Biological activity of persimmon (Diospyros kaki) peel extracts

PHYTOTHERAPY RESEARCH Phytother. Res. 17, 495 – 500 (2003) Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/ptr.1183 John Wiley & Sons, Ltd.

Biological Activity of Persimmon (Diospyros kaki) Peel Extracts Masami Kawase1*, Noboru Motohashi2, Kazue Satoh3, Hiroshi Sakagami4, Hideki Nakashima5, Satoru Tani1, Yoshiaki Shirataki1, Teruo Kurihara6, Gabriella Spengler7, Krisztina Wolfard7 and Joseph Molnár7

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Faculty of Pharmaceutical Sciences, Josai University, Sakado, Saitama, Japan Meiji Pharmaceutical University, Kiyose, Tokyo, Japan 3 School of Pharmaceutical Sciences, Showa University, Shinagawa-ku, Tokyo, Japan 4 Department of Dental Pharmacology, Meikai University School of Dentistry, Sakado, Saitama, Japan 5 Department of Microbiology, St. Marianna University School of Medicine, Kanagawa, Japan 6 Faculty of Science, Josai University, Sakado, Saitama, Japan 7 Faculty of Medicine, Institute of Medical Microbiology, Albert Szent-Györgyi Medical Centrum, University of Szeged, Szeged, Hungary 2

Fractionated extracts of persimmon (Diospyros kaki) peels were studied for cytotoxic activity, multidrug resistance (MDR) reversal activity, anti-human immunodeficiency virus (HIV) activity and anti-Helicobacter pylori (H. pylori) activity. The potent cytotoxic activity against human oral squamous cell carcinoma cells (HSC-2) and human submandibular gland tumor (HSG) cells was found in the acetone fractions (A4 and A5) with IC50 ranging from 21 to 59 µg/mL. However, the cytotoxic activity was not correlated with the radical intensity of the fractions. Three 70% MeOH extract fractions (70M2-4) produced radical and efficiently scavenged the O2− produced by hypoxanthine and xanthine oxidase reaction. All of the fractions tested were not effective for anti-H. pylori and anti-HIV. Fractions H3 and H4 of hexane extract, and M2 and M3 of ±)-verapamil (a positive MeOH extract showed a remarkable MDR reversal activity comparable with that of (± control). These results indicate the therapeutic value of persimmon peel extracts as potential antitumor and MDR-reversing agents. Copyright © 2003 John Wiley & Sons, Ltd. Keywords: Diospyros kaki; Ebenaceae; persimmon peel; cytotoxic activity; MDR; radical intensity.

INTRODUCTION Diospyros kaki Thunb. (Ebenaceae) has been cultivated in Japan for several centuries. It is thought to have originated in Southern China. The fruits of kaki are very astringent until fully ripened, at which time the tannin content is completely transformed into insoluble crystals. The riped orange-red fruits are then sweet and palatable. Persimmon contains many medicinally bioactive compounds, such as carotenoids, tannins, flavonoids, terpenoids, steroids, naphthoquinones, sugars, amino acids, minerals, and lipids (Mallavadhani et al., 1998). Both fruits and leaves are used to treat coughs, hypertension and dysponea. Calyces are used both in Japanese folk medicine and in Chinese medicine for the treatment of hiccough (Matsuura and Iinuma, 1985). The main constituent of D. kaki, plumbagin is reported to exhibit the antifeedant, insecticidal, insect growth regulator and sterilant activities (Gujar, 1990). The leaf extract contains antiallergic substrates such as astragalin, which inhibit the histamine release from human basophilic cell line KU812 (Kotani et al., 2000). Persimmon is also a good source of nutritional antioxidant vitamins, polyphenols, and dietary fiber (Gorinstein et al., 1994; 1998). Furthermore, the concentration of carotenoids and polyphenols * Correspondence to: Dr M. Kawase, Faculty of Pharmaceutical Sciences, Josai University, Sakado, Saitama 350-0295, Japan. E-mail: [email protected]

Copyright © 2003 John Wiley & Sons, Ltd.

