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Protection of hippocampal slices against hypoxia/hypoglycemia injury by a Gynostemma pentaphyllum extract.

Abstract

In transverse hippcampus slices a short period of hypoxia/hypoglycemia induced by perfusion with [O.sub.2]/glucose-free medium caused early loss and incomplete restoration of evoked held potentials to only 50% in the [CA.sub.1] region. We report about a study investigating the effect of an ethanolic Gynostemma pentaphyllum extract in this system. When given with reperfusion the extract completely protected the cells of the slices from functional injury. The extract also protected at the subcellular level isolated mitochondria which had been subjected to hypoxia/reoxygenation in combination with elevated extramitochondrial [Ca.sup.2+] concentration from functional injury. In isolated mitochondria the extract protected from [Ca.sup.2+] -induced opening of the mitochondrial permeability transition pore and reduced lipid peroxidation. Our data demonstrate that the ethanolic extract of Gynostemma pentaphyllum has a high potential to protect from ischemia/reperfusion injury. It should be beneficial as prophylactic nutrition supplement and during revascularization of arterial blood vessels from stroke and other ischemic events such as coronary occlusion.

[c] 2009 Elsevier GmbH. All rights reserved.

Keywords: Gynostemma pentaphyllum:, Hypoxia/hypoglycemia; .Mitochondria; Mitochondrial permeability transition pore

Introduction

In the course of ischemia/reperfusion several cellular processes are initiated that mediate functional injury and cell death in the infarct area. Among them are calcium overload (Murphy and and Steenbergen 2008), oxidative and nitrosive stress (Halestrap et al. 2007), decrease in mitochondrial ATP production (Halestrap et al, 2007), induction of the intrinsic pathway of programmed cell death (Lang and McCuIlough 2008), and membrane permeabilization (Kroemer et al. 2007). Attempts to target cellular processes during reperfusion by applying antioxidants, inhibitors of calcium transport or inhibitors of apoptosis were only partially beneficial (Perlmann 2006). Moreover, treatments targeting such cellular processes did not reach clinical approval. Consequently, the clinical intervention after ischemic stroke or heart attack is actually restricted to enable reperfusion by administering tissue plasminogen activator which stimulates the degradation of fibrin (Stroke Study Group 1995).

It is very unlikely that one single substance can completely protect against cellular injury because of the complex scenario during ischemia/reperfusion. To improve the outcome of the treatment targeting cellular processes, the combination of different substances is a promising strategy. In this context, the application of multi component extracts of special herbs could be an attractive approach.

In fact, complex mixtures derived from herbs had been used in traditional Chinese medicine to treat patients in stroke therapy. More than 100 Chinese medicines had been used for stroke prevention and therapy. The broad spectrum of effects includes antithrombotic and/or thrombolytic activity, improvement of blood circulation, acceleration of blood flow and microcirculation, inhibition of lipid peroxidation, and protection during ischemia/reperfusion. Some of the ingredients and their effects had been characterized. Inhibition of NF-kB signalling, decrease in intracellular [Ca.sup.2 +] concentration by ginsenosides due to increased ATPase activity, and inhibition of free-radical generation by ginsenosides had been demonstrated (for review of these effects see Gong and Sucher 2002). Although it is known that Gynostemma pentaphyllum also contains ginsenosides, this particular herb had not yet been used in stroke therapy.

Gynostemma pentaphyllum is a wild growing plant that had been used in Asian countries in traditional medicine. Main components of extracts from Gynostemma pentaphyllum are as much as 82 different gypenosides, several amino acids and vitamins, and trace elements (Deng et al. 1994). A broad spectrum of beneficial effects had been reported including anti-oxidative activity (Li et al. 1993; Shang et al. 2006), regulation of blood pressure (Tanner et al. 1999), immune regulatory activity (Hou et al. 1991; Huang et al. 2007a, b), adhesion inhibition (Huang et al. 2007a, b), anti allergic activity (Huang et al. 2008), inhibition of the microsomal [Na.sup.+]and [K.sup.+]-ATPase in brain and heart (Han et al. 2007), anti cancer activity (Han et al. 1995; Chen et al. 2008; Lu et al. 2008; Wang et al. 2002), anti-hyperlipidemic and hypoglycemic activity (Megalli et al. 2006), and regulation of nitric oxide metabolism (Aktan et al. 2003; Tanner et al. 1999).

