Content analysis and drug safety evaluation of four furanocoumarins in six pomelos produced in China

Abstract The content of 4 6',7'-dihydroxybergamottin (DHB), bergamottin, isoimperatorin and epoxybergamottin of six pomelos produced in China were detected by High-performance liquid chromatography-diode array detection and their safety of related medicines was evaluated by inhibition of medium concentration (IC50) of cytochrome oxidases CYP450-like. The results showed that the total content of the four furanocoumarins in these pomelo juices from high to low in order was Guanximi pomelo > Liangping pomelo > Pinghemi pomelo > grapefruit > Huyou > Shatian pomelo. The concentration of isoimperatorin in grapefruit, DHB, bergamottinand and isoimperatorin in Liangping, bergamottin and epoxybergamottin in Pinghemi and all the four furanocoumarins in Guanximi were exceeded the corresponding IC50; although Huyou and Shatian contained some furanocoumarins, they did not exceed IC50. Therefore, when taking drugs metabolised by CYP450-like enzymes, Guanximi, Liangping, Pinghemi, and grapefruit should be avoided to consume, but it is relatively safe to eat Huyou and Shatian. Graphical Abstract


Introduction
Pomelo (Citrus grandis Osbeck) is an important species of the genus Citrus in the family Rutaceae.China is one of the origins of pomelo, which was cultivated as early as 4000 years ago (Zhou and Ye 2010).In 2019, the planting area has reached 93.3 thousand hectares, with an annual output of 5.08 million tons, ranking the first in the world (Ministry of Agriculture and Rural Affairs, https://www.sohu.com/a/443150228_747096).Pomelo fruit has a unique and pleasant flavor and is very nutritious, and is an important fruit favored by consumers in China.According to the geography, climate, and variety of characteristics of the place of origin, there are two major types of pomelo in China.One is mainly native to Zhejiang, Fujian, Guangdong, and Taiwan on the southeast coast, represented by Wendan, including Guanximimi pomelo and Siji pomelo, Yuhuan pomelo, etc., the quality characteristics are moderately sweet and sour or more sour, juicy with a few or no seeds; the other is mainly native to inland areas such as Guangxi, Guangdong, Sichuan, Chongqing, Jiangxi, Yunnan, and Guizhou.Shatian pomelo is represented, including Rongxian Shatian pomelo, Meizhou Shatian pomelo, Changshou Shatian pomelo, Nankang pomelo, etc.The quality characteristics are sweet and low acid, less juice, and more seediness.Others include pomelo hybrids such as Huyou and grapefruit which are from abroad.Pomelo fruit has high nutritional value.A large number of studies have shown that pomelo fruit is rich in naringin, naringenin, vitamins, and other functional components, which have antibacterial, anti-inflammatory, antiviral, anticoagulant, prevention and treatment of cardiovascular diseases, neurodegenerative diseases, cancer, diabetes, etc. (Zeng et al; Jin et al. 2018;Tang et al. 2019;Varughese et al. 2022).Some metabolites in grapefruit pulp may interact with drugs.For example, Bailey et al. (2013) reported that grapefruit juice significantly increased the blood concentration of the antihypertensive prescription drug felodipine, thereby reducing its efficacy.Subsequent studies have found that more than 100 drugs related to antibiotics, antiviral, antifungal, antipsychotic, antidepressant, antiarrhythmic, antiatherosclerotic, antitumor, antihistamine, analgesia, hypnosis, hormones, etc., that be concomitantly used of grapefruit and other pomelo juices can produce similar side effects (Forrester et al.1992;Spatzenegger and Jaeger 1995;Kenworthy et al. 1999).Zhao et al. (2003) pointed out that these drugs have one thing in common, they are all metabolised by drug enzymes (CYP3A4, a subtype of cytochrome P450) in the body.The studies confirmed that grapefruit juice did inhibit CYP3A4 enzymes, their active effect would slow down the metabolism of the drug and increase the blood drug concentration.Further research found that some of the furanocoumarins contained in grapefruit juice worked, and grapefruit juice without furanocoumarins did not have this inhibitory effect of grapefruit.The UK Medicines Agency (MHRA) issued an update on contraindications for simvastatin use in December 2014, which included the avoidance of drinking grapefruit juice while taking simvastatin (https://www.gov.uk/drug-safety-update/simvastatin-updated-adviceon-drug-interactions).Therefore, doctors advise patients taking this type of drug to be cautious about eating pomelo fruit or juice.Many studies have shown that there are five furanocoumarins, namely berganmottin, 6 0 , 7 0 -dihydroxybergamottin (DHB), isoimperatorin, epoxybergamottin, and paradisins.which have strong inhibitory effects on P450 and its subtypes CYP1A2, CYP2A6, CYP3A4, CYP3A5, CYP1B1, CYP2B6, CYP2C9, CYP2C19, CYP2D6 and CYP2E1 ( Edwards and Bernier 1996;Edwards et al. 1996;Dresser et al. 2000;Goosen et al. 2000;Garvan et al. 2000;Kane et al. 2000;Frerot and Decorzant 2004;Goosen et al. 2004;Mertens-Talcott et al. 2006;Fujita et al. 2008;Chen et al. 2011;Cao et al. 2013).The chemical structures of the first four furanocoumarins are shown in Figure 1, and they have a common feature that an oxypsoralen group is linked at the 5th position of the furanocoumarin structure.Up to now, there are few relevant articles to evaluate the safety of taking the drug with the inhibition of medium concentration (IC 50 ) of furanocoumarin substance.Therefore, this study started with the analysis of furanocoumarins that have inhibitory effects on drugs in the main pomelo varieties in China and then compared them with the corresponding drug inhibitory doses to evaluate their drug safety.This study intends to evaluate the safety of taking the medicine based on finding out the distribution of specific furanocoumarins in the fruits of the main pomelo varieties in China, which is of great significance for avoiding risks and scientifically guiding the consumption of nutritious pomelo fruits and juices.

