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Keywords:

  • Pentylenetetrazol;
  • Kainic acid;
  • Pilocarpine;
  • Ginsenosides;
  • HSP72

Abstract

  1. Top of page
  2. Abstract
  3. METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgments
  7. REFERENCES

Summary: Purpose: To test the anticonvulsant activity of three preparations of American ginseng: whole root extract, whole leaves/stems extract, and a partially purified extract that concentrates the Rb ginsenosides (Rb extract).

Methods: One hour after treatment with normal saline, or one of the three ginseng preparations, seizures were induced in adult, male, Sprague–Dawley rats with kainic acid (KA; 10 mg/kg), pilocarpine (300 mg/kg, preceded by methylscopolamine, 1 mg/kg, s.c.), or pentylenetetrazol (PTZ, 50 mg/kg). Time to onset of seizure activity, duration of seizure activity for PTZ, seizure severity, and weight change for KA and pilocarpine were determined for each animal. The brains from animals who had received KA or pilocarpine were examined for severe neuronal stress, by using immunoreactivity for heat-shock protein (HSP)72.

Results: The Rb extract had a dose-dependent anticonvulsant effect in all three models of chemically induced seizures: increasing the latency to the seizures; decreasing the seizure score, weight loss, and subsequent neuronal damage after pilocarpine; and shortening the seizure duration and reducing mortality after PTZ. The Rb extract also significantly reduced the effects of KA, including completely blocking behavioral seizures. The root preparation increased the mortality rate after administration of pilocarpine, but had no other significant effects. The leaves/stems preparation, at 120 mg/kg, reduced the weight loss after pilocarpine, but had no other significant effects.

Conclusions: Ginseng extract made from either the root or leaves/stems is ineffective against chemically induced seizures. A partial purification of the whole extract that concentrates the Rb1 and Rb3 ginsenosides has significant anticonvulsant properties.

Used medicinally by Asians for 2,000 years, ginseng is now used regularly by ∼6 million Americans (1). Seven major species of ginseng are known, but the three most commonly used are Panax ginseng (Asian), Panax quinquefolius (American), and Panax japonicus (Japanese). Although some other constituents of the plant extract may have some activity, the ginsenosides are considered to have the most activity (2). Over 28 ginsenosides have been isolated from American ginseng, including: the panaxadiols (Rb1, Rb2, Rc, Rd, Rg3, Rh2, and Rh3) and the panaxatriols (Re, Rf, Rg1, Rg2, and Rh1). Pharmacologic effects attributed to ginsenosides have been shown in the central nervous system, the cardiovascular system, the endocrine system, and the immune system. They are thought to have antineoplastic, antistress, and antioxidant properties. The possibility that ginseng may have anticonvulsant activity was previously suggested by Lee et al. (3). It was reported that the seizures after administration of kainic acid (KA) were shorter when the animals were pretreated with ginseng, but the details of this effect were not provided, and the experiments were done by using a mixture of ginsenosides. It is not known which of the ginsenosides in the mixture contribute to the antiepileptic effect, but another study reported that one of the ginsenosides, Rd, has no effect on KA–induced neurotoxicity in the hippocampus (4).

The root, leaves, and stems of the ginseng plant all contain ginsenosides. Interestingly, American ginseng generally contains a lower Rg1/Rb1 ratio than does Asian ginseng. Given the reports that Rb1 is a central nervous system depressant and Rg1 is a stimulant (5), a lower Rg1/Rb1 ratio would be predicted to be more effective as a potential anticonvulsant agent. Therefore, for this project, American ginseng was used. The leaves, stems, and roots of both American and Asian ginseng are rich in the Rb ginsenosides, and an extract can be prepared that is enriched in the Rb ginsenosides (termed Rbextract) relative to the whole extract. This study tested whether ginseng, including an extract enriched in panaxadiols, may have activity as an anticonvulsant.