Here we report about the effect of an ethanolic extract from Gynostemma pentaphyllum applied in an ischemia/ reperfusion in vitro model. Therefore, hippocampal slices were treated with the extract after reversible hypoxia/hypoglycemia and evoked field potentials were measured as functional parameter. Additionally, isolated mitochondria were subjected to hypoxia/reoxygenation in combination with elevated [Ca.sup.2+] concentrations in the presence of the Gynostemma pentaphyllum extract and respiration was analysed to document mitochondrial function. Furthermore, the antioxidative potential of the extract against oxidative stress was separately evaluated in a mitochondrial model of iron-ascorbate-induced lipid peroxidation; and the ability of the extract to prevent [Ca.sup.2+]-induced opening of the mitochondrial permeability transition pore was tested by swelling experiments with isolated mitochondria.

Materials and methods

Materials

The standardized Gynostemma pentaphyllum extract powder was obtained by extraction of dried aerial parts with 75% ethanol/25% water and was generously received from Herbasin (Shenyang) Co., Ltd. (China). All chemicals were of analytical grade.

TLC- and HPLC- fingerprint-analysis

TLC-fingerprint analysis (Fig. 1 A)

Extraction: 5 mg of the dried ethanolic extract (75% ethanol/25% water) were dissolved in 1 ml of ethanol and filtered over Millipore filtration unit-type 0.45 [mu]n. Reference compound: 0.5 mg of Ginsenoside Rbl was dissolved in 0.5 ml methanol. Separation parameters: Plate: HPTLC Silica gel 60 F254, (Merck). Applied amount: Gynostemma pentaphyllum extract: 20 (mu)l, reference compound: 10 (mu)l. Solvent system: Chloroform/ acetic acid/methanol/water 60/32/12/8. Detection: ani-saldehyd-sulphuric acid reagent: 0.5 ml anisaldehyd is mixed with 10 ml glacial acetic acid, followed by 85 ml methanol and 5 ml concentrated sulphuric acid, in that order. The TLC-plated is sprayed with about 10 ml, heated at 100[degrees]C for 10 min, then evaluated in VIS. The reagent has only limited stability and is no longer useable when the colour has turned to red-violet.

HPLC-fingerprint analysis (Fig. 1 B)

[FIGURE 1 OMITTED]

Sample preparation: 5 mg of the dried ethanolic extract (75% ethanol/25% water) were dissolved in l ml of ethanol, filtered over Millipore[R] filtration unit, type 0.45 (mu)m, and injected into the HPLC apparatus. Injection volume: Gynostemma pentaphyllum extract: 20.0 (mu)l, HPLC parameter: Apparatus: MERCK HITACHI D-6000 A Interface, MERCK HITACHI L-4500 A Diode Array Detector, MERCK HITACHI AS-2000 Autosampler, MERCK HITACHI L-6200 A Intelligent Pump. Separation column: LiChroCART[R] 250-4 Li-Chrospher[R] 100 RP-18 (5(mu)m) (Merck), Precolumn: LiChroCART[R] 4-4 LiChrospher[R] 100 RP-18 (5 (mu)m) (Merck). Solvent: A: dist. Water (Millipore Ultra Clear UV plus[R] filtered), B: acetonitrile (Fa. VWR). Gradient: 5-100% B in 60 min, total runtime: 60 min. Flow: 0.8 ml/min. Detection: 203 nm.

Animals

The experiments were conducted with 8 weeks old male Wistar rats (Tierzucht Schonwalde, Germany). The animals were kept under controlled laboratory conditions (light regime of 12 h light/12 h dark, temperature 20[+ or -]2[degrees]C, humidity 55-60%). The animals were housed in groups of four animals and had free access to commercial pellets and water. For all experiments, ethical approval was sought prior to the experiments, according to the requirements of the National Act on the Use of Experiments. Animals and the experiments were performed in accordance with the European Communities Directive 86/609/EEC. All possible steps were taken to avoid animals suffering at each stage of the experiments.

