Antioxidant Activity of Saponins Isolated from Ivy: α-Hederin, Hederasaponin-C, Hederacolchiside-E and Hederacolchiside-F

• Abstract
• Materials and Methods
• References

Abstract

The antioxidant activities of α-hederin and hederasaponin-C from Hedera helix, and hederacolchisides-E and -F from Hedera colchica were investigated, in this study. The antioxidant properties of the saponins were evaluated using different antioxidant tests: 1,1-diphenyl-2-picryl-hydrazyl (DPPH·) free radical scavenging, total antioxidant activity, reducing power, superoxide anion radical scavenging, hydrogen peroxide scavenging, and metal chelating activities. α-Hederin, hederasaponin-C, as well as hederacolchisides-E and -F exhibited a strong total antioxidant activity. At the concentration of 75 μg/mL, these saponins showed 94, 86, 88 and 75 % inhibition on lipid peroxidation of linoleic acid emulsion, respectively. These various antioxidant activities were compared with model antioxidants such as α-tocopherol, butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT).

Many antioxidant compounds, naturally occurring in plant sources, have been identified as free radical or active oxygen scavengers. Recently, interest has considerably increased in finding naturally occurring antioxidants for use in foods or medicinal materials to replace synthetic antioxidants, which are being restricted due to their side effects such as carcinogenecity. Natural antioxidants can protect the human body from free radicals and retard the progress of many chronic diseases as well as lipid oxidative rancidity in foods [1]. Hence, studies on natural antioxidants have increasingly gained importance.

Fresh leaves and fruits of Hedera helix L. (Araliaceae) (ivy) were reported to be toxic, i. e., to cause gastrointestinal irritation, bloody diarrhoea and death. The plant itself can also cause contact dermatitis. Additionally, antibacterial, antihelmintic, leishmanicidic, antitussive, antispasmodic and antifungal effects of Hedera helix extracts have been reported [2]. On the other hand, Hedera colchica is a less known member of this family and only antifungal and antiprotozoal activities were investigated up to now. Also, hederacolchiside A1 was tested against proliferation of human carcinoma and melanoma, recently [2]. α-Hederin, known to deplete intracelular GSH and protein thiols, leads to increased production of reactive oxygen species in vitro [3]. It stimulates nitric oxide release and is able to upregulate nitric oxide synthase expression through NF-kappa B transactivation [4]. Also, α-hederin has protective effects against H₂O₂-mediated DNA damage by scavenging free radicals or by enhancing catalase activity [5]. In addition, it was reported that it decreases the expression and activities of P450 2E1 enzyme, reduces biotransformation of CCl₄ and diminishes CCl₄-induced liver injury [6]. It seems that α-hederin enhances some non-enzymatic antioxidant components in the liver that may account for its protective role against chemical-induced hepatotoxicity [7].

In the test with DPPH, the antioxidants were able to reduce the stable radical DPPH to the yellow-coloured diphenyl-picrylhydrazine. Table [1] illustrates a significant (P < 0.05) decrease in the concentration of DPPH radical due to the scavenging ability of the extracts of saponins and standards. We used BHA, BHT and α-tocopherol as reference radical scavengers. The scavenging effect of saponins and standards on the DPPH radical decreased in the following order: BHA > α-tocopherol > BHT > α-hederin > hederacolchiside-E > hederasaponin-C, which were 79, 78, 76, 54, 51, 46, and 39 %, at the concentration of 75 μg/mL, respectively. The scavenging activity of DPPH increasing with increasing concentration.

The total antioxidant activity of saponins was determined by the thiocyanate method. The four compounds isolated from ivy exhibited effective antioxidant activity. The effects of α-hederin, hederasaponin-C, hederacolchiside-E and hederacolchiside-F at 75 μg/mL concentration on peroxidation of linoleic acid emulsion are shown in Table [1]. The percentage inhibitions caused by these compounds in the linoleic acid system were 94, 86, 88, and 75 %, respectively, and greater than α-tocopherol (67 %), but close to BHA (90 %) and BHT (95 %) at the same concentration (P > 0.05).

Like the antioxidant activity, at the same concentrations, the reducing power of the saponins increased with increasing concentrations of the compounds. At different concentrations, α-hederin, hederasaponin-C, hederacolchiside-E and hederacolchiside-F showed higher activities than control (P < 0.05). The reducing power of saponins and positive standards exhibited the following order: BHA > BHT > α-tocopherol > α-hederin > hederasaponin-C > hederacolchiside-E > hederacolchiside-F.