METHODS

  1. Top of page
  2. Abstract
  3. METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgments
  7. REFERENCES

Ginseng from the root and from the leaves/stems of American ginseng were purchased from Jilin Ginseng in China (http://www.ginseng99.com). The levels of total ginsenosides in each were provided in an analysis from the company (Table 1). The levels of the Rb1, Rb3, and Rd ginsenosides (5) were determined by high-performance liquid chromatography (Column: Adsorbosphere XL- C-18B 90A 5μ, 250 × 4.6 mm id; flow phase, CH3CN: 0.06% TFA in H2O; flow rate, 0.3 ml/min; detection, UV at 203 nm). As can be seen from the data in Table 1, the levels of ginsenosides are higher in the leaves/stems preparation for both American and Asian ginseng. In addition, the levels of ginsenosides in the Rb group are highest in the leaves/stems from American ginseng. Therefore for this study, the partial purification to concentrate the Rb ginsenosides was done with leaves/stems from American ginseng. The results with this partial purification were then compared with those of the root preparation (total ginsenosides from roots: TG – Rt) and leaves/stems preparation (total ginsenosides from leaves and stems: TG – LS) also from American ginseng.

Table 1. Analysis of ginseng products
 Total ginsenosidesRb1Rb3RdTotal Rb1, Rb3, Rd% that is in Rb group (by weight)
  1. The final column shows the calculation of the percentage of each gram of powder that is Rb1, Rb3 or Rd (i.e., the major components of the Rb extract). Except for the Rb extract, analysis was provided by Jilin Ginseng Co.

American root30.2% 2.4%14.6% 9.9%26.9% 8.1%
American leaves/stems85.2% 1.9%19.2%14.7%35.8%30.5%
Rb extract94.7%24.8%46.4%13.1%84.3%79.8%
Asian root30.2% 1.9% 3.2% 7.1%12.1% 3.7%
Asian leaves/stems85.1% 3.9% 6.8%11.9%22.6%19.2%

To purify the leaves/stems partially, the powdered product was extracted with 95% ethanol 3 times under reflux. After removal of solvent, the extract was suspended in water and then partitioned in ethyl acetate 5 times. The water partition was then freeze-dried to give a dry powder. This powder was dissolved in water and passed over an open octadecal silica column eluting with 30%, then with 55%, and finally with 75% methanol. The 75% methanol elution was evaporated and freeze-dried to give the final Rb extract used in this study. High-performance liquid chromatography analyses indicate that this partial purification contains three major ginsenosides (Fig. 1): Rb1 (24.8%), Rb3 (46.4%), and Rd (13.1%). No other single component was >10%. For reference, this partial purification will be termed Rbextract.

image

Figure 1. Chemical analysis of the ginseng extract. The chemical structures of the three component ginsenosides in the Rb extract are shown.

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All animal experiments were carried out in accordance with the National Institutes of Health guide for the care and use of laboratory animals (NIH publication 8023, revised 1996) and with the approval of the local Animal Use Committee. Adult male Sprague–Dawley rats weighing 190–230 g were used throughout this study. These animals were housed in a room with controlled temperature (22 ± 1°C) and humidity (50 ± 5%) under a 12:12-h light/dark cycle.

KA (Ocean Produce Int., Shelburne, Nova Scotia, Canada), pentylenetetrazol (PTZ; Sigma Chemical Co., St. Louis, MO, U.S.A.) and pilocarpine (Sigma Chemical) were dissolved in normal saline, and the pH adjusted to 7.4. Convulsants were administered intraperitoneally. Methylscopolamine (Sigma Chemical) was administrated to animals (1 mg/kg, subcutaneously) 10 min before the pilocarpine. Preliminary experiments demonstrated that 10 mg/kg KA, 50 mg/kg PTZ, or 300 mg/kg pilocarpine was the minimal dose that resulted in behavioral seizure activity in all naive animals. Therefore these doses of the convulsants were used to test the anticonvulsant activity of the ginseng products. The time from injection of each chemical convulsant to the first appearance of seizure activity was measured for each animal and is referred to as the seizure latency. After administration of KA, this was the time to the first wet-dog shake. After pilocarpine, this was defined as the time to the first appearance of forelimb clonus.

For the PTZ-induced seizures, the total duration of the behavioral seizure activity also was measured for each animal. For pilocarpine and KA-induced seizures, the behavioral seizures were scored according to the scale of Racine et al. (6), and each animal was assigned the score of the most severe seizure observed. Animals were weighed before administration of the convulsants and again 24 h later. The change in body weight was then determined for each animal after pilocarpine and KA.