is higher in the peel than in the pulp. A diet fortified with dry persimmon peel is more efficient than the same diet fortified with dry persimmon pulp (Gorinstein et al., 1998; 2001). Therefore, it is proposed that the persimmon peel showing the antioxidant activity can be used by individual consumers and in industrial processing. We have investigated for new pharmaceutical agents from edible and/or exhaused parts of cacao (Motohashi et al., 1999), grape (Shirataki et al., 2000), feijoa (Motohashi et al., 2000), kiwifruit (Motohashi et al., 2000), and sweet pepper (Motohashi et al., 2001), which have been continuously consumed worldwide. In general, the peel was regarded as a waste matter, although, based on our recent studies, the peel contains biologically and nutritionally important compounds. Therefore, this raw material can be considered as a source of various medicines. Based on the above, we here investigated the cytotoxic activity, anti-Helicobacter pylori (H. pylori) activity, anti-human immunodeficiency virus (HIV) activity and multidrug resistance (MDR) reversal activity of persimmon peel extracts.

MATERIAL AND METHODS Plant material. Persimmon, Diospyros kaki Thunb. ‘Aodanshi’ (Ebenaceae), peels were supplied from Kohyama farm garden in Ibi, Gifu, Japan. A voucher specimen was deposited in the Herbarium of Josai University. Received 23 January 2002 Accepted 1 March 2002

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Preparation of persimmon peel extracts. Persimmon peel (850 g) was cut into small pieces and successively extracted with hexane, acetone, MeOH and 70% MeOH at room temperature. The solvent was concentrated in vacuo and the hexane extract [H0](2.5 g), acetone extract [A0](29.3 g), MeOH extract [M0](405 g) and 70% MeOH extract [70M0](61 g) were obtained, respectively. First, the aliquot of hexane extract [H0](2.2 g) was applied to silica gel column chromatography, which was then eluted with a hexane-acetone gradient. The hexane fraction [H1](220 mg), hexane-acetone (9:1) fraction [H2](650 mg), hexane-acetone (4:1) fraction [H3](600 mg) and [H4](40 mg) were eluted stepwise. Second, the acetone extract [A0](18 g) was applied to silica gel column chromatography, which was then eluted with a benzene-AcOEt gradient. The benzene fraction [A1](200 mg), benzeneAcOEt (10:1) fraction [A2](200 mg), [A3](50 mg), benzene-AcOEt (1:1) fraction [A4](4.1 g), AcOEt fraction [A5](2.5 g), and AcOEt-EtOH (5:1) fraction [A6](2.8 g) were eluted stepwise. Third, the MeOH extract [M0] (30 g) was applied to silica gel column chromatography, which was then eluted with a CH2Cl2-MeOH gradient. The CH2Cl2 fraction [M1](150 mg), CH2Cl2-MeOH (50:1) fraction [M2](240 mg), CH2Cl2-MeOH (9:1) fraction [M3](6.2 g), [M4](21.6 g), and CH2Cl2-MeOH (4:1) fraction [M5](1.3 g) were eluted stepwise. Finally, the 70% MeOH extract [70M0](30 g) was applied to octadecylsilane (ODS) column chromatography, which was then eluted with a H2O-MeOH gradient. The H2O-MeOH (2:1) fraction [70M1](19 g), [70M2](12.3 g), H2O-MeOH (1:1) fraction [70M3](700 mg), and MeOH fraction [70M4](1.0 g) were eluted stepwise.

3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) to a blue formazan product (Nakashima et al., 1992). All data represent the mean values of triplicate measurements. The selectivity index (SI) was defined as follows: SI = CC5 0 /EC50.

Bacterials. Helicobacter pylori (ATCC43504) was purchased from the American Type Culture Collection (Rockville, MD, USA).

Radical intensity. Radical intensity was determined at 25 °C using electron spin resonance (ESR) spectroscopy (JEOL JES REIX, X-band, 100 kHz modulation frequency). Instrument settings: center field, 336.0 ± 5.0 mT; microwave power, 8 mW; modulation amplitude, 0.1 mT; gain, 630; time constant, 0.03 s; scanning time, 2 min. Radical intensity was determined in 0.1 M NaHCO3/Na2CO3 buffer (pH 10.5) or 0.1 N KOH (pH 12.5) containing 50% DMSO and the radical intensity was defined as the ratio of peak heights of these radicals to that of MnO (Satoh et al., 1997).