Recording of evoked field potentials on hippocampal slices

Preparation of hippocampal slices and recording of evoked field potentials in the CA1 region in response to constant stimulation of Schaffer's collaterals was performed as previously described (Ruthrich and Krug 1999). In brief, 400 [mu]m transverse hippocampal slices maintained submerged in a perfusion chamber with perfusion medium containing (mM) NaCl 124, KCl 4.9, [KH.sub.2][PO.sub.4] 1.2, [MgSO.sub.4] 1.3, [CaCl.sub.2] 2.5,[ NaHCO.sub.3] 25.6, gluclose 10.6, 95% [O.sub.2] and 5% [CO.sub.2] at 32.5[degrees] C. After 2h of equilibration extracellular recording of evoked field potentials (population spike amplitude, mV) was performed in response to constant current stimulation of Schaffer's collaterals with biphasic rectangular pulses of 0.1 ms and input/output (I/O) curves were determined by stimulating with four pulses of increased stimulation intensities/threshold intensity for population spike amplitude multiplied by 1.5, 2 and 2.5). Baseline values of population spike amplitudes after equilibration were set as 100%. Hypoxia/hypoglycemia was induced by perfusion with an [O.sub.2]/glucose-free medium bubbled with 95% [N.sub.2] and 5% [CO.sub.2]. After the population spike amplitude reached zero (usually after 2-3 min), the slices were kept for 7 min in the hypoxic/hypoglycemic state, thereafter they were reperfused with medium containing glucose, 95% [O.sub.2] and 5% [CO.sub.2]. Population spike amplitudes were recorded every 10 min before hypoxia/ hypoglycemia, every minute until population spike amplitude reached zero after initiation of hypoxia/hypoglycemia and at the end of hypoxia/hypoglycemia. During reperfusion, recording was performed every 10-15 min until the end of the experiment. At the end of the experiment, I/O curves were determined again.

Administration of ethanolic Gynostemma pentaphyttum extract

For this experiment, hippocampi of extract-naive rats were prepared. After equilibration, hypoxia/hypoglycemia was achieved by perfusion with [O.sub.2]/glucose-free medium. Afterwards, the extract with DMSO as solvent was added to the perfusion medium at a final concentration of 240 [mu]g/ml as outlined in the legends of the figures.

Isolation of mitochondria

Liver mitochondria were prepared from 220-240 g male Wistar-rats in ice-cold medium containing 250 mM sucrose, 20 mM Tris (pH 7.4), 2mM EGTA, and 1% (w/v) bovine serum albumin using a standard procedure (Johnson and Lardy 1967). After the initial isolation, Percoll was used for purification of mitochondria from a fraction containing some endoplasmatic reticulum, golgi apparatus and plasma membranes (McCormack and Denton 1998).

Incubation of mitochondria

Mitochondria (1-2 mg protein per ml) were incubated in a medium containing l0 mM sucrose, 120 mM KC1, 20 mM Tris, 15 mM potassium phosphate, 0.5 mM EGTA and 1 mM free [Mg.sup.2+] at pH 7.4. When [Ca.sup.2+] was present in the incubations, extramitochondrial [Ca.sup.2+] concentrations were adjusted by using [Ca.sup.2+]/EGTA buffers. After preparation of the buffers, the free [Ca.sup.2+] concentration was checked by means of a [Ca.sup.2+]-selective electrode. The actual concentration of [Ca.sup.2+] in the incubations was calculated considering the complex formation with other constituents of the medium such as [Mg.sup.2+] and adenine nucleotides. For the calculation, the complexing constants were used according to Fabiato et al. (Fabiato and Fabiato 1979). Hypoxia was produced by bubbling 2 ml of incubation medium with [N.sub.2] until an oxygen content of less than 1% of air saturation was reached. Afterwards, mitochondria were added to this oxygen-free medium and the incubation chamber was closed. The mitochondria themselves consumed most of the remaining oxygen resulting in very low oxygen concentrations reflected by collapse of the mitochondrial membrane potential (not shown). Reoxygenation was achieved by adding another 2 ml of incubation medium, which was air saturated, to the incubation chamber (Schild et al. 1997).