Doses of the whole root and leaves/stems preparations were calculated to provide equivalent doses of the ginsenosides in the Rb extract. Each gram of the Rb extract contains 800 mg of the ginsenosides of interest (Rb1, Rb3, and Rd). To determine the doses of root and leaves/stems preparations for use in this study, the relative concentrations of total ginsenosides and ginsenosides in the Rb group were taken into account (Table 1). For the ginseng root preparation (TG – Rt), doses of 450 and 600 mg/kg include Rb ginsenosides at 36 and 48 mg/kg, respectively. For the leaves/stems preparation (TG – LS), doses of 120 and 160 mg/kg include Rb ginsenosides at 36 and 48 mg/kg, respectively. Ginseng products were dissolved in normal saline and administered intraperitoneally 60 min before the chemical convulsant.

Severe neuronal stress in the brain was assessed by using immunohistochemistry for HSP72 (7). Twenty-four hours after administration of pilocarpine or KA, animals were deeply anesthetized and perfused through the heart with 4% buffered paraformaldehyde. The brains were removed and fixed overnight. Coronal sections (50 μm) were cut with a Vibratome (Technical Products, Int, Inc. O'Fallon, MO, U.S.A.). For immunostaining, free-floating sections were blocked in 4% normal horse serum, 1% bovine serum albumin, and 0.3% Triton-100 in phosphate-buffered saline (PBS; 2 h at room temperature). Then the sections were processed for immunolabeling with anti-HSP72 (1:2,000, mouse monoclonal antibody; Oncogene Research Products, Boston, MA, U.S.A.) overnight at 4°C, followed by biotinylated goat anti-mouse (1:200; Vector Laboratories, Burlingame, CA, U.S.A.). Sections were then incubated with avidin–peroxidase complex (ABC kit; 1 h at room temperature; Vector Laboratories), followed by 3-3′-diaminobenzidine for visualization (Vector Laboratories). Adjacent sections were stained with cresyl violet. Naïve animals do not express HSP72 in the brain, but after status epilepticus, widespread expression of HSP72 occurs, particularly in the cortex and hippocampus. For these experiments, the brains were scored as positive or negative for HSP72. In brains scored as negative, no neurons were positive for HSP72 in any stained section. In brains scored as positive, neurons positive for HSP72 were seen throughout the brain, and the stain was clearly positive without the need for magnification (Fig. 2).

image

Figure 2. Nissl stain and immunoreactivity for heat-shock protein (HSP)72. Each pair of photographs was obtained from two adjacent sections from a single brain. Left: Section stained with cresyl violet. Right: Section stained for HSP72 immunoreactivity. The pair of sections on the left is from a control, untreated, animal. The middle pair is from an animal that had received 300 mg/kg of pilocarpine 24 h earlier. The sections on the right (Rb/Pilocarpine) are from an animal that received 40 mg/kg of the Rb extract 1 h before the pilocarpine and was killed 24 h later. The control and Rb/Pilocarpine sections were scored as negative for HSP72, whereas the animal treated with pilocarpine alone (middle) was scored as positive.

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The latency to seizure onset, seizure duration, change in body weight, and seizure score were averaged across animals in the same treatment group. Comparisons between groups were done with an analysis of variance (ANOVA) with nonparametric measures (Kruskal–Wallis) and post hoc test (Dunn's) with comparison with chemical convulsant alone. The mortality and HSP72 immunoreactivity were analyzed with a contingency table by using χ2 analysis. A statistical difference was determined by a value of p < 0.05.

