Anticancer and Antihypertensive Effects of Small Fruit Juices

Chapter 2

Anticancer and Antihypertensive Effects of Small Fruit Juices Downloaded by UNIV MASSACHUSETTS AMHERST on July 26, 2012 | http://pubs.acs.org Publication Date: December 1, 2003 | doi: 10.1021/bk-2004-0871.ch002

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Yuko Yoshizawa , Satoru Kawaii , Takao Sato , Noboru Murofushi , and Hiroyuki Nishimura 1

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1

Laboratory of Bio-organic Chemistry, Akita Prefectural University, Akita 010-0195, Japan Laboratory of Bio-organic Chemistry, Tokyo Denki University, Hatoyama, Saitama 350-0394, Japan Hokkaido Forestry Research Institute, Bibai 079-0198, Japan Department of Bioscience and Technology, Hokkaido Tokai University, Sapporo 005-8601, Japan 2

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Epidemiological studies indicate that high intakes offruitsand vegetables are associated with a reduced risk of hypertension and cancer. Since only a small part of the flora has been tested for those bioactivities, we chose small fruits as sources of inhibitory activity of angiotensin I-converting enzyme (ACE), differentiation-inducing activity against HL-60 leukemia cells, and antiproliferative activity toward several cancer cell lines. Among 43 juices, prepared from small fruits mainly grown in northern part of Japan, six exhibited ACE inhibition, while four demonstrated potent differentiation-inducing and antiproliferative activities to cancer cell lines, and yet low cytotoxicity toward normal cell lines. The results demonstrated the feasibility of the physiological screening on the small fruits in order to identify plants rich in anti-hypertensive and/or anticancer substances.

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© 2004 American Chemical Society

In Nutraceutical Beverages; Shahidi, F., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2003.

Downloaded by UNIV MASSACHUSETTS AMHERST on July 26, 2012 | http://pubs.acs.org Publication Date: December 1, 2003 | doi: 10.1021/bk-2004-0871.ch002

9 There is an increasing interest in health-promoting agents of plant origin. It was suggested that lifestyle changes in activity (walking etc.) and diet can reduce risk for vascular disease and cancer. Hypertension is a highly prevalent risk factor for vascular disease, therefore prevention of hypertension is essential for the reduction of cardiovascular morbidity and mortality. Intensive cohort investigation have indicated that a healthy diet of fresh fruits, vegetable and whole grains is beneficial, because it improves the lipid levels and provides high levels of natural antioxidants. Some active components from foods of plant origin have been found as the candidates to lower blood pressure (7, 2). There is also epidemiological evidence that high intakes of fruits and vegetables are associated with a reduced risk of cancer (5). The association is generally most marked for epithelial cancer, apparently stronger for those of the digestive and respiratory tracts, and somewhat weaker for hormone-related cancer (4). Several plant-derived drugs have been developed in medical oncology for the following reasons. First, administration of fruits to rodents protects against chemical carcinogenesis. Second, many seemingly unrelated compounds including flavonoids, coumarins, cinnamates, and other phenolics, can protect rodents against chemical carcinogens. Since only a small part of the flora has been tested for any kind of bioactivity, we chose small fruits as sources of anti-hypertensive and anti-cancer activities. We have prepared juices from various small fruit plants, which grow primarily in the northern part of Japan, especially on Hokkaido Island. These juices were subjected to screening for anti-hypertensive activity by inhibition of angiotensin I-converting enzyme (ACE) and anti-cancer activity by HL-60 differentiation-inducing activity.

Fruit Sample Preparation All fruits were harvested from trees at the Hokkaido Forestry Research Institute, Bibai, Hokkaido, Japan in July - September 1996. Fruit was homogenized in ethanol and the ethanolic extract was filtrated, concentrated in vacuo, and dissolved in distilled water (concentration: 10 mg/mL) for ACE inhibitory assay, and dissolved in RPMI1640 medium (concentration: 10 mg/mL) for anticancer assay.

