The New Lemon Cultivar ‘Yellow Bell’ Increases Serum Adiponectin Levels in Rats Fed a High Fat Diet

Abstract

This study investigated the flavonoid contents of the new lemon cultivar ‘Yellow Bell’ (YB) and its effects on fat metabolism in rats. YB contains neoeriocitrin, hesperidin, rhoifolin, neodiosmin and aurapten, and is high in neohesperidin (1020 – 1270 mg/100 g whole dry weight) and naringin (178 – 270 mg/100 g whole dry weight). The flavonoid pattern of YB differs from that of the conventional lemon cultivar. In experiment 1, rats were fed a high fat diet (HF) or HF containing 5% dry whole YB or conventional lemon cultivar Villafranca (Vil). Serum adiponectin was significantly increased by YB intake compared with control at 2 and 5 weeks (p < 0.05, p < 0.01, respectively). In experiment 2, rats were fed HF or HF containing 5% dry whole YB or dry peel YB. Serum adiponectin was significantly increased by whole YB intake compared with control at 5 weeks (p < 0.05). We revealed the novel effect of YB on increasing serum adiponectin.

Introduction

In Japan, Hiroshima prefecture produces the largest amount of lemons, accounting for 51% of the 2010 national yield. The new lemon cultivar ‘Yellow Bell’ (YB) was bred by the Agricultural Technology Research Center, Hiroshima Prefectural Technology Research Institute (Kaneyoshi et al., 2014). ‘Yellow Bell’ (registration number 21709 i)) is a natural hybrid cultivar of Michitani-line Villafranca (lemon). It was estimated that the pollen parent is Summer Fresh (Citrus L., registration number 299 ii)) by SSR (Nakano et al., 2012). Villafranca (Vil) is a conventional lemon. Summer Fresh (SF) is a hybrid cultivar of hassaku (Citrus hassaku hort. ex Tanaka) and natsudaidai (Citrus natsudaidai Hayata ii)). YB is characterized as triploid, seedless, round shape and mildly acidic (Kaneyoshi et al., 2014). Thus, the features of YB differ from conventional lemon.

Citrus fruits have different patterns of flavonoid and aurapten contents (Nogata et al., 2006; Takahashi et al., 2002). Flavonoids are characteristic constituents of fruits (Nogata et al., 2006). Aurapten is a kind of coumarin, found in large amounts in hassaku and natsudaidai (Takahashi et al., 2002). Further, flavonoids and aurapten have biological activities (Jung et al., 2003; Gorinstein et al., 2007; Murakami et al., 1997; Tanaka et al., 1997).

The biological activities of lemon or hassaku have been reported previously. Administration of flavonoid solutions prepared from lemon juice was observed to lower systolic blood pressure in SHR rats (Miyake et al., 1998). Administration of an ethanolic extract from hassaku improved blood fluidity in a rat model of LPS-induced disseminated intravascular coagulation (Itoh et al., 2010). In these studies, extracts of citrus fruits were used. In this study, we investigated the flavonoid and aurapten concentrations of YB whole fruit or peel and their effects on fat metabolism in rats.

Materials and Methods

Fruits The examined Vil and YB were grown and harvested at the Agricultural Technology Research Center, Hiroshima Prefectural Technology Research Institute (Hiroshima, Japan). The ripe Vil was harvested from the Mihara field (Hiroshima, Japan) in November and December 2011. The ripe YB was harvested from the Akitsu field (Hiroshima, Japan) in December 2011 and 2012.

Determination of flavonoid and aurapten contents Flavonoid contents were determined based on the method of Nogata et al. (Nogata et al., 2006). The aurapten content was determined based on the method of Ando et al. (Ando et al., 2009). Authentic standards used for identifying flavonoids and aurapten were purchased from Funakoshi Co., Ltd. (Tokyo, Japan). All fruits were lyophilized and ground using an ultracentrifugal mill (ZM1000; Retsch, Germany) and passed through a 0.5 mm filter. Each ground sample (100 mg) was extracted for 16 hr with 5 mL of methanol (HPLC grade): DMSO (HPLC grade) (1:1,v/v) at 20°C. After centrifugation at 3,000 × g for 10 min, the extract was collected and the residue was extracted twice more with 1 mL of the same solution. The combined extract was adjusted to 10 mL with the same solution, and filtered through a membrane filter (0.45μm) before HPLC analysis. The HPLC system (Waters, USA) consisted of two pumps, photodiode array detector, column oven and C18 column (Lichrospher 100-RP, 250 × 4.0 mm; Merck, Germany). A two-solvent gradient system was used for the assays, aqueous 10 mmol/L phosphoric acid (A) and methanol (B), as follows: (1) 0 – 65 min, 10 – 45% B, (2) 65 – 105 min, 45 – 100% B, (3) 105 – 110 min, 100% B. The flow rate was 0.6 mL/min, with a column oven temperature of 40°C and an injection volume of 10μL. The detection wavelength was 285 nm; spectra were measured from 200 to 360 nm. Flavonoids and aurapten were identified by comparing their retention times and spectra with those of standards. The concentrations of flavonoids and aurapten were calculated from each standard curve.