Determination of mitochondrial respiration

Oxygen consumption of mitochondria was measured in an incubation chamber equipped with a Clark-type electrode. The experimental approach was calibrated using the oxygen content of air saturated medium of 435 ng-atoms/ml at 30 [degrees]C (Reynafarje et al. 1985).

Measurement of mitochondrial swelling

The absorption of mitochondrial incubations was measured with a Varian spectrophotometer (Cary 1E) at 546 nm. To promote swelling mitochondria were incubated in a hypotonic medium containing 5mM sucrose, 120mM KC1, 15mM NaCl, l0mM Tris, 2mM [MgCl.sub.2], 5 mM [NaH.sub.2][PO.sub.4], and 0.5 mM EGTA (pH 7.4). Calcium-dependent swelling caused by opening of the mitochondrial permeability transition pore was induced by adding 200 [mu]M [CaCl.sub.2]

Iron/ascorbate-induced peroxidation

Peroxidation was initiated in a medium containing 100 mM KC1, 10 mM Tris buffer (pH 7.7) and about 5 mg mitochondrial protein/ml, by 40 [mu]M ferrous sulphate and 500 [mu]M ascorbic acid at 30 [degrees]C in an open air atmosphere. At timed intervals, samples were taken for the immediate determination of malondialdehyde.

Determination of malondialdehyde

Malondialdehyde formation was measured according to the thiobarbituric acid method (Buege and Aust 1978). The colored complex was extracted by pyridine/butanol (1:15) and measured at 532 nm.

Determination of protein

The protein content of the mitochondrial suspension was measured according to the method of Bradford (Bradford 1976) using BSA as the standard.

Statistics

Statistical analysis was performed by the Student's t-test or by one way analysis of variance (ANOVA). Actual p-values are given in the legends of the figures. Data are presented as mean [+ or -] SEM.

Results

Effect of the ethanolic extract from Gynostemma pentaphyllum on evoked field potentials in hippocampal slices during transient hypoxia/hypoglycemia

In rat control slices, a brief hypoxia/hypoglycemia of 7 min duration led to a loss of field potentials, monitored at 1.5 fold threshold stimulation intensity, followed by an impaired recovery (about 50% of baseline) during reperfusion. In contrast, the recovery was improved (complete restoration to 100%) when 240 [mu]g/ml gynostemma extract was administered with reoxygenation (left part in Fig. 2A). The protective effect of the gynostemma extract was also seen in the I/O curve determined at the end of the experiment (right part in Fig. 2A2).

[FIGURE 2 OMITTED]

In contrast, no difference to control slices which were treated with transient hypoxia/hypoglycemia was observed when the gynostemma extract was administered 30 min after reoxygenation (Fig. 2B). At that stage, no protection against hypoxia/hypoglycemia induced functional injury could be achieved.

Effect of the ethanolic extract from Gynostemma pentaphyllum on isolated mitochondria during hypoxia/reoxygenation and elevated [Ca.sup.2+]concentrations

The drop in evoked field potentials after hypoxia/hypoglycemia mirrors injury of individual neuronal cells that may be mediated by the impairment of mitochondria. We therefore treated isolated mitochondria with 10 min hypoxia followed by 5 min reoxygenation alone or in combination with 3 [mu]M extramitochondrial [Ca.sup.2+] and analyzed the effect of the Gynostemma pentaphyllum extract. The rate of active respiration with glutamate plus malate as substrates and 200 [mu]M ADP was determined as parameter for mitochondrial function. In comparison to a normoxic control (set to 100%), hypoxia/reoxygenation caused decrease of active respiration to 65% of control. This moderate loss of mitochondrial function was not significantly affected by an ethanolic Gynostemma pentaphyllum extract at concentrations up to 240 [mu]M (Fig. 3A).

[FIGURE 3 OMITTED]

The combination of hypoxia/reoxygenation with 3 [mu]M extramitochondrial [Ca.sup.2+] led to a dramatic loss of mitochondrial function (from 100% to 18% of control). Under this condition, respiration could not be stimulated by ADP indicating the disturbance of oxidative ATP generation. This dramatic loss of mitochondrial function was by about 80% prevented in the presence of the ethanolic Gynostemma pentaphyllum extract even at low concentrations (Fig. 3B).

