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PHYTOTHERAPY RESEARCH Phytother. Res. 18, 280–284 (2004) Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/ptr.1380 S. M. SUBOH ET AL.
Protective Effects of Selected Medicinal Plants against Protein Degradation, Lipid Peroxidation and Deformability Loss of Oxidatively Stressed Human Erythrocytes S. M. Suboh1, Y. Y. Bilto1*, T. A. Aburjai2 1 2
Department of Biological Sciences, University of Jordan, Amman, Jordan Department of Pharmaceutical Sciences, Faculty of Pharmacy, University of Jordan, Amman, Jordan
The effects of seven medicinal plants including Artemisia herba-alba, Ferula hermonis, Hibiscus sabdariffa, Nigella sativa, Teucrium polium, Trigonella foenum-graecum, and Allium sativum on protein degradation, lipid peroxidation, erythrocyte deformability and osmotic fragility of erythrocytes exposed in vitro to 10 mM H2O2 for 60 min at 37 °C have been examined. Preincubation of erythrocytes with Nigella sativa and Allium sativum protected erythrocytes against protein degradation, loss of deformability and increased osmotic fragility caused by H2O2, while the other plants failed to protect erythrocytes against these damages. Artemisia herba-alba did not protect erythrocytes against lipid peroxidation, while Trigonella foenum-graecum unexpectedly increased lipid peroxidation of erythrocytes exposed to H2O2. Ferula hermonis, Hibiscus sabdariffa, Nigella sativa, Teucrium polium and Allium sativum protected erythrocytes against lipid peroxidation. The results indicate the importance of oxidatively damaged cellular proteins in compromising the rheologic behaviour of the erythrocytes, and that the medicinal plants which have anti-protein-oxidant activity (e.g. Nigella sativa and Allium sativum) could be rheologically useful, particularly in pathological conditions related to free radicals. Copyright © 2004 John Wiley & Sons, Ltd. Keywords: erythrocyte deformability; protein degradation; lipid peroxidation; oxidative stress; H2O2; medicinal plants.
INTRODUTION Oxidative damage as a result of an increase in the free radical load and/or decrease in the efficiency of the antioxidant systems has been implicated in many human diseases (Saltman, 1989; Halliwell, 1991). As plants could represent a source of natural compounds with antioxidant activities, many studies have been conducted searching for the antioxidant activities of many plant extracts and their constituents (Tseng et al., 1996; Duh and Yen, 1997; Kim et al., 1997; Tseng et al., 1997; Asai et al., 1999; Duh et al., 1999; Aniya et al., 2000). Erythrocytes are particularly susceptible to oxidation by oxygen radicals, because they are very rich in Fe2+-containing molecules, primarily hemoglobin. Oxidative damage of external origin can be induced in erythrocytes upon incubation with H2O2. This compound is known to cross the erythrocyte membrane and reacts rapidly with hemoglobin, generating a very reactive radical species, e.g. hydroxyl radical (Van den Berg et al., 1992). Treatment of erythrocytes with H2O2 has been observed to bring about lipid peroxidation, protein degradation and a progressive loss of deformability in a concentration and time dependent manner (Srour et al., 2000). This loss of deformability appears to be related to oxidation
* Correspondence to: Prof. Y. Y. Bilto, Department of Biological Sciences, University of Jordan, Amman 11942, Jordan. E-mail:
[email protected] Contract/grant sponsor: University of Jordan. Copyright © 2004 John Wiley & Sons, Ltd. Copyright © 2004 John Wiley & Sons, Ltd.
of heme proteins resulting in their cross-linking to skeletal proteins, i.e. spectrin and actin, and in addition, to the cytoplasmic component of band 3 (Snyder et al., 1985; Sato et al., 1995). Previous studies in our laboratory found that this loss of deformability could be completely prevented by prior exposure of the erythrocytes to carbon monoxide (known to inhibit heme-protein oxidation) (Srour et al., 2000). Thus it is noteworthy that protein oxidation and consequent degradation may prove to be of practical significance in the loss of erythrocyte deformability following exposure to H2O2. The present study was therefore undertaken to screen selected medicinal plants for protective activities against protein degradation, lipid peroxidation and deformability loss of human erythrocytes after exposure to H2O2.