Animals and diets Male Sprague-Dawley rats (4 weeks old) were purchased from Hiroshima Laboratory Animal Center (Hiroshima, Japan) and maintained according to the Guidelines for the Care and Use of Laboratory Animals established by the Food Technology Research Center, Hiroshima Prefectural Technology Research Institute. Animals were individually housed in an air-conditioned room at 23 – 26°C with a 12 hr light cycle (light 5:00 – 17:00, dark 17:00 – 5:00), and had free access to the experimental diets and water. Rats were fed a basal diet (MF powder, Oriental Yeast Co., Ltd., Tokyo, Japan) for 6 days and were then assigned to one of three groups, each consisting of 8 rats. The average initial body weight in experiments 1 and 2 was 134 g and 136 g, respectively. For experiments 1 and 2, the chemical composition of the administered lemon is presented in Tables 1 and 2, and the components of each experimental diet are presented in Tables 3 and 4. YB contains more carbohydrate and less dietary fiber than Vil; also, whole YB has more carbohydrate and less dietary fiber than peel YB. Because of these differences, the dietary levels of each experimental diet were adjusted. Each experimental diet contained 20% beef tallow. In experiment 1, 5% of lyophilized and ground (0.5 mm filter) whole Vil (harvested in November and December, 2011) or whole YB (harvested in December, 2011) was contained. In experiment 2, 5% of lyophilized and ground (0.5 mm filter) whole YB or peel YB (harvested in December, 2012) was contained. These experimental diets were placed in individual food cups at 4:50 p.m. Food intake was controlled to ensure identical caloric intake. All diets were consumed by the next day at 9:00 a.m. Rats were fed the experimental diets for 5 weeks. Blood samples were collected at 2 and 5 weeks later. Food was removed from the cages at 8:30 a.m., and 5 hours later blood samples were collected from the caudal vein (at 2 weeks) or from the ventral aorta under diethyl ether anesthesia (at 5 weeks). Serum was isolated by centrifugation at 1,500 × g for 10 min. The liver was immediately removed and stored at −80°C until analysis. Epididymal and perirenal fat pads were immediately removed and weighed.

Table 1. Components of lemon (experiment 1)

	% (lyophilized)
	whole Vil	whole YB
Water	10.4	11.5
Protein	6.2	7.4
Fat	3.4	4.0
Ash	3.7	3.2
Dietary fiber	34.3	27.1
Carbohydrate	42.0	46.8

Vil, Villafranca; YB, Yellow Bell

Table 2. Components of lemon (experiment 2)

	% (lyophilized)
	whole YB	peel YB
Water	8.8	3.8
Protein	4.9	3.8
Fat	2.4	2.1
Ash	3.0	2.7
Dietary fiber	27.7	41.4
Carbohydrate	53.2	46.2

YB, Yellow Bell

Table 3. Components of experimental diets (experiment 1)

	Control (%)	whole Vil (%)	whole YB (%)
whole Vil	-	5.0	-
whole YB	-	-	5.0
Milk casein	24.0	23.6	23.6
α-Cornstarch	26.3	23.7	23.3
Beef tallow	20.0	19.8	19.8
Sucrose	20.0	20.0	20.0
Cellulose	5.0	3.2	3.6
L-cystine	0.2	0.2	0.2
Mineral mixture*	3.5	3.5	3.5
Vitamin mixture*	1.0	1.0	1.0

Vil, Villafranca; YB, Yellow Bell

*  AIN-93G mixture. Vitamin mixture contained 20% choline bitartrate. The diet consisited of protein, 19.8%; fat, 20%; fiber, 5.4%; calories, 453 – 454 kcal/100 g.

Blood and liver analyses The frozen liver was homogenized and extracted with chloroform : methanol (2:1, v/v) by the method of Folch et al. (Folch et al., 1957); extracts were evaporated and reconstituted with isopropyl alcohol containing 10% TritonX100. Serum adiponectin and leptin were determined using ELISA kits (adiponectin kit, Otsuka Pharmaceutical Co., Ltd., Tokyo, Japan; leptin kit, Morinaga Institute of Biological Science, Inc., Kanagawa, Japan). The other serum concentrations were measured by the contract research organization, Japan Clinical Laboratories, Inc. (Hiroshima, Japan). Triacylglycerol, total cholesterol, HDL-cholesterol and LDL-cholesterol were determined by enzymatic methods. The liver triacylglycerol and total-cholesterol were measured by using enzymatic kits (Wako Pure Chemical Industries, Ltd., Osaka, Japan).

