In Vivo Antioxidant Action of a Lignan-Enriched Extract of Schisandra Fruit and an Anthraquinone-Containing Extract of Polygonum Root in Comparison with Schisandrin B and Emodin

• Abstract
• Abbreviations
• Introduction
• Materials and Methods
• Chemicals
• Preparation of herbal extracts
• Animal treatment
• Biochemical analysis
• Statistical analysis
• Results
• Discussion
• References

Abstract

The in vivo antioxidant action of a lignan-enriched extract of the fruit of Schisandra chinensis (FS) and an anthraquinone-containing extract of the root of Polygonum multiflorum (PME) was compared with their respective active constituents schisandrin B (Sch B) and emodin by examining their effect on hepatic mitochondrial glutathione antioxidant status in control and carbon tetrachloride (CCl₄)-intoxicated mice. FS and PME pretreatments produced a dose-dependent protection against CCl₄ hepatotoxicity, with the effect of FS being more potent. Pretreatment with Sch B, emodin or α-tocopherol (α-Toc) also protected against CCl₄ hepatotoxicity, with the effect of Sch B being more potent. The extent of hepatoprotection afforded by FS/Sch B and PME/emodin pretreatment against CCl₄ toxicity was found to correlate well with the degree of enhancement in hepatic mitochondrial glutathione antioxidant status, as evidenced by increases in reduced glutathione level and activities of glutathione reductase, glutathione peroxidase as well as glutathione S-transferases, in both control and CCl₄-intoxicated mice. α-Toc, which did not enhance mitochondrial glutathione antioxidant status, seemed to be less potent in protecting against CCl₄ hepatotoxicity. The ensemble of results indicates that FS/PME produced a more potent in vivo antioxidant action than α-Toc by virtue of their ability to enhance hepatic mitochondrial glutathione antioxidant status and that the differential potency of FS and PME can be attributed to the difference in in vivo antioxidant potential between Sch B and emodin.

Abbreviations

ALT:alanine aminotransferases

CCl₄:carbon tetrachloride

FS:lignan-enriched extract of Schisandra fruit

GRD:glutathione reductase

GSH:reduced glutathione

GSH-Px:Se-glutathione peroxidase

GST:glutathione S-transferases

mt:mitochondrial

MDA:malondialdehyde

PME:anthraquinone-containing fraction of Polygonum root

Sch B:schisandrin B

SDH:sorbitol dehydrogenase

α-Toc:α-tocopherol

Introduction

The ”Free Radical Theory of Aging” provides a practical approach for understanding the aging process as well as exploring effective ”anti-aging” agents [1]. Free radical scavenging antioxidants such as α-tocopherol (α-Toc) and plant-derived polyphenolics have been implicated in pharmacological interventions aiming at retarding the aging process and reducing the risk of aging-related degenerative diseases [2]. In this regard, a number of commonly used Chinese tonifying herbs, including the fruit of Schisandra chinensis (Turcz.) Baill. (Schisandraceae) and the root of Polygonum multiforum Thunb. (Polygonaceae), have been shown to possess potent superoxide radical and hydroxyl radical scavenging activities in vitro [3]. Previous studies in our laboratory have demonstrated that a lignan-enriched extract of Schisandra fruit (FS) and an anthraquinone-containing extract of Polygonum root (PME) produced in vivo antioxidant action in protecting against carbon tetrachloride (CCl₄) hepatotoxicity [4], [5] and myocardial ischemia-reperfusion injury [6], [7] in rodents. Schisandrin B (Sch B, Fig. [1] a) and emodin (Fig. [1] b), which are active constitutents isolated from FS and PME, respectively, were found to enhance hepatic/myocardial glutathione antioxidant status [7], [8]. Mitochondrion possesses its own glutathione antioxidant system, which comprises mainly reduced glutathione (GSH), Se-glutathione peroxidase (GSH-Px), glutathione S-transferases (GST), and glutathione reductase (GRD). In essence, the GRD-catalyzed regeneration of GSH from its oxidized form can sustain the GSH-dependent free radical scavenging activity of GSH-Px and GST in decomposing hydrogen peroxide or other organic hydroperoxides [10]. Recent studies have revealed the crucial antioxidant action of Sch B in enhancing hepatic mitochondrial glutathione redox status [9]. However, whether or not the in vivo antioxidant action of FS/PME can be attributed to the antioxidant properties of Sch B/emodin has yet to be determined. In the present study, we aimed to compare the in vivo antioxidant action of FS/Sch B and PME/emodin by examining their effect on hepatic mitochondrial glutathione antioxidant status in control and CCl₄-intoxicated mice. α-Toc, a lipid-soluble free radical scavenging antioxidant, was used as a positive control.