RESULTS

  1. Top of page
  2. Abstract
  3. METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgments
  7. REFERENCES

Effects of ginseng extracts on pilocarpine-induced seizures

Administration of 300 mg/kg pilocarpine (n = 9) caused status epilepticus in every animal, with a mean latency to seizure onset of 18 ± 1 min (Fig. 3). The most severe seizures were scored stage 4 or 5 in eight (90%) of nine animals. The remaining animal had stage 2 seizures. The mean seizure score in these animals was 4.3 ± 0.2 (mean ± SEM, n = 9). Two of the animals with stage 5 seizures died (22% death rate). In the animals that survived 24 h, HSP72 immunoreactivity was found in five (71%) of the seven animals. Animals lost an average of 27 ± 3.2 g over the first 24 h after administration of pilocarpine.

image

Figure 3. Effects of ginseng on the seizures induced by pilocarpine. Each measured parameter is graphed as a function of the treatment group (TG-Rt, total ginsenosides from root of American ginseng; TG-LS, total ginsenosides from leaves and stems of American ginseng). The number of animals was seven to 10 for each experimental group, except that four animals were in the Rb, 20-mg/kg, group. A: The mean time from the injection of the pilocarpine to the first observable behavioral seizure (latency) is presented. B: The most severe seizure recorded for each animal was determined and averaged across animals in each treatment group. C: The weight loss in each animal that survived 24 h was determined and averaged across animals in each treatment group. D, E: Percentage of animals that survived 24 h and had immunoreactivity for heat-shock protein (HSP)72 are presented for each treatment group. No data for the TG-Rt at 450 mg/kg appear in graphs D and E because none of the animals in these groups survived 48 h. Statistical analysis (ANOVA) determined whether differences existed between the treatment groups. The results of the comparison between each drug group compared with the pilocarpine alone group are indicated on graphs A–C. *p < 0.05; **p < 0.01. Statistical analysis of the mortality rate and presence of immunoreactivity for HSP72 was done with a χ2 test.

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One set of animals was pretreated with the Rb extract at 20 (n = 4), 40 (n = 10), and 60 (n = 8) mg/kg, which corresponds to 16, 36, and 48 mg/kg of the Rb ginsenosides. The 20-mg/kg dose did not appear to be effective, because the latency (28 ± 4 min), the seizure score (3.6 ± 0.5), weight loss (25 ± 2 g), and the mortality rate (50%) were not significantly different from those of the pilocarpine-treated group. However, the 40- and 60-mg/kg doses had significant effects on the pilocarpine-induced seizures. The latency to seizure onset was 48 ± 9 min and 38 ± 7 min; the seizure scores were 2.7 ± 0.5 and 2.6 ± 0.4 in the 40- and 60-mg/kg groups, respectively; and the weight loss was 14 ± 3 g in both groups. These values are significantly different from those for pilocarpine-treated animals. Only two of 10 animals pretreated with 40 mg/kg died, and three of eight animals pretreated with 60 mg/kg died. In the surviving animals, three of eight had HSP72 immunoreactivity in the 40-mg/kg pretreatment group, whereas two of five were positive in the 60-mg/kg group. The mortality rate and number of animals with HSP72 immunoreactivity did not reach statistical significance by using the χ2 analysis for a trend.

To test the effect of the extract from the root of American ginseng, three animals were injected with 600 mg/kg of the root extract (which corresponds to 48 mg/kg of the Rb ginsenosides) and then given 300 mg/kg pilocarpine 1 h later. All of these animals died. An additional three animals were tested with a dose of 450 mg/kg (which corresponds to 36 mg/kg of the Rb ginsenosides). Only one animal survived, and this animal had a seizure score of 5. HSP72 immunoreactivity was found throughout the brain of this animal. A χ2 analysis of the mortality in these animals compared with that in the animals treated with pilocarpine alone showed that the TG – Rt product was significantly more toxic than pilocarpine alone at these doses. Seven additional animals were tested with 150 mg/kg, which corresponds to 12 mg/kg of the Rb ginsenosides. The mean latency to seizure in this group was 24 ± 6 min, and the mean seizure score was 4.1 ± 0.4, neither of which was statistically different from those of the pilocarpine-treated animals. Three of the seven animals died before 24 h. Of the remaining four animals, all had HSP72 immunoreactivity throughout the brain. The mean weight loss in these surviving animals was 27 ± 3.8 g, which is not different from the loss in animals receiving pilocarpine alone.

