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Characterization of chemical ingredients and anticonvulsant activity of American skullcap (Scutellaria lateriflora).

Abstract

American skullcap (the aerial part of Scutellaria lateriflora L.) has been traditionally used by Native Americans and Europeans as a nerve tonic, sedative, and anticonvulsant. However, despite some previous studies, the quality and safety, the bioactive ingredients, and the pharmacological properties of American skullcap are not fully understood. The aims of this study were to characterize the chemical ingredients of American skullcap and to evaluate its anticonvulsant activity. Twelve phenolic compounds including 10 flavonoids and two phenylethanoid glycosides were isolated and identified from American skullcap and used as marker compounds. An HPLC analytic method for analyzing these marker compounds in commercial American skullcap products from different sources was established and validated. The anticonvulsant activity of American skullcap was determined in rat models of acute seizures induced by pilocarpine and pentylenetetrazol. The results from this study indicate that (1) phenolic compounds, especially flavonoids, are the predominant constituents in American skullcap; (2) American skullcap products have similar constituents, but the content and relative proportions of the individual constituents varies widely; and (3) American skullcap has anticonvulsant activity in rodent models of acute seizures.

[C] 2008 Elsevier GmbH. All rights reserved.

Keywords: American skullcap; Scutellaria lateriflora; Flavonoids; Phenylethanoid glycosides; Anticonvulsant activity; HPLC

Introduction

The genus Scutellaria L. (Family: Lamiaceae) has approximately 200 species, but only two are the officially recognized sources of the herbal products: American skullcap from the aerial part of S. lateriflora L. (North America and Europe) (Wolfson and Hoffmann, 2003) and Baikal skullcap from the root of S. baicalensis Georgi (Asia) (Editorial Committee of Chinese Herbal, 1999). Baikal skullcap is one of the most commonly prescribed traditional medicines for the treatment of inflammatory-related disorders, hyperlipidaemia, and atherosclerosis in China and Japan (Wolfson and Hoffmann, 2003; Editorial Committee of Chinese Herbal, 1999). Recently, Baikal skullcap and its major flavonoids (baicalin, baicalein, and wogonin) have attracted attention for their diverse activities, including anti-inflammatory (Huang et al., 2006), antibacterial (Hahm et al., 2001), antiviral (Ahn et al., 2001), antioxidative (Huang et al., 2006), antitumor (Zhang et al., 2003), neuroprotective (Cho and Lee, 2004; Liu et al., 2005), anticonvulsant (Park et al., 2007), and anxiolytic (Liao et al., 2003) activities.

American skullcap is a perennial herb indigenous to North America. It has been recognized as one of nature's most treasured nervines and has been traditionally used medicinally by Native Americans and Europeans (Millspaugh, 1974). As a nerve tonic, sedative, and anticonvulsant, American skullcap has been traditionally used for patients with epilepsy and other neurological disorders such as nervous tension states, hysteria, chorea, anxiety, and insomnia (Wolfson and Hoffmann, 2003; Awad et al., 2003). Previous chemical investigations on the raw plant materials of American skullcap indicated the presence of essential oil (Yaghmai, 1988), diterpenoids (Bruno et al., 1998), amino acids (Awad et al., 2003), and flavonoids (Awad et al., 2003; Gafner et al., 2003; Bergeron et al., 2005). Recent studies indicated that the contents of major biomarker flavonoids (baicalein, baicalin, wogonin, and wogonoside) of the commercial American skullcap products used by herbal practitioners in UK (Gao et al., 2008) and Japan (Makino et al., 2008) varied widely. Results of an animal study suggested that an extract of raw plant material of American skullcap has anxiolytic property, and that the flavonoids, and possibly amino acids, are responsible for this activity (Awad et al., 2003). Two flavone-glycosides, scutellarein and ikonnikoside I, isolated from American skullcap have been found to bind to one of the serotonin receptors, the 5-[HT.sub.7] receptor, in the brain (Gafner et al., 2003). The 5-[HT.sub.7] receptor is thought to play a role in both the pathogenesis and preventive treatment of various ailments such as sleep disorders; depression, and migraine (Gafner et al., 2003). A double blind, placebo-controlled clinical trial strongly suggested that commercial American skullcap products have clinical benefits as a relaxing nervine with no overt evidence of toxicity or side effects (Wolfson and Hoffmann, 2003).