Measurement of anti-H. pylori activity. The micro-dilution broth method was used to determine the minimum inhibitory concentration (MIC). Mueller-Hilton broth containing 5% fetal bovine serum (FBS) (JRH Biosciences, Lenexa, KS, USA) was used as the medium, and was cultured in a jar conditioned with Campylo Pack (Dia Iatron) for 48 h. Briefly, H. pylori strains were inoculated on Brucella agar plate containing 10% horse serum, and cultured at 37 °C for 48 h. The collected bacterial colonies were diluted to 107 colony forming unit (CFU)/ mL with 0.9% saline. The fractions were dissolved in DMSO, and then diluted with Mueller-Hilton broth. To the solution of the fractions, a suspension of bacteria was added to make 106 CFU/100 mL/well. The mixture was incubated at 37 °C for 48 h. The MIC values of the fractions tested were determined by observation. Assay for anti-HIV activity. Human T cell leukemia virus 1 (HTLV1)-bearing CD4 positive human T cell lines, MT-4 cells, were infected with HIV-1IIIB at a multiplicity of infection (m.o.i.) of 0.01. HIV- or mockinfected MT-4 cells (1.5 × 105/mL, 200 µL) were placed into 96-well microtiter plates in RPMI 1640 medium supplemented with 10% heat-inactivated fetal calf serum (FCS) and incubated in the presence of varying concentrations of the fractions tested. After incubation for 5 days at 37 °C in a CO2 incubator, cell viability was quantified by a colorimetric assay (at 540 nm and 690 nm), monitoring the ability of viable cells to reduce Copyright © 2003 John Wiley & Sons, Ltd.

Cell culture. Human squamous cell carcinoma (HSC-2) cells and human submandibular gland tumor (HSG) cells were maintained as a monolayer culture at 37 °C in Dulbecco’s modified Eagle medium (DMEM) (Gibco BRL, Grand Island, NY, USA) supplemented with 10% heat-inactivated FBS in a humidified 5% CO2 atmosphere, and subcultured by trypsinization. Human gingival fibroblasts (HGF) were isolated from healthy gingival biopsy of a 10-year-old female, as described previously (Sakagami et al., 2000). Cells between the fifth and seventh passages were used. Cytotoxic activity. Cells were incubated for 24 h with the indicated concentrations of test samples in culture medium, and the viable cell number was determined by MTT method (Sakagami et al., 2000). In brief, the cells were washed with phosphate buffered saline (PBS), and incubated for 4 h with fresh culture medium containing 0.2 mg/mL MTT (Sigma Chem. Ind., St. Louis, MO, USA). After removing the medium, cells were lysed with 100 µL DMSO and the absorbance at 540 nm of the cell lysate was measured with Labsystems MutiskanR (Biochromatic) with Star/DOT Matrix printer JL-10. The A540 values of control HSC-2, HSG and HGF cells were 1.519, 0.884, and 0.283, respectively. The 50% cytotoxic concentration (CC50) was determined from the dose-response curve.

Radical scavenging activity against superoxide anion (O2−). O2− was generated by hypoxanthine (HX) and xanthine oxidase (XOD) reaction (total volume: 200 µL) [2 mM HX in 0.1 M phosphate buffer (PB) (pH 7.4) 50 µL, 0.5 mM DETAPAC 20 µL, DMPO (5%) 50 µL, sample (in DMSO) 30 µL, H2O or SOD 20 µL, XOD (0.5 U/mL in PB) 30 µL]. The gain and time constant were changed to 400 and 0.1 s, respectively. The radical intensity was determined by ESR spectroscopy after 1 min mixing of them. The O2− scavenging activity was expressed as SOD unit/mg sample, by calibration with standard curve of SOD. Cell and fluorescence uptake. The MDR1/A expressing cell lines were selected by culturing the infected cells with 60 ng/mL colchicine to maintain the expression of the MDR phenotype (Kessel, 1989). The L5178 MDR cell line and the L5178 Y parent cell line were grown in McCoy’s 5A medium supplemented with 10% heatinactivated horse serum, L-glutamine and antibiotics. The cells were adjusted to a concentration of 2 × 106/mL Phytother. Res. 17, 495 –500 (2003)

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and resuspended in serum-free McCoy’s 5A medium, and 0.5 mL aliquot of the cell suspension were distributed into each Eppendorf centrifuge tube. Then, 10 µL of 2 mg/mL test fractions were added and incubated for 10 min at room temperature. Then, 10 µL rhodamine 123 (R123) as indicator of drug accumulation was added to the samples (5.2 µM final concentration) and the cells were incubated for a further 20 min at 37 °C, washed twice and resuspended in 0.5 mL phosphate buffered saline (PBS) (pH 7.4) for analysis. The fluorescence of cell population was measured by flow cytometry using Beckton Dickinson FACScan instrument. (±)-Verapamil (VP) was used as the positive control in R123 accumulation experiments (Weaver et al., 1993). The R123 accumulation was calculated from fluorescence of one height values. Then, the percentage of mean fluorescence intensity was calculated in treated MDR1 and parental cell lines, compared to untreated cells. The fluorescence activity ratio was calculated by the following equation (Kessel, 1989; Weaver et al., 1993): MDR1 reversal activity = (MDR1 treated/MDR1 control)/(parental treated/parental control).