To test the effect of the extract from the leaves/stems of American ginseng, three animals were injected with 160 mg/kg of the root extract (which corresponds to 48 mg/kg of the Rb ginsenosides) and then given 300 mg/kg pilocarpine 1 h later. Two of these animals died, so the dose was reduced for subsequent testing. An additional seven animals were tested with a dose of 120 mg/kg (which corresponds to 36 mg/kg of the Rb ginsenosides). Two of these animals died. The animals that survived had a mean seizure score of 3.2 ± 0.7 and a mean weight loss of 10 ± 2 g. This weight loss was significantly decreased compared with that of animals that received pilocarpine alone. HSP72 immunoreactivity was found in two of the six surviving animals that received either 120 or 160 mg/kg of the TG-LS extract. The mean survival rate, latency to seizure onset, and seizure score were not different from those in the animals treated only with pilocarpine. Seven additional animals were treated with 60 mg/kg, which corresponds to 18 mg/kg of the Rb ginsenosides. The mean latency to seizure in this group was 18 ± 4.0 min, and the mean seizure score was 3.8 ± 0.5. Four of the seven animals died before 24 h. Of the remaining three animals, two had HSP72 immunoreactivity throughout the brain. The mean weight loss in the surviving animals was 12 ± 9 g. The latency to seizure onset, seizure score, death rate, weight loss, and neuronal damage rate were not different from those of the pilocarpine-treated animals.

To determine whether any of the ginseng preparations altered the degree of neuronal stress, the stained sections from animals with the same seizure score and same weight change (±5 g) were compared. No difference was noted in the presence or absence of immunoreactivity for HSP72 in animals with the same degree of seizure. Animals with stage 1 or 2 seizures had no HSP72 immunoreactivity, whereas animals with stage 3–5 seizures had HSP72 immunoreactivity throughout the cortex and hippocampus.

Effects of ginseng extracts on pentylenetetrazol-induced seizures

All animals (n = 11) administered 50 mg/kg of PTZ had generalized seizures (Fig. 4). The latency to the seizure activity was 89 ± 7 s, and the seizure activity had a mean duration of 159 ± 62 s. Three of the 11 animals did not survive the seizures.

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Figure 4. Effect of ginseng on the seizures induced by pentylenetetrazol (PTZ). A: The latency to the first observable seizure activity was measured and averaged across animals in each treatment group. B: The duration of the observable seizure activity was measured for each animal and averaged across animals in each treatment group. C: The mortality rate in each treatment group is presented. Statistical analysis (ANOVA) determined whether differences existed between the treatment groups (n = 6–10). The results of the comparisons between each drug group compared with the PTZ-alone group are indicated on graphs A–C. *p < 0.05; **p < 0.01. Statistical analysis of the mortality rate was done with a χ2 test.

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The Rb extract was tested at doses of 20 (n = 10), 40 (n = 10), and 60 (n = 10) mg/kg, which contain 16, 36, and 48 mg/kg of the Rb ginsenosides, respectively. All animals pretreated with the Rb extract had generalized seizures. The latency to seizure onset was significantly increased by all three doses of the Rb extract (198 ± 53 s for 20 mg/kg, 193 ± 51 s for 40 mg/kg, and 198 ± 51 s for 60 mg/kg). In addition, the seizure duration was significantly shortened by the 40-mg/kg dose (14 ± 4 s), and a trend was seen toward a decrease in seizure duration after the 20-mg/kg (66 ± 30 s) and 60-mg/kg (71 ± 40 s) doses. Two animals (of 10) pretreated with 20 mg/kg died, but no animals in either the 40- or 60-mg/kg groups died. The decrease in mortality was statistically significant by using the χ2 analysis for trend.

The root extract of American ginseng was tested at 133 (n = 10) and 450 (n = 6) mg/kg, which contain 10.6 and 36 mg/kg of the Rb ginsenosides, respectively. Higher doses were not tested because of the increased toxicity seen with higher doses in the pilocarpine model. All animals that were pretreated with the root preparation had generalized seizures. For both doses, the latency to seizure, seizure duration, and mortality rate were not different from those of the group of animals treated with PTZ alone. The latency to onset of the seizure activity was 121 ± 13 s for the 133-mg/kg dose and 110 ± 8 s for the 450-mg/kg dose. The seizure duration was 62 ± 45 s for the 133-mg/kg dose and 151 ± 72 s for the 450-mg/kg dose. In the 133-mg/kg pretreated group, two of the 10 animals died, whereas one of six died in the group pretreated with 450 mg/kg.

