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The antidepressant effects and mechanism of action of total saponins from the caudexes and leaves of Panax notoginseng in animal models of depression.

ARTICLE INFO

Keywords: Saponins

Panax notoginseng

Antidepressant

Monoamine neurotransmitter

ABSTRACT

Total saponins extracted from the caudexes and leaves of Panax notoginseng (SCLPN) have been used in the clinic for improving mental function, treating insomnia, and alleviating anxiety. The present study evaluated the potential antidepressant activity of SCLPN in rodent models of depression and the mechanism of action of SCLPN. Mice were received SCLPN at doses of 10-1000 mg/kg daily for 1, 7, and 14 days and then were subjected to the forced swim test and locomotor activity test. The results showed that SCLPN decreased immobility time in the forced swim test, with little effect on locomotion. In the chronic mild stress model, chronic SCLPN treatment (70 mg/kg) reversed the rats' depression-like behavior. Furthermore, SCLPN exerted its antidepressant-Uke effect by increasing the levels of 5-hydroxytryptamine, dopamine, and noradrenaline. Additionally, SCLPN treatment reduced intracellular [Ca.sup.2+] in cultured neurons. The present study suggests that SCLPN may function as an antidepressant, and the antidepressant-like effects of SCLPN may be mediated by modulation of brain monoamine neurotransmitters and intracellular [Ca.sup.2+] concentration.

[c] 2010 Elsevier GmbH. All rights reserved.

Introduction

Panax notoginseng (Burk) F.H. Chen (Araliaceae) (PN) is a commonly used Chinese medicine, the root of which (Sanqi orTianqi in Chinese) has been used for the treatment of hemoptysis, hemostatic, and hematoma in China for more than 400 years (Wang et al. 2006; Wan et al. 2008). Its main ingredients are similar to those present in the two other well-known species of the same genus herb: Panax ginseng (Asian ginseng) and Panax quinquefolium (American ginseng) (Wang et al. 2006). The main bioactive compounds in PN are saponins, commonly referred to as ginsenosides and notoginsenosides, which are present in the root, rootlet, corm, leaf, flower, and berry of the plant (Li et al. 2000). Modern pharmacological research on PN has found that PN exerts various effects on the cardiocerebral vascular system (Cicero et al. 2000), central nervous system, endocrine system, and inflammation (Ng 2006). It also displayed hypoglycemic, hypolipidemic, immunostimulatory, antitumor, antrinflammatory, analgesic, antioxidant, hemostatic, antithrombotic, antiatherosclerotic, fibrinolytic, antiarrhythmic, hypotensive, estrogen-like, and sperm motility-enhancing effects (Ng 2006).

The portion of PN commonly used in remedies is the root. Compared with the long growth cycle required for harvesting the roots, annual recovery of PN caudexes and leaves could be a feasible alternative source of saponins. Saponins have been isolated from the aerial parts of PN (e.g., leaves, flower buds, fruit pedicels, etc.), and the activity and safety are similar to that of the root (Wan et al. 2006). However, the chemical characteristics of the underground and aerial parts of P. notoginseng are significantly different. The saponins from the caudexes and leaves of PN (SCLPN) also contain more than 20 ginsenosides and notoginsenosides.The ginsenosides Rb3, Rbi, Rc and the notoginsenoside Fc were found in high concentrations in SCLPN. The ginsenosides Rc, Rh2, and Rb3 are rare in the underground parts but rich in the aerial parts of PN (Li et al. 2005; Wan et al. 2006; Liu et al. 2007).

SCLPN has been developed for the treatment of insomnia, and many studies have reported that SCLPN can be used for the treatment of anxiety and neurasthenia neurosis (Zhao and Lan 2005; Tan and Tang 1999; Gong and Liu 1998). SCLPN treatment has also been suggested to promote neuronal plasticity (Guo et A1 2003) and enhance the expression of Nestin and brain-derived neurotrophic factor (BDNF; Wang et al. 2007) following focal cerebral ischemia. BDNF and other neurotrophic or growth factors were shown to be decreased in postmortem hippocampal tissue from depression patients, and antidepressant administration can reverse or block this effect (Schmidt and Duman 2007). SCLPN can increase the proliferation and differentiation of neural progenitor cells (Cheng et al. 2005) and exert protective effects on the central nervous systems partially by reducing free radical damage. Specifically, ginsenoside [Rb.sub.3], with a high concentration in SCLPN, has been shown to exhibit strong neuroprotective effects (Xu et A1 2005). These studies indicate that Panax notoginseng has many pharmacological effects and may improve mental function, have antiinsomnia effects, alleviate anxiety, and decrease neural network excitation.

