Free radicals have been regarded as the fundamental cause of different kinds of diseases, including aging, coronary heart disease, inflammation, stroke, diabetes mellitus, rheumatism, liver disorders, renal failure and cancer.1—3)Reactive oxygen
species (ROS) comprise free radicals such as · O2
(superox-ide anion), · OH (hydroxyl radical), H2O2 (hydrogen
perox-ide) and 1O
2 (singlet oxygen), which can cause cellular
in-juries and also initiate peroxidation of polyunsaturated fatty acids in biological membranes.4,5) The tissue injury caused by ROS include DNA damage,5,6) protein damage7) and oxi-dation of enzymes8)in the human body. Antioxidants such as
a-tocopherol is capable of mitigating free radical damage and scavenging ROS.9,10)
Enzymatic antioxidants include superoxide dismutase (SOD), catalase and glutathione peroxidase (GPX), whereas non-enzymatic antioxidants comprise a-tocopherol, b-carotene, carotenoids, chlorophylls, flavonoids, tannin and certain micronutrients (e.g. zinc and selenium).11—13) The present of these antioxidants in the cellular system are known to prevent oxidative damage. Thus, a number of antioxidants derived from plants such as Ginkgo biloba,14) Prunella vul-garis,15) Therminalia chebula,16) Cinnamomum cassia.17) have been widely studied. Dietary intake of antioxidants has been increasingly accepted as a strategy for maintaining a healthy life.
P. peruviana is a medicinal plant widely used in folk medi-cine as anticancer, antimycobacterial, antipyretic, im-munomodulatory, and for treating diseases such as malaria, asthma, hepatitis, dermatitis, diuretic and rheumatism.18—20) Hot water extract of P. peruviana (HWEPP) is often used to prepare health beverages. However, it remains unknown if HWEPP and extracts prepared from different concentrations of ethanol possess antioxidant activity. In this study, our ob-jective was to evaluate the antioxidant activities of HWEPP and extracts prepared from 20, 40, 60, 80 and 95% ethanol of whole plant of P. peruviana. Its antioxidant activities were
also compared with a well known natural antioxidant, a-to-copherol.
MATERIALS AND METHODS
Chemicals L-()-Ascorbic acid, thiobarbituric acid (TBA), xanthine, xanthine oxidase and cytochrome c were purchased from Sigma Chemical Co. (St. Louis, MO, U.S.A.). a-Tocopherol, dimethylsulphoxide (DMSO) and ferrous chloride were obtained from Wako Pure Chemical In-dustries (Osaka, Japan). All other chemicals used were of an-alytical grade.
Animals Male Wistar rats, age 4—6 weeks old, were ob-tained from the animal center of the National Cheng Kung University, Tainan, Taiwan. They were housed in an air-con-ditioned room with temperature maintaining at 223 °C and humidity at 555%. Animals were fed a standard laboratory diet and tap water ad libitum throughout the experimental pe-riod. They were used for FeCl2–ascorbic acid induced lipid peroxidation study.
Plant Materials The plant of Physalis peruviana L. (PP) of the family Solanaceae was obtained from Tainan District Agricultural Research and Extension Station, Taiwan. The authenticity of the plant species was confirmed by Professor CC Lin of Graduate Institute of Natural Products, Kaohsiung Medical University, Taiwan. The whole plants were dried and ground to sawdust form, which was then kept in an air-tight brown bottle until use.
Preparation of Aqueous Extract One hundred grams
of sample was extracted with 1 l of boiling water for 1 h. The extract was filtered while the residue was re-extracted under the same conditions twice. The filtrates collected were com-bined and evaporated to dryness under vacuum. The average yield obtained for HWEPP was 20.7%.
Preparation of Ethanol Extract One hundred grams of
P. peruviana sawdust was soaked with 1 l of 20, 40, 60, 80
June 2005 Biol. Pharm. Bull. 28(6) 963—966 (2005) 963
∗ To whom correspondence should be addressed. e-mail: [email protected] © 2005 Pharmaceutical Society of Japan
Antioxidant Activities of Physalis peruviana
Sue-Jing WU,a,bLean-Teik NG,cYuan-Man HUANG,dDoung-Liang LIN,dShyh-Shyan WANG,d
Shan-Ney HUANG,dand Chun-Ching LIN*,a
aGraduate Institute of Natural Products, Kaohsiung Medical University; Kaohsiung, Taiwan: bDepartment of Health and
Nutrition, Chia-Nan University of Pharmacy and Science; Tainan, Taiwan: cDepartment of Biotechnology, Tajen Institute
of Technology; Pingtung, Taiwan: and dTainan District Agricultural Research and Extension Station; Taiwan.