Anti-Hypertensive Activity ACE Inhibitory Assay. The procedure was following the method described in the literature (J) with minor modification. One unit of ACE from rabbit lung



10 (Sigma Chemical Co., St. Louis, MO, USA) was dissolved in 8 mL of 125 mM borate buffer (pH 8.3) containing 1 M NaCl. To 50 of the enzyme solution was added 50 \xL offruitsample solution (10 mg/mL), and it was incubated for 5 min at 37°C. Synthetic substrate Bz-Gly-His-Leu (Peptide Institute Inc., Osaka, Japan) solution, 3.5 mM in the same buffer, of 150 μΜ was added to the mixture, then it was further incubated for 20 min. The reaction was stopped by adding 50 μ ί of 1 M HC1 and the mixture was directly injected to HPLC equipped with ODS column [mobile phase: 80% 20 mM phosphate buffer (pH 3.0) in acetonitrile; flow rate: 1 mL/min] , and hippuric acid produced was detected at 254 nm. The inhibition of enzyme activity was calculated by the amount of hippuric acid based on that of negative control experiment where water was added to the enzyme reaction mixture instead offruitsample. Anti-Hypertensive Effects. Among 40 small fruit samples, juices of Elaeagunus multiflora Thunb. (Japanese name: Natsugumi; IC : 1.4 χ 10 μg/mL), Elaeagnus umbellate Thunb. (Japanese name: Akigumi; IC : 6.9 10 μg/mL), Rubus idaeus L. (Japanese name; Yoroppa Ki-ichigo; e.g. European raspberry; IC : 2.3 χ 10 μg/mL), Ribes latifolium Janczewski (Japanese name: Ezosuguri; IC : 4.0 10 μg/mL), Ribes nigrum Linn. (Japanese name: Kurosuguri; IC : 4.7 χ 10 μg/mL), mdAlonia melanocarpa (Japanese name: Melanocarpa; IC : 1.2 χ 10 μg/mL), as shown in Table I. To test anti-hypertensive activity, one methods that we could employ was feeding experiment using a spontaneously hypertensive rat (SHR) as a hypertension model. However, such an animal experiment is expensive and time- and labor-consuming. For screening of large number of samples and for activity-guided fractionation of active principles from natural source, in vitro ACE inhibitory assay is more preferable than SHR animal experiment. As to the small fruits judged to active in this study, nothing has been reported on their ACE inhibitory activity and/or anti-hypertensive activity. A C E inhibitory activity has been demonstrated from other plant sources, such as seeds from Acacia plant (7) and Ashitaba leaves (2), aerial part of Jasminum plants (9), wheat germ hydrolysate (70), fermented soybean (77), and soy sauce (72). Nicotianamin was found and/or suggested as an active principle in some plants. Although the active plants reported herein do not include those plants reported before, some of them may contain the same active compound. Isolation of active principles from active smallfruitjuices is undergoing. 2

50

χ

2

50

2

50

x

2

50

50

2

50

3

Anticancer Activity Cell differentiation assay. The differentiated HL-60 phenotype is characterized by nitro blue tetrazolium (NBT) reducing, non-specific esterase, specific


11 esterase, and phagocytic activities. NBT reduction, non-specific esterase, and phagocytosis is positive in HL-60 cells, which are induced to monocytes/macrophages, whereas HL-60 cells, differentiated to monocyte/granulocytes, increase NBT reducing and specific esterase activities.


Cell proliferation assay. The level of cell proliferation was measured by using alamar Blue (Biosource International, Lewisville, TX, U . S. Α.), an oxidationreduction indicator. The level of proliferation was measured for the cancer and normal cell lines grown in 96-well microtiter plates. Analysis of Total Phenolics. The total phenolics were determined with FolinCiocalteu reagent primarily according to the method described in the literature (6, 7). We used 100 μΐ of 1/10 or 1/20 diluted sample or 50, 40, 30, 20, 10 mg/L and a 0-blank of standard series of gallic acid solutions plus 500 of 1/10 diluted Folin-Ciocalteu stock reagent, followed after 5 min by addition of 400 μ ί of 7.5% (w/v) Na C0 solution, and after 2 h at room temperature reading the absorbance at 765 nm. Results were expressed as milligrams of gallic acid equivalent per 10 g of ethanol extract. 2