MATERIALS AND METHODS Plant material. The selected plants were purchased from the local market (Table 1). The taxonomic identity of these plants was confirmed by comparing them with those of known identity in the herbarium of the Department of Biological Science, Faculty of Science, University of Jordan with the assistance of Professor. Barakat Abu-Irmaileh, Faculty of Agriculture, University of Jordan. A voucher specimen has been deposited in the author’s research laboratory at the Department of Pharmaceutical Sciences, Faculty of Pharmacy, University of Jordan. October(2004) 2002 Phytother.Received Res. 18,31 280–284 Accepted 19 May 2003
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Table 1. List of studied plant species
No. 1 2 3 4 5 6 7
Botanic name
English Name
Arabic Name
Voucher specimen
Family
Yield (g/Kg)
Part(s) extracted
Artemisia herba-alba Asso. Ferula hermonis Boiss. Hibiscus sabdariffa L. Nigella sativa L. Teucrium polium L. Trigonella foenum-graecum L. Allium sativum Linne’
Herba-alba Wormwood Ferula hermonis Hibiscus Black cumin Germander Fenugreek Garlic
Shieh Zallouh Karkade Kezha Jeada Hilba Thoum
T.A 02-8 Y.B 99–2 T.A 02-17 Y.B 02-20 T.A 02-3 SS-02-1 SS-02-11
Compositae Umbelliferae Malvaceae Ranunculaceae Labiatae Leguminosae Liliaceae
50 135.2 136.7 90 112.9 132.9 –
Leaves Roots Calyx Seeds Leaves Seeds Bulb
Preparation of the crude extracts. 500 g of air dried and finely powdered plant materials were extracted in a Soxhlet with 1000 ml MeOH for 24 h except for Allium sativum. After filtration, the filtrate was evaporated in a vacuum below 40 °C on a rotary evaporator. The final dry weight of the solid extracts were used to estimate the yield (g/Kg) of each plant, the mean values of which are presented in Table 1, and also to prepare stock solutions from these plant extracts by dissolving the dried solid extracts in dimethylsulfoxide (DMSO) (final concentration not exceeding 0.2%), prior to being diluted with phosphate buffered saline (PBS). All solutions were stored in a refrigerator at 4 °C until use. Preparation of Allium sativum extract. One gram of air dried and finely powdered bulb was extracted with 10 ml of distilled water at 37 °C for 2 h and then filtered. The filtrate obtained was used as the original herbal extract. Ratios of 1:1 and 1:2 dilutions were prepared from this filtrate before being used in this study (Aniya et al., 2000). Exposure of erythrocytes to H2O2. Washed erythrocyte suspensions were prepared by centrifugation of heparinized whole blood from adult volunteers to remove the buffy cout layer and then washing the packed cells three times with cold phosphate buffered saline (PBS) as described by Dacie and Lewis (1995). Washed erythrocyte suspensions were pre-incubated with 2 mM sodium azide for 60 min at 37 °C in a shaking water-bath to inhibit catalase. Next, equal volumes of cell suspension and 20 mM H2O2 were mixed and incubated for a further 60 min at 37 °C. Controls contained PBS instead of H2O2. Following the incubation period, the suspensions were mixed and used for alanine and MDA determinations (Srour et al., 2000). A given plant extract was added to erythrocyte suspensions at 30 min of the pre-incubation period with sodium azide. Alanine determination. Erythrocyte alanine concentration was determined as a measure of protein degradation according to Davies and Goldberg method (1987) as modified by Srour et al. (2000). All alanine concentrations were expressed as nmol/gHb. The Hb was measured by the cyanmethemoglobin method as described by Dacie and Lewis (1995). Malonyldialdehyde (MDA) determination. Erythrocyte MDA was determined as a measure of lipid peroxidation according to Stocks and Dormandy’s method (1971) as modified by Srour et al. (2000). All MDA concentrations were expressed as nmol/gHb. The Hb Copyright © 2004 John Wiley & Sons, Ltd.