Received July 20, 2004; accepted March 7, 2005
Physalis peruviana (PP) is a widely used medicinal herb for treating cancer, malaria, asthma, hepatitis,
der-matitis and rheumatism. In this study, the hot water extract (HWEPP) and extracts prepared from different con-centrations of ethanol (20, 40, 60, 80 and 95% EtOH) from the whole plant were evaluated for antioxidant activi-ties. Results displayed that at 100mmg/ml, the extract prepared from 95% EtOH exhibited the most potent
inhibi-tion rate (82.3%) on FeCl2–ascorbic acid induced lipid peroxidation in rat liver homogenate. At concentrations 10—100mmg/ml, this extract also demonstrated the strongest superoxide anion scavenging and inhibitory effect on
xanthine oxidase activities. In general, the ethanol extracts revealed a stronger antioxidant activity than a
a-toco-pherol and HWEPP. Compared to aa-tocopherol, the IC50value of 95% EtOH PP extract was lower in thiobarbi-turic acid test (IC5023.74 mmg/ml vs. 26.71 mmg/ml), in cytochrome c test (IC5010.40 mmg/ml vs. 13.39 mmg/ml) and in xanthine oxidase inhibition test (IC508.97 mmg/ml vs. 20.68 mmg/ml). The present study concludes that ethanol ex-tracts of PP possess good antioxidant activities, and the highest antioxidant properties were obtained from the 95% EtOH PP.
and 95% ethanol at room temperature for 6 d. The extract was filtered with filter paper (Advantec No. 1, Japan) while the residue was further extracted under the same conditions twice. The filtrates collected from three separate extractions were combined and evaporated to dryness under vacuum. The average yield obtained for 20% (20EtOH-PP), 40% (40EtOH-PP), 60% (60EtOH-PP), 80% (80EtOH-PP) and 95% (90EtOH-PP) ethanol extracts (EEPP) were 7.3, 17.7, 20.7, 24.1 and 24.0%, respectively.
Preparation of Liver Homogenate Liver homogenate
was prepared according to a previously described method with minor modification.21)Briefly, rats weighing 180—230 g
were sacrificed and the liver was quickly removed. The liver sample was then cut into small pieces and homogenized in 150 mMTris–HCl buffer (pH 7.2) with a disperser (PT1300D, Kinematica) at 20500g for 3 min to give a 20% (w/v) liver homogenate. The liver homogenate was further centrifuged at 500g for another 10 min. Supernatant of the liver ho-mogenate was collected and the amount of protein was deter-mined by the method of Lowry et al.22)
FeCl2-Ascorbic Acid Stimulated Lipid Peroxidation in
Rat Liver Homogenate The effect of PP extracts on
FeCl2–ascorbic acid induced lipid peroxidation in rat liver
homogenate was conducted according to the modified meth-ods as described by Kimura et al.23) and Wong et al.24) A mixture containing 0.5 ml of liver homogenate, 0.1 ml of Tris–HCl buffer (pH7.2), 0.05 ml of 0.1 mM ascorbic acid, 0.05 ml of 4 mMFeCl2and 0.05 ml of various concentrations of EEPP, HWEPP or a-tocopherol. It was then incubated for 1 h at 37 °C. After incubation, 0.9 ml of distilled water and 2 ml of 0.6% TBA were added and then shaken vigorously. The mixture was heated for 30 min in a boiling water bath at 100 °C. After cooling, 5 ml of n-butanol was added and the mixture was then shaken vigorously. The n-butanol layer was separated by centrifugation at 3000g for 10 min. The su-pernatant was collected and measured spectrophotometrically at 532 nm.
Cytochrome c Test Enzymatic formation of superoxide anions was assayed by the reduction of cytochrome c method.25)Ten milligrams of samples were dissolved in 1 ml
of distilled water or DMSO and then diluted with 50 mM phosphate buffer (pH=7.8) to various concentrations (10 to 100mg/ml), followed by adding 0.07 units/ml of xanthine ox-idase, 100mMof xanthine and 50mMof cytochrome c to the samples. All samples were incubated for 3 min at room tem-perature and then measured spectrophotometrically at 550 nm.
