Furanocoumarins (FCs) are plant defense compounds that function as toxins against pathogens, insects and other plant pests [1] [2] . Insects exposed to linear FCs suffer decreased larval weight, extended generation time and increased mortality [3] . High doses of some FCs are also toxic to humans; among maladies caused in humans by FCs are “celery picker’s itch” and “bartender’s itch”, caused by contact with FCs-containing celery plants or oil of lime peels, respectively. Also, FCs can cause temporary or even permanent blindness if introduced into the eyes. Some FCs are also carcinogenic and teratogenic, i.e. they can cause cancer and birth defects, respectively [3] - [5] .

FCs are found in several plant families, including edible plant species such as the citrus species pummelo and grapefruit, but the levels of mutagenic FCs (such as psoralen) in pummelo and grapefruits are rather low, and therefore there is no risk associated with consumption of their fruit and juice for healthy individuals [3] . Citrus is one of the most important fruit crops worldwide with total annual production of 115.5 million tons, to which grapefruit contributes about 5.5% [6] . However, in recent years, there has been a decline in grapefruit consumption, especially in the US, as a result of the “grapefruit juice effect”, attributed to FCs: FC-containing grapefruit juice negatively affects the body’s degradation of medicines, including statins, leading to overdose effects.

The mechanism of the “grapefruit juice effect” is based on FCs that inhibit the intestinal drug catabolic enzyme CYP3A, thereby leading to accumulation of medicines, which in turn can lead to harmful effects ranging from relatively mild hypotension and dizziness in the case of some calcium-channel blockers, to potentially severe nephrotoxicity in the case of some immunosuppressant drugs [5] [7] - [16] .

Drugs that interact with FCs [17] include:

ANTICANCER

Dasatinib (leukaemia)

Erlotinib (lung cancer and pancreatic cancer)

Everolimus (kidney cancer)

Lapatinib (breast cancer)

Nilotinib (leukaemia)

Pazopanib (kidney cancer)

Sunitinib (kidney/gastrointestinal cancer)

Vandetanib (thyroid cancer)

Venurafenib (skin cancer)

ANTI-INFECTIVE

Erythromycin (antibiotic)

Halofantrine (antimalaria)

Maraviroc (antiHIV)

Primaquine (antimalaria)

Quinine (antimalaria)

Rilpivirine (antiHIV)

ANTICHOLESTEROL

Atorvastatin

Lovastatin

Simvastatin

CARDIOVASCULAR

Amiodarone (heart rhythm disorders)

Apixaban (anticlotting)

Dronedarone (heart rhythm disorders)

Eplerenone (heart failure)

Felodipine (high blood pressure/angina)

Nifedipine (high blood pressure/angina)

Quinidine (heart rhythm disorders)

Rivaroxaban (blood anticlotting)

Ticagrelor (blood anticlotting after heart attack)

CENTRAL NERVOUS SYSTEM

Oral alfentanil (painkiller)

Oral fentanyl (painkiller)

Oral ketamine (painkiller, sedative)

Lurasidone (schizophrenia/mental health problems)

Oxycodone (painkiller)

Pimozide (schizophrenia/other mental health problems)

Ziprasidone (schizophrenia, mania, bipolar disorder)

GASTROINTESTINAL

Domperidone (antinausea)

IMMUNOSUPPRESSANTS

Cyclosporine (post organ transplant, rheumatoid arthritis, psoriasis)

Sirolimus (post organ transplant)

Tacrolimus (post organ transplant)

URINARY TRACT

Solifenacin (frequent urination/incontinence)

Silodosin (enlarged prostate)

Tamsulosin (enlarged prostate)

The FC-biosynthesis pathway starts with the amino acid phenylalanine (Figure 1) via umbelliferone to obtain linear or angular FCs. In citrus, only the linear FC-biosynthesis pathway is present which leads to formation of demethyl suberosin by alkylation of umbelliferone (Figure 1). Three FCs―6,7-dihydroxybergamottin (6,7- DHB), bergamottin and paradisin C, appear to co-segregate genetically, which suggests that these three compounds are tightly linked metabolically as co-products of the “bergamottin pathway” [19] . Ranked in descending order of their inhibitory potency, the FCs found in grapefruit are: paradisin C > 6,7-DHB > bergamottin > isoimperatorin > bergapten > bergaptol [9] [20] .

In the present study, we report on variations in the biosynthesis levels of various FCs in diverse varieties of grapefruit and pummelo. Twelve citrus cultivars (Table 1), including four pummelos, two grapefruits, one mandarin, one orange and three newly selected grapefruit-like varieties, were tested for juice and leaf levels of five furanocumarins (FCs)―bergamottin, bergaptol, epoxy bergamottin, 6,7-dihydroxybergamottin and isoimperatorin- and two FC precursors-psoralen and umbelliferone. The results show that the orange and the mandarin do not contain FCs; moreover, the lack of FC precursor compounds suggests that the biosynthesis pathway is entirely not active in mandarins and oranges. We also observe great diversity in the accumulation of FCs and their precursor compounds in various grapefruit and pummelo cultivars. Finally, we describe two new low-FC and seedless grapefruit-like cultivars, “Aliza” and “Cookie” that were selected in our breeding program by induced mutagenesis [21] .