Depression is expected to be the second highest cause of morbidity (Kessler et al. 2005). There are currently four classes of antidepressant drugs and atypical antidepressants (Erica 2008). However, these drugs have undesirable side effects, with high relapse rates and a long onset of therapeutic action. Therefore, critically important is the development of efficient and safe drugs for the treatment of depression. In Oriental society, herbal preparations are widely used and have a long history of use as medications. Based on this premise, we investigated whether Panax notoginseng (SCLPN) has a favorable side-effect profile while exerting antidepressive-like effects in rodent models of depression. We also sought to elucidate SCLPN's mechanism of action.

Materials and methods

Preparation of Panax notoginseng (SCLPN)

Total saponins were extracted and purified from the caudexes and leaves of P. notoginseng (SCLPN) by Guangdong Medi-World Pharmaceutical Co. Ltd. (Foshan, China) according to the criterion of the Pharmcopoeia of the People's Republic of China 2005. SCLPN (total saponins: 78.5%) contained ginsenosides [Rb.sub.3] 15.62%, Rc 7.62%, [Rb.sub.1] 2.98%, [Rb.sub.2] 3.83%, Fl 0.75%, F2 10.52%, notoginsenoside Fa 3.27%, Fc 6.83%, gypenosides 10.52%, determined by high performance liquid chromatography (HPLC, Fig. 1 A), and the structure formulas of the main ginsenosides [Rb.sub.l], Re and [Rb.sub.3] were shown in Fig. 1B. Additionally, the total flavonoid content was 0.34% determined by ultraviolet spectrophotometry. Alkaloids, polysaccharides, starches, tannins, proteins, amino acids, and peptides were not detected by the typical colorimetric method. SCLPN was dissolved in distilled water, and dosages are expressed as milligrams per kilogram body weight.

Animals

Male KM mice, weighting 20-24g (SCXK(Yue)2003-0002), and male SD rats, weighting 180-220g (SCXK(Yue)2008-0002), were all purchased from the Experimental Animal Center of Guangdong Province. Animals were housed under standard experimental conditions (room temperature, 24 [+ or -] 2 C; 12h/12h light/dark cycle) with free access to commercial food and water. Animals were acclimatized for at least 3 days before the experiments began and were exposed only once to every experimental manipulation. The experimental protocol was approved by the Institutional Review Committee for the use of Human or Animal Subjects, and the procedures were in compliance with the Declaration of Helsinki for human subjects, National Institutes of Health Guide for the Care and Use of Laboratory Animals (publication no. 85-23, revised 1985), and regulations of the Animal Welfare Committee of Sun Yat-Sen University. All efforts were made to minimize the number of animals used and their suffering.

Drug treatments and chemicals

Maprotiline hydrochloride tablets (Novartis, Beijing, China), Anafranil (clomipramine hydrochloride tablets, Novartis, Beijing, China), Seroxat (paroxetine hydrochloride tablets, SmithKline, Tianjin, China), Prozac (fluoxetine hydrochloride capsules, Patheon, Bourgoin-Jallieu, France), and Neurostan (extract of St. John's Wort tablets, Karlsurhe, German) were purchased from Guangdong South of China Pharmacological Corp. (Guangdong, China). 5-hydroxytryptaphan (5-HTP) and L-3,4-dihydroxyphenylalanine (l-DOPA) were obtained from Nanjing Adobe Fournier Biological Technology Co., Ltd. Clonicline hydrochloride was produced by Hong Kong Advanced Technology and Industry Company. Before the experiment, the reference drugs used in this study were weighed according to the active ingredient, grinded and then suspended with distilled water.

Forced swim test for mice

The forced swim test (FST) paradigm has been described previously (Porsoitetal. 1977; Detkeetal. 1997; Siuciaketal. 1997). Mice were randomly separated into nine groups (n = 9 per group) and for 2 weeks orally received distilled water, SCLPN (10,30,100, 300, and 1000mg/kg), Paroxetine (6mg/kg), Maprotiline (18 mg/kg), or St. John's wort (100 mg/kg). All treatments were administered in a volume of 10 ml/kg body weight. Doses of antidepressants and 30 mg/kg SCLPN were selected based on their clinic dosage to achieve antidepressant effects or therapeutic effects on insomnia. The test was performed 1 h after administration of SCLPN or the positive control drugs on days 1, 7, and 14. A mouse was placed in a Plexiglas cylinder (18 cm diameter x 30 cm height) with water at a depth of 18 cm (22-24 C) for 6 mm video-taped. The software, ZH-QPT Analytic System, was used to count the cumulative immobility time in the last 4 min of the trial, which purchased from Huaibei Zhenghua bioinstrumentation limited company. Immobility refers to the cessation of struggling and remaining motionless in the water, making only those movements needed to keep the animal's head above the water (Porsolt et ai. 1977).