Xanthine Oxidase Inhibition Test Xanthine oxidase ac-tivity was estimated by the formation of uric acid from xan-thine.26) Ten milligrams of samples were dissolved in 1 ml distilled water or DMSO, and then diluted with 50 mM KH2PO4 buffer (pH7.8) to various concentrations (10 to 100mg/ml). After 100 mM of xanthine and 20ml of xanthine oxidase (0.4 units) were added, samples were incubated for 3 min at room temperature. Superoxide formation was counted by spectrophotometric measurement of uric acid production at 295 nm.
Statistical Analysis Data are presented as meansS.D. IC50value of each sample was calculated. Values were
evalu-ated by one way analysis of variance (ANOVA), followed by post hoc Duncan’s multiple range tests.
RESULTS
Anti-Lipid Peroxidation Activity The inhibitory effect of extracts on FeCl2–ascorbic acid induced malondialdehyde (MDA) production in rat liver homogenate is shown in Table 1. Results showed that the inhibition of MDA formation in-creases with increasing concentrations of PP extracts and a-tocopherol. At concentrations 10—100mg/ml, all extracts displayed an anti-lipid peroxidation activity, with an inhibi-tion rate varies from 2.0 to 82.3%. At concentrainhibi-tion 100mg/ml, the formation of MDA was inhibited by 82.3% for 95EtOH-PP and 25.0% for HWEPP.
In the thiobarbituric acid test, the IC50values of
80EtOH-PP and 95EtOH-80EtOH-PP were 23.11 and 23.74mg/ml, respec-tively, which were lower than a-tocopherol (26.71 mg/ml) (Table 2). The potency of anti-lipid peroxidation activity was in the order of 80EtOH-PP95EtOH-PPa-tocopherol 40EtOH-PP, 60ETOH-PP20EtOH-PPHWEPP. Based on the IC50 values, 80EtOH-PP and 95EtOH-PP demonstrated
the best inhibitory effect against lipid peroxidation.
Free Radical Scavenging Activity The results on the ef-fect of different concentrations of PP extracts on superoxide anions are shown in Table 3. The scavenging effect was found to increase with increasing concentration of PP ex-tracts. At concentrations 10—100mg/ml, they were shown to possess a scavenging activity ranging from 0.0 to 100.0%. The 95EtOH-PP exhibited a scavenging effect of 49.2% at 10mg/ml, 99.3% at 50 mg/ml, and 100.0% at 100 mg/ml, which were more active than a-tocopherol at the same con-centrations.
964 Vol. 28, No. 6
Table 1. Inhibitory Effect of Hot Water Extract (HWEPP) and Extracts of Different Ethanol Concentrations (20, 40, 60, 80 and 95% EtOH) Prepared from P. peruviana (PP) on FeCl2–Ascorbic Acid Induced MDA Production
in Rat Liver Homogenate
Samples Concentration MDA
a) Inhibition rateb) (mg/ml) (nM/mg protein) (%) FeCl2-AA — 50.01.5 — Control — 20.00.8 — HWEPP 10 49.01.0 2.0g 20EtOH-PP 10 42.60.9 24.7f 40EtOH-PP 10 36.22.3 46.0b 60EtOH-PP 10 38.01.5 40.0d 80EtOH-PP 10 35.51.0 48.3a 95EtOH-PP 10 37.03.0 43.3c a-Tocopherol 10 38.50.5 38.3e HWEPP 50 45.01.0 16.7f 20EtOH-PP 50 38.00.7 40.0e 40EtOH-PP 50 35.01.2 50.0d 60EtOH-PP 50 35.02.5 50.0d 80EtOH-PP 50 33.81.3 54.0c 95EtOH-PP 50 31.21.0 62.7b a-Tocopherol 50 30.00.1 66.7a HWEPP 100 42.50.9 25.0f 20EtOH-PP 100 36.01.5 46.7e 40EtOH-PP 100 34.00.5 53.3d 60EtOH-PP 100 33.62.0 54.7d 80EtOH-PP 100 27.51.8 75.0c 95EtOH-PP 100 25.30.7 82.3b a-Tocopherol 100 21.20.9 96.0a
a) MDA data were presented as meansS.D. (n5). b) The inhibitory rates within
the column of same concentration with the different superscript letters were signifi-cantly different at p0.05 as analyzed by Duncan’s multiple range tests.
In the cytochrome c test, the 95EtOH-PP (IC50 value 10.40mg/ml) exhibited the highest scavenging effect on su-peroxide anions (Table 2). On the other hand, HWEPP and 20EtOH-PP exhibit a weak superoxide radical scavenging ac-tivity.