All saponins tested had strong superoxide radical scavenging activity. The inhibition of superoxide radical generation by saponins and standards was found to be similar. The inhibition percentages of superoxide generation by 75 μg/mL concentrations of α-hederin, hederasaponin-C, hederacolchiside-E and hederacolchiside-F were found to be as follows: 74, 82, 81 and 82 %. On the other hand, at the same concentration, BHA, BHT and α-tocopherol exhibited 70, 82 and 75 % superoxide radical scavenging activity, respectively (Table [1]).

The formation of the Fe²⁺-ferrozine complex is not complete in the presence of saponins, indicating that saponins chelate the iron. The absorbance of the Fe²⁺-ferrozine complex was linearly decreased, concentration dependently (from 25 to 75 μg/mL). Controls contained Fe²⁺ and ferrozine, as complex forming molecules. The difference between saponins and the control was statistically significant (p < 0.01). The percentages of metal scavenging capacity of 75 μg/mL concentrations of α-hederin, hederasaponin-C, hederacolchiside-E and hederacolchiside-F, α-tocopherol, BHA, and BHT were found to be 73, 71, 53, 62, 75, 69 and 66 %, respectively. The iron scavenging effect of these samples decreased in the order of α-tocopherol > α-hederin > hederasaponin-C >BHA > BHT > hederacolchiside-F > hederacolchiside-E.

The ability of saponins to scavenge H₂O₂ is shown in Table [1] and compared with that of the reference compounds. The saponins were capable of scavenging H₂O₂ in a concentration-dependent manner; 75 μg/mL of α-hederin, hederasaponin-C, hederacolchiside-E and hederacolchiside-F exhibited 83, 63, 91, and 56 % scavenging activity on H₂O₂, respectively. On the other hand, BHA, BHT, and α-tocopherol exhibited 88, 97, and 93 % H₂O₂ scavenging activity at the same concentration. These results showed that all saponins had stronger H₂O₂ scavenging activity. There was statistically significant differences between these values and control (P < 0.01). Controls consisted of H₂O₂ and buffer solutions. The percentage of each antioxidant activity was calculated by the following equation: % Inhibition = [(A_o - A₁)/A_o] × 100, where A_o was the absorbance of the control and A₁ was the absorbance in the presence of the sample.

As a result, the present work indicates that α-hederin, hederasaponin-C, hederacolchiside-E and hederacolchiside-F showed effective antioxidant activities in vitro when compared to different reference antioxidants such as BHA, BHT, and α-tocopherol. These saponins can be used as accessible sources of natural antioxidants.

Materials and Methods

Chemicals: 1,1-Diphenyl-2-picrylhydrazyl (DPPH·), 3-(2-pyridyl)-5,6-bis(4-phenylsulfonic acid)-1,2,4-triazine (Ferrozine), nicotinamide adenine dinucleotide (NADH), nitroblue tetrazolium (NBT), N-methylphenazonium methyl sulfate (PMS), butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) were obtained from Sigma (Sigma-Aldrich GmbH, Sternheim, Germany). All other chemicals used were analytical grade and obtained from either Sigma-Aldrich or Merck (Darmstadt, Germany).

Plant materials, isolation and identification of saponins: Plant material, purification procedure of α-hederin, hederasaponin-C from Hedera helix, and hederacolchisides-E and -F from Hedera colchica have been described in previous reports [8], [9]. The following optical rotations were found for α-hederin: + 14.5° (MeOH), hederasaponin-C: + 78.8 (EtOH), hederacolchiside-E: -22.28 (MeOH), hederacolchiside-F: 0 (MeOH) at 25 °C. Purities of the isolated compounds were determined by HPLC: α-hederin and hederasaponin-C have 99 % purity, while hederacolchiside-E and hederacolchiside-F have 98 % of purity. The qualitative and quantitative analyses of saponins were carried out using HPLC (Waters): μ Bondapak C-18, 10 μm, column size 3.9 × 300 mm, (Pomp 510, photodiode array detector 996), Millenium 32, version 3.05 as a software [4].

Antioxidant assays: The 1,1-diphenyl-2-picrylhydrazyl free radical (DPPH) scavenging activities of α-hederin, hederasaponin-C, hederacolchiside-E and hederacolchiside-F were measured by the method proposed by Blois [10]. The radical scavenging effect of antioxidants on DPPH· was thought to be due to their hydrogen donating ability. DPPH· is a stable free radical and accepts an electron or hydrogen radical to become a stable diamagnetic molecule. The total antioxidant activities of α-hederin, hederasaponin-C, hederacolchiside-E and hederacolchiside-F were determined according to the thiocyanate method. In this method, peroxide formation occurred during the oxidation of linoleic acid and these compounds oxidized Fe²⁺ to Fe³⁺. The latter form complexes with SCN^- and this complex has a maximum absorbance at 500 nm. The reducing power of the saponins was determined by the method of Oyaizu [11]. The principle of this method is the determination of Fe³⁺-Fe²⁺ transformation ability in the presence of antioxidant compounds. Measurement of superoxide anion scavenging activity was based on the method described by Liu [12]. Superoxide anion derived from dissolved oxygen by PMS-NADH coupling reaction reduces NBT. The decrease of the absorbance at 560 nm in the reaction mixture with antioxidants indicates the consumption of superoxide anions. The chelating ability of ferrous ions by saponins and standards was estimated by the method of Dinis. The ability of the saponin to scavenge H₂O₂ was determined according to the method of Ruch. These different antioxidant assays were described in our previous studies [13], [14].