RESULTS Anti-H. pylori activity The current medical consensus shows that H. pylori is the primary causative agent of acute gastritis (Blaser, 1992). Antibacterial agents from a regularly consumed fruit may be superior as H. pylori control agents, when compared to many non-natural products. Persimmon peel was successively extracted with hexane, acetone, MeOH and 70% MeOH, and the each extract was further separated by column chromatography. However, all of fractions did not exhibit potent anti-H. pylori activity (MIC > 100 µg/mL), in contrast to three effective agents: metronidazole (MIC = 74 µg/mL), clarithromycin (MIC = 1.9 µg/mL) and erythromycin (MIC = 1.8 µg/mL). Anti-HIV activity Plant-derived substances are considered as important tools for the development of antiviral drugs against viral infections caused by HIV (Vlietinck et al., 1998). The inhibition of HIV-induced cytopathic effects by persimmon extracts was studied. However, there was not a significant inhibition by any fractions of the cytopathic effects on HIV infection in MT4 cells using effective concentrations of >500 µg/mL (Selectivity Index (SI) < 1), compared with the four positive controls – curdlan sulfate (SI > 1074), dextran sulfate (SI > 1072), AZT (SI > 5322) and ddC (SI > 157). Cytotoxic activity It is acceptable that in cancer cells, many anticancer agents act as apoptosis-inducers (Hickman, 1992). The cytotoxicity of all fractions was evaluated in two human oral tumor cell lines (HSG-2, HSG) and one human gingival fibroblasts (HGF), using the microculture plates Copyright © 2003 John Wiley & Sons, Ltd.

Figure 1. Effect of 70M0-4 fractions, gallic acid and quercetin on the radical intensity of sodium ascorbate (3 mM) (A) and the stability of ascorbate radical (B) in 0.1 M Tris-HCl, pH 8,0.

stained with methyltetrazolium (MTT) assay (Table 1). Two fractions of acetone extracts, A4 and A5, were the highest cytotoxic activity against two tumor cell lines (HSC-2 and HSG) and the normal fibroblast (HGF) among total 23 extracts and fractions. The 50% cytotoxic concentration (CC50) against MT4 mock infected cells varied greatly (Table 1); values of A0, A4, A5, gallic acid or quercetin were correlated to their cytotoxic activity against other three cell lines (Table 1). Radical The extracts and fractions of hexane, acetone and MeOH did not produce radicals at pH 10.5 and 12.5. On the other hand, three 70% MeOH extract fractions [70M2, 70M3, 70M4] produced broad peak of radical at pH 10.5 and 12.5 (Table 1). The fractions 70M3 and 70M4 slightly reduced the radical intensity of sodium ascorbate at lower concentration (0.1 mg/mL) (Fig. 1(A)). The radical enhancing effect of 70M3 and 70M4 was much stronger, whereas that of gallic acid and quercetin was much less. Phytother. Res. 17, 495 –500 (2003)

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Table 1. Biological activity and radical modulation effect of persimmon peel extracts Cytotoxic activity (CC50, µg /mL)

Fraction HSC-2

HSG

HGF

MT4

a

Radical intensityb SI (HGF/HSC-2)

O2− scavenging activity (SOD unit/mg)

pH 10.5

pH 12.5

>1.3 >1.0 > 200 51 127 >200 >200

>1.1 > 1.0

1.9 >1.0 >1.2 >1.4 >1.7

0.27 75 1.3 – – – –

1.31 5.29 – – – –

14.10 1.98 – – – –

8090 1205 – – – –

70M0 70M1 70M2 70M3 70M4 Gallic acid Quercetin CRDS DS AZT (µM) ddC (µM) a b

6.1 31 – – – –

Mock-infected T4 cells. Final concentration of the sample except gallic acid (0.03 mg/mL) was 3 mg/mL. Radical intensity was determined from the ESR chart.