Inhibitory Effect On Xanthine Oxidase Activity All
extracts exhibit an inhibitory effect on the xanthine oxidase-induced superoxide formation in a concentration-dependent manner (Table 4). At concentrations 10—100mg/ml, PP ex-tracts inhibited the xanthine oxidase activity with an inhibi-tion rate ranging from 6.5 to 100.0 % (p0.05). At concen-trations 10—100mg/ml, the xanthine oxidase inhibition ef-fect of all samples was in the order of 95EtOH-PP a-toco-pherol 80EtOH-PP60EtOH-PP40EtOH-PP20EtOH-PPHWEPP.
In the xanthine–xanthine oxidase system, the IC50value of
95EtOH-PP was 8.97mg/ml, which is 2.4 fold more active
than a-tocopherol (20.68 mg/ml) (Table 2), suggesting that 95EtOH-PP was a potent xanthine oxidase inhibitor.
DISCUSSION
Excessive ROS can react with biological molecules such as DNA, proteins, phospholipids, and eventually cause oxida-tive damage in tissue and resulted in free radical-related dis-eases such as inflammation, heart disease, diabetes, gout and cancer.27)Aerobic organisms are protected from ROS toxicity
by their natural antioxidant defense system involving enzy-matic and non-enzyenzy-matic mechanisms. An imbalance be-tween the amount of ROS and the antioxidant defense system could lead to an appearance of health problem. Thus, it is why the daily intake of foods or medicated diet or heath drink with antioxidant activity is important.6,28)In this study,
six extracts of PP and a-tocopherol were examined for their antioxidant activities. EEPP was shown to possess an excel-lent antioxidant activity in enzymatic and nonenzymatic liver tissue oxidative systems.
It was reported that Fe2and ascorbic acid stimulated lipid peroxidation in rat liver microsomes and mitochondria.23)
a-Tocopherol is a natural antioxidant, which functions as a free-radical quencher in biological cells and localizes within the phospholipid bilayer of cell membranes to protect against biological lipid peroxidation.29) Tocopherols were also
re-ported to decrease atherosclerosis and delay death from my-ocardial infarction, this therapeutic benefit was presumably derived from the inhibition of lipid peroxidation.30) The health benefits of certain natural medicines have been ex-plained by their ability in preventing free radical damage to cell membranes through reducing the level of lipid peroxides.
June 2005 965
Table 2. IC50 Values of Antioxidant Activities of Hot Water Extract
(HWEPP) and Extracts of Different Ethanol Concentrations (20, 40, 60, 80 and 95% EtOH) Prepared from P. peruviana (PP)
IC50values (mg/ml)
Samples
Thiobarbituric Cytochrome c Xanthine oxidase acid test test inhibition test
HWEPP 100 100 100 20EtOH-PP 100 100 63.93 40EtOH-PP 50.00 55.85 30.02 60EtOH-PP 50.00 52.52 37.79 80EtOH-PP 23.11 27.34 26.00 95EtOH-PP 23.74 10.40 8.97 a-Tocopherol 26.71 13.39 20.68
Table 3. Superoxide Scavenging Activity of Hot Water Extract (HWEPP) and Extracts of Different Ethanol Concentrations (20, 40, 60, 80 and 95% EtOH) Prepared from P. peruviana (PP)
Samples Concentration Scavenging rate
(mg/ml) (%)a) HWEPP 10 0.00.0f 20EtOH-PP 10 0.00.0f 40EtOH-PP 10 8.30.2e 60EtOH-PP 10 20.10.5d 80EtOH-PP 10 38.00.9c 95EtOH-PP 10 49.20.1a a-Tocopherol 10 47.20.3b HWEPP 50 23.80.6g 20EtOH-PP 50 30.21.0f 40EtOH-PP 50 43.60.1e 60EtOH-PP 50 48.01.9d 80EtOH-PP 50 65.71.2c 95EtOH-PP 50 99.30.1a a-Tocopherol 50 80.20.6b HWEPP 100 49.00.5e 20EtOH-PP 100 48.70.8e 40EtOH-PP 100 98.31.0b 60EtOH-PP 100 87.72.5d 80EtOH-PP 100 95.73.1c 95EtOH-PP 100 100.00.1a a-Tocopherol 100 100.00.0a
a) Data were presented as the percentage of free radical scavenging, meansS.D. (n3); values within the column of same concentration with the different superscript letters were significantly different at p0.05 as analyzed by Duncan’s multiple range tests.