Psychostimulants have been reported to be clinically ineffective as antidepressants but show antidepressant-like effects m the FST (Sherman et al. 1982). To discount the possibility of false positive results of SCLPN in the FST, we evaluated its acute effects in the open-field test of locomotor activity before the FST. Spontaneous locomotor activity was measured in the open-field test in a square arena (35 cm x 24cm x 17 cm; Boissier and Simon 1965). A mouse was placed in a corner of the apparatus 1 h after the last drug administration, and its behavior was videotaped during a 5 min session and the travelled distance of one was analyzed by ZH-QPT Analytic System. The apparatus was cleaned between tests. Decreased distances travelled were interpreted as decreased locomotor activity.

Chronic mild stress experiment for rats

Stress protocol, animal groups, and drug administration

Forty-two male rats (180-220 g) were used in the chronic mild stress (CMS) experiment. According to a modification of the methods reported in previous studies (Willner et al. 1987; D'Aquila et al. 1997; Sowa-Kucma et al. 2008; Przegalinski et al. 1995), animals were first trained to consume 1% (w/v) sucrose solution and then subjected to a baseline sucrose preference test before the start of the CMS protocol. After the baseline test, the animals were assigned to two groups matched on the basis of sucrose preference: control group (n = 10) and stressed group. Stressed animals were housed in individual cages (one rat per cage) and exposed to CMS for 3 weeks. The weekly stress regimen consisted of food and water deprivation, 45 cage tilt, intermittent overnight illumination, a soiled cage (i.e., 250 ml water in sawdust bedding), paired housing, and background noise. All of the stressors were applied continuously during the day and night. During the CMS experiment, rats in the control group were left undisturbed in their home cages in a separate room, with the exception of general handing (i.e., for regular cage cleaning and body weight measurements) that matched the CMS groups.

[FIGURE 1 OMITTED]

The sucrose preference test was performed for both the control animals and CMS animals. Based on their sucrose preference results, the CMS rats were divided into three groups: CMS/vehicle (n = 10), CMS/Fluoxetine (1.8mg/kg, n=10), and CMS/SCLPN (70 mg/kg, n = 12). Drug treatments were administered orally once per day beginning in week 4 for 4 weeks, and the CMS schedule was performed continuously. The control group and CMS/control group were given 10 ml/kg distilled water daily.

Sucrose preference test

Intake and preference for sucrose solutions are the exact measures of anhedonia commonly used and accepted in the CMS literature (Gronli et al. 2006; Baker et al. 2006; Jayatissa et al. 2006). In the present study, we used the one-bottle sucrose intake test (with only sucrose solution available). Before each test, the animals were deprived of food and water for 14h. The rats were presented with a 1% (w/v) sucrose solution for 1 h, and consumption was measured by comparing the bottle weight before and after the test (Willner et al. 1987). The sucrose preference test consisted of training and testing trials (An et al. 2008). The training procedure also served as the baseline test before the start of the CMS protocol. Subsequently, sucrose consumption was monitored weekly under similar conditions throughout the entire experiment.

Measurement of locomotor activity

Locomotor activity was assessed by the open field test 2 weeks after drug administration (the fifth week of the entire experiment). Rats were individually placed in the center of the box (50 cm x 55 cm x 50 cm), and the travelled distance of 5 min by the rats was scored by ZH-QPT Analytic System.

Hippocampus weight analysis

After the final sucrose preference test, all animals were decapitated according to Ren et al. (2008). The hippocampus and the remaining cerebrum were rapidly removed, frozen with liquid nitrogen, put into a centrifuge tube, and weighed; the percentage of the hippocampus relative to the cerebral tissue was calculated. Possible antidepressant mechanism of SCLPN involving monoamine neurotransmitters

L-DOPA-induced running behavior in mice

The L-DOPA-induced locomotor activity was assessed according to the method of Serra et al. (1990). Male mice were divided into three groups and orally received distilled water (n = 19) or SCLPN (30 mg/kg, n =19 and 100 mg/kg, n = 17) every day for 1 week. Thirty minutes after the last drug administration, the mice were intraperitoneally treated with l-DOPA (200 mg/kg) and were then placed in the open-field box. Locomotor activity was videotaped for 5 min to determine the distance travelled.