Materials and Methods

Chemicals

Assay kits for measuring plasma alanine aminotransferases (ALT) and sorbitol dehydrogenase (SDH) activities, emodin, (±)-α-Toc and 1,1,3,3-tetramethoxypropane were purchased from Sigma Chemical Co. (St. Louis, MO, USA). The purity of emodin and (±)-α-Toc are ≥ 90 % and 95 %, respectively. Sch B was purified from the petroleum ether extract of Schisandra fruit, with its chemical structure being confirmed by comparing the TLC and spectral characteristics with an authentic standard as described in [9]. The purity of Sch B, as determined by HPLC analysis, was higher than 95 %. All other chemicals were of analytical grade. Solvents used for HPLC were filtered and degassed prior to use.

Preparation of herbal extracts

Dried Schisandra fruit and Polygonum root were imported from mainland China. They were authenticated and supplied by a commercial dealer (Lee Hoong Kee Limited) in Hong Kong. Voucher specimens (FS9507 and PM9612) have been deposited in the Department of Biochemistry, Hong Kong University of Science & Technology (HKUST), Hong Kong, China. A lignan-enriched extract of Schisandra fruit (FS), which was shown to protect against CCl₄ hepatotoxicity [4], was prepared by petroleum ether extraction of the powdered herb, as described in [4], at a yield of 13 % (w/w). Activity-directed fractionation of FS enabled to obtain lignan-containing antioxidant-active fractions at a total yield of ˜30 % [4], and HPLC analysis of FS (Waters μ-Bondpak C₁₈ 300 mm × 3.9 mm i. d. column, eluted with 80 % methanol [v/v in Mini-Q water), flow rate: 1 ml/min, detected by UV 220 nm] indicated the presence of Sch B at 38 mg/g. Activity-directed fractionation of Polygonum root has shown the presence of antioxidant activity in the ethyl acetate fraction [5]. Granulated Polygonum root (300 g) was extracted with 1.2 l of ethyl acetate at 77 °C for 2 h. The extraction was repeated twice. The pooled extract was dried under reduced pressure by rotavaporation to obtain PME extract at a yield of 1.3 % (w/w). After chromatographic fractionation of PME, the anthraquinone-containing fractions were obtained at a total yield of ∼20 % (unpublished data), and HPLC analysis of PME indicated the presence of emodin at 2.6 mg/g [7].

Animal treatment

Animals were supplied by the Animal Care Facilities at HKUST; they were maintained on a 12-h dark/light cycle at about 22 °C and allowed food and water ad libitum. Experimental protocols have been approved by the University Committee on Research Practice in HKUST. Female Balb/c mice (20 - 24 g) were randomly divided into groups of 5 animals in each. For the drug pretreatment groups, mice were administered orally with increasing daily doses of FS or PME ranging from 0.5 to 1.5 g/kg b. w. for three days. Sch B and emodin were administered at a daily dose of 1 mmol/kg b. w. (i. e., 400 mg/kg and 280 mg/kg, respectively) for three days. The doses have been adjusted to the total amount of lignan-containing antioxidant-active fraction (∼450 mg) present in 1.5 g FS and anthraquinone-containing fraction (∼300 mg) present in 1.5 g PME, respectively. α-Toc was administered at a daily dose of 6 mmol/kg (∼2.5 g/kg) for three days to produce hepatoprotection against CCl₄ toxicity to the extent comparable to those of the herbal extracts. All drugs were suspended/dissolved in olive oil. Control animals were given the olive oil only. For the CCl₄-treated groups, twenty-four hours after the last dosing of drug, mice were administered orally with CCl₄ (1 % in olive oil, v/v) at 0.1 ml/kg. Control animals were given the vehicle (olive oil, 10 ml/kg) only. Twenty-four hours after the challenge, heparinized blood sample was drawn from ether-anesthetized animal by cardiac puncture, and the animal was sacrificed thereafter. Liver tissue sample was taken and subjected to biochemical analyses as described below.