was measured by the cyanmethemoglobin method as described by Dacie and Lewis (1995). Deformability studies. Leukocyte-depleted and plateletdepleted erythrocyte suspensions were prepared by prefiltration of heparinized whole blood from adult volunteers through Imugard IG500 cotton wool (Termo corporation, Tokyo, Japan) as described by Bilto et al. (1987). The cotton wool filtered erythrocytes were resuspended in PBS at a hematocrit of 7%. Erythrocyte deformability was measured by filtration of erythrocyte suspension through 5 µm pore diameter polycarbonate membranes (Nuclepore corporation, Pleasanton, USA) using a temperature controlled Hemorheometre MK1 (Hanss, 1983) at 37 °C. A small batch of 12 membranes was used and reused after cleaning by ultrasonication in aqueous sodium dodecylsulfate (1%, w/v) for 10 sec (Bilto and Stuart, 1985). Results were expressed as an index of filtration (IF) of the flow time for the erythrocyte suspension relative to buffer and corrected for hematocrit (Bilto et al., 1987). An increase in IF indicates loss of deformability. Osmotic fragility measurements. Aliquots (0.2 ml) of erythrocyte suspension (2.5% hematocrit) were added to 1.8 ml of buffered saline solutions of decreasing concentrations, pH 7.4, (NaCl range of 9.0–1.0 g/L). The suspensions were allowed to stand for 30 min at room temperature, mixed again and then centrifuged for 5 min at 1200 rpm. The supernatants were removed and the amount of lysis was determined spectrophotometrically at 540 nm. The percentage of hemolysis was calculated from the ratios of the absorbance (Dacie and Lewis, 1995). Statistical analysis. The results presented are means ± SD of several separate experiments with duplicate tubes. Statistical significance was determined using one-way analysis of variance followed by student’s t-test for paired samples. Differences were considered significant when p < 0.05.
RESULTS Incubation of erythrocytes with H2O2 for 60 min caused a significant increase in intracellular alanine (i.e. an increase in protein degradation) from a range of 369.2–513.7 nmol/gHb without H2O2 to a range of 2817.9–4741.0 nmol/gHb with H2O2 (Table 2). When the erythrocytes were preincubated with different Phytother. Res. 18, 280–284 (2004)
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Table 2. Alanine and MDA concentrations, and IF of normal erythrocytes when incubated at 37 °C for 60 min with or without 10 mM H2O2 or with H2O2 plus different concentrations of tested medicinal plant extracts. Values are presented as a mean ± SD of 5 experiments with duplicate tubes Alanine (nmol/g Hb) Plant
Artemisa herba-alba
Ferula hermonis
Hibiscus sabdariffa
Nigella sativa
Teucrium polium
Trigonella foenum-graecum
Allium sativum
Conc. (mg/ml) 0.0 0.2 0.4 0.6 0.8 1.0 0.0 0.2 0.4 0.6 0.8 1.0 0.0 0.2 0.4 0.6 0.8 1.0 0.0 0.2 0.4 0.6 0.8 1.0 0.0 0.2 0.4 0.6 0.8 1.0 0.0 0.2 0.4 0.6 0.8 1.0 0.0% 25% 50% 100%
