid
chromatograms of these pomelo and grapefruit cultivars (supplemen­
tary Fig. S1). Fig. 2. ORAC and CAA values of different pomelo and grapefruit cultivars. Bars
with no letters in common are significantly different (p < 0.05).

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M. Deng et al. Food Chemistry: X 15 (2022) 100368

determined cultivars except for Guanximiyu-R, which also showed molecular structure to a more non-planar and flexible stereochemical
similar activity to Guanximiyu-W. Correlation analysis showed that the structure, forming steric hindrance to reduce the ability of flavonoids to
Pearson coefficient r between the ORAC and CAA values of the tested bind with the enzymes (Sun, Warren and Gidley, 2019). The main fla­
pomelos and grapefruit was 0.66 (p = 0.15), indicating that the anti­ vonoids in pomelo and grapefruit analyzed in the present study were
oxidant activities reflected by these 2 methods were not completely flavanones, while flavonols and flavonons were important components
consistent. Compared with ORAC, a chemical antioxidant determination of many other fruits apart from flavanones (Balasuriya and Rupasinghe,
method, CAA assay was conducted in a more physiological reaction 2012; Pérez-Navarro et al., 2021).
system involving cells, which might give more indicative information for
the in vivo activity of the tested samples. In addition to phenolic con­ 3.6. Inhibitory activity to pancreatic lipase
tents, the CAA activity of the samples is also influenced by their phenolic
compositions since the uptake and metabolism of the antioxidants The pancreatic lipase inhibitory activity of different pomelo and
depended on their structures (Wolfe and Liu 2007). Our previous study grapefruit cultivars are presented in Table 3. The IC50 values of
found that naringin contributed the least to the CAA activity of phenolic pancreatic lipase inhibitory activity varied from 11.4 to 240 mg fruit/
extracts of Shatianyu pulp among the 11 isolated flavonoids (Deng et al., mL. Grapefruit, Liangpingyu and Guanximiyu-W presented the highest
2021). Coincidentally, the percentage of naringin in the TFC of grape­ pancreatic lipase inhibitory activity, followed by Guanximiyu-R and
fruit (52.3%) was higher than all the tested pomelo. These might explain Yuhuanyu (p < 0.05). Shatianyu showed the lowest inhibitory activity to
why grapefruit showed the lowest CAA value despite its relatively high pancreatic lipase, and its IC50 value was 10 to 21 times that of the former
phenolic content. Liangpingyu showed higher CAA activity than cran­ 3 cultivars (p < 0.05). In the tested grapefruit and pomelo, strong cor­
berry, plum and cherry (27.4–47.9 μmol QE/100 g FW) (Wolfe et al., relations were observed between the IC50 values of pancreatic lipase
2008), which were considered as good sources of natural antioxidants. inhibitory activity and the content of cigranoside A (r = 0.92, p < 0.01),
Although Shatianyu and Yuhuanyu had much lower CAA values than cigranoside B (r = 0.91, p < 0.05), cigranoside D (r = 0.97, p < 0.01),
Liangpingyu, they exhibited higher CAA activity than apple, red grape, cigranoside E (r = 0.89, p < 0.05), bergamjuicin (r = 0.95, p < 0.01) and
kiwifruit, mango, pineapple and orange (13.7–21.9 μmol QE/100 g FW) melitidin (r = 0.94, p < 0.01), respectively. These above-mentioned
(Wolfe et al., 2008). Moreover, the CAA activity of all tested pomelo and flavonoids all had a 3-hydroxy-3-methylglutaryl (HMG) substitution at
grapefruit was higher than lemon, peach, pear, cantaloupe and banana 7-O-neohesperidoside of the A ring, indicating that the presence of HMG
(3.2–12.3 μmol QE/100 g FW) (Wolfe et al., 2008). Therefore, pomelo, moiety in the structure of flavonoids might weaken their inhibitory
especially Liangpingyu and Shatianyu, are excellent sources of natural activity to pancreatic lipase. The lowest inhibitory activity to pancreatic
antioxidants. lipase of Shatianyu might be attributed to its highest content of HMG
substituting flavonoids, which was 13 to 75 times higher than those of
3.5. Inhibitory activity to α-amylase, α-glucosidase other pomelo and grapefruit cultivars. Huang et al. (2020) revealed that
hesperidin could interact with pancreatic lipase through hydrogen
The inhibitory activity to α-amylase and α-glucosidase of different bonds and van der Waals forces to change the secondary structure of
grapefruit and pomelo cultivars are presented in Table 3. The IC50 values pancreatic lipase, making itself the key pancreatic lipase inhibitor in
of α-amylase and α-glucosidase inhibitory activity varied from 707.1 to citrus peel extracts. The highest content of hesperidin in grapefruit
1788 and 1053 to 2514 mg fruit/mL, respectively. Shatianyu, grapefruit might explain its strongest pancreatic lipase inhibitory activity among
and Liangpingyu with proximate IC50 values showed higher α-amylase the tested pomelo and grapefruit cultivars. Grapefruit, Liangpingyu and
inhibitory activity than Guanximiyu-W, Guangximiyu-R and Yuhuanyu Guanximiyu-W showed comparable pancreatic lipase inhibitory activity
(p < 0.05). However, Yuhuanyu and Liangpingyu showed the highest with blackberry, strawberry, cherry, plum and apple (5.7–14 mg fruit/
inhibitory activity to α-glucosidase, followed by grapefruit, mL) and stronger inhibition of pancreatic lipase than pear, peach, ba­
Guanximiyu-W and Shatianyu (p < 0.05), and Guanximiyu-R presented nana and mandarine (30–135 mg fruit/mL) (Podsedek et al., 2014).
the lowest inhibitory activity to α-glucosidase (p < 0.05). IC50 values of Therefore, grapefruit and some pomelo cultivars were effective in­
commonly consumed fruits, such as sour cherry, strawberry, apple, ba­ hibitors of pancreatic lipase and had anti-obesity potential.
nana, bilberry, peach and pomegranate, ranged from 18.18 to more than
200 mg fruit/mL against α-amylase and from 156.4 to 399.1 mg fruit/ 4. Conclusion
mL against α-glucosidase (Podsedek et al., 2014). Evidently, the tested
pomelo and grapefruit presented much weaker inhibition to the sac­ Significant varietal differences were observed in flavonoid profiles
charides hydrolyzing enzymes. Generally, flavonols and flavones and in vitro bioactivity among different pomelo and grapefruit cultivars.
showed stronger inhibition to these enzymes than flavanones and fla­ Liangpingyu, Shatianyu and grapefruit had higher phenolic and flavo­
vanols (Spínola et al., 2020; Sun, Warren and Gidley, 2019), in which noid contents than other 3 pomelo cultivars. Fourteen flavonoid com­
the hydroxylated 2,3-double bond transformed the near-planar pounds were identified in pomelo and grapefruits. Naringin was the
major flavonoid in grapefruit, Guanximiyu-W, Guanximiyu-R, and
Liangpingyu, while melitidin and rhoifolin was the predominant flavo­
Table 3
noid in Shatianyu and Yhuanyu, respectively. Cigranoside C, D and E
The inhibitory activities of different pomelo and grapefruit cultivars to
α-amylase, α-glucosidase and pancreatic lipase. Values with no letters in com­ were firstly quantified in pomelo and grapefruit since they were isolated
mon in each column are significantly different (p < 0.05). from Shatianyu as new compounds. Furthermore, cigranoside C was one
of the main flavonoid compounds in Liangpingyu, Guanximiyu-W,
Cultivars Enzyme inhibition IC50 (mg of fresh fruit/mL)
Guanximiyu-R and grapefruit, ranking second or third place among all
α-amylase α-glucosidase pancreatic lipase the detected flavonoids. Cigranoside A, cigranoside B, bergamjuicin and
inhibition inhibition inhibition
neoeriocitrin were also firstly quantified in pomelo and grapefruit pulp.
Guanximiyu- 1558 ± 33b 1274 ± 18b 23.7 ± 2.1 a The total contents of the 7 flavonoids ranged from 1114.7 (Yuhuanyu) to
W
8941 (Shatainyu) μg/100 g FW, accounting for 5.5% to 25.6% of the
Guanximiyu- 1788 ± 208b 2514 ± 72 d 58.1 ± 1.4b
R total contents of 14 detected flavonoids. Pomelo and grapefruit
Shatianyu 707.1 ± 11.2 a 1988 ± 10c 240 ± 15c possessed strong antioxidant activity, especially CAA activity. Despite
Yuhuanyu 1693 ± 99.3b 1058 ± 62 a 72.6 ± 6.3b their weak α-amylase and α-glucosidase inhibitory activity, the deter­
Liangpingyu 798.3 ± 39.6 a 1053 ± 73 a 12.8 ± 0.3 a mined pomelo and grapefruit cultivars presented strong inhibition of
Grapefruit 877.8 ± 49.9 a 1255 ± 37b 11.4 ± 1.1 a
pancreatic lipase, especially grapefruit, Liangpingyu and Guanximiyu-