Biochemical analysis

Plasma sample was obtained by centrifuging the whole heparinized blood at 2000 g at 4 °C for 10 min, and ALT and SDH activities were measured as described in [11]. Liver tissue sample was rinsed with ice-cold isotonic buffer (0.25 mM sucrose, 0.1 mM EDTA, 50 mM Tris/HCl, pH 7.4) and used for preparing mitochondrial fraction as described in [9]. The mitochondrial pellet was resuspended in 1 ml of homogenizing buffer (50 mM Tris, 0.1 mM EDTA, pH 7.6). Aliquots (500 μl and 250 μl) of the mitochondrial fraction were taken for measuring mitochondrial reduced glutathione (mtGSH) and malondialdehyde (mtMDA) levels, respectively, by HPLC methods using GSH and acid hydrolyzed 1,1,3,3-tetramethoxypropane as standards, as described in [9]. An aliquot (100 μl) of mitochondrial fraction was mixed with 2.8 ml Triton X-100 solution [0.3 %, (v/v) in homogenizing buffer] and sonicated for 2 min on ice. The mixture was then subjected to the measurement of mitochondrial GRD (mtGRD), GSH-Px (mtGSH-Px) and GST (mtGST) activities by spectrophotometric methods, as described [8]. Protein concentration of the mitochondrial fraction was determined using Bio-Rad protein assay kit.

Statistical analysis

Data were analyzed by one-way analysis of variance followed by Duncan’s multiple range test to detect inter-group differences. Significant difference was determined when p < 0.05.

Results

As shown in Table [1], treating mice with FS or PME at a daily dose of 0.5 - 1.5 g/kg for three days produced dose-dependent decreases in plasma ALT and SDH activities as well as hepatic mtMDA level, with the effect of FS being more potent. Sch B (1 mmol/kg × 3), emodin (1 mmol/kg × 3) or α-Toc (6 mmol/kg × 3) treatment also reduced plasma enzyme activities, but only Sch B treatment produced a significant decrease in hepatic mtMDA level in mice. CCl₄ treatment caused hepatocellular damage in mice, as indicated by drastic increases in plasma ALT (567-fold) and SDH (130-fold) activities as well as hepatic mtMDA level (3.3-fold). The same pretreatment regimen of FS and PME produced a dose-dependent protection against CCl₄ hepatotoxicity, as evidenced by significant decreases in plasma enzyme activities and hepatic mtMDA level, with the effect of FS being more potent. Pretreatment with Sch B, emodin or α-Toc also protected against CCl₄ hepatotoxicity, with the effect of Sch B being more potent (Table [1]).

As shown in Fig. [2] a, treating mice with FS or PME caused significant and dose-dependent increase in hepatic mtGSH level, with the value being significantly increased by 120 % and 55 %, respectively, at a dose of 1.5 g/kg. Pretreatments with Sch B and emodin also increased hepatic mtGSH level by 79 % and 44 %, respectively. However, α-Toc treatment did not produce any detectable change in hepatic mtGSH level in mice. Both FS and PME treatments caused dose-dependent increases in hepatic mtGRD, mtGSH-Px and mtGST activities, with the effect of FS being more potent (Fig. [2] b - d). Sch B and emodin treatments also increased hepatic mitochondrial glutathione antioxidant enzyme activities, with the effect of Sch B being more potent (Fig. [2] b - d). In contrast, α-Toc treatment only slightly increased hepatic mitochondrial GST activity (Fig. [2] d). CCl₄ hepatotoxicity was associated with a 50 % decrease in mtGSH level and impairment in mitochondrial glutathione antioxidant enzymes, with activities of mtGRD, mtGSH-Px and mtGST being decreased by 15 - 18 % (Fig. [2] b - d). The same pretreatment regimen of FS and PME caused a dose-dependent increase in hepatic mtGSH level in CCl₄-intoxicated mice, with the value being elevated by 282 % and 77 %, respectively, in FS and PME pretreated animals at a dose of 1.5 g/kg, when compared with the CCl₄ control (Fig. [2] a). FS and PME pretreatments also significantly increased hepatic mitochondrial glutathione antioxidant enzyme activities in CCl₄-intoxicated mice, with the effect of FS being more potent (Fig. [2] b - d). Pretreatment with Sch B or emodin also significantly increased hepatic mtGSH level in CCl₄-intoxicated mice, with the value being elevated by 163 % and 100 %, respectively, when compared with the CCl₄ control (Fig. [2] a). α-Toc treatment produced a slight, but insignificant, increase in hepatic mtGSH level in CCl₄-treated mice. Both Sch B and emodin pretreatments increased hepatic mitochondrial glutathione antioxidant enzyme activities in CCl₄-treated mice, with the effect of Sch B being much more potent (Fig. [2] b - d). α-Toc pretreatment also caused significant increases in activities of hepatic mtGRD and mtGST, but not mtGSH-Px, when compared with the CCl₄ control (Fig. [2] b - d).