Clonidine-induced aggressive behavior in mice

Clonidine-induced aggression in mice was assessed according to Morpurgo (1968). This procedure was used to reveal whether the noradrenergic system is involved in the antidepressant-like effects of SCLPN. Briefly, male mice were divided into four groups (n = 12) and orally treated with distilled water, Maprotiline (18 mg/kg), and SCLPN (30 and 100 mg/kg) for 1 week. Thirty minutes after the last administration, clonidine (50 mg/kg) was intraperitoneaHy administered to each animal. Every two mice of the same sex from the same group were then paired and placed in a 30 cm x 18 cm x 16 cm plastic cage. A blind observer recorded the biting/fighting episodes during a 30 min period.

5-HTP-induced head-twitch test

To investigate the possible involvement of serotonergic mechanisms in the antidepressant-like effects of SCLPN, we performed the 5-HTP-induced head-twitch test (Corne et al. 1963). Mice orally received distilled water (n = 9), SCLPN (30 mg/kg, n = 9 and 100 mg/kg, n = 10), and Anafranil (30mg/kg, n = 9) for 1 week. Thirty minutesafterthe lastadmtrxistration, 5-HTP (100 mg/kg) was intraperitoneally administered to animals. Immediately after the injection, mice were placed into cages, and the cumulative number of head twitches was recorded by a blind observer during a 30 min period.

Possible antidepressant mechanism of SCLPN involving cytoplasmic free intracellular [Ca.sup.2+] levels

Intracellular [Ca.sup.2+] ([[Ca.sup.2+]] i) measurements were performed on cultured cortical cells using the fluorescent dye Fluo-3/AM as previously reported (Kwan et al. 2000). Briefly, the cells were grown in culture medium on 12-well tissue plates at 37[degrees]C. After 7-11 days in culture, the cells were washed in normal physiological saline solution (NPSS; 140 mM NaCI, 1 mM KC1, 1 mM Mg[Cl.sub.2], 1 mM Ca[Cl.sup.2], 5mM HEPES, 10 mM glucose, and 0.1% bovine serum albumin, pH 7.4) and incubated in NPSS containing 10 uM Fluo-3/AM for 1 h in the dark at room temperature. The cells were then washed three times in new NPSS and placed in a special chamber containing NPSS for [[Ca.sup.2+]] i measurement. The fluorescence signal was monitored and recorded by a Laser Scanning Confocal Imaging System (TCS SP2; Leica Microsystems, Wetzlar, Germany) with excitation at 488 nm and acquirement at 525-545 nm. The change in fluorescence intensity after drug treatments was normalized to the initial intensity.

Data acquisition and analysis

All data were analyzed by Origin 7.5 software (Microcal Software, Piscataway, NJ, USA). The results are expressed as mean [+ or -] SD. Significant differences between groups were evaluated using oneway analysis of variance (ANOVA) followed by the Tukey post hoc test. Values of p < 0.05 were considered statistically significant.

Results

Effect ofSCLPN treatment in mice in the forced swim test

Effect ofSCLPN on locomotor activity in mice

The effects of SCLPN (10, 30, 100, 300, and '1000 mg/kg) on locomotor activity on days 1, 7, and 14 are depicted in Table 1. One-way ANOVA indicated no significant differences in locomotor activity between the experimental groups on day 1, However, on day 7, SCLPN at doses of 30, 100,300, and 1000 mg/kg significantly decreased locomotor activity by 26.5%, 21.2%, 26.1%, and 15.6%, respectively, compared with the control group (p < 0.05). This effect was comparable to that of the positive control drug, St. John*s Wort (100 mg/kg), which reduced locomotor activity by 33.7% compared with controls (p < 0.05). On day 14, one-way ANOVA revealed that only the 300 mg/kg dose ofSCLPN significantly reduced locomotor activity (by 32.9%) compared with the control group (p < 0.05). The other doses ofSCLPN and the positive control drug had no effect in the open-field test.
Table 1

Effect of SCLPN at different concentration (10,30.100,300,1000mg/kg) on
locomotor activity and the forced swim test in mice.The data are
presented as mean [+ or -] SD. * p > 0.05, n = 9 per group, compared
with corresponding control (one-way ANOVA followed byTukey post hoc
test).