However, despite its extensive uses and some previous studies, the quality and safety, the bioactive constituents, and the pharmacological properties of American skullcap, especially the commercial products used in USA, are not fully understood. The aims of this study were (1) to characterize the chemical profiles of the commercial American skullcap products by identifying the chemical constituents and determining the concentration of each individual constituent and (2) to assess its anticonvulsant activity using rodent models of acute seizures.

Materials and methods

American and Baikal skullcap samples and materials

Ten batches of commercial American skullcap products (termed samples A--J) were purchased from different natural product companies or local herbal product stores in Texas. All samples were sold as extracts (samples A--F) or fine plant powders (samples G--J) in capsules. The roots of Baikal skullcap were obtained from Yan on Ginseng Co. (Houston, USA) and ground to fine powders (sample K). Octadecyl-functionalized silica gel (ODS) and HPLC-grade methanol were purchased from Sigma-Aldrich (St. Louis, MO). Deionized water was prepared using a Barnstead B-Pure water system (Barnstead/Thermolyne, Dubuque, Iowa). All other organic solvents met the standards needed for the experiments. Pilocarpine and pentylenetetrazole (PTZ) were obtained from Sigma-Aldrich (St. Louis, MO).

Preparation of whole extract and fraction A

A commercial American skullcap product (sample A, 200 g) was first refluxed at 80[degrees]C with 70% ethanol twice and then with water twice (American skullcap/solvent: 1/30; 30min each time). The combined ethanol and water solution was concentrated under vacuum and then freeze-dried to make the whole extract (70.88 g, 35.44% yield). Part of the whole extract (15.11 g) was fractioned by a column (500 x 45 mm) of ODS (500g) eluting with 65% and 100% methanol (each 1500 ml). The 65% MeOH elution was concentrated under vacuum and freeze-dried to furnish fraction A (11.35g, yield 75.12%). The whole extract and fraction A were used for chemical investigation and animal testing.

Isolation and identification of individual chemical constituents in American skullcap

The whole extract (36.06 g) was dissolved in 100 ml DMSO and then applied on a column (600 x 70 mm) of ODS (1000g) eluting with water, 45%, 65%, and 90% MeOH (each 2000 ml) to give water, 45%, 65%, and 90% MeOH fractions, The 45% MeOH fraction was separated by preparative HPLC (MeOH/[H.sub.2]O/HoAc: 470/525/5) to give compounds 1-4. Similarly, the 65% MeOH fraction was isolated by preparative HPLC (MeOH/[H.sub.2]O/HoAc: 650/345/5) to furnish compounds 5-8, while compounds 9-12 were isolated and purified by preparative HPLC (MeOH/[H.sub.2]O/HoAc: 760/235/5) from the 90% MeOH fraction. Preparative HPLC was performed with an Acuflow Series III pump connected with an Acutect 500 UV/vis detector using an Econosil ODS column (250 x 22 mm, 10[mu]m, Alltech; detection: 278 nm; flow rate: 3.0 ml/min). The structures of these compounds were determined by extensive NMR analyses and by comparison of the reference data.

NMR analysis

The [.sup.1]H and [.sup.13] C NMR, [.sup.1]H-[.sup.1]H COSY, HMQC, and HMBC experiments were performed on a Bruker 600MHz NMR instrument. NMR data were reported as [delta] (ppm) values and referenced to the solvent used.

Preparation of sample solution for HPLC analysis

Accelerated solvent extraction (ASE, Dionex model ASE 200) was applied to prepare the sample solution. Five different solvents of water, 30%, 50%, 70%, and 95% ethanol, were used to evaluate the ability to extract the compounds. To prepare a single extraction cartridge, about 20 g of 30-40-mesh sand SX0075-3 (EM Sciences, Gibbstown, NJ) was mixed with 1.0 g of herb product. Each herb product was taken from a mixture of the botanic extracts (or fine plant powers) of 20 capsules. Extractions were conducted at temperature 85[degrees]C with a pressure of 800 psi and 120 s nitrogen purges for a total of 30 min with three 10 min cycles. The solution of each extraction was transferred into a 50 ml volumetric flask and diluted with DMSO to make a final solution of 20 mg/ml. The solution of each sample was filtered through a 0.2[mu]m nylon cartridge before HPLC analysis. Extraction for each sample was performed in triplicate. Both the whole extract and fraction A were made final solution of 2.0 mg/ml in MeOH for HPLC analysis.