Discussion

The in vivo antioxidant action of herbal extracts (FS and PME) together with their active constituents (Sch B and emodin) was assessed by examining the effect on hepatic mitochondrial glutathione antioxidant status in control and CCl₄-intoxicated mice. The lignan-containing antioxidant fractions of FS and the anthraquinone-containing fractions of PME were found to constitute 30 % and 20 %, respectively, by weight of the crude extract. Using HPLC analysis, FS and PME were found to contain 38 mg/g Sch B and 2.6 mg/g emodin, respectively. Experimental findings indicated that the enhancement of hepatic mitochondrial glutathione antioxidant status, as evidenced by increases in mtGSH level and activities of mtGRD, mtGSH-Px and mtGST, by FS/Sch B or PME/emodin treatment was associated with the decrease in hepatic mtMDA level, indicative of decrease in tissue oxidative stress (Table [1]). On the other hand, α-Toc did not enhance hepatic mitochondrial glutathione antioxidant status or decrease mtMDA level in control mice (Table [1]). The beneficial effect of FS/Sch B and PME/emodin on hepatic mitochondrial glutathione antioxidant status became more apparent after CCl₄ intoxication. It is well established that the pathogenesis of CCl₄ hepatotoxicity involves free radical-mediated processes [12], [13]. CCl₄ can undergo reductive metabolism in the mitochondrion, with resultant formation of toxic oxidant species [14]. This is consistent with our findings that the CCl₄-induced hepatocellular damage was associated with the increase in hepatic mtMDA level, an indirect index of lipid peroxidation and that α-Toc, an inhibitor of lipid peroxidation, could protect against CCl₄ hepatotoxicity (Table [1]). Both FS/Sch B and PME/emodin pretreatments protected against CCl₄ hepatotoxicity, with the effect of FS/Sch B being more potent as assessed by both plasma ALT and SDH activities (Table [1]). The leakage of hepatic housekeeping enzymes such as ALT and SDH is commonly used as an indirect biochemical index of hepatocellular damage. ALT and SDH, while being present in most tissues, vary considerably in concentrations [15] [16]. Presumably, the relatively lower enzyme level of SDH in the liver results in a lower detection sensitivity of hepatocellular damage, as observed in the present study (Table [1]). However, the larger extent of decrease in plasma ALT activity than that of SDH activity by FS pretreatment in CCl₄-intoxicated mice is likely due to the specific suppression of hepatic ALT level by FS treatment (unpublished data). In the present study, the extent of hepatoprotection afforded by FS/Sch B and PME/emodin pretreatment against CCl₄ toxicity should be therefore better compared on the basis of plasma SDH activity.