3

Analysis of Total Anthocyanin. The total anthocyanin was estimated by a pH differential method (5). Absorbance was measured at 510 nm and 700 nm in buffers at pHl.O and pH4.5, using A = (^ IO-^7OO) HI.O - (^5io-^7oo)H4.5> and a molar extinction coefficient of cyanidin-3-glucoside of 29600. Results were expressed as milligrams of cyanidin 3-glucoside equivalent per 10 g of ethanol extract. 5

p

P

HL-60 differentiation-inducing effects. Among 43 samples, juices of Actinidia polygama Maxim. (Japanese name: Matatabi), Ribes nigrum (Japanese name: Kurosuguri, the small fruit-producing line), Rosa rugosa (Japanese name: Hamanasu), Rubus parvifolius (Japanese name: Nawashiroichigo), Sorbus sambucifolia M . Roem. (Japanese name: Takanenanakamado), and Vaccinium smallii Roem. (Japanese name: Obasunoki) demonstrated potent differentiationinducing activity towards HL-60, as shown in Table II. The relationship between NBT-reducing activity and the total phenolics or total anthocyanin content was investigated. A positive correlation was observed between NBT-reducing activity and the total phenolic or total anthocyanin content. The correlation coefficient was much higher between NBT reducing activity and the total phenolics (Figure 1, r - 0.4292) compared to NBT reducing activity and the total anthocyanin (Figure 2, r = 0.1352). Samples, which contained relatively high content of phenolics, namely Λ. polygama, Rosa rugosa, Rubus parvifolius, S. sambucifolia, and V. smallii, demonstrated potent HL-60 differentiating activity.


In Nutraceutical Beverages; Shahidi, F., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2003. Kokuwa Issaikokuwa Miyamamatatabi Hasukappu Hasukappu Hasukappu Hasukappu Hasukappu Keyonomi Keyonomi Keyonomi Hyoutanboku Kanboku Miyamagamazumi Natsugumi Akigumi Iwatsutsuji Obasunoki Marusuguri Marusuguri Komagatakesuguri Ezosuguri Kurosuguri Kurosuguri Fusasuguri

Actinidia arguta Planch. Actinidia arguta Planch, cv. Issaikokuwa Actinidia kolomikta Maxim. Lonicera caerulea var. emphyllocalyx Nakai Lonicera caerulea var. emphyllocalyx Nakai Lonicera caerulea var. emphyllocalyx Nakai Lonicera caerulea var. emphyllocalyx Nakai Lonicera caerulea var. emphyllocalyx Nakai Lonicera caerulea var. edulis Turczaninov Lonicera caerulea var. edulis Turczaninov Lonicera caerulea var. edulis Turczaninov Lonicera morrowii A. Gray Viburnum opulus var. calvescens H ara Viburnum wrightii Miquel Elaeagnus multiflora Thunb. Elaeagnus umbellata Thunb. Vaccinium praestans Lamb. Vaccinium smallii A. Gray Ribes grossularia Linn. Ribes grossularia Linn. Ribes japonicum Maxim. Ribes latifolium Janczewski Ribes nigrum Linn. Ribes nigrum Linn. Ribes rubrum Linn.

Actinidaceae

Hydrangeaceae

Ericaceae

Elaeagnaceae

Caprifoliaceae

Japanese Name

Scientific Name

Plant family

46.5 17.9 49.1 71.4 26.6 78.8 29.6 65.3 12.6 61.7 31.2 63.2 39.1 17.7 88.1 48.7 98.5 57.2 47.9 115.9 50.2 38.9 36.9 58.8 28.1

(mg/gfreshfruit)