HPLC analysis

1. Marker compounds: A total of 12 phenolic compounds (purity: 96-98%) isolated and purified from American skullcap were used as marker compounds. 2. Preparation of marker compound solution: Each marker compound (10.0-50.0 mg) was accurately weighed into a 10 ml volumetric flask and dissolved in methanol to make a stock solution 1.0-5.0 mg/ml of the 12 components. The final solution for HPLC analysis was prepared from the stock solution. 3. Calibration equation and calibration curve: The calibration curve and calibration equation were investigated between peak area (y) and the quantity (x, [mu]g) of each component. Five injections were performed to obtain the absorption plots ranging from 10 to 500 [mu]g/ml in five increments. 4. Apparatus: HPLC analysis was performed on an Agilent 1100 HPLC system with an Agilent 1100 diode array detector using a Hypersil ODS column (250 x 4.6 mm, 5 [mu]m, Supelco). The water with 0.05% acetic acid was employed as mobile phase A, and methanol as mobile phase B. The gradient procedure was 0-25 min with 10-30% B, 25-39 min with 30-40% B, 39-40 min with 40% B, 40-60 min with 40-65% B, 60-80 min with 65-90% B, 80-85 min with 90% B. 85-86 min with 90-10% B, and 86-90 min with 10% B. The flow rate was l.0 ml/min. The column temperature was set at 36[degrees]C, and detection was carried out at 278 nm.

Reproducibility of the HPLC method

The precision and accuracy of the HPLC method were assessed by within and between run validations. The variation was evaluated by three injections of a mixture of 12 marker compounds each day on 3 consecutive days after the preparation of marker compound solution. By substituting the peak area into the calibration curve equation from the same run, the measured concentrations were obtained. The coefficient of variance RSD% was calculated by comparing the measured concentrations. The relative error RE% was obtained by comparing calculated and theoretical concentrations.

Recovery of 12 marker compounds

A certain amount of each marker compound was added into American skullcap product (sample E), and the mixtures were extracted by ASE using 70% ethanol as solvent as described previously. The extract solution was transferred into a 50 ml volumetric flask, diluted with DMSO to make a final solution of 20mg/ml, and then filtered through a 0.2 [mu]m nylon cartridge prior to HPLC analysis. For comparison, a blank sample without spiking with marker compounds was prepared and similarly analyzed.

Seizure induction and treatment with American skullcap

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 200-250 g (n = 59) were used throughout this study. Animals were housed in a room with controlled temperature 22 [+ or -]1[degrees]C and humidity 50 [+ or -] 5%. Pilocarpine (300mg/kg ip), preceded by methylscopolamine (l mg/kg, sc) and pentylenetetrazol (PTZ, 50 mg/kg ip) were used to induce seizures as described previously (Lian et al., 2005). For each animal, the latency to the onset of the behavioral seizure activity (forelimb clonus for pilocarpine and tonic extension for PTZ) and the score for the most severe seizure observed were determined. The behavioral seizures induced by pilocarpine were scored according to a modified version of the scale of Racine (Bough et al., 2002): stage 1, head nodding after pilocarpine; stage 2, head bobbing and stereotypies; stage 3, unilateral forelimb clonus; stage 4, bilateral forelimb clonus; stage 5, rearing and falling; stage 6, jumping and/or running followed by falling. When at least 1 h had passed without any head bobbing, seizures were considered over. Status epilepticus lasting longer than 5 h for pilocarpine was assigned 5 h for seizure duration. For animals receiving PTZ, the seizure duration was defined as the period of tonic--clonic seizures. All measurements of behavioral seizures were done by an investigator blinded to the treatment group of the animal under observation.