The leaves and stems extract of American ginseng was tested at 60 (n = 9) and 120 (n = 8) mg/kg, which contain 18 and 36 mg/kg of the Rb ginsenosides, respectively. As with the root preparation, higher doses were not tested because of the increased toxicity seen in the pilocarpine model. All animals pretreated with the leaves/stems preparation had generalized seizures. The latency to seizure, seizure duration, and mortality rate were not different from those in the group of animals treated with PTZ alone for either dose. The latency to onset of the seizure activity was 230 ± 78 s for the 60-mg/kg dose and 102 ± 4 s for the 120-mg/kg dose. The seizure duration was 39 ± 20 s for the 60-mg/kg dose and 104 ± 48 s for the 120-mg/kg dose. In the 60-mg/kg pretreated group, one of the nine animals died, whereas one of eight died in the group pretreated with 120 mg/kg.

Neuronal stress was not systematically examined in these animals because PTZ-induced seizures are quite short and do not induce HSP72 (ref. 8 and personal observation). In addition, none of the animals in the ginseng-treated groups had seizures that were significantly longer than those of the control group.

Effects of ginseng extracts on kainic acid–induced seizures

To test whether these results with the Rb extract would generalize to another model of status epilepticus, KA was used to induce seizures in an additional set of animals (Fig. 5). The root and leaves/stems preparations were not used in the KA model because they were, for the most part, ineffective in the pilocarpine and PTZ models. Administration of 10 mg/kg KA caused status epilepticus with stage 3–5 motor seizures in nine (of 10 animals, 90%). The remaining animal had intermittent seizures for several hours that reached stage 1 (10%). The mean seizure score for the entire group was 3.5 ± 0.4, and the mean weight loss was 30 ± 4 g. HSP72 immunoreactivity was found in all of the animals that had stage 3–5 seizures, but not the in animal with stage 1 seizures.

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Figure 5. Effects of ginseng on the seizures induced by kainic acid. Data are presented as in Fig. 2. A: The mean time from the injection of the kainic acid to the first wet-dog shake (latency). B: The most severe seizure recorded for each animal was determined and averaged across animals in each treatment group. C: The change in body weight in each animal was determined and averaged across animals in each treatment group. D: The percentage of animals that had immunoreactivity for heat-shock protein (HSP)72 are presented for each treatment group. Statistical analysis (ANOVA) determined whether differences existed between the treatment groups (n = 10 for each group). The results of the comparison between each drug group compared with the kainic acid–alone group are indicated on graphs A–C. *p < 0.05; **p < 0.01; ***p < 0.001. Statistical analysis for the presence of immunoreactivity for HSP72 was done with a χ2 test.

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The Rb extract was tested in the KA model in doses of 10, 20, 40 and 60 mg/kg, which contain 8, 16, 36, and 48 mg/kg of the Rb ginsenosides. All animals that received 10 mg/kg had seizures after administration of KA (mean seizure score, 2 ± 0.3, n = 10), but only 60% of animals that received 20 mg/kg (mean seizure score, 1.5 ± 0.5, n = 10) and 50% of animals that received 40 (mean seizure score, 0.8 ± 0.2, n = 10) and 60 mg/kg (mean seizure score, 0.8 ± 0.3, n = 10) had behavioral seizures. The decrease in seizure score was statistically significant for all doses of the Rb extract. None of the animals that were pretreated with Rb extract died. In all of the animals pretreated with the Rb extract, the initiation of the seizure activity was delayed. The latency to the first wet-dog shake was 35 ± 4 min with no pretreatment, 61 ± 3 after pretreatment with 10 mg/kg, 55 ± 5 after 20 mg/kg, 55 ± 5 after 40 mg/kg, and 49 ± 4 after pretreatment with 60 mg/kg of the Rb extract. The slowing of the onset of the seizures was statistically significant for the 10-, 20-, and 40-mg/kg groups. In parallel with the decrease in seizure score, pretreatment with the Rb extract significantly reduced the number of animals with immunoreactivity for HSP72. The weight loss also decreased in a dose-dependent manner. After 10 mg/kg of the Rb extract, the animals (n = 10) lost 15 ± 6 g. After 20 mg/kg, the animals lost 10 ± 5 g (n = 10), whereas after the 40- and 60-mg/kg doses, the animals actually gained some weight (3 ± 1 g for 40 mg/kg, n = 10; 2 ± 2 g for 60 mg/kg, n = 10).