Extract

ACE

100 100 17 39

—

11

— —

12 29 59 3 60 6 16 100 91 2

—

(%) 15 52 51 46 5 25

activity

a )

inhibitory

Table I. A C E Inhibitory Effects of Small Fruit Juices


smallfruit-producingline largefruit-producingline

collected on 7/27/96 collected on 8/8/96

origin; Kiritappu origin; Taiki-cho origin; Tomato, highbush origin; Tomato, lowbush origin; China origin; Taisetsu origin; Yokotsu-dake origin; Bihoro-toge

remarks


Schisandraceae Taxaceae

Lardizabalaceae Rosaceae

AkebiatrifoliataKoidz. Alonia melanocarpa Chaenomeles japonica Lindl. Cydonia oblonga Miller Malus baccata var. mandshurica C. K. Schn. Prunus salicina Linn. Rosa rugosa Thunb. Rubus idaeus L. Rubus mesogaeus Focke Rubus parvifolius Linn. Rubus phoenicolasius Maxim. Rubus phoenicolasius Maxim. Sorbus sambucifolia Roem. Schisandra chinensis Baill. Taxus baccata L.

Mitsuba-akebi Melanocarpa Kusaboke Marumero Ezonokoringo Sumomo Hamanasu Yoroppa Ki-ichigo Kuroichigo Nawashiroichigo Ebigaraichigo Ebigaraichigo Takanenanakamado Tyosengomishi Yoroppa Ichii

30.7 52.4 27.9 32.4 89.4 66.9 44.4 59.2 58.6 60.3 22.9 41.4 92.1 169.4 108.1 100 100 32 5 7

3 76 5


collected on 8/8/96 collected on 8/27/96

In Nutraceutical Beverages; Shahidi, F., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2003. >70 >70 >70 66.1 >70 >70 >70 >70 >70 >70 >70 >70

>70 >70 >70 >70 >70 >70 >70 >70 >70 >70 >70 >70

>70 >70 >70 >70 >70 >70 >70 >70 >70 >70 >70 >70

>70 >70 >70 >70 >70 >70 >70 >70 >70 >70 >70 >70

8.3 ± 1 . 2 45.3 ± 6 . 7 50.8 ± 7 . 6 17.0 ± 7 . 9 7.0 ±7.1 10.3 ± 9 . 9 30.3 ± 5 . 3 33.8 ± 5 . 6 24.7 ± 4 . 2 28.8 ± 8 . 5 23.7 ± 8.2

Ampélopsis brevipedunculata Trautv.

Chaenomeles japonica Lindl.

Cydonia oblonga Miller

Elaeagnus multiflora Thunb.

Elaeagnus umbellata Thunb.

Gaultheria Miqueliana Takeda

Lonicera caerulea (Kiritappu)

Lonicera caerulea (Taiki-cho)

Lonicera caerulea (Tomato-tate)

Lonicera caerulea (Tomato-yoko)

Alonia melanocarpa

33.2 ± 9 . 4

Akebia trifoliata Koidz.

5.9

Actinidia polygama Maxim.

58.9

20.5

77.7 ± 5 . 0

8.7

52.7

49.3 ± 7 . 4

Actinidia Kolomikta Maxim.

58.1

>70

>70

7.3 ± 3.7

Actinidia arguta Planch, cv. Issaikokuwa

13.4

>70

>70

19.3 ± 5 . 4

Actinidia arguta Planch.

39.8

CCRF-HSB-2 TGBCJITKB

>70

BJ6

69.2

A549

Antiproliferative Activity

>70

NBT reducing cell (%)

HL-60 Differentiation

>70

Scientific Name

Table II. Anticancer Effects of Small Fruit Juices


In Nutraceutical Beverages; Shahidi, F., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2003. 27.5 ± 9 . 2 10.3 ± 4 . 9 15.2 ±4.1 21.3 ± 6 . 5 26.3 ± 2 . 5 38.3 ±10.4 10.0 ± 5 . 9 8.0 ± 2 . 9

Lonicera morrowii A. Gray

Malus baccata var. mandshurica C. K. Schn.

Prunus salicina Lindl.

Ribes grossularia L.

Ribes grossularia L.

Ribes idaeus L.

Ribes japonicum Maxim.

Ribes latifolium Jancz.

64.7 ±10.8 31.3 ± 6 . 8 29.5 ±10.4

Rubus parvifolius L.

Rubus phoenicolasius Maxim.

Rubus phoenicolasius Maxim.

7.3 ± 1 . 2

77.7 ± 9 . 9

Rosa rugosa Thunb.