FCs were found at varied concentrations in different cultivars, but were completely absent from mandarins, oranges and were close to absent in the new grapefruit-like varieties “Aliza” and “Cookie” (Figure 2). In grapefruit and pummelo, Bergamottin and 6,7-DHB were found at higher concentrations than other FCs, probably because they are final products that accumulate over time. In correlation, the FC precursors are present in very low concentrations in grapefruit and pummelo (Figure 2), as they are utilized for biosynthesis of the final products. Interestingly, although the FC biosynthesis pathway of grapefruit (Figure 1) was inherited from pummelo, the accumulated concentrations of bergamottin and 6,7-DHB in the grapefruit cultivars “Duncan” and “Marsh” (a seedless mutation of “Duncan”) were much higher than those observed in the pummelo cultivars that we examined (Figure 2).

The complete absence of FCs and FC precursors, from umbelliferone to psoralen in oranges and mandarins (Figure 2), suggests that the entire pathway for biosynthesis of FC is absent or inactive in these species, and that only a null allele for FC biosynthesis can be inherited from these species. The new selection “Aliza”, a hybrid of “Chandler” pummelo and “Orah” mandarin is almost completely void of FCs, suggesting the pummelo parent is heterozygous for FC biosynthesis (i.e. contains one active copy and one inactive copy, presumably inherited by “Aliza”). Furthermore, the finding that the pummelo hybrid “Hana” (cross between “Chandler” and “Tahitian” pummelos) has no FCs (Figure 2) indicates that “Chandler” probably also contributed its inactive allele for FC biosynthesis to “Hana”. In contrast, “Chandler” contributed its active allele for FC biosynthesis to the pummelo “Flamingo” (a different cross between “Chandler” and “Tahitian” pummelos) (Figure 2); the rather higher level of 6,7-DHB in “Flamingo” might be a result of the “release” of the isoimperatorin that accumulates in “Tahitian”, by the active allele of “Chandler”.

The concentrations of bergamottin and 6,7-DHB observed in 4- to 5-month-old leaves were extremely low relative to those observed in the fruits. Pummelo cultivars have much lower concentrations of epoxy bergamottin in their fruits versus other FCs compounds (Figure 2(a)), but the accumulated amounts of this FC in their leaves were equivalent to the highest concentrations observed among all of the examined cultivars (Figure 2). We note that, during commercial processing, epoxy bergamottin may be hydrolyzed to 6,7-DHB, which has been identi-

fied as a potentially important inhibitor of CYP3A4 [17] [22] .

In all of the fruit juices of the examined cultivars, bergaptol and isoimperatorin were found at significantly lower concentrations than the other FCs, reflecting the fact that they are intermediate products in the pathway. However, pummelo “Tahitian” contained an extraordinarily high concentration of isoimperatorin; this pummelo also had very low concentrations of bergamottin and 6,7-DHB, suggesting that the pathway in this variety is partially blocked and it accumulates isoimperatorin instead of bergamottin and 6,7-DHB.

We have selected two new seedless grapefruit-like cultivars, “Aliza” and “Cookie” (Table 1), which do not accumulate FCs. At the start of ripening “Aliza” fruits have green skin and yellow flesh, but the former turns

yellow later in the season, and when fully ripe these fruits have orange skin and orange flesh (Figure 3). By inducing mutations, we selected seedless types of “Aliza” and “Cookie” (Figure 3 and Figure 4). Both of these selections have unique and favorable characteristics: they are juicy and, because they contain naringin (Table 2), they have been classified as grapefruit-like cultivars; “Aliza” and “Cookie” are also easy to peel.

FC composition and concentrations are highly variable in citrus, suggesting that selection for new grapefruit like cultivars with low FCs is a feasible objective. Indeed, we describe the selection of two new low FC grapefruit-like varieties that are potentially safe for consumption by humans using statins and other drugs. The corre-

lation of FC accumulation between leaves and fruits provides a potential marker for screening young plantlets for additional FC-free grapefruit-like varieties.

LenaFidel,MiraCarmeli-Weissberg,YosefYaniv,FelixShaya,NirDai,EranRaveh,YoramEyal,RonPorat,NirCarmi, (2016) Breeding and Analysis of Two New Grapefruit-Like Varieties with Low Furanocoumarin Content. Food and Nutrition Sciences,07,90-101. doi: 10.4236/fns.2016.72011