Comparison of the contents of four identical furanocoumarins in different pomelo juices
The six different pomelo juices were detected by HPLC-DAD for bergamottin, DHB, isoimperatorin, and epoxybergamottin (Figure S1).A quantitative analysis of the four target substances in pomelo juice listed in Table S1 was carried out.Table 1 shows the SPE and LSE contents of the four target substances in different pomelo juices, as well as their summed total content.Judging from the content of DHB in different varieties, the content of Guanximimi pomelo was the highest, which was 6546.04 lg/ L. Others from high to low are Liangping pomelo > grapefruit > Pinghemi pomelo > Huyou, it was not detected in Shatian pomelo juice, the difference  between these 6 varieties is several times to hundreds, even thousands of times (compared with Shatian pomelo).Among the six pomelo varieties, the trend of the content of bergamottin, was the same as that of DHB, and it was still the Guanximi pomelo with the highest content at 3822.90 lg/L, which was ten to several thousand times that of other varieties.However, neither the grapefruit juice nor the Shatian pomelo juice was detected with bergamottin.The content trends of isoimperatorin in these six pomelo varieties are different.Liangping pomelo had the highest content, up to 36064.23 lg/L, followed by Guanximi pomelo, which was 268.14 ± 10.56 lg/L, and the others from high to low (grapefruit > Shatian pomelo > Huyou), and it was not detected in Pinghemi pomelo; the Guanximi pomelo had the highest epoxybergamottin content of 448.19 ± 78.55 lg/L, followed by Pinghemi pomelo, was 207.84 lg/L, and the substance was not detected in Liangping pomelo, Huyou, Shatian pomelo, and grapefruit.Judging from the total content of these four furanocoumarins, Guanximi pomelo > Liangping pomelo > Pinghemi pomelo > grapefruit > Huyou > Shatian pomelo.

Comparison of the contents of four different furanocoumarins in the same pomelo juice
The contents of four furanocoumarins in the juice of each pomelo variety from high to low are as follows: Guanximi pomelo: DHB > bergamottin > epoxybergamottin > isoimperatorin; the content of DHB is about 1.7 times that of bergamottin, about 15 times that of epoxybergamottin and 24 times that of isoimperatorin.This is quite different from the 1.61 lg/g of DHB measured by Ma (2013), some difference from 0.13 lg/g of epoxybergamottin, and a few difference of 4.01 lg/g of bergamottin.This difference is related to the authors' determination of furanocoumarins in both the juice and the pulp precipitate.
Liangping pomelo: Isoimperatorin > DHB > bergamottin, no epoxybergamottin was detected.The content of isoimperatorin is more than 3 times that of DHB, and is about 94 times that of bergamottin; Pinghemi pomelo: bergamottin > epoxybergamottin > DHB, no isoimperatorin was detected, the content of bergamottin was 1.8 times that of epoxybergamottin, and is about 2 times that of DHB.
Grapefruit: DHB > Isoimperatorin, no bergamottin and epoxybergamottin were detected, and the content of DHB was 2.6 times that of isoimperatorin.
Huyou: DHB > Bergamottin > Isoimperatorin, no epoxybergamottin was detected.The content of DHB is about 2 times that of bergamottin, and 10 times that of isoimperatorin.The content of DHB was very close to the 0.06 lg/g measured by Ma (2013), and the content of bergamottin is lower than the 0.04 lg/g measured by Ma (2013).However, the total content of these three furanocoumarins detected in this study was only 81.74 lg/L, which was 0.77% of the 10637.08lg/L in Guanximi pomelo and 0.22% of the 37643.01lg/L in Liangping pomelo.
Shatian pomelo: Only a small amount of isoimperatorin was detected, and none of the three others were detected.Ma (2013) detected 0.06 lg/g and 9.41 lg/g of epoxyberganmottin in different seasons.