The anticonvulsant activity of the whole extract of American skullcap was determined by ip administration of the extract dissolved in 3% DMSO in water followed 1 h later by the convulsant. Four doses of the whole extract (30, 60, 90, and 150 mg/kg) were tested in the pilocarpine model. The most effective dose (90 mg/kg) was then tested in the PTZ model. Fraction A (67.5 mg/kg) prepared from the whole extract was also tested in the pilocarpine model. The dose for fraction A was chosen to provide ingredients at concentrations that are equivalent to 90 mg/kg of the whole extract. Data were expressed as means [+ or -] SEM for each treatment group. Seizure parameters for the dose--response curve with the whole extract in the pilocarpine model were compared using a one-way ANOVA followed by a Dunnett's multiple comparison analysis to the control group. Seizure parameters for the PTZ model and for fraction A were analyzed using the Student's grouped t-test. Statistical difference was defined as p<0.05.

Results and discussion

Chemical ingredients of American skullcap

From the whole extract of a commercial American skullcap product (sample A), a total of 12 phenolic compounds including 10 flavonoids and two phenylethanoid glycosides were isolated and purified. On the basis of extensive NMR analyses including [.sup.1]H, [.sup.13]C, [.sup.1]H-[.sup.1]H COSY, HMQC and HMBC spectra and comparison of the reference data, the structures (Fig. 1) of these compounds were determined as viscidulin III-2'-O-[beta]-D-glucopyranoside (1) (Zhang et al., 1994), chryin-6-C-[alpha]-L-arabionopyranosyl-8-C-[beta]-D-glucopyranoside (2) (Takagi et al., 1981), trans-verbascoside (acteoside, 3) (Numata et al, 1987), viscidulin III (4) (Liu et al., 1984), baicalin (5) (Wu et al., 2005), trans-martynoside (6) (Sasaki et al., 1978), oroxylin A-7-O-[beta]-D-glucopyranoside (7) (Kikuchi et al., 1991), wogonoside (8) (Wu et al., 2005), baicalein (9) (Chen et al., 2003), wogonin (10) (Xiao et al., 2003), chryin (11) (Shen et al., 1993), and oroxylin A (12) (Xiao et al., 2003). Compounds 1-12 had purity of 96-98%, which were determined by HPLC, and were used as the marker compounds for chemical analysis of American and Baikal skullcap, the whole extract and the fraction A.

[FIGURE 1 OMITTED]

Preparation of sample solution for HPLC analysis

Accelerated solvent extraction (ASE) was used for the preparation of sample solution for HPLC analysis in this study. We compared five different solvents of water, 30%, 50%, 70%, and 95% ethanol to prepare the sample solution from the same product (sample F). The results showed that 70% ethanol extract had the highest content of total phenolic compounds (20.55 [+ or -] 0.38%), following by 50% ethanol extract (19.78[+ or -]0.60%), 30% ethanol extract (12.69 [+ or -]0.05%), 95% ethanol extract (10.45[+ or -]0.13%), and water extract (3.31[+ or -]0.05%). The recovery of the 12 marker compounds extracted by using ASE with 70% ethanol as solvent was also investigated. The results indicated that the recovery of the analyzed compounds 1-12 was 101.23 [+ or -] 0.59%, 98.87 [+ or -] 0.40%, 102.27 [+ or -] 2.53%, 101.77 [+ or -] 0.35%, 100.90 [+ or -] 1.68%, 102.67 [+ or -] 2.99%, 99.57 [+ or -] 0.42%, 101.57 [+ or -] 0.23%, 101.50[+ or -]1.90%, 99.27 [+ or -] 2.28%, 99.80 [+ or -] 0.35%, and 101.00 [+ or -] 3.34%, respectively. All these data indicated that using ASE with 70% ethanol as extract solvent is a validated method to prepare the sample solution for HPLC analysis.

HPLC analysis

An HPLC method for simultaneous determination of 12 marker compounds in American and Baikal skullcaps has been developed and validated. Fig. 2 is the HPLC chromatograph of the 12 marker compounds.