                  Group      Day 1      Day 7    Day 14

Locomotor       Control    74.3 [+    75.6 [+   70.5 [+
activity                        or         or        or
                           -]21.78    -]15.32   -]18.29

               SCLPN l0    68.7 [+    70.3 [+   65.0 [+
                  mg/kg      or -]         or        or
                             20.32    -]11.52   -]22.46

               SCLPN 30    65.3 [+    55.6 [+   52.3 [+
                  mg/kg      or -]         or     or -]
                             19.11   -]29.87'     18.7)

              SCLPN l00    66.4 [+    59.6 [+   54.5 [+
                  mg/kg      or -]         or        or
                             13.82   -]25.38'   -]14.94

              SCLPN 300    63.3 [+    55.9 [+   47.3 [+
                  mg/kg      or -]         or     or -]
                             11.97  -]24.33 *    15.68'

             SCLPN l000    69.4 [+     63.9[+   65.0 [+
                  mg/kg      or -]         or     or -]
                             21.91  -]25.10 *     12.43

             St. John's    65.9 [+    50.1 [+   60.4 [+
               wort 100       or -]         or     or -]
                  mg/kg       23.92   -]20.98'     13.33

The             Control   122.0 [+   136.3 [+  152.9 [+
immobility                   or -]         or     or -]
time (s)                     10.67    -]11-62     11.36

               SCLPN 10   132.3 [+    113.2[+  125.1 [+
                  mg/kg      or -]      or -]        or
                             16.63      12.81    -]2.78

               SCLPN 30   116.8 [+  1 13.1 [+  130.4 [+
                  mg/kg      or -]         or        or
                             15.55   -] 17.28    -]9.51

                  SCLPN    97.7 [+    95.7 [+  115.7 [+
              100 mg/kg      or -]      or -]     or -]
                             11.41    10.75 *   10.57 *

                  SCLPN    88.4 [+    96.9 [+  114.8 [+
              300 mg/kg         or      or -]     or -]
                         -]15.02 *    10.81 *   13.06 *

                  SCLPN  89.3[+ or    89.8 [+  107.8 [+
             1000 mg/kg         -]      or -]     or -]
                             8.07'    10.93 *   15.26 *

            Maprotiline    83.9 [+    99.0 [+  106.1 [+
               18 mg/kg      or -]         or     or -]
                            9.79 *   -]9.42 *   11.81 *

             Paroxetine    93.1 [+    96.2 [+  114.1 [+
                5 mg/kg         or         or     or -]
                          -]7.51 *  -]10.27 *    6.83 *

             St. John's  92.5[+ or   101.6 [+  120.2 [+
               wort 100         -]      or -]     or -]
                  mg/kg     13.14'      5.7 *    6.63 *


Effect ofSCLPN on immobility time in the forced swim test

The antidepressant effects ofSCLPN administration once daily for 1, 7, and 14 days were evaluated in the FST in mice. As shown in Table 1, on day 1, SCLPN at doses of 300 and 1000 mg/kg significantly reduced the immobility time, resulting in a 27.6% and 26.8% immobility reduction compared with the control group (p < 0.05). The three antidepressants Maprotiline (18 mg/kg). Paroxetine (6 mg/kg) and St. John's Wort (100 mg/kg), reduced the duration of immobility by 31.3%, 23.7%, and 24.2%, respectively (p < 0.05). After 7 days of SCLPN administration (100, 300, and 1000 mg/kg), immobility time in the FSTdecreased by 29.8%, 28.9%, and 34.1%, respectively, compared with controls (p < 0.05). The three positive control drugs reduced immobility time by 27.3%, 29.4%, and 25.5%, respectively (p < 0.05). On day 14, SCLPN at doses of 100, 300, and 1000 mg/kg also significantly decreased immobility time by 24.3%, 24.9%, and 29.5%, respectively, compared with controls (p < 0.05), which was similar to the effects of the three classical antidepressants (30.6%, 25.4%, and 21.4%, respectively; p < 0.05 compared with controls).

[FIGURE 2 OMITTED]

Effect ofSCLPN treatment in the chronic mild stress model in rats

During the 3 consecutive weeks of CMS, the body weights of the rats were measured weekly. The average body weight of the CMS groups was less than that of the control group, but did not showed significantly different. However, CMS exposure resulted in behavioral changes related to major depression, including decreased aggressive and investigative behaviors. Control rats did not exhibit such behaviors.

Effect ofSCLPN on sucrose consumption

As shown in Fig. 2A, no difference in sucrose consumption was observed among groups before the CMS procedure. However, after 3 weeks of CMS (week 0), the reduction in sucrose consumption was considerably greater in the CMS group (28.8%) than in the control group (p < 0.05). After one week treatment with 70 mg/kg SCLPN, sucrose consumption increased by 27.4% compared with the CMS/vehicle group (p < 0.05). Treatment with 1.8mg/kg fluoxetine increased sucrose consumption by 22.7% compared with the CMS/vehicle group (p < 0.05). After two weeks treatment with 70 mg/kg SCLPN and 1.8 mg/kg fluoxetine, sucrose preference increased by 29.0% and 32.5%, respectively, compared with the CMS/vehicle group (p < 0.05). Four weeks of treatment with 70 mg/kg SCLPN increased sucrose consumption by 16.5%, while fluoxetine (1.8 mg/kg) increased sucrose consumption by 19.5% compared with the CMS/vehicle group (p < 0.05).