There is a large number of furanocoumarins in the juice precipitation
The four furanocoumarins detected in the sample centrifugal precipitation are more complete than the supernatant, and some even have higher contents.The reason may be that the furanocoumarins exist in the pulp cells and the cell wall space, and the precipitate was residual.cells and cell wall fragments, containing considerable amounts of furanocoumarins.Therefore, when measuring these components by centrifugation, it is necessary to measure the content both of the supernatant and the precipitate.

The moderate amount of inhibition is the basis for the safety evaluation of the drug
A large number of studies have shown that furanocoumarins have strong inhibitory effects on cytochrome enzymes CYP450 and its subtypes CYP3A, CYP3A4, CYP1A1, CYP1A2, CYP2B6, CYP2D6, and CYP2C9, which are involved in drug metabolism.This inhibitory effect is carried out by inhibiting the metabolic activity of these enzymes on related drugs and the operation of P-glycoprotein, thus inhibiting the oxidative metabolism of related drugs, resluting in increased the blood drug concentration and reduced the efficacy.See Table S2 for the median inhibitory effect (IC 50 ) of these four furanocoumarins on cytochrome enzymes.Different scholars have studied the IC 50 of a certain furanocoumarin against the same drug enzyme, which is even very different.For example, the IC 50 of bergamottin against CYP3A4 is the lowest at 0.41 lmol/L (Zsid o et al. 2020), and the highest is 6.78 ± 0.091 lmol/L (Girennavar et al. 2006).Another example is the IC 50 of DHB for CYP3A4, the lowest is 0.3 lmol/L (Cesar et al. 2009), the highest is 3.26 ± 0.107 lmol/L (Girennavar et al. 2006), and the lowest IC50 of epoxybergamottin to CYP3A4 is 0.33 lmol/L (Schmiedlin-Ren et al. 1997), the highest is 4.2 lmol/L (Wangensteen et al. 2003), they differ by more than ten times.From the perspective of safety, this study selected the lowest known IC 50 value to evaluate its food and drug safety.Judging from the test results (Table S3), Shatian pomelo juice contained only 0.1624 lmol/L of isoimperatorin, which was lower than any IC 50 .Although Huyou juice contained DHB, bergamottin, and isoimperatorin, their concentration was lower than the corresponding drug enzyme IC 50 ; the concentration of DHB in grapefruit juice exceeded the IC 50 of 3 drug enzymes, and the concentration of isoimperatorin exceeded 1 drug enzyme IC 50 ; the DHB content in Liangping pomelo juice exceeded the IC 50 of 5 medicinal enzymes, and the concentration of isoimperatorin exceeded the IC 50 of 3 medicinal enzymes; the bergamottin content in Pinghemi pomelo juice exceeded the IC 50 of 6 medicinal enzymes, it's DHB exceeded the IC 50 of 2 medicinal enzymes, and the concentration of epoxybergamottin was higher than the corresponding IC 50 of 1 medicinal enzyme; the concentrations of bergamottin and DHB in Guanximi pomelo juice were much higher than the IC 50 of the discovered medicinal enzymes, and the concentrations of isoimperatorin and epoxybergamottin were both higher than the IC 50 of 1 medicinal enzyme.Health and food safety has always been important issues that cannot be ignored, because furanocoumarins have side effects against more than 100 kinds of drugs, such as tinctures, dihydropyridines, midazolam, cyclosporine, carbamazepine, and warfarin, etc. that treat for more than 10 types of diseases such as cardiovascular disease, allergies, and mental illness, etc.Therefore, for patients taking these drugs, it is best to avoid eating the fruits and juices of Guanximi pomelo, Liangping pomelo, Pinghemi pomelo, and grapefruit, while Huyou and Shatian pomelo are relatively safe.However, so far, no authoritative organisation has unified the inhibitory amount of furanocoumarins on cytochrome enzymes, and the content of furanocoumarins that have inhibitory effects in the fruits of different pomelo varieties in different producing areas is also different.Therefore, taking the medicine during the period, try not to eat pomelo fruits and their products at the same time, so as not to affect the curative effect.On the other hand, it is necessary to strengthen the research on the safety of pomelo fruits and medication to ensure the edible safety of pomelo fruits and juices.Israeli scientists are cultivating new grapefruit varieties with low furanocoumarin content and high naringin and naringenin content (Shaya et al. 2016;Guttman et al. 2020).Therefore, while avoiding the side effects of furanocoumarin on drugs, it could also give full play to the therapeutic effects of naringin and naringenin, which are rich in pomelo fruits, on diseases, this is a solution to the food and drug safety of pomelo fruits.