[FIGURE 2 OMITTED]

This HPLC method was validated for linearity and reproducibility. The linearity of the standard curves was studied for compounds 1-12. The linearity is expressed in terms of the correlation coefficient ([gama]). The correlation coefficient was found to be better than 0.999 for most of the marker compounds in the range of 20-500 [mu]g/ml. The calibration equation of each compound was Y = 1632.68331 X -2.76701 (1, [gamma] = 0.9995), Y = 1424.11273X -4.35683 (2, [gamma] = 0.9994), Y = 560.20170X --7.98712 (3, gamma = 0.9932), Y = 1199.47410X-13.26095 (4, [gamma] = 0.9996), Y = 1616.64913X -44.96556 (5, [gamma] = 0.9996), Y = 542.34345 X -8.16791 (6, [gamma] =0.9945), Y = 1685.87667 X -10.65231 (7, [gamma] = 0.9995), Y = 1768.26367 X -10.03832 (8, [gamma] = 0.9995), Y = 1317.66260 X -147.31166 (9, [gamma] = 0.9906), Y = 2437.59643 X -187.68362 (10, [gamma] = 0.9999). Y = 2307.81848 X -216.29561 (11, [gamma] = 0.9992), and Y = 1970.31826 X-18.62120 (12, [gamma] = 0.9991), respectively.

The reproducibility of the method was assessed by within and between run validations. The RSD% and RE% were found to be 0.13-2.25% and 0.03-5.56%, respectively.

Chemical profiles of American skullcap

The chemical profiles of 10 batches of commercial American skullcap products and one Baikal skullcap were characterized by defining and verifying the chemical ingredients and determining the concentration of total and individual phenolic ingredients using the established and validated HPLC method. Twelve phenolic compounds isolated and identified from American skullcap were used as the marker compounds. Fig. 3 is the typical HPLC chromatograph of American and Baikal skullcap products. Table 1 presented the concentration of individual and total phenolic compounds. The results showed that nine American skullcap products (samples A--I) contained most of the analyzed ingredients and the flavonoids are the predominant ingredients with baicalin (5) as the major one (43.1-78.5% of total analyzed compounds). But the total analyzed ingredients varied widely from 1.11% for sample E to 20.55% for sample F. Also, the relative proportions of ingredients in each sample were significantly different. For example, the ratios of baicalin (5)/wogonoside (8)/baicalein (9)/wogonin (10) for samples A-I were 20/6/4/1, 139/13/11/1, 17/4/4/1, 94/11/7/1, 48/16/21/1, 202/70/26/1/, 100/8/10/1, 120/8/9/1, and 385/20/25/1, respectively. In addition, it was noted that sample J (Fig. 3) seemed to have a different chemical profile. The major compound baicalin (5) found in samples A--I had been not detected in sample J, which had wogonoside (8) as the major ingredient instead. Previously studies showed that the concentrations of these flavonoids also varied widely in the commercial products of American skullcap used by herbal practitioners in UK (Gao et al., 2008) and Japan (Makino et al., 2008). In this study, chemical variability of American skullcap products was observed not only in the extracts, but also in the fine plant powders. These data suggest that the chemical variability of the commercial American skullcap products might come from the plant raw materials that had variable chemical compositions.

[FIGURE 3 OMITTED]

The chemical variability of herbal medicines will result in inconsistent results and uncertain efficacy. Thus, there is a need for standardization of chemical ingredients in plant raw materials. Therefore, it is very important to establish the good agriculture practice (GAP) standard to grow medicinal plants to obtain stable and consistent chemical ingredients and assure efficacy for herbal products.