Effect ofSCLPN on locomotor activity

Locomotor activity was assessed using the open-field test (Fig. 2B). The travelled distance by the CMS/vehicle group was significantly reduced to 650 [+ or -] 95.6 cm after 4 weeks of CMS exposure, whereas rats treated with 70 mg/kg SCLPN travelled 1020 [+ or -] 130.5 cm, indicating that SCLPN increased locomotor activity by 56.9% compared with the CMS/vehicle group (p < 0.05). However, The CMS/Fluoxetine group (1.8mg/kg) only showed a 50.2% increase in locomotor activity compared with the CMS/vehicle group (p < 0.05).

Effect ofSCLPN on hippocampal atrophy

CMS induced a significant decrease in the percentage of the hippocampus relative to cerebrum tissue (8.9 [+ or -] 0.55% compared with 10.3 [+ or -] 0.62% in control rats). Four weeks of SCLPN treatment returned the hippocampal percentage to 9.6 [+ or -] 0.46%, whereas fluoxetine treatment returned the percentage to 10.2 [+ or -] 0.42%, which was shown a significant increase compared with the CMS/vehicle group (p < 0.05).

Effect of SCLPN on three behavioral models involving monoamine neurotransmitters

Effect of SCLPN on t-DOPA-induced locomotor activity

The effects of SCLPN on L-DOPA-induced locomotor activity are shown in Fig. 3A. One-way ANOVA revealed that 1 week treatment with SCLPN (30 and l00mg/kg) increased locomotor activity induced by l-DOPA (200 mg/kg) by 65.1% and 64.6%, respectively, compared with control mice (p < 0.05), indicating that SCLPN increased hyperactivity induced by l-DOPA.

Effect of SCLPN on clonidine-induced aggressive behavior

The number of fighting bouts in the control group was 19.8 [+ or -] 3.28, while the SCLPN at 30 mg/kg and 100 mg/kg decreased the fighting number to 14.0 [+ or -] 2.22 and 8.2 [+ or -] 2.81, respectively. Maprotiline (18 mg/kg) had the more effective, which decreased the number to 7.5 [+ or -] 0.76. One-way ANOVA revealed significant differences in the number of bouts between experimental groups. SCLPN (100 mg/kg) significantly decreased the number of bouts by 58.8% compared with controls. Maprotiline (18 mg/kg) decreased the number of bouts by 62.2% compared with controls (p < 0.05). Thus, the antidepressant activity of SCLPN may also involve the adrenergic system.

Effect of SCLPN on the 5-HTP-induced head-twitch response

Fig. 3B illustrates the effects of SCLPN and Anafranil on 5-HTP-induced head twitches in mice. Treatment with 30 and 100 mg/kg SCLPN induced bidirectional effects on head twitches. The 30 mg/kg dose increased the number of head twitches by 101.8% significantly compared with controls (p < 0.05). The 100 mg/kg dose decreased the number of head twitches by 52.5% compared with controls (p > 0.05). Anafranil no selectively inhibited 5-hydroxytryptamine (5-HT) reuptake at the presynaptic membrane and significantly increased 5-HTP-induced head twitches by 80.0% compared with controls (p < 0.05).

[FIGURE 3 OMITTED]

Effect ofSCLPN on [[Ca.sup.2+]] i

After bathing cortical neurons in NPSS, the basal levels of neurons were normalized to 100%. SCLPN (0,125 mg/ml) significantly decreased basal [[Ca.sup.2+]] i; from 100% to 85%. The addition of sodium glutamate (100 p.,M) significantly increased [[Ca.sup.2+]] i, (Fig. 4). A previous report demonstrated that the components of SCLPN blocked calcium over-influx into neuronal cells and reduced glutamate excitotoxicity in neurons (Radad et al. 2004). These results suggest that SCLPN may be able to reduce basal [[Ca.sup.2+]] i, levels but cannot fully antagonize the excitotoxicity influx of calcium induced by glutamate.

[FIGURE 4 OMITTED]

Discussion

SCLPN, the total saponins extracted and purified from the caudexes and leaves of P. notoginseng, consists of ginsenosides and notoginsenosides. In the current study, we assessed the possible antidepressant-like effects of SCLPN in the FST and CMS model in mice and rats.