Table 4. Xanthine Oxidase Inhibition Activity of Hot Water Extract (HWEPP) and Extracts of Different Ethanol Concentrations (20, 40, 60, 80 and 95% EtOH) Prepared from P. peruviana (PP)
Samples Concentration Scavenging rate
(mg/ml) (%)a) HWEPP 10 6.50.5f 20EtOH-PP 10 20.00.0e 40EtOH-PP 10 31.12.0d 60EtOH-PP 10 20.10.5e 80EtOH-PP 10 38.00.9c 95EtOH-PP 10 59.10.8a a-Tocopherol 10 40.60.2b HWEPP 50 40.00.8e 20EtOH-PP 50 40.01.5e 40EtOH-PP 50 68.92.0c 60EtOH-PP 50 63.11.0d 80EtOH-PP 50 68.02.5c 95EtOH-PP 50 100.00.0a a-Tocopherol 50 75.90.6b HWEPP 100 46.01.0e 20EtOH-PP 100 75.92.9d 40EtOH-PP 100 78.01.5c 60EtOH-PP 100 77.40.8c 80EtOH-PP 100 90.72.0b 95EtOH-PP 100 100.00.0a a-Tocopherol 100 100.00.0a
a) Data were presented as the percentage of inhibition on xanthine oxidase activity,
meansS.D. (n3); values within the column of same concentration with the different superscript letters were significantly different at p0.05 as analyzed by Duncan’s multi-ple range tests.
In this study, the 80EtOH-PP and 95EtOH-PP exhibited the best antioxidant action in the rat liver homogenate model system. Although the bioactive components present in these extracts are unknown, it was reported that the trihydroxy steroidal lactone (i.e. withaperuvin E) isolated from the roots of PP possess protective effect against lipid peroxidation.31)
Compared to SOD, the inhibitory rate of xanthine oxidase activity of 10mg/ml 95EtOH-PP (49.20%) and 10 mg/ml a-tocopherol (47.20%) was stronger than 10 units/ml SOD (13.00%) but inferior than 100 units/ml (88.46%) and 300 units/ml (93.90%) of SOD. It has reported that xanthine oxidase inhibitors may be useful for the treatment of hepatic disease and gout.32) In this study, the 95EtOH-PP displayed the most potent activity in inhibitory the xanthine oxidase ac-tivity, suggesting that this extract is an excellent xanthine ox-idase inhibitor.
Enzymatic formation of superoxide anions was estimated by reduction of cytochrome c. Xanthine oxidase converts hy-poxanthine to xanthine and then xanthine to uric acid in the presence of molecular oxygen to yield superoxide anion and hydrogen peroxide. These free radicals can directly reduced ferri-cytochrome c to ferro-cytochrome c.14) Hence, phyto-chemicals or extracts with inhibitory effect on superoxide anion regeneration by the enzymatic pathway would be bene-ficial in preventing ischaemia and edema.33)
The inhibitory effect of 95EtOH-PP on the lipid peroxida-tion, superoxide anion regeneration and xanthine oxidase ac-tivity were more potent than other extracts. This observation suggests that ethanol PP extracts, particularly the extract of 95EtOH-PP, could be prepared into wines or Chinese med-icated diets and be consumed daily in life for maintaining good health. The strong antioxidant activities demonstrated by the ethanol extracts of PP may be contributed by the flavonoids and other yet to be discovered antioxidative com-pounds.34,35) Plants such as Caesalpinia sappan heartwood
and Vaccinium berries, containing flavonoids and phenolics are known to possess strong antioxidant properties.36,37)
In conclusion, extracts of PP prepared with different sol-vent conditions possessed a different magnitude of antioxi-dant potency. Compared to the aqueous extract, ethanol ex-tracts exhibited a better antioxidant activity, with 95EtOH-PP showed the most potent antioxidant and free radical scaveng-ing activities. The 95EtOH-PP also exhibited the higher an-tioxidant action in the rat liver homogenate model system. Therefore, ethanol extracts may be used as a potential dietary antioxidant to retard aging and preventing diseases caused by ROS or ameliorating oxidative damage in tissue. The active components and antioxidative mechanism(s) of action of PP ethanol extracts warrant further studies in both in vitro and in vivo models.
REFERENCES
1) Bulkley G. B., Surgery, 94, 407—411 (1983). 2) Dormandy T. L., Lancet, 11, 1010—1014 (1983).
3) Cheng H. Y., Lin T. C., Yu K. H., Yang C. M., Lin C. C., Biol. Pharm.