Without H2O2 439.8 ± – – – – 435.3 ± 513.7 ± – – – – 453.4 ± 465.1 ± – – – – 436.0 ± 435.6 ± – – – – 435.3 ± 369.2 ± – – – – 409.4 ± 398.4 ± – – – – 426.7 ± 445.8 ± – – 380.4 ±
73
18 66
25 191
225 189
224 34
37 40
17 199
215
MDA (nmol/g Hb)
With H2O2 3322.8 3164.1 3213.5 3401.4 3300.7 3309.1 3241.8 3176.4 3113.9 3472.3 3285.0 3125.5 2817.9 2963.6 2719.1 3027.3 3027.3 2763.0 3033.7 2403.5 1777.5 1399.3 943.8 674.0 3352.8 3257.5 3238.2 3428.1 3378.7 3362.0 3412.3 3112.5 3310.8 3459.0 3356.1 3490.0 4741.0 3790.3 3055.4 2621.6
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
Without H2O2
284.7 356.9 217.9 364.7 217.4 433.1 261.1 73.0 233.8 349.8 152.0 482.9 434.9 444.1 452.0 436.2 564.5 689.2 395.4 455.8* 425.9* 386.8* 268.9* 158.3* 262.7 145.0 210.3 337.2 145.0 380.6 308.2 335.1 159.7 452.3 195.2 470.9 576.0 246.6* 714.5* 399.8*
17.6 ± – – – 12.9 ± – 17.6 ± – – – 12.9 ± – 9.9 ± – – – 9.9 ± – 10.2 ± – – – 8.2 ± – 17.6 ± – – – 12.9 ± – 13.8 ± – – – 16.0 ± – 9.6 ± – – 9.0 ±
4.7
3.1 4.7
3.1 4.4
3.3 0.5
1.7 3.0
3.0 2.9
3.7 3.3
2.9
IF
With H2O2 ± ± ± ± ± – 327.0 ± 198.8 ± 187.3 ± 183.5 ± 184.8 ± – 381.2 ± 351.4 ± 303.9 ± 268.2 ± 255.4 ± – 300.7 ± 263.6 ± 226.4 ± 203.7 ± 206.0 ± – 363.6 ± 294.6 ± 248.1 ± 243.0 ± 236.7 ± – 305.6 ± 327.1 ± 387.9 ± 429.9 ± 462.4 ± – 310.3 ± 250.3 ± 216.4 ± 208.7 ± 327.0 336.2 327.0 336.2 327.0
Without H2O2
13.0 24.0 13.0 24.0 13.0 13.0 15.0* 10.0* 10.0* 10.0* 24.0 20.0* 23.0* 22.0* 14.4* 27.3 32.5* 37.4* 22.4* 28.4* 68.0 57.0* 34.0* 33.0* 38.0* 20.0 21.0* 25.0* 28.0* 30.0* 42.0 29.5* 31.7* 22.5*
14.4 ± – – – 15.4 ± – 13.2 ± – – – 14.5 ± – 13.0 ± – – – 12.8 ± – 13.2 ± – – – – 13.0 ± 9.1 ± – – – 11.5 ± – 12.1 ± – – – 12.7 ± – 15.3 ± – – 13.1 ±
3.0
2.5 2.8
3.4 2.1
0.9 1.8
2.2 1.0
2.8 1.6
0.9 1.4
0.7
With H2O2 76.2 ± 4.7 – – 76.1 ± – 81.5 ± – – – 95.6 ± – 80.0 ± – – – 87.2 ± – 136.2 ± 73.6 ± 43.6 ± 33.7 ± 25.6 ± 19.2 ± 66.5 ± – – – 61.2 ± – 107.1 ± – – – 104.6 ± – 163.3 ± 130.9 ± 98.7 ± 75.0 ±
2.8 8.1
25.6 11.3
9.7 19.1 8.3* 5.0* 5.5* 5.8* 3.8* 10.6
8.4 9.8
7.4 19.8 18.0* 10.2* 5.7*
– not determined * p < 0.05 compared to control erythrocytes with H2O2 alone.
concentrations of tested plant extracts only Nigella sativa and Allium sativum caused a significant inhibition of alanine production in a concentration dependent manner. Nigella sativa at 1.0 mg/ml showed a great level of inhibition reaching 91%, whereas original extract of Allium sativum caused 48% inhibition of alanine production. Incubation of erythrocytes with H2O2 for 60 min also caused a significant increase in intracellular MDA (i.e. an increase in lipid peroxidation) from a range of 9.0–17.6 nmol/gHb without H2O2 to a range of 300.7– 381.2 nmol/gHb with H2O2 (Table 2). When the erythrocytes were preincubated with different concentrations of screened plant extracts, Artemisia herba-alba had no significant effect on MDA production, whereas Trigonella foenum-graecum unexpectedly increased the production of MDA (i.e. increased lipid peroxidation) Copyright © 2004 John Wiley & Sons, Ltd.