Table 4. Components of experimental diets (experiment 2)

	Control (%)	whole YB (%)	peel YB (%)
whole YB	-	5.0	-
peel YB	-	-	5.0
Milk casein	24.0	23.7	23.8
α-Cornstarch	26.3	23.1	23.7
Beef tallow	20.0	19.9	19.9
Sucrose	20.0	20.0	20.0
Cellulose	5.0	3.6	2.9
L-cystine	0.2	0.2	0.2
Mineral mixture*	3.5	3.5	3.5
Vitamin mixture*	1.0	1.0	1.0

YB, Yellow Bell

*  AIN-93G mixture. Vitamin mixture contained 20% choline bitartrate. The diet consisted of protein, 21.0%; fat, 20.4%; fiber, 5.2%; calories, 457 kcal/100 g.

Statistical analysis The experimental data are expressed as the mean ± standard error (SE). The statistical analysis was performed using Tukey's multiple-range test.

Results and Discussion

Flavonoid and aurapten contents Whole YB fruit was used. Because the features of YB differ from conventional lemon (Kaneyoshi et al., 2014), we may be able to use YB in novel ways for cooking and eating, for example, cooking and eating whole fruit.

The individual flavonoid and aurapten concentrations of whole Vil, whole YB and peel YB are shown in Tables 5 and 6. Vil contains diosmin and is rich in eriocitrin (744 mg/100 g whole dry weight) and hesperidin (1200 mg/100 g whole dry weight). This result is similar to that previously reported (Nogata et al., 2006). YB contains neoeriocitrin, hesperidin, rhoifolin, neodiosmin, and aurapten, and is rich in neohesperidin (1020 – 1270 mg/100 g whole dry weight) and naringin (178 – 270 mg/100 g whole dry weight). Peel YB has 1.1 – 2.1 times the flavonoids of whole YB. SF is a hybrid cultivar of hassaku and natsudaidai ii). Hassaku and natsudaidai contain naringin and neohesperidin (Nogata et al., 2006). It is suggested that YB's naringin and neohesperidin components originate from its grandparents. YB has a different flavonoids pattern compared with Vil and conventional lemon (Nogata et al., 2006). Flavonoids have various biological activities. For example, naringin was shown to lower plasma total-cholesterol and LDL-cholesterol and increase erythrocyte superoxide dismutase and catalase activities (Jung et al., 2003). Diosmin and hesperidin were shown to inhibit the development of neoplasms and aberrant crypt foci (Tanaka et al., 1997), and hesperetin lowered plasma total-cholesterol and triglyceride levels (Kim et al., 2003). These results suggest that YB may have different biological activities compared with conventional lemon.

Table 5. Flavonoid and aurapten concentrations in lemon (harvested in November – December 2011)

	Concentration (mg/100g-dryweight)
	ERC	NER	NRG	HSP	NHP	RFN	DSM	NDM	AUR
whole Vil	744	-	-	1200	-	-	196	-	-
whole YB	-	-	178	51.1	1020	46.7	-	117	-

Vil, Villafranca; YB, Yellow Bell

- : Not detected

ERC, eriocitrin; NER, neoeriocitrin; NRG, naringin; HSP, Hesperidin; NHP, neohesperidin; RFN, rhoifolin; DSM, diosmin; NDM, neodiosmin; AUR, aurapten

Table 6. Flavonoid and aurapten concentrations in whole YB and peel YB (harvested in December 2012)

	Concentration (mg/100g-dryweight)
	ERC	NER	NRG	HSP	NHP	RFN	DSM	NDM	AUR
whole YB	-	16.0	270	55.9	1270	45.8	-	141	4.04
whole YB	-	29.0	493	60.3	1860	88.7	-	173	8.62

YB, Yellow Bell

- : Not detected

ERC, eriocitrin; NER, neoeriocitrin; NRG, naringin; HSP, Hesperidin; NHP, neohesperidin; RFN, rhoifolin; DSM, diosmin; NDM, neodiosmin; AUR, aurapten