Bull., 26, 1331—1335 (2003).
4) Compori M., Lab. Invest., 53, 599—620 (1985). 5) Halliwell B., Nutr. Rev., 55, S44—S52 (1997). 6) Halliwell B., Nutr. Rev., 1, 253—265 (1994).
7) Bartold P. M., Wiebkin O. W., Thonard J. C., J. Periodontol. Res., 19, 390—400 (1984).
8) Varani J., Fligiel S. E. G.., Till G. O., Kunkel R. G.., Ryan V. S., Ward P. A., Lab. Inves., 53, 656—663 (1985).
9) Burton G. W., Proc. Nutr. Soc., 53, 251—262 (1994).
10) Buettner G. R., Arch. Biochem. Biophys., 300, 535—543 (1993). 11) Lee S. E., Shin H. T., Hwang H. J., Kim J. H., Phytother. Res., 17,
1041—1047 (2003).
12) Polidori M. C., Stahl W., Eichler O., Niestroj I., Sies H., Free Radic.
Biol. Med., 30, 456—462 (2001).
13) Ho K. Y., Huang J. S., Tsai C. C., Lin T. C., Hsu Y. F., Lin C. C., J.
Pharm. Pharmacol., 51, 1075—1078 (1999).
14) Gohil K., Moy R. K., Farzin S., Maguire J. J., Packer L., Free Radic.
Res., 33, 831—849 (2000).
15) Psotova J., Kolar M., Sousek J., Svagera Z., Vicar J., Ulrichova J.,
Phy-tother. Res., 17, 1082—1087 (2003).
16) Lin C. C., Cheng H. Y., Yang C. M., Lin T. C., J. Biomed. Sci., 9, 656—664 (2002).
17) Lin C. C., Wu S. J., Chang C. H., Ng L. T., Phytother. Res., 17, 726— 730 (2003).
18) Pietro R. C., Kashima S., Sato D. N., Januario A. H., Franca S. C.,
Phytomedicine, 7, 335—338 (2000).
19) Soares M. B. P., Bellintani M. C., Ribeiro I. M., Tomassini T. C. B., Santos R. R., Eur. J. Pharmacol., 459, 107—112 (2003).
20) Perry L. M., “Medicinal Plants of East and Southeast Asia,” The MIT Press, Cambridge, Massachusetts, 1980, p. 393.
21) Masaol H. M., Yang X. W., Miyashiro H., Namba T., Phytother. Res., 7, 395—401 (1993).
22) Lowry O. H., Rosebrough N. J., Farr A. L., Randall R. J., J. Biol.
Chem., 193, 265—275 (1951).
23) Kimura K., Kubo M., Tani T., Arich S., Okuda H., Chem. Pharm.
Bull., 29, 2610—2617 (1981).
24) Wong S. H., Knight J. A., Hopfer S. M., Zaharia O., Leach C. N., Sun-derman F. W., Clin. Chem., 33, 214—220 (1987).
25) McCord J. M., Fridovich I., J. Biol. Chem., 244, 6049—6055 (1969). 26) Chang W. S., Chang Y. H., Lu F. J., Chiang H. C., Anticancer Res., 14,
501—506 (1994).
27) Slater T. F., Biochem. J., 222, 1—15 (1984).
28) Schinella G. R., Tournier H. A., Prieto J. M., Mordujovich de Buschi-azzo P., Rios J. L., Life Sci., 70, 1023—1033 (2002).
29) Hafeman D. G., Hoekstra W. G., J. Nutr., 107, 666—672 (1997). 30) Byers T., Nutr. Rev., 51, 333—345 (1993).
31) Tripathi Y. B., Shukla S., Ind. J. Biochem. Biophys., 32, 308—309 (1995).
32) Lin C. C., Huang P. C., Lin J. M., Am. J. Chin. Med., 28, 87—96 (2000).
33) Lin C. C., Yen M. H., Lo T. S., Lin J. M., J. Ethnopharmacol., 60, 9— 17 (1998).
34) Fischer G., Ebert G., Ludders P., Acta Hortic., 531, 263—267 (2000). 35) Hassanien M. F. R., Moersel J. T., Fluessiges Obst., 70, 398—402
(2003).
36) Shrishailappa B., Sudheer M., Sujay R. R., Elango K., Suresh B., Biol.
Pharm. Bull., 26, 1534—1537 (2003).
37) Todd G. T., Dany L. B., Jerry E., J. Agric. Food Chem., 52, 3169— 3176 (2004).