The CCl₄ hepatotoxicity was associated with the impairment in hepatic mitochondrial glutathione antioxidant status, as evidenced by the decreases in mtGSH level and activities of mtGRD, mtGSH-Px and mtGST (Fig. [2]). Reactive oxidant species arising from CCl₄ metabolism in mitochondria can deplete mtGSH level and inactivate the activity of glutathione antioxidant enzymes [14]. The finding of the ability of α-Toc pretreatment in preventing the CCl₄-induced decreases in mitochondrial glutathione antioxidant enzyme activities may be related to the free radical scavenging of α-Toc (Fig. [2]). Glutathione plays a pivotal role in mitochondrial antioxidant defense, and the depletion of mtGSH was found to increase the sensitivity of hepatic tissue to free radical-mediated damage caused by xenobiotics metabolism [17]. In this regard, CCl₄ hepatotoxicity has been shown to be strongly related to mitochondrial functional changes secondary to alterations in mitochondrial thiols [18]. Because of the GSH-mediated reduction of protein thiols in the mitochondrion is critical for cell survival [19], the maintenance of mitochondrial GSH level is therefore important to protect against xenobiotic-induced hepatic damage. The interplay among mitochondrial glutathione antioxidant enzymes, namely, mtGRD, mtGSH-Px and mtGST, helps to regenerate mtGSH from its oxidized form and remove reactive hydrogen peroxide or lipid hydroperoxides [20], as well as conjugate electrophilic xenobiotics and their reactive intermediates with GSH for excretion [21]. Results obtained from the present study indicated that α-Toc treatment, which could not enhance hepatic mitochondrial glutathione antioxidant status, was less effective than that of FS/Sch B or PME/emodin in protecting against CCl₄ hepatotoxicity (Table [1] and Fig. [2]). The greater degree of hepatoprotection afforded by FS/Sch B, when compared with that of PME/emodin, was paralleled by the larger extent of enhancement in mitochondrial glutathione antioxidant status, particularly in terms of mtGSH level, mtGRD and mtGST activities. Furthermore, the difference in potency of hepatoprotection between FS and PME was found to resemble that between Sch B and emodin, both of which were tested at a dose being adjusted to the total amount of lignan-containing antioxidant-active fractions of FS (30 %, w/w) and anthraquinone-containing fractions of PME (20 %, w/w), respectively (vida infra). Since Sch B and emodin pretreatments at the adjusted dose produced hepatoprotection at a similar degree as compared with those of FS and PME at 1.5 g/kg, respectively, the results suggest that the in vivo antioxidant capacity of FS and PME can be accounted for by their respective active lignan and anthraquinone constituents. On the other hand, the laxative effect of high doses of anthraquinone-containing PME and emodin [22], as also observed in the present study, will limit their use as antioxidant. However, synergistic interactions among anthraquinones and other herbal constituents, as in the case of multi-component herbal formula [23], may greatly reduce the effective dose for producing antioxidant action, thereby alleviating the laxative side effect.

In conclusion, the ensemble of results indicates that FS/PME produced a more potent in vivo antioxidant action than α-Toc by virtue of their ability to enhance hepatic mitochondrial glutathione antioxidant status and that the differential potency of FS and PME can be attributed to the difference in in vivo antioxidant potential between Sch B and emodin.