To determine whether the Rb extract altered the degree of neuronal stress independent of the change in seizure severity, the stained sections from animals with the same seizure score and same weight change (±5 g) were compared. No difference was noted in the presence or absence of immunoreactivity for HSP72 in animals with the same degree of seizure. Animals with stage 1 or 2 seizures had no HSP72 immunoreactivity, whereas animals with stage 3–5 seizures had HSP72 immunoreactivity throughout the cortex and hippocampus.

DISCUSSION

  1. Top of page
  2. Abstract
  3. METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgments
  7. REFERENCES

This study compared the anticonvulsant effects of a preparation of root, a preparation of leaves and stems, and a partially purified preparation from American ginseng. The possibility that ginseng may have anticonvulsant activity was previously suggested by Lee et al. (3), but those experiments were done by using a mixture of ginsenosides, and the details of the anticonvulsant activity were not provided. In this study, the partially purified preparation (Rb extract), had significant anticonvulsant activity in all three models of chemically induced seizures, whereas the preparations from leaves/stems and root had little or no anticonvulsant activity. These results suggest that some ginsenosides are anticonvulsant, but that other components of the whole extract from roots or leaves/stems are proconvulsant, or alternatively contain components that neutralize the anticonvulsant effects of the active components. Whether the Rb extract or a purified ginsenoside would be clinically effective for patients with epilepsy will require further testing. In the current study, the root preparation had no effect on the seizures produced by PTZ or pilocarpine, but did significantly increase mortality after pilocarpine. The increased mortality rate after administration of the root preparation and the fact that most of the ginseng available in the American market is made from the root of American ginseng suggests that people with epilepsy, especially those with a history of status epilepticus, should not take ginseng.

The optimal dose of Rb extract appeared to be 40 mg/kg, with 60 mg/kg being roughly equivalent, but less effective on some of the measured parameters, including latency to seizures after pilocarpine and seizure duration after PTZ. This inverted U-shaped dose–response relation is relatively common with complex herbal products and also was reported by Lee et al. (3). It suggests that other constituents in the herbal complex have opposite or toxic effects. Thus the effect of the mixture is a sum of the relative doses and maximal effects of the individual components within the mixture.

The latency to seizure onset appeared to be the most sensitive to the effects of the Rb extract. The latency was increased in every model, and as the dose of the Rb extract was increased, it was the effect seen at the lowest doses. This suggests that the most potent effect of the active ginsenoside(s) is to slow, or block, synchronization or speed of spread of focal epileptiform activity. This is supported by the observation that the Rb extract could completely block seizures induced by KA, but not those induced by pilocarpine or PTZ. The seizures induced by KA begin slowly with hippocampal/limbic system seizures with a latency to the first wet-dog shake (a limbic seizure) of 35 min. These seizures then gradually spread to the cortex and other brain areas (9). In contrast, the seizures induced by pilocarpine, which are also partial seizures with secondary generalization, have a latency of <20 min to the first forelimb clonus (a cortical seizure). PTZ induces generalized seizures with a latency of <2 min. Thus the seizures induced by KA have a more gradual onset. The differential effect of the Rb extract in the different models also could due to the Rb extract being more effective in the limbic system compared with the cortex. With increasing doses, the next effect of the Rb extract was a decrease in seizure score and decrease in the weight loss after pilocarpine and KA. This suggests that the Rb extract is reducing the severity of the seizure activity and reducing the duration of the most severe seizures. The exact mechanism by which the ginsenosides produce the anticonvulsant action remains to be determined.