For comparison, the chemical constituents of Baikal skullcap (sample K) were also analyzed by HPLC. The results (Table 1 and Fig. 3) indicated eight of the 12 marker compounds isolated from American skullcap were also found in Baikal skullcap with baicalin as the major one, but the relative proportions of ingredients in both skullcaps were different.
Table 1. Percentage of total and individual marker compounds in
commercial American skullcap products (S. A-J), Baikal skullcap (S. K),
whole extract (W.E.) and fraction A (Fr. A)(%, mean + s.d., n = 3)

Comp.  S. A            S. B        s. c        S. D        S. E

1      0.10 [+ or -]   0.17 [+ or  0.04 [+ or  0.39 [+ or  -
       0.01            -] 0.01     -] 0.01     -] 0.01

2      0.39 [+ or -]   0.13 [+ or  0.27 [+ or  0.26 [+ or  0.03 [+
       0.02            -] 0.01     -] 0.01     -] 0.01     or -]
                                                           0.00

3      0.08 [+ or      0.17 [+ or  -           0.20 [+ or  0.05 [+
       -]0.02          -] 0.01                 -] 0.01     or -]
                                                           0.00

4      1.04 [+ or -]   0.13 [+ or  0.77 [+ or  0.02 [+ or  0.04 [+
       0.03            -] 0.00     -] 0.02     -] 0.00     or -]
                                                           0.00

5      6.72 [+ or -]   2.77 [+ or  4.60 [+ or  3.75 [+ or  0.48 [+
       0.15            -] 0.05     -] 0.08     -] 0.07     or -]
                                                           0.01

6      0.85 [+ or -]   0.31 [+ or  0.39 [+ or  0.55 [+ or  0.04 [+
       0.05            -] 0.01     -] 0.03     -] 0.02     or -]
                                                           0.00

7      1.46 [+ or -]   0.14 [+ or  0.84 [+ or  0.16 [+ or  0.10 [+
       0.02            -] 0.01     -] 0.01     -] 0.01     or -]
                                                           0.01

8      1.87 [+ or -]   0.25 [+ or  1.18 [+ or  0.43 [+ or  0.16 [+
       0.09            -] 0.01     -] 0.01     -] 0.01     or -]
                                                           0.00

9      1.35 [+ or -]   0.22 [+ or  1.07 [+ or  0.29 [+ or  0.21 [+
       0.06            -] 0.01     -] 0.02     -] 0.02     or -]
                                                           0.01

10     0.34 [+ or -]   0.02 [+ or  0.27 [+ or  0.04 [+ or  0.01 [+
       0.01            -] 0.00     -] 0.01     -] 0.00     or -]
                                                           0.00

11     0.02 [+ or -]   0.03 [+ or  0.06 [+ or  0.03 [+ or  0.002 [+
       0.00            -] 0.00     -] 0.00     -] 0.00     or -]
                                                           0.01

12     0.12 [+ or -]   0.01 [+ or  0.07 [+ or  0.01 [+ or  0.003 [+
       0.01            -] 0.00     -] 0.01     -] 0.00     or -]
                                                           0.00

Total  14.26 [+ or -]  4.25 [+ or  9.56 [+ or  6.12 [+ or  1.11 [+
       0.15            -] 0.01     -] 0.10     -] 0.10     or -]
                                                           0.06

Comp.  S. F         S. G        S. H        S. I         S.J

1      0.22 [+ or   -           -           -            0.98 [+ or -]
       -] 0.02                                           0.01

2      0.59 [+ or   -           0.10 [+ or  0.10 [+ or   0.71 [+ or -]
       -] 0.01                  -] 0.00     -] 0.01      0.01

3      0.09 [+ or   0.15 [+ or  0.17 [+ or  0.34 [+ or   0.09 [+ or -]
       -] 0.01      -] 0.01     -] 0.01     -] 0.01      0.00

4      1.22 [+ or   -           -           -            0.08 [+ or -]
       -] 0.04                                           0.00

5      10.10 [+ or  2.99 [+ or  3.61 [+ or  7.70 [+ or   -
       -] 0.26      -] 0.02     -] 0.01     -] 0.01

6      0.73 [+ or   0.21 [+ or  0.20 [+ or  0.48 [+ or   0.35 [+ or -]
       -] 0.08      -] 0.01     -] 0.00     -] 0.01      0.00

7      2.10 [+ or   0.22 [+ or  0.19 [+ or  0.25 [+ or   0.62 [+ or -]
       -] 0.10      -] 0.01     -] 0.01     -] 0.01      0.00

8      3.49 [+ or   0.25 [+ or  0.23 [+ or  0.40 [+ or   1.10 [+ or -]
       -] 0.01      -] 0.01     -] 0.00     -] 0.01      0.01