in a concentration dependent manner. However, the following plants Ferula hermonis, Hibiscus sabdariffa, Nigella sativa, Teucrium polium, Allium sativum inhibited significantly the production of MDA, in a concentration dependent manner, with the Ferula hermonis being the most potent (Table 2). Incubation of erythrocytes with H2O2 for 60 min also caused a significant increase in IF (i.e. loss of deformability) from a range of 9.1–15.3 without H2O2 to a range of 66.5–163.3 with H2O2 (Table 2). Preincubation of erythrocytes with Artemisia herba-alba, Ferula hermonis, Hibiscus sabdariffa, Teucrium polium and Trigonella foenum-graecum showed no effect on deformability loss (Table 2). Whereas, preincubation of erythrocytes with Nigella sativa showed almost complete prevention (i.e. about 96%) of the deformability loss caused by H2O2. Allium sativum, however, also prevented significantly (56.6%) Phytother. Res. 18, 280–284 (2004)
PROTECTIVE EFFECTS OF SELECTED MEDICINAL PLANTS
Figure 1. Osmotic fragility curves (mean of 5 experiments) for heparinized erythrocytes incubated at 37 °C for 30 min (A) without (–䉫–) and with 10 mM H2O2 (–䉬–) or in the presence of 0.6 mg/ml F. h. without (–䉭–) and with 10 mM H2O2 (–䉱–); (B) without (–䊐–) and with 10 mM H2O2 (–䊏–) or in the presence of 0.6 mg/ml N. s. without (–䊊–) and with 10 mM H2O2 (–䊉–) or in the presence of A. s. original extract without (–䉭–) and with 10 mM H2O2 (–䉱–). * p < 0.05 as compared with erythrocytes treated with H2O2 alone.
the deformability loss of erythrocytes exposed to H2O2 in a concentration dependent manner (Table 2). In an attempt to explain the observed effects of H2O2 and the tested plants on erythrocyte deformability, the osmotic fragility of erythrocytes exposed to H2O2 and to tested plant extracts was studied. As shown in Fig. 1, erythrocytes exposed to H2O2 showed an increase in osmotic fragility when compared to control erythrocytes incubated similarly, but in the absence of H2O2. However, Ferula hermonis which exhibited the highest inhibition of MDA production in erythrocytes exposed to H2O2, but which did not have any effect on either alanine production or the erythrocyte deformability, showed no effect on the osmotic fragility of erythrocytes before and after exposure to H2O2 (Fig. 1A). In contrast, Nigella sativa and Allium sativum improved the fragility of erythrocytes exposed to H2O2 (Fig. 1B). This would suggest that, by protecting the erythrocyte against oxidative damage, both Nigella sativa and Allium sativum make the cytoskeleton of the erythrocyte more resistance to mechanical insult.
DISCUSSION The present study evaluated the antioxidant properties of seven medicinal plants using human erythrocytes exposed to 10 mM H2O2 (Table 2). Preincubation of erythrocytes with Artemisia herba-alba, Ferula hermonis, Hibiscus sabdariffa, Teucrium polium and Trigonella foenum-graecum showed no effect on alanine production (i.e. no effect on protein degradation), nor on IF Copyright © 2004 John Wiley & Sons, Ltd.