Which of the components in the Rb extract is likely to be the active component(s)? The extract contains 24.8% Rb1, 46.4% Rb3, and 13.1% Rd. It is least likely that Rd had anticonvulsant activity. First, it is not concentrated to a significant degree in the Rb extract (from 14.7% in the leaves/stems starting material to 13.1% in the Rb extract), and there is little anticonvulsant activity of the starting material compared with the Rb extract. Second, it has been reported (Lee et al., 2003) that the isolated ginsenoside Rd has no effect on pyramidal cell death in CA1 or CA3 in the hippocampus of mice treated with KA. By contrast, evidence indicates that Rb1, and possibly Rb3, have anticonvulsant activity. The ginseng preparation used by Lee et al. (3) contained 18.3% Rb1 (levels of Rb3 were not reported). The effective doses in that study correspond to doses of 9.2–18.3 mg/kg of Rb1, which is similar to the doses of Rb ginsenosides that were active against KA-induced seizures in the present report. In addition, the relative activity of the ginseng preparations in the present study correlates with increasing concentrations of Rb1 and Rb3. Studies with individual ginsenosides are needed to determine the exact contribution of each component to the total effectiveness of the extract.

Although ginseng has been used by millions of people for thousands of years, the exact effects of the components within the product are not known. Some evidence exists of an effect in the central nervous system for both the whole extract and some of the individual ginsenosides, particularly a neuroprotective action. Pretreatment with whole ginseng extract has been shown to reduce the neuronal loss after prolonged seizures induced with KA (3). This attenuation of KA-induced damage was confirmed by Lee et al. (4). However, these studies do not separate the anticonvulsant effect from a neuroprotective effect. In the present study, no evidence was seen of an additional neuroprotective effect, beyond the reduction in seizure activity.

Additional evidence exists of a neuroprotective effect of the individual panaxadiol ginsenosides, most notably Rb1, which demonstrates that Rb1 has effects in the central nervous system and supports the hypothesis that Rb1 may be one of the active components in the Rb extract. Pretreatment with the isolated ginsenoside Rb1 reduces the loss of neurons in the hippocampus in gerbils after bilateral occlusion of the carotid arteries (10,11). This action is thought to be, in part, mediated by the scavenging of free radicals by Rb1 (12). More recent studies have examined the effects of panaxadiols, including Rb1, in some in vitro systems. Rb1 protects against glutamate-induced excitotoxicity in neuronal cultures (13,14). In additional work, Rb1 has been shown to have anxiolytic effects (15), but the mechanism behind this effect is not known. Rb1, injected into the lateral ventricle, has no effect on basic synaptic transmission (16). This suggests that Rb1 is not anxiolytic by suppression of excitatory transmission. Other measures of γ-aminobutyric acid (GABA)ergic function in vivo that might explain the reported anxiolytic and anticonvulsant actions have not been tested.

How do ginsenosides work? Ginsenosides are amphiphilic and have the ability to intercalate into the plasma membrane. This leads to changes in membrane fluidity, which can alter membrane function and secondarily alter the function of receptors in that membrane. Evidence exists of interactions with membrane-bound receptors and the possibility of binding to steroid receptors in the cytoplasm, leading to changes in gene expression (2), but this action has not been demonstrated in the central nervous system. The most accepted theory about the mechanism of action is that the ginsenosides (at least some of them) have antioxidant properties and the ability to scavenge free radicals (12,17). These actions might explain the neuroprotective effects of ginseng, but do not explain the anticonvulsant action reported here.

In the present experiments, the changes in body weight that were calculated for each animal appear to be a very sensitive and accurate measure of the total seizure severity and duration. In general, as young-adult animals, male Sprague–Dawley rats, fed ad lib, will gain 10–15 g/day (http://www.harlan.com/strain%20details/rats/sd.html). Animals that have status epilepticus will actually lose weight in the 24 h after convulsant administration. Over the years of using KA to induced prolonged seizures, a correlation was noted between the severity of the seizure activity and the amount of weight lost by the animals in the first 24 h. The weight loss is multifaceted, with contributions from the motor seizures, reduced food intake, and general distress. Thus the weight change gives an indication of the overall well-being of the animal. In contrast, the seizure score simply records the most severe seizure in each animal, with no indication of the duration of the most severe seizures. Therefore both seizure score and weight change were reported here.

Acknowledgments

  1. Top of page
  2. Abstract
  3. METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgments
  7. REFERENCES

Acknowledgment:  This study was supported by NS39941 to J.L.S.

REFERENCES

  1. Top of page
  2. Abstract
  3. METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgments
  7. REFERENCES