5
M. Deng et al. Food Chemistry: X 15 (2022) 100368

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The authors declare that they have no known competing financial Pérez-Navarro, J., Izquierdo-Caas, P. M., Mena-Morales, A., Martínez-Gascuea, J., &
interests or personal relationships that could have appeared to influence Gómez-Alonso, J. (2021). Genotypic variation in phenolic composition of novel
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This study was supported by the Guangdong Special Support Pro­ α-glucosidase and pancreatic lipase by phenolic compounds of Rumex maderensis
gram (2019BT02N112), the Guangdong Provincial Special Fund for (Madeira sorrel). Influence of simulated gastrointestinal digestion on
Modern Agriculture Industry Technology Innovation Teams hyperglycaemia-related damage linked with aldose reductase activity and protein
glycation. LWT - Food Science and Technology, 118, 108727.
(2021KJ117), the Special fund for Scientific Innovation Strategy- Salahuddin, M. A. H., Ismail, A., Kassim, N. K., Hamid, M., & Ali, M. S. M. (2020).
Construction of High-level Academy of Agriculture Science (R2020PY- Phenolic profiling and evaluation of in vitro antioxidant, α-glucosidase and
JG011, 202108TD), and Guangzhou Science and Technology Planning α-amylase inhibitory activities of Lepisanthes fruticosa (Roxb) Leenh fruit extracts.
Food Chemistry, 331, 127240.
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Polyphenols as inhibitors of alpha-amylase. Trends in Food Science & Technology, 91,
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