Effect of the ethanolic Gynostemma pentaphyllum extract on [Ca.sup.2+]-induced swelling of isolated mitochondria

In this series of experiments we tested the ability of the ethanolic Gynostemma pentaphyllum extract to affect [Ca.sup.2+]-stimulated opening of the mitochondrial permeability transition pore. The absorption of the mitochondrial suspension at 546 nm was measured as parameter of mitochondrial volume. Time-dependent decrease in absorption was induced by 200 [mu]M [CaCl.sub.2] indicating opening of the mitochondrial permeability transition pore. The presence of 240 [mu]g/ml Gynostemma pentaphyllum extract remarkably prevented mitochondrial swelling caused by opening of the pore (Fig. 4A). In Fig. 4B the slopes of decrease in absorption, expressed in percent of control, are presented. Even at the low concentration of 24 [mu]g/ml gynostemma extract, about 50% of protection was observed. It could not further improved by elevating the gynostemma concentration.

[FIGURE 4 OMITTED]

Effect of ethanolic Gynostemma pentaphyllum extract on iron/ascorbate-induced lipid peroxidation in isolated mitochondria

We tested the antioxidative effect of the ethanolic Gynostemma pentaphyllum extract within a biological system that generates reactive oxygen species. In the presence of oxygen, iron and ascorbate, mitochondria form the reactive hydroxyl radical that oxidizes mitochondrial lipids. Under this control condition a lag phase of about 40 min duration was followed by massive increase in products of lipid peroxidation (measured as thiobarbituric acid reactive substances). Finally, plateau levels of thiobarbituric acid reactive substances were reached (Fig. 5A). The presence of the ethanolic Gynostemma pentaphyllum extract in this system delayed increase in lipid peroxidation products and decreased the plateau level in a concentration dependent manner. We determined the time of half maximal concentration of thiobarbituric acid reactive substances in order to quantify the delay. The data is presented in Fig. 5B. Concentration of the extract equal or higher than 120[mu]g/ml caused a clear delay of lipid peroxidation. The data of the effect of the ethanolic Gynostemma pentaphyllum extract on the plateau level is summarized in Fig. 5C. Again, a clear decrease was obtained at concentrations equal or higher than 120[mu]g/ml.

[FIGURE 5 OMITTED]

Discussion

From numerous studies it became clear that besides the duration of the ischemic period reperfusion is crucial for the outcome of an ischemic insult (Powers et al. 2007). Several strategies have been developed targeting cellular processes to protect from ischemic injury. The administration of distinct foods or drugs prior to the ischemic insult has been performed to achieve prevention by preconditioning (Riess et al. 2004). Furthermore, drugs have been applied during reperfusion (Chacon et al. 2008). In this context, antioxidants were used to improve the outcome. However, antioxidants administered in vivo failed to significantly protect from ischemia-induced injury (Hicks et al. 2007). Likewise, attempts to prevent [Ca.sup.2+] overload by [Ca.sup.2+] antagonists were not convincing (Dirksen et al. 2007). The same is true for compounds that target steps of cellular cell death (Renoleau et al. 2007). Another concept of intervention is to support regeneration after ischemia/reperfusion injury.

In this study we focused on intervention during reperfusion. Subjects of our investigations were hippo-campal slices from the CA1-region which were treated with temporary hypoxia/hypoglycemia. Evoked field potentials were measured as functional parameter of neuronal cells. This in vitro model of ischemia/reperfusion reflects the interplay of different cell types in the CA1 region of the hippocampus since the electrical stimulus was applied in the distance of several cell-lengths to the detection of the evoked field potential. The population spike amplitude is a very sensitive parameter for cellular function of the investigated area (Ruthrich and Krug 2001). Our reference experiments (control) were designed to cause a dramatic loss of cellular function by transient hypoxia/hypoglycemia. Under this condition, the potential recovered only to 50% of initial during reperfusion. The ethanolic extract from Gynostemma pentaphyllum completely prevented functional injury when given with reperfusion. The extract is a mixture of numerous components. Essential increments are gypenosides. The diversity of the components has been attributed to the numerous biological effects of the gynostemma extract. From the observed beneficial effect of the gynostemma extract it is concluded that the main part of hypoxia/hypoglycemia-induced injury occurred during reperfusion.