Rubes mesogaeus Focke

20.7 ± 9 . 6

Ribes rubrum L.

9.3 ± 1 . 7

19.7 ±3.1

Lonicera caerulea (Bihoro-toge)

Ribes nigrum L. (large fruits)

49.0 ± 0

Lonicera caerulea (Yokotsu-dake)

65.0 ± 9 . 8

17.7 ± 0 . 5

Lonicera caerulea (Taisetsu)

Ribes nigrum L. (small fruits)

25.7 ± 8 . 0

Lonicera caerulea (China)

>70

>70

>70

>70

49.7

>70

>70

>70

>70

>70

>70

>70

>70

>70

>70

>70

>70

>70

>70

>70

>70

>70

65.0

>70

21.2

>70

>70

26.5

>70

>70

>70

>70

>70

>70

>70

>70

>70

>70

>70

>70 >70

>70

>70

47.6

>70

17.2

>70

>70

30.7

>70

>70

>70

>70

>70

>70

>70

>70

>70

>70

>70

>70

>70

>70

46.2

>70

12.6

>70

>70

22.3

>70

>70

>70

>70

>70

>70

>70

>70

>70

>70

>70

Continued on next page.


In Nutraceutical Beverages; Shahidi, F., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2003. 48.3 >70 100

50.2 >70 100

68.0 >70 100

>70 >70 100

52.8 ± 5 . 6 52.7 ± 7 . 0 16.3 ± 4 . 6 14.5 ± 5 . 0

Vaccinium praestans Lamb.

Viburnum opulus var. sargentii Takeda

Viburnum Wrightii Miq.

Blank

5.2 >70

14.0 >70

22.5

Vaccinium smallii A Gray

>70

53.5 >70

75.5 ± 5 . 4

Taxus baccata L.

>70 >70

>70

14.0 ± 4 . 3

Sorbus sambucifolia M. Roem.

>70

24.8

36.7

6.6

>70

>70

>70

TGBCJITKB

15.4

CCRF-HSB-2 >70

B16

65.3 ± 8 . 6

A549

Antiproliferative Activity

44.8 ± 7 . 4

NBT reducing cell (%)

HL-60 Differentiation

Schizandra chinensis Baill.

Scientific Name

Table Π. Continued



17

160 I

»

140 h

1

ι

1

r|

1

Γ

- y = 7.472 + 0.55412x R= 0.42922

120

S. sambucifolia . V. smallii A. polygama R, fugosa

0

10

20

30

40

50

60

70

80

NBT reducing celle (%) Figure 1. Relation between HL-60 differentiation-inducing activity and amount of total phenolics



160

140 h

- y = 7.5157 + 0.141X R= 0.13519

120

V. smallii 100

80

60

S. sambucifolia and R. rugosa

40

A. polygama

20

0 0

10

20

30

40

50

60

70

80

NBT reducing cells (%) Figure 2. Relation between HL-60 differentiation-inducing activity and amount of total anthocyanin



19 The results of phenolic assay shows the difference of the total phenolic content between the small-fruit producing and large-fruit producing lines of Ribes nigrum, thus suggesting the possible role of phenolics for the NBT reducing activity. On the other hand, the total phenolic or total anthocyanin content, however, could not explain the difference of NBT reducing activity of Lonicera caerulea var. emphyllocalyx Nakai (Japanese name: Keyonomi), which had been originally collected at different sites. Concentration-response effect of A. polygama, Rosa rugosa, V. smallii, and 5. sambucifolia, which exhibited the potent HL-60 differentiating activity, was examined, and the results are shown in Figure 3. The differentiation-inducing activity was monitored by NBT reducing activity, specific and nonspecific esterase activities, and phagocytic activity, in order to determine the direction of HL-60 cellular differentiation. These juices appeared to induce monocyte/macrophage characteristics, since HL-60 cells treated with these compounds showed NBT reducing activity, non-specific esterase activity, and phagocytic activity in a concentration-dependent manner, whereas they did not express any naphthyl AS-D chloroacetate esterase activity.