Materials and reagents
The test materials were randomly collected from six pomelo varieties or hybrids in Chongqing, Guangdong, Fujian and Zhejiang in China, respectively, at the commercial maturity stage, intact fruits with the characteristics of the variety were peeled and squeezed to get juice, filtrated through an 80-mesh sieve, and stored at À20 C for later use.See Table S4 for sample details.The main reagents are methanol, acetonitrile, and ethyl acetate (chromatographically pure) from Honeywell Company in the United States.DHB and isoimperatorin standards were purchased from Shanghai Yuanye Biotechnology Co., Ltd in China.Bergamottin and epoxybergamottin standards were purchased from ChromaDex.lnc,USA.

Instruments and equipment
The instrumentation detail was provided in the supplemental material S1 3.3.Experimental method

Sample pretreatment method
The samples frozen were completely dissolved with cold water, and Brix and pH were detected.The results are shown in Table S4.The sample processing and detection methods refer to Li et al. (2019), with only slight improvements.Accurately remove 9 mL of each sample and transfer it to a 10 mL centrifuge tube, adjust to 12,000 rpm, and centrifuge at 4 C for 15 min to obtain juice supernatant and pulp pellet.In order to completely obtain the content of these furanocoumarins in fruit juice, not only the supernatant liquid is subjected to solid-phase extraction (SPE), but also the precipitate is also subjected to liquid-solid extraction (LSE).The method details of SPE and LSE were shown as supplemental material S2.

Preparation of standard reference solution
Accurately weigh an appropriate amount of the standard, dissolve it in methanol and dilute to 5 mL to prepare a standard stock solution, which is then diluted step by step to obtain 7 mixed standard solutions with different mass concentrations.

Data analysis
The four furanocoumarins were quantified by the external standard method, and double-identified by the retention time and characteristic ultraviolet absorption spectrum of the standard product on HPLC.As shown in Figure S1.The data of this experiment are all from 3 parallels.The average value, standard deviation, and charting are calculated by ChemDraw and Office Excel 2016 (Microsoft, USA) software; Open LAB CDS Chem Station workstation is used to collect information, data acquisition, and simple analysis of HPLC-PDA.

Conclusion
In this experiment, the contents of four furanocoumarins in six varieties of pomelo juices produced in China were determined, and the results showed that their total contents in order were Guanximi pomelo > Liangping pomelo > Pinghemi pomelo > grapefruit > Huyou > Shatian pomelo.The concentration of isoimperatorin in grapefruit juice exceeded the corresponding drug-metabolising enzyme inhibitory level (IC 50 ); the concentrations of DHB, bergamottin, and isoimperatorin in Liangping pomelo juice all exceeded the corresponding IC 50 ; Pinghemi pomelo juice had higher concentrations of the corresponding IC 50 of bergamottin and epoxybergamottin; the concentrations of these four furanocoumarins in Guanximi pomelo juice were much higher than IC 50 ; Huyou juice contained bergamottin, DHB and isoimperatorin, but their concentration were very low, which did not exceed IC 50 ; Shatian pomelo only contained a small amount of isoimperatorin, and the concentration was below its IC 50 .When taking drugs metabolised by CYP450 enzymes, it would avoid eating Guanximi pomelo, Liangping pomelo, Pinghemi pomelo, and grapefruit, but it is relatively safe to consume Huyou and Shatian pomelo.

Table 1 .
The contents of four furanocoumarins in six different pomelos juices produced in China (lg/L).