SCLPN has antidepressant-like effects in the forced swim test and chronic mild stress model

SCLPN significantly reduced immobility time in the FST, reduced sucrose preference and reversed the decrease in locomotor activity in rats subjected to CMS. Based on the behavioral results above with both the acute and chronic models, we can conclude that SCLPN has antidepressant-like activity. Interestingly, previous studies with P. ginseng and our findings with P. notoginseng have produced different results. Einat (2007) reported that a ginseng extract (500mg/kg/day) showed no significant effect in the FST but reduced spontaneous locomotor activity. However, Dang et al. (2009) confirmed that the ginseng total saponins at doses of 25-100 mg/kg had a marked antidepressant-like action in the FST but did not produce significant effects on exploratory activity in normal mice in the open-field test. This discrepancy may be because that the biological effects of saponins are related to their structures when isolated from American ginseng ( Panax quin-quefolius), Asian ginseng (Panaxginseng), and Sanqi ginseng (Panax notoginseng). The panaxadiols (Rbi, Rb2, Re, Rd, Rg3, Rh2, and Rh3) are sometimes referred to as the Rb group and show tranquiliz-ing effects on the central nervous system (Sengupta et al. 2004). The panaxatriols (Re, Rf, [Rg.sub.1], [Rg.sup.2], and [Rh.sup.1] ) are dominated by the presence of Rg1, which possesses excitation effects on the central nervous system, antifatigue effects, and hemolysis (Li and Fitzloff 2002; Shen 2000; Lian et al. 2006). However, the samples from each report contained the different percent of ginsenosides and components. Correspondingly, oral administration of P. notoginseng dry root extract showed a significant increase in total locomotor activity, but the observed effects did not appear to be dose-related (Cicero et al. 2000), in contrast to our results for locomotor activity. Therefore, chemical characteristics are very important for the pharmacological action of the different ginsengs, and separating the different types of saponins is valuable for elucidating their respective pharmacological effects.

SCLPN may exert its antidepressant effect through a monoaminergic mechanism

Although the biochemical basis of depression is still uncertain, most of the currently available antidepressant drugs act through monoamines, such as norepinephrine, dopamine, and serotonin (Hamon and Bourgoin 2006).

Serotonin is involved in the regulation of food intake, mood, fear, sleep, reproduction, and hypothalamic-pituitary-adrenal axis activity, all of which are known to be disturbed in psychiatric disorders (Linthorst and Reul 2008). In the present study, pretreatment with 30 mg/kg SCLPN for 7 days significantly increased the number of head twitches, similar to the positive control drug, indicating an increase in serotonergic activity in vivo. However, SCLPN at a higher dose (100 mg/kg) decreased the number of head twitches by nearly half. The high concentration of SCLPN may exert its effects on the serotonergic system through a more complicated mechanism compared with the lower concentration. A previous study reported that duloxetine at doses of 3.8, 7.7, and 15.3 mg/kg increased extracellular levels of both serotonin and norepinephrine. The maximal increase in serotonin release was achieved at lower doses (Sanchez et al. 2007). These results reflect that serotonin involves a complex system of 16 receptor subtypes (Jans et al. 2007), and the precise regulation of serotonin release remains unclear.

Moreover, selective dopamine and norepinephrine reuptake inhibitors have proven antidepressant efficacy in the treatment of depression (Clausius et al. 2009). In the present study, chronic administration of SCLPN at a dose of 100 mg/kg for 1 week significantly reduced clonidine-induced aggressive behaviors, indicating increased activity of the noradrenergic system. SCLPN at doses of either 30 or 100 mg/kg enhanced locomotor activity in mice, indicating an effect of SCLPN on the dopaminergic system. These results are consistent with previous reports demonstrating that dopamine and norepinephrine levels were increased by ginsenosides in the cerebral cortex in rodents (Itoh et al. 1989; Dang et al. 2009). The antidepressant-like effect of SCLPN might also involve functional stimulation of dopaminergic pathways ^s well as noradrenergic systems.