Antiproliferative Effects toward several cancer and normal cell lines (14 Antiproliferative effects of the small fruit juices were examined on cellular growth of lung carcinoma (A549), melanin pigment-producing mouse melanoma (B16 melanoma 4A5), T-cell leukemia (CCRF-HSB-2), and gastric cancer cell, and lymph-node metastasized (TGBC11TKB), as shown in Table II. The cellular growth was monitored by reduction of an oxidation-reduction dye, alamar Blue, 3 days after the addition of samples. Among 43 samples examined in this report, A. polygama Maxim., V. smallii A . Gray, Ribes nigrum, Rosa rugosa Thunb., and Sorbus sambucifolia Roem. demonstrated a potent antiproliferative activity, while a weak activity was found in Actinidia kolomikta Maxim. And Rubus parvifolius L. Among the cell lines examined, the growth of TGBC11TKB and CCRFHSB-2 cells were more sensitive to the small fruit samples and A549 and B16 melanoma 4A5 showed a higher degree of resistance to the antiproliferative activity. The rank order of potency for TGBC11TKB cells was V. smallii, A. polygama, S. sambucifolia, Rosa rugosa, and Ribes nigrum (the small fruitproducing line). The antiproliferative activity of Ribes nigrum was influenced by the morphological variety of fruits. We have separately examined the fruits from two different lines of Ribes nigrum-, namely the smallfruit-producingline and the large fruit-producing one. There was a marked difference between these fruits. The small fruit inhibited cellular growth of Β16 melanoma 4A5, CCRFHSB-2, and TGBC11TKB approximately 70%, whereas the large fruit did not show any activity.




01


20

0.

40 S

****

20

40

60

80

100

Cone. ^g/ml)

Figure 3. HL-60 Differentiation-inducing activity

0,

(Sorbus sambucifolia)

Takanenanakamado

HL-60 proliferation ( Δ ) , nitro blue tetrazolium reducing activity « non-specific esterase activity ( • ) , specific esterase a c t i v i t y ( É ) , phagocytic activity ( O ) .

Cone, (ng/ml)

100 ~

120

140

ο co ο ο

(Vaccinium smallii)

iteration ι

Obasunoki


Differenli ation

Is)

22


The juices that showed strong activity to cancer cell lines, namely V. smallii, A. polygama, Rosa rugosa, and S. sambucifolia, were also examined for their cytotoxicity against normal human cell lines, human foreskin keratinocytes (HFK) and human umbilical vein endothelial (HUVE) cells. These samples, by contrast, are substantially less cytotoxic toward normal human cell lines; the sample did not show any cytotoxicity toward normal human cell lines at a concentration of 5 mg/mL.

Conclusions The screening on 43 fruit juices from small fruits growing in northern part of Japan exhibited that anticancer and/or anti-hypertensive biological activity. A massive screening for anti-hypertensive and anti-cancer substances in foods, would lead to discovery and development of new agents with less side-effect.

Acknowledgements This work was supported in part by a Grant-in-Aid for scientific research (09680051) to Y . Y . from the Ministry of Education, Science, and Culture, Japan. We thank Ms. Mimako Urashima for her excellent experimental support.

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23 10. Matsui, T.; L i , C. H.; Tanaka, T.; Maki, T.; Osajima, Y.; Matsumoto, K. Biol. Pharm. Bull. 2000, 23, 427-431. 11. Okamoto, Α.; Hanagata, H.; Kawamura, Y.; Yanagida, F. Plant Foods Hum. Nutr. 1995, 47, 39-47. 12. Kinoshita, E.; Yamakoshi, J.; Kikuchi, M . Biosci. Biotechnol. Biochem 1993, 57, 1107-1110. 13. Yoshizawa, Y.; Kawaii, S.; Urashima, M . ; Fukase, T.; Sato, T.; Murofushi, N.; Nishimura, H. J. Agric. Food Chem. 2000, 48, 3177-3182. 14. Yoshizawa, Y.; Kawaii, S.; Urashima, M . ; Fukase, T.; Sato, T.; Tanaka, R.; Murofushi, N.; Nishimura, H. Anticancer Res. 2000, 20, 4285-4289.


Anticancer and Antihypertensive Effects of Small Fruit Juices

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