9      1.32 [+ or   0.30 [+ or  0.28 [+ or  0.49 [+ or   0.08 [+ or -]
       -] 0.02      -] 0.01     -] 0.01     -] 0.01      0.00

10     0.05 [+ or   0.03 [+ or  0.03 [+ or  0.02 [+ or   0.11 [+ or -]
       -] 0.03      -] 0.01     -] 0.00     -] 0.00      0.01

11     0.02 [+ or   0.02 [+ or  0.03 [+ or  0.003 [+ or  0.01 [+ or -]
       -] 0.00      -] 0.00     -] 0.00     -] 0.01      0.00

12     0.14 [+ or   0.06 [+ or  0.01 [+ or  0.004 [+ or  -
       -] 0.00      -] 0.00     -] 0.00     -] 0.00

Total  20.55 [+ or  4.24 [+ or  4.75 [+ or  9.81 [+ or   3.55 [+ or -]
       -] 0.38      -] 0.05     -] 0.02     -] 0.01      0.02

Comp.  S. K                 W.E.                 Fr. A

1      -                    0.28 [+ or -] 0.01   0.36 [+ or -] 0.03

2      0.65 [+ or -] 0.00   1.09 [+ or -] 0.02   1.49 [+ or -] 0.05

3      -                    1.01 [+ or -] 0.01   1.38 [+ or -] 0.03

4      0.51 [+ or -] 0.00   2.93 [+ or -] 0.05   3.95 [+ or -] 0.08

5      10.27 [+ or -] 0.05  18.95 [+ or -] 0.16  25.30 [+ or -] 0.30

6      -                    2.40 [+ or -] 0.08   3.19 [+ or -] 0.08

7      -                    4.12 [+ or -] 0.07   5.50 [+ or -] 0.17

8      2.82 [+ or -] 0.00   5.27 [+ or -] 0.09   7.03 [+ or -] 0.13

9      4.33 [+ or -] 0.01   3.81 [+ or -] 0.06   -

10     1.29 [+ or -] 0.01   0.96 [+ or -] 0.03   -

11     0.13 [+ or -] 0.00   0.10 [+ or -] 0.06   -

12     0.62 [+ or -] 0.00   0.35 [+ or -] 0.01   -

Total  20.62 [+ or -] 0.06  41.06 [+ or -] 0.35  47.20 [+ or -] 0.40


Anticonvulsant activity of American skullcap

The anticonvulsant activity of the whole extract (contained total marker compounds of 41.06%, Table 1) of American skullcap product (sample A) was first determined in rat model of acute seizures induced by pilocarpine (300 mg/kg ip, preceded by 1 mg/kg methylscopolamine sc, n = 35). Four doses of the whole extract (30, 60, 90, and 150 mg/kg, n = 4-8 for each dose) were tested. The results (Table 2) indicated that the effective dose range was between 60 and 150 mg/kg with 90 mg/kg being the best dose. There is a suggestion of an inverted U-shaped dose response curve with the extract. The whole extract, at 90 mg/kg, significantly decreased the total seizure duration and there was a trend towards an increase in the latency to stage 3 seizures and decrease in the maximal seizure score. The anticonvulsant activity of fraction A (67.5 mg/kg, n = 8) prepared from the whole extract was also determined in the model of pilocarpine. This fraction concentrated eight of the marker compounds (1-8) with the total marker compounds being 47.20% of the total weight (Table 1). Fraction A significantly reduced seizure severity (by all measures), compared to vehicle control animals and animals treated with 90 mg/kg of the whole extract.
Table 2. Anticonvulsant effect of American skullcap extract and
fraction A on seizures induced by pilocarpine

Treatment     Animal  Latency to stage  Maximal seizure  Total
(dose)        number  3 seizures        score            seizure
              (n)     (min)                              duration
                                                         (min)

Vehicle       12      14 [+ or -] 2     5.1 [+ or -]     275 [+ or
                                        0.3              -] 41

Extract (30   4       14 [+ or -] 2     4.8 [+ or -]     303 [+ or
mg/kg)                                  0.8              -] 32