Experiment 1 Total food intake was the same among control, whole Vil, and whole YB groups (547 g, respectively), since caloric intake was controlled. Body weight gain did not differ among all treatments (control, 225.5 ± 4.3; whole Vil, 213.1±7.0; whole YB, 215.2 ± 5.3 g). Intake of YB or Vil did not inhibit growth in rats. YB and Vil groups showed significantly higher serum HDL-cholesterol (p < 0.05) compared with the control group at 5 weeks (control, 25.3 ± 1.7; whole Vil, 31.0 ± 1.6; whole YB, 32.6±1.5 mg/100 mL). Vil group showed significantly lower liver triacylglycerol (p < 0.05) compared with control (control, 13.6±1.6; whole Vil, 8.6 ± 1.2; whole YB, 10.7 ± 1.1 mg/liver g). Supplementation with hesperidin and naringin or tangerine-peel extract in rats fed a high cholesterol diet lowered liver triglycerides (Bok et al., 1999). Hesperidin contained in Vil may have contributed to the amelioration of cholesterol values. The mitigating effect on liver lipids only appeared in the Vil group. There are differences in flavonoid concentrations. Vil contains hesperidin at 1200 mg/100 g dry weight, YB contains hesperidin at 51.1 mg and naringin at 178 mg/100 g dry weight (Table 5), which might account for the effects. There was no significant difference in serum triacylglycerol, serum total-cholesterol, serum LDL-cholesterol, liver weight, liver total-cholesterol, and fat pad weight. The YB group showed significantly higher serum adiponectin compared with the control group at 2 and 5 weeks (p < 0.05, p<0.01, respectively; Fig. 1). This effect was not observed in Vil. Serum leptin concentration was not affected. This obvious effect of increasing serum adiponectin has not been reported previously in lemon. We revealed this novel effect in lemon. In previous studies (Miyake et al., 1998; Itoh et al., 2010; Bok et al., 1999), extracts from fruits were used. However, we found this effect using whole lemon, and the supplementation rate (5%) was low. Adiponectin is known to have various health effects. Adiponectin-deficient mice showed increased neointimal formation and insulin resistance, and glucose intolerance (Kubota et al., 2002), while adiponectin reduced atherosclerotic lesion formation (Yamauchi et al., 2003). Our results suggest that intake of whole YB increases serum adiponectin and serum HDL- cholesterol.

Fig. 1.

Effect of YB on serum adiponectin in rats fed a high fat diet (in experiment 1, at 2 and 5 weeks). Values are expressed as the mean ± SE. Means with different letters are significantly different at the same week (ab, p < 0.05; xy, p < 0.01).

Experiment 2 Total food intake was the same among the control, whole YB and peel YB groups (543 g, respectively), since caloric intake was controlled. Body weight gain did not differ among all treatments (control, 227.3 ± 5.2; whole YB, 218.5±4.7; peel YB, 224.4 ± 5.8 g). Intake of whole YB or peel YB did not inhibit growth in rats. The increasing effect on HDL-cholesterol shown in experiment 1 was not observed in experiment 2; thus, this effect requires clarification at present. The whole YB group showed significantly higher serum adiponectin compared to the control group at 5 weeks (p < 0.05, Fig. 2). The increasing effect on serum adiponectin in the whole YB group was reconfirmed in experiment 2. Although peel YB contains more flavonoids than whole YB (Table 6), this effect did not appear in the peel YB group. The effect of increasing serum adiponectin was reported previously for the silk protein sericin (Okazaki et al., 2010). The reason for the different effects on adiponectin of whole YB and peel YB is unknown at present, but it is suggested that ingredients other than flavonoids may have contributed. Adiponectin has various effects, such as improved insulin sensitivity and decreased atherosclerotic lesion formation (Kubota et al., 2002; Yamauchi et al., 2003). Further examination is necessary under different conditions, for example a high glucose or high cholesterol diet, in order to further reveal the biological activities of YB.

Fig. 2.

Effect of YB on serum adiponectin in rats fed a high fat diet (in experiment 2, at 0, 2 and 5 weeks). Values are expressed as the mean ± SE. Means with different letters are significantly different at the same week (ab, p < 0.05).

Conclusion

YB contains neohesperidin, naringin, neodiosmin, hesperidin, rhoifolin, neoeriocitrin, and aurapten, and its pattern differs from that of conventional lemon. The intake of whole YB increased serum adiponectin levels in rats fed a high fat diet.

Acknowledgements The authors thank Dr. Norihisa Kato (Professor, Hiroshima University) for his helpful suggestions.

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• i)  http://www.hinsyu.maff.go.jp/vips/CMM/apCMM112.aspx?TOUROKU_NO=21709&LANGUAGE=Japanese (Feb.23,2014)
• ii)http://www.hinsyu.maff.go.jp/vips/CMM/apCMM112.aspx?TOUROKU_NO=299&LANGUAGE=Japanese (Feb.23,2014)