Statistical analysis: All analyses were performed in triplicate. The data were recorded as mean ± SD and analysed by SPSS (version 9.0 for Windows 98, SPSS Inc.). One-way analysis of variance was performed by ANOVA. Significant differences between means were determined by Duncan’s Multiple Range tests. The data presented in percentage were arranged by the correct mathematical transformation (Arcsin) before ANOVA [14].

References

• 1 Gülçin İ, Oktay M, Kireçci E, Küfreviogõlu Ö İ. Screening of antioxidant and antimicrobial activities of anise (Pimpinella anisum L.) seed extracts.  Food Chem. 2003;  83 371-82
  CrossrefPubMedSearch in Google Scholar
• 2 Süleyman H, Mshvildadze V, Gepdiremen A, Elias R. Acute and chronic anti-inflamatory profile of the ivy plant, Hedera helix, in rats.  Phytomedicine. 2003;  10 370-4
  CrossrefPubMedSearch in Google Scholar
• 3 Swamy S M, Huat B T. Intracellular glutathione depletion and reactive oxygen species generation are important in α-hederin-induced apoptosis of P388 cells.  Mol Cell Biochem. 2003;  245 127-39
  CrossrefPubMedSearch in Google Scholar
• 4 Jeonga H G, Choi C Y. Expression of inducible nitric oxide synthase by α-hederin in macrophages.  Planta Medica. 2002;  68 392-6
  Thieme ConnectPubMedSearch in Google Scholar
• 5 Mba Gachou C, Laget M, Guiraud-Dauriac H, De Meo M, Elias R, Dumenil Mutation G. The protective activity of α-hederine against H₂O₂ genotoxicity in HepG2 cells by alkaline comet assay.  Research. 1999;  445 9-20
  PubMedSearch in Google Scholar
• 6 Jeonga H G, Lee S S. Suppressive effects of α-hederin on 2,3,7,8-tetrachlorodibenzo-p-dioxin-mediated murine Cyp1a-1 expression in the mouse hepatoma Hepa-1c1c7 cells.  Cancer Lett. 1999;  138 131-7
  CrossrefPubMedSearch in Google Scholar
• 7 Liu Y P, Liu J. Effect of α-hederin on hepatic detoxifying systems in mice.  Zhongguo Yao Li Xue Bao. 1997;  18 33-6
  PubMedSearch in Google Scholar
• 8 Mshvildadze V ., Elias R ., Baghdikian B ., Ollivier E ., Dekanosidze G ., Kemertelidze E ., Balansard G. Qualitative and quantitative HPLC of dry extracts from the leaves of Hedera colchica and H. pastuchowii. 5th International Symposium on the Chemistry of Natural compounds. May 20 - 23, 2003 Tashkent, Uzbekistan;
  Search in Google Scholar
• 9 Elias R, Diaz-Lanza A M, Vidal-Ollivier E, Balansard G, Faure R, Babadjamin A. Triterpenoid saponins from the leaves Hedera helix .  J Nat Prod. 1991;  54 98-103
  CrossrefPubMedSearch in Google Scholar
• 10 Blois M S. Antioxidant determinations by the use of a stable free radical.  Nature. 1958;  26 1199-200
  PubMedSearch in Google Scholar
• 11 Oyaizu M. Studies on product of browning reaction prepared from glucose amine.  Jpn J Nutr. 1986;  44 307-15
  CrossrefPubMedSearch in Google Scholar
• 12 Liu F, Ooi V EC, Chang S T. Free radical scavenging activity of mushroom polysaccharide extracts.  Life Sci. 1997;  60 763 - 71
  PubMedSearch in Google Scholar
• 13 Gülçin I, Büyükokurogõlu M E, Oktay M, Küfreviogõlu Ö İ. Antioxidant and analgesic activities of turpentine of Pinus nigra Arn. subsp. pallsiana (Lamb.) Holmboe.  J Ethnopharmcol. 2003;  86 51-8
  PubMedSearch in Google Scholar
• 14 Gülçin İ, Beydemir Ş, Alici H A, Elmastaş M, Büyükokurogõlu M E. In vitro antioxidant properties of morphine.  Pharmacol Res. 2003;  49 59-66
  PubMedSearch in Google Scholar