Extract (60   4       18 [+ or -] 4     4.8 [+ or -]     252 [+ or
mg/kg)                                  1.6              -] 70

Extract (90   8       20 [+ or -] 3     4.0 [+ or -]     144 [+ or
mg/kg)                                  0.7              -] 55 (a)

Extract (150  7       18 [+ or -] 2     5.9 [+ or -]     318 [+ or
mg/kg)                                  0.5              -] 46

Fraction A    8       26 [+ or -] 2     2.9 + 0.7 (a)    123 [+ or
(67.5 mg/kg)          (a)                                -] 50 (a)

Means [+ or -] SEM are presented.
(a) p<0.05 compared to vehicle control group.


The anticonvulsant activity of the extract was further determined after administration of pentylenetetrazol (PTZ). In this model, injection of 50 mg/kg (ip) PTZ results in a generalized tonic, followed by clonic, seizure in all animals (n = 9). The latency to the first tonic extension is quite reproducible with a mean of 76[+ or -]6s. Two of the nine animals died within 20 min. When pretreated with the whole extract of American skullcap (90 mg/kg) l h before the PTZ, five of seven tested animals had a generalized seizure. Two animals had no seizures and no animals died. The latency to seizure onset was significantly increased (127 [+ or -] 6s, p <0.05 compared to vehicle control group).

The data from this study showed that the whole extract of American skullcap product has modest anticonvulsant activity in two rodent models of acute seizures, substantiating the claims for the use of American skullcap as an anticonvulsant. The flavonoids isolated from Baikal skullcap have been previously shown to have high affinity for the benzodiazepine binding site of [GABA.sub.A] receptor (Wang et al., 2002) and neuroprotective effects by anti-oxidation (Cho and Lee, 2004; Liu et ah, 2005), anti-inflammation (Lee et al., 2003), and anti-excitotoxicity (Cho and Lee, 2004; Liu et al., 2005). Interestingly, these active flavonoids in Baikal skullcap were also found in American skullcap. Since excessive stimulation of NMDA receptors (Lipton and Rosenberg, 1994), a deficiency of [GABA.sub.A] neurons (Wu et al., 2006), inflammation (Vezzani and Granata, 2005), and oxidative stress (Patel, 2004) have been proposed to underlie seizures, and agonists at the [GABA.sub.A] receptor are widely prescribed as antiepileptic drugs (Schwartz et al., 1995), these flavonoids may be the active ingredients responsible for the anticonvulsant activity of American skullcap. Indeed, wogonin has recently been shown to have anticonvulsant effects on chemically induced seizures and electroshock seizures in animals (Park et al., 2007). Whether American skullcap has anticonvulsant activity in other animal models and which ingredients are responsible for the anticonvulsant activity of American skullcap will need to be further investigated.

Interestingly, this study also suggested that, at an equivalent dose, fraction A is more effective as an anticonvulsant agent than the whole extract. Further fractionation and isolation using activity-guided procedure may lead to the design of a product from American skullcap with the active constituents but less, or none, of the unnecessary ingredients, resulting in a safer and more effective anticonvulsant agent for patients with epilepsy.

Acknowledgments

The authors would like to thank the Keck/IMD NMR Center, which is funded by the W. M. Keck Foundation and the University of Houston, and Dr. Youlin Xia for NMR analysis assistance.

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Zhizhen Zhang (a), *, Xiao-yuan Lian (b), (c), Shiyou Li (a), Janet L. Stringer (b)

(a) National Center for Pharmaceutical Crops. College of Forestry and Agriculture, Stephen F. Austin State University, Nacogdoches, TX 75961, USA

(b) Department of Pharmacology, Baylor College of Medicine, Houston, IX 77030, USA

(c) Institute of Biomedical Sciences, School of Life Sciences, East China Normal University Shanghai 200241, China

* Corresponding author. Tel.: + 19364681738; fax: + 19364687058.

E-mail address: zzhang@sfasu.edu (Z. Zhang).

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doi:10.1016/j.phymed.2008.07.011
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Author:Zhang, Zhizhen; Lian, Xiao-yuan; Li, Shiyou; Stringer, Janet L.
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
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Date:May 1, 2009
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