Table 2. Effect of persimmon peel extracts on the multidrug resistance of L-5178 cells Fraction Par (control)a MDR (control)b (±)-Verapamil H0 H1 H2 H3 H4 A0 A1 A2 A3 A4 A5 A6

a b

Concentration (µg/mL) Fluorescence activity ratio – – 10 4 40 4 40 4 40 4 40 4 40 4 40 4 40 4 40 4 40 4 40 4 40 4 40

29.85 – 5.91 5.31 20.23 4.30 12.51 1.38 3.15 27.09 128.94 22.61 54.16 2.70 5.46 0.67 0.60 0.67 0.85 0.89 1.12 4.10 9.01 1.08 3.66 5.11 15.93

Fraction M0 M1 M2 M3 M4 M5 70M0 70M1 70M2 70M3 70M4 Quercetin Gallic acid DMSO

Concentration (µg /mL) Fluorescence activity ratio 4 40 4 40 4 40 4 40 4 40 4 40 4 40 4 40 4 40 4 40 4 40 4 40 4 40 40

0.82 1.18 1.48 1.49 46.14 103.24 8.80 63.13 1.88 1.65 1.62 1.28 1.35 3.17 0.98 5.68 0.86 8.73 0.77 0.95 0.79 0.84 1.02 5.65 1.00 6.61 0.65

Par: a parental cell without transfection of MDR1 gene. MDR: a parental cell transfected with human MDR1 gene.

Copyright © 2003 John Wiley & Sons, Ltd.

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Gallic acid was rather inhibitory. The ascorbate radicals in the presence of gallic acid and quercetin were stable for 10 min, however, in the case of 70M3 or 70M4, the radical intensity reduced to 1/4–1/5 (Fig. 1(B)). The fractions [70M2-4] also efficiently scavenged the O2− produced by hypoxanthine-xanthine oxidase reaction. The scavenging activity was roughly increased with the water solubility (Table 1). The O2− scavenging activity of 70M fractions suggests a highly possible application of compounds found in the fraction for cancer prevention and a protective action against oxidative stress. MDR reversal on tumor cells The emerging drug resistance is known among viruses, bacteria, protozoa and cancer cells including retroviruses such as the HIV (Szabo et al., 2000). The development of potent MDR inhibitors has become a desirable goal for the treatment of drug resistance cancer to test the MDR-reversal hypothesis in the clinical practice. MDR-inhibitors from a regularly consumed fruit or vegetable may be considered for the treatment of non-curable cancer and these MDR modulators can be compared to many known chemicals (Lehne, 2000). Rhodamine 123 assay has been widely accepted as a direct and reproducible assay for measuring P-glycoprotein (Pgp)-dependent efflux (Lee et al., 1994). We measured the ability of the fractions to inhibit the Pgp-mediated R-123 efflux in the MDR mouse T cell lymphoma L5178 transfected by human MDR1 gene, which was cultured in colchicine-containing medium (Table 2). Six fractions

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H3, H4, A4, A6, M2, M3 showed exceptionally promising levels of MDR-reversing activity. These fractions might improve the drug accumulation of cancer cells due to the inhibition of Pgp-mediated efflux pump (Szabo et al., 2000).

DISCUSSION Persimmon species (Ebenaceae) are characterized by the presence of flavonoids, carotenoids, terpenoids, naphthoquinones, saponins, tannins and steroids (Mallavadhani et al., 1998). Persimmon tannin is composed of epicatechin, epicatechin-3-O-gallate, epigalocatechin and epigallocatechin-3-O-gallate and a proanthocyanidin polymer whose molecular weight is about 10 000 (Matsuo and Ito, 1978). Persimmon tannin has a potent scavenging action toward active oxygen free radicals. The radical enhancing effect and O2− scavenging activity of 70M fractions may be due to their content of various condensed tannins. Significant tumor-specific cytotocity was found in two acetone fractions (A4, A5) at a concentration of 21–59 µg/mL. Additionally, six fractions (H3, H4, A4, A6, M2, M3) exhibited significant MDRmodulating activity by the R123 assay. The above results suggest that persimmon peel fractions might play a role of MDR reversal effects by carotenoids, flavonoids and other functional components in its contents. The results at this study apparently indicate the existence of therapeutically useful substances and the therapeutic value of persimmon peel extracts. Further purification of these fractions is required to identify the MDR reversalrelated compounds.

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