Dr. Ilhami Gülçin

Atatürk University

Science and Arts Faculty

Department of Chemistry

25240 Erzurum

Turkey

Phone: +90-442-2314-444

Fax: +90-442-2360-948

Email: igulcin@atauni.edu.tr

References

• 1 Gülçin İ, Oktay M, Kireçci E, Küfreviogõlu Ö İ. Screening of antioxidant and antimicrobial activities of anise (Pimpinella anisum L.) seed extracts.  Food Chem. 2003;  83 371-82
  CrossrefPubMedSearch in Google Scholar
• 2 Süleyman H, Mshvildadze V, Gepdiremen A, Elias R. Acute and chronic anti-inflamatory profile of the ivy plant, Hedera helix, in rats.  Phytomedicine. 2003;  10 370-4
  CrossrefPubMedSearch in Google Scholar
• 3 Swamy S M, Huat B T. Intracellular glutathione depletion and reactive oxygen species generation are important in α-hederin-induced apoptosis of P388 cells.  Mol Cell Biochem. 2003;  245 127-39
  CrossrefPubMedSearch in Google Scholar
• 4 Jeonga H G, Choi C Y. Expression of inducible nitric oxide synthase by α-hederin in macrophages.  Planta Medica. 2002;  68 392-6
  Thieme ConnectPubMedSearch in Google Scholar
• 5 Mba Gachou C, Laget M, Guiraud-Dauriac H, De Meo M, Elias R, Dumenil Mutation G. The protective activity of α-hederine against H₂O₂ genotoxicity in HepG2 cells by alkaline comet assay.  Research. 1999;  445 9-20
  PubMedSearch in Google Scholar
• 6 Jeonga H G, Lee S S. Suppressive effects of α-hederin on 2,3,7,8-tetrachlorodibenzo-p-dioxin-mediated murine Cyp1a-1 expression in the mouse hepatoma Hepa-1c1c7 cells.  Cancer Lett. 1999;  138 131-7
  CrossrefPubMedSearch in Google Scholar
• 7 Liu Y P, Liu J. Effect of α-hederin on hepatic detoxifying systems in mice.  Zhongguo Yao Li Xue Bao. 1997;  18 33-6
  PubMedSearch in Google Scholar
• 8 Mshvildadze V ., Elias R ., Baghdikian B ., Ollivier E ., Dekanosidze G ., Kemertelidze E ., Balansard G. Qualitative and quantitative HPLC of dry extracts from the leaves of Hedera colchica and H. pastuchowii. 5th International Symposium on the Chemistry of Natural compounds. May 20 - 23, 2003 Tashkent, Uzbekistan;
  Search in Google Scholar
• 9 Elias R, Diaz-Lanza A M, Vidal-Ollivier E, Balansard G, Faure R, Babadjamin A. Triterpenoid saponins from the leaves Hedera helix .  J Nat Prod. 1991;  54 98-103
  CrossrefPubMedSearch in Google Scholar
• 10 Blois M S. Antioxidant determinations by the use of a stable free radical.  Nature. 1958;  26 1199-200
  PubMedSearch in Google Scholar
• 11 Oyaizu M. Studies on product of browning reaction prepared from glucose amine.  Jpn J Nutr. 1986;  44 307-15
  CrossrefPubMedSearch in Google Scholar
• 12 Liu F, Ooi V EC, Chang S T. Free radical scavenging activity of mushroom polysaccharide extracts.  Life Sci. 1997;  60 763 - 71
  PubMedSearch in Google Scholar
• 13 Gülçin I, Büyükokurogõlu M E, Oktay M, Küfreviogõlu Ö İ. Antioxidant and analgesic activities of turpentine of Pinus nigra Arn. subsp. pallsiana (Lamb.) Holmboe.  J Ethnopharmcol. 2003;  86 51-8
  PubMedSearch in Google Scholar
• 14 Gülçin İ, Beydemir Ş, Alici H A, Elmastaş M, Büyükokurogõlu M E. In vitro antioxidant properties of morphine.  Pharmacol Res. 2003;  49 59-66
  PubMedSearch in Google Scholar

Dr. Ilhami Gülçin

Atatürk University

Science and Arts Faculty

Department of Chemistry

25240 Erzurum

Turkey

Phone: +90-442-2314-444

Fax: +90-442-2360-948

Email: igulcin@atauni.edu.tr