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values (i.e. no effect on erythrocyte deformability). However, Nigella sativa and Allium sativum protected human erythrocytes against H2O2-induced protein degradation and loss of deformability in a concentration dependent manner (Table 2). It is interesting to note that Nigella sativa at 1.0 mg/ml resulted in a remarkable protection against loss of deformability as compared with erythrocytes treated with H2O2 alone (Table 2). This improvement might be due to its ability to fully suppress protein degradation (Table 2). This protection reached 96% in the case of deformability and 91% in the case of protein degradation (Table 2). Thus, from this study, it was confirmed that the protection against loss of deformability was parallel to the inhibitory activity of protein degradation. The effects of these plant extracts on lipid peroxidation were studied, and the results (Table 2) showed that Artemisia herba-alba had no significant effect on MDA production, whereas, Trigonella foenum-graecum increased significantly the MDA production in H2O2-treated erythrocytes without causing any effect on non-H2O2treated erythrocytes. This could be explained by the ability of this extract to interfere with erythrocytes antioxidant system either by inhibiting the antioxidant enzymes or by increasing the consumption of antioxidant compounds such as vitamin E and glutathione that are present in erythrocyte membrane. And hence, this would open up the opportunity for H2O2 to induce more lipid peroxidation in H2O2-treated erythrocytes. In line with this result, Ravikumar et al. (1999) reported that supplementation of Trigonella foenum-graecum seeds in the diets of alloxan diabetic rats significantly lowered blood vitamin E content compared with those control animals which were fed commercial rat chow. Ferula hermonis, Hibiscus sabdariffa, Nigella sativa and Teucrium polium inhibited the MDA production in a concentration dependent manner in H2O2-treated erythrocytes, with the Ferula hermonis being the most potent (Table 2). These observations are consistent with those reported for the effects of Hibiscus sabdariffa and its constituent protocatechuic acid and the fixed oil of Nigella sativa and its constituent thymoquinone on lipid peroxidation of rat primary hepatocytes and OX brain phospholipids liposomes, respectively (Houghton et al., 1995; Tseng et al., 1996; Tseng et al., 1997). Furthermore our results with Nigella sativa are also in line with the results of Burits and Bucar (2000) who found that the essential oil of Nigella sativa and its constituents thymoquinone, carvacrol, t-anethole, 4-terpinol possessed variable antioxidant activity when tested for non-specific hydrogen atom or electron donating property, these agents were also found to have an effective hydroxyl radical scavenging activity when tested for non-enzymatic lipid peroxidation in liposomes. From these reports it seems likely that the strong anti-protein-oxidant activity of Nigella sativa extract, observed for the first time in the present study, was due to the richness of this plant with many radical scavenging compounds such as the ones mentioned above and others such as 2-(2-methoxypropyl)-5methyl-1,4-benzenediol, thymol, C20:2 unsaturated fatty acids, which may be acting synergetically (Houghton et al., 1995; Ghosheh et al. 1999; Burits and Bucar 2000; Enomoto et al., 2001). Allium sativum decreased significantly the MDA production with increasing the original extract concentration (Table 2). This protective Phytother. Res. 18, 280–284 (2004)
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action of Allium sativum may be attributed to the organosulfur compounds, particularly to allicin (thio-2propene-1-sulfinic acid S-allyl ester), which were found to inhibit lipid peroxidation and possess anti-oxidant and radical scavenging activities (Prasad et al., 1996; Rabinkov et al., 1998). The osmotic fragility test is a commonly used technique to detect changes in the erythrocyte content, shape and membrane flexibility. Regidification of the erythrocyte membrane (i.e. decreased membrane flexibility) is therefore supposed to be responsible for the increased osmotic fragility of oxidant exposed erythrocytes in this study (Fig. 1). It also supposed to be responsible for the loss of deformability in these cells (Table 2), since the flexibility of cell membrane is also considered to be one of the determinant factors of erythrocyte deformability (Stuart and Nash, 1990). The failure of Ferula hermonis to diminish either protein degradation or loss of deformability, may explain the inability of this extract to preserve the osmotic fragility of H2O2-treated erythrocytes (Fig. 1A). From this result it could be concluded that lipid peroxidation is not a determinant factor of erythrocyte osmotic fragility, since Ferula hermonis extract protected, to a high level, H2O2-treated erythrocytes against lipid peroxidation
(Table 2). However, preincubation of erythrocytes with Nigella sativa or Allium sativum decreased significantly the osmotic fragility of oxidant exposed erythrocytes (Fig. 1B), presumably this is the result of their capabilities to inhibit protein degradation, indicating that the osmotic fragility is consistent with protein degradation. The results of the present study showed a strong agreement between protein degradation, loss of deformability and increased osmotic fragility. The marked efficiency of Nigella sativa and Allium sativum in protecting erythrocytes against protein degradation, lipid peroxidation, loss of deformability and increased osmotic fragility may offer new possibilities in the therapy of pathological conditions related to free radicals, where, besides the classical therapeutic intervention, selected medicinal plants with anti-protein-oxidant and anti-lipidperoxidant activities can be used as adjuvant therapy (Halliwell et al., 1992).
Acknowledgements The authors are grateful to the University of Jordan for supporting the MSc thesis of S. M. Suboh.
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