References

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Dr. Robert Ko

Department of Biochemistry

Hong Kong University of Science & Technology

Clear Water Bay, Hong Kong, China

Email: bcrko@ust.hk

Fax: (852) 2358 1552

References

• 1 Harman D. Free-radical theory of aging.  Annual of New York Academy of Sciences. 1994;  717 1-15
  PubMedSearch in Google Scholar
• 2 Ames B N, Shigenaga M K, Hagen T M. Oxidants, antioxidants, and the degenerative diseases of aging.  Proceedings of National Academy of Sciences USA. 1993;  90 7915-22
  CrossrefPubMedSearch in Google Scholar
• 3 Ohsugi M, Fan W, Hase K, Xiong Q, Tezuka Y, Komatsu K. et al . Active-oxygen scavenging activity of traditional nourishing-tonic herbal medicines and active constituents of Rhodiola sacra .  Journal of Ethnopharmacology. 1999;  67 111-9
  CrossrefPubMedSearch in Google Scholar
• 4 Ko K M, Ip S P, Poon M KT, Wu S S, Che C T, Ng K H, Kong Y C. Effect of a lignan-enriched fructus schisandrae extract on hepatic glutathione status in rats: Protection against carbon tetrachloride toxicity.  Planta Medica. 1995;  61 134-7
  Thieme ConnectPubMedSearch in Google Scholar
• 5 Ip S P, Tse S M, Poon M KT, Ko K M. Antioxidant activities of Polygonum multiforum Thunb., in vivo and in vitro .  Phytotherapy Research. 1997;  11 42-4
  CrossrefPubMedSearch in Google Scholar
• 6 Li P C, Mak D HF, Poon M KT, Ip S P, Ko K M. Myocardial protective effect of Sheng Mai San (SMS) and a lignan-enriched extract of fructus schisandrae in vivo and ex vivo .  Phytomedicine. 1996;  III 217-21
  PubMedSearch in Google Scholar
• 7 Yim T K, Wu W K, Mak D HF, Ko K M. Myocardial protective effect of an anthraquinone-containing extract of Polygonum multiflorum ex vivo .  Planta Medica. 1998;  64 607-11
  PubMedSearch in Google Scholar
• 8 Ip S P, Wu S S, Poon M KT, Che C T, Ng K H, Kong Y C, Ko K M. Effect of schisandrin B on hepatic glutathione antioxidant system in mice: Protection against carbon tetrachloride toxicity.  Planta Medica. 1995;  61 398-401
  PubMedSearch in Google Scholar
• 9 Ip S P, Ko K M. The crucial antioxidant action of schisandrin B in protecting against carbon tetrachloride hepatotoxicity in mice. A comparative study with butylated hydroxytoluene.  Biochemical Pharmacology. 1996;  52 1687-93
  CrossrefPubMedSearch in Google Scholar
• 10 Meister A. Glutathione metabolism and its selective modification.  Journal of Biological Chemistry. 1988;  263 17 205-8
  PubMedSearch in Google Scholar
• 11 Ip S P, Yiu H Y, Ko K M. Differential effect of schisandrin B and dimethyl diphenyl bicarboxylate (DDB) on hepatic mitochondrial glutathione redox status in carbon tetrachloride intoxicated mice.  Molecular and Cellular Biochemistry. 2000;  205 111-4
  CrossrefPubMedSearch in Google Scholar
• 12 Wolf C R, Harrelson WG J r, Nastainczyk W M, Philpot R , Kalyanaraman B, Mason R P. Metabolism of carbon tetrachloride in hepatic microsomes and reconstituted monoxygenase systems and its relationship to lipid peroxidation.  Molecular Pharmacology. 1980;  18 553-8
  PubMedSearch in Google Scholar
• 13 Yamamoto H, Nagano T, Hirobe M. Carbon tetrachloride toxicity on Escherichia coli exacerbated by superoxide.  Journal of Biological. Chemistry1988;  263 12 224-7
  PubMedSearch in Google Scholar
• 14 Tomasi A, Albano E, Banni S, Botti B, Corongiu F, Dessi M A, Iannone A. et al . Free-radical metabolism of carbon tetrachloride in rat liver mitochondria.  Biochemical Journal. 1987;  246 313-7
  PubMedSearch in Google Scholar
• 15 Zilva J F, Pannall P R, Mayne P D. 15. Plasma Enzyme in Diagnosis. Clinical Chemistry in Diagnosis and Treatment 5^th Edition. London, Melbourne, Auckland; Edward Arnold 1988: 307-22
  Search in Google Scholar
• 16 Kirchain W R, Rendall M S. Aldose reductase inhibitors.  Pharmacotherapy. 1990;  10 326-36
  PubMedSearch in Google Scholar
• 17 Shan X, Jones D P, Hashmi M, Anders M W. Selective depletion of mitochondrial glutathione concentrations by (R,S)-3-hydroxy-4-pentenoate potentiates oxidative cell death.  Chemical Research in Toxicology. 1993;  6 75-81
  CrossrefPubMedSearch in Google Scholar
• 18 Pascoe G A, Olafsdottir K, Read D J. Vitamin E protection against chemical-induced cell injury. I. Maintenance of cellular protein thiols as a protective mechanism.  Archives of Biochemistry and Biophysics. 1987;  256 150-8
  PubMedSearch in Google Scholar
• 19 Brattin W J, Walker R L. Effect of halomethanes on intracellular calcium distribution in hepatocytes.  Life Sciences. 1984;  35 1231-40
  CrossrefPubMedSearch in Google Scholar
• 20 Griffith O W, Meister A. Origin and turnover of mitochondrial glutathione.  Proceedings of the National Academy of Sciences USA. 1985;  82 4468-72
  PubMedSearch in Google Scholar
• 21 Morgenstern R, Meijer J, Depierre J W, Ernster L. Characterization of rat liver microsomal glutathione S-transferase activity.  European Journal of Biochemistry. 1980;  104 167-74
  CrossrefPubMedSearch in Google Scholar
• 22 Garcia-Villar R, Leng-Peschlow E, Ruckebusch Y. Effect of anthraquinone derivatives on canine and rat intestinal motility.  Journal of Pharmacy & Pharmacology. 1980;  32 323-9
  PubMedSearch in Google Scholar
• 23 Yim T K, Wu W K, Pak W F, Mak D HF, Liang S M, Ko K M. Myocardial protection against ischaemia-reperfusion injury by a Polygonum multiflorum extract supplemented ‘Dang-Gui Decoction for enriching blood’. A compound formulation, ex vivo .  Phytotherapy Research. 2000;  14 195-9
  CrossrefPubMedSearch in Google Scholar

Dr. Robert Ko

Department of Biochemistry

Hong Kong University of Science & Technology

Clear Water Bay, Hong Kong, China

Email: bcrko@ust.hk

Fax: (852) 2358 1552