It has been shown by many investigators that mitochondria become injured in the course of ischemia/reperfusion. In the presented study we simulated the deleterious effect of ischemia/reperfusion in isolated mitochondria by hypoxia/reoxygenation in combination with elevated extramitochondrial [Ca sup.2+] concentration This stress was adjusted to a level that was associated with the completely loss of the ability for ATP synthesis Also in this system, the Gynostemma pentaphyllum extract was highly protective. It is reasonable to assume that, at least, components of the lipophilic gynostemma extract are able to penetrate biological membranes since evoked field potentials are generated by cellular processes. In this context it is very likely that components of the extract directly interact with mitochondria in cellular systems. It has been shown that essential factors of hypoxia/reoxygenation-dependent injury in isolated mitochondria are [Ca sup.2+] overload and oxidative stress (Schild et al. 2001). Both stimulate opening of the mitochondrial permeability transition pore (Schild et al 2003). This process leads to collapse of oxidative phosphorylation and to mitochondrial disruption. Recent experiments have demonstrated that opening of the mitochondrial permeability transition pore occurs during ischemia/reperfusion (Javadov and Karmazyn 2007). In our swelling experiments we provoked the opening of the mitochondrial permeability transition pore by high extramitochondrial [Ca sup.2+] concentration. In the presence of the ethanolic Gynostemma pentaphyllum extract the mitochondrial swelling was remarkably reduced. Thus, the Gynostemma pentaphyllum extract has the potency to protect mitochondria from opening of the permeability; transition pore.

Gypenosides posses antioxidative activity (Shang et al. 2006). However, a biological environment may reduce the antioxidative effect. In the course of iron ascorbate induced lipid peroxidation mitochondria generate high amounts of the reactive hydroxyl radical with short live span which initiate the chain reaction of lipid peroxidation. The Ethanolic Gynostemma pentaphyllum extract significantly hindered this process. Since the hydroxyl radical is generated within the mitochondria the antioxidative effect of the extract requires the penetration of gypenosides through the mitochondrial membrane system.

Our experiments demonstrate that the ethanolic extract from Gynostemma pentaphyllum effectively protects cellular systems such as brain slices from hypoxia/hypoglycemia-induced injury. The extract acted immediately within a short period of time. Because of the complex composition of the extract it combines antioxidative with protective effects against opening of the mitochondrial permeability transition pore. From our investigations it may be suggested that the ethanolic extract from Gynostemma pentaphyllum is a superior nutrition supplement for individuals with high risk of stroke or heart attack. The prophylactic intake of the extract may protect those persons from ischemia-induced cellular damage. A second line of application could be the combination with strategies of revascularization of blood vessels in stroke and heart attack patients in order to protect from reperfusion injury.

Acknowledgement

We grateful acknowledge Mrs. D. Stolze and Mrs. S. Niemann for excellent technical assistance. We also wish to express our special gratitude to Herbasin, Shenyang, PR China, (http://www.herbasin.com) for generously providing the extract. We are indebted to Dr. Martin Schmitt, Bad Kreuznach, for supporting this investigation and for helpful discussions. The TLC- and HPLC-fingerprint analysis were performed by Mrs. Dana Marotel (Inst. of Pharmaceutical Biology, laboratory of Prof. Dr. H. Wagner, University Munich).

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L. Schild (a), *, A. Roth (a), G. Keilhoff (b), A. Gardemann (a), R. Brodemann (c)

(a) Department of Pathological Biochemistry, Otto-von-Guericke-University, Magdeburg D-39I20, Germany

(b) Institute of Medical Neurobiology, Otto-von-Guericke-Universtity, Magdeburg, Germany

(c) Institute of Pharmacology and Toxicology, Otto-von-Guericke-University. Magdeburg. Germany

*Corresponding author. Tel.: +49 3916713644; fax: +4939167290176.

E-mail address: lorcnz.schild@med.ovgu.de (L. Scluld).

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doi:10,1016/j.phymed.2009.03.006
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Author:Schild, L.; Roth, A.; Keilhoff, G.; Gardemann, A.; Brodemann, R.
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Geographic Code:4EUGE
Date:Aug 1, 2009
Words:5037
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