The antidepressant-like effect of SCLPN may involve regulation of [[Ca.sup.2+]]i

Evidence has also accumulated demonstrating the involvement of [[Ca.sup.2+]]i in the pathophysiology of mood disorders (Galeotti et al. 2006). The regulation of free [[Ca.sup.2+]]i is a complex, mul-tifaceted process regulated by various mechanisms related to physiological function. Our results with the fluorescence dying of cultured cortical neurons indicated that SCLPN dawnregulates [[Ca.sup.2+]]i concentration, implying that the mechanisms underlying SCLPN's effects may involve neurotransmitter mediation of calcium concentration. Ginsenoside Rb3, the main component of SCLPN in the present study, was recently shown to inhibit the influx of calcium current in rat hippocampal neurons induced by N-methyl-D-aspartate (NMDA; Sheng et al. 2008). Ginsenoside Rb3 also protected ischemic neurons by inhibiting persistent sodium currents in rat hippocampal neurons (Jiang et al. 2007). Previous research demonstrated that SCLPN was a calcium channel blocker in neurons and can block [[Ca.sup.2+]]i overload and [[Ca.sup.2+]]i caImodulin complex production in nerve cells after cerebral injury, thereby protecting the injured brain (Han et al. 1999; Ma et al. 1997; Xu et al. 2003). SCLPN-mediated inhibition of [[Ca.sup.2+]]i elevations could be the basis of in vitro or in vivo protection against excitatory amino acid - or neurotoxin-induced neuronal cell damage.

The antidepressant-like effect ofSCLPN may involve neurotrophy and neurogenesis

Many reports have suggested that alterations in hippocampal function and reductions in hippocampal volume may be involved in the etiology of depression (Saylam et al. 2006; Warner-Schmidt and Duman 2006; Iritani et al. 2006). Several factors can contribute to this atrophy, including stress, with mechanisms involving glucocorticoids, serotonin, and excitatory amino acids (Dhikav and Anand 2007). According to the present results, SCLPN may significantly recover hippocampal volume, an effect that is similar to that of fluoxetine. Previous studies found that SCLPN increased the expression of BDNF, which plays a critical role in hippocampal neurogenesis (Cheng et al. 2005; Amaral and Pozzo-Miller 2007; Slikker et al. 2007). The reduction in hippocampal volume is attributable to decreased neurogenesis, death or atrophy of existing neurons, changes in neuropil, the number of synapses, and synaptic bulk (Sapolsky 2000; Kempermann 2001). We therefore speculate that the recovery of hippocampal atrophy by SCLPN involves BDNF. Panax Notoginseng treatment can promote neuronal plasticity (Guo et al. 2003) and enhance the expression of Nestin and BDNF (Wang et al. 2007) following focal cerebral ischemia. In in vitro studies, gin-senosides have been reported to increase the survival of cultured neuronal cells and enhance neurite outgrowth (Radad et al. 2004). Panax notoginseng and Panax ginseng have been used for nourishment and tonicity (Shen 2000; Ng 2006), and this functional aspect may be related to SCLPN's antidepressant-like effects.

In summary, the present study confirmed the antidepressant-like effects of SCLPN in the FST and CMS model and demonstrated that SCLPN may exert these effects through a monoamine neurotransmitter mechanism. SCLPN, the total saponins from Panax notoginseng, may be an effective antidepressant through neurotransmitter modulation and its neuroprotective effects on neurotrophy and neurogenesis via multiple pathways. Future studies should clarify the distinct functions of each component of SCLPN on the central nervous system and identify the neuronal receptors responsible for its effects.

Acknowledgements

This work was supported by grants from the Guangdong Science-Tech Program (2001331004202516 and 2007B031400001), Cooperation of Industry, Education and Academy between Gongdong Province Government - Education Department of Chinese Government (2008B090500028) National Talented Person Foundation of China (J0730638), Science Foundation of Life Sciences School, and Opening Laboratory Fund, Sun Yat-Sen University.

0944-7113/$ - see front matter[C] 2010 Elsevier GmbH. All rights reserved.

doi: 10.1016/j.phymed.2010.11.014

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Hui Xiang (a), *, Yingxue Liu (a) (1), BaibingZhang (a) (1), Junhao Huang (a) (b), Yi Li (a), Bing Yang (b), Zhangxin Huang (b), Feijun Xiang (b), Hualin Zhang (b)

* Corresponding author. Tel.: +86 20 84115583; fax: +86 20 84115583. E-mail addresses: malhoorP163.com, lssxh@mail.sysu.edu (H. Xiang).

(1) These authors contributed equally to this work.

(a) Department of Biological Science and Technology, School of Life Sciences, Sun Yat-Sen University, 135 XingangXi Road, Guangzhou 510275, Guangdong Province, People's Republic of China

(b) Guangdong Medi-World Pharmaceutical Co, Ltd, Foshan 528305, Guangdong Province, People's Republic of China
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Author:Xiang, Hui; Liu, Yingxue; Zhang, Baibing; Huang, Junhao; Li, Yi; Yang, Bing; Huang, Zhangxin; Xiang,
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Geographic Code:9CHIN
Date:Jun 15, 2011
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