Effects of extracts and neferine from the embryo of Nelumbo nucifera seeds on the central nervous system.

Abstract

The effects of embryos of the seeds of Nelumbo nucifera on the central nervous system were studied in mice. MeOH extracts of embryos of Nelumbo nucifera seeds significantly inhibited locomotor activity in mice. The MeOH extract was successively partitioned between[H.sub.2]O and n-hexane, between [H.sub.2]O and CH[Cl.sub.3], and between [H.sub.2]O and n-BuOH. CH[Cl.sub.3] extracts strongly inhibited locomotor activity in mice, although other extracts had no effect on locomotor activity. The main alkaloid of CH[Cl.sub.3] extracts, neferine, dose-dependently inhibited locomotor activity in mice. Neferine induced hypothermia in mice and apparently potentiated thiopental-induced sleeping time. An anxiolytic, diazepam, decreased locomotor activity, rectal temperature and enhanced sleep elicited by thiopental, similar to neferine. In addition, neferine and diazepam showed anti-anxiety effects in the elevated plus maze test. Neferine did not affect muscle coordination by the rota-rod test. Neferine did not affect strychnine- nor picrotoxin-induced seizure. In contrast, diazepam had apparent muscle relaxant and anti-convulsant effects. These results suggest that neferine has several central effects and that neferine may participate in the efficacy of the sedative effects of embryos of the seeds of Nelumbo nucifera. The mechanisms of the sedative effects of neferine are not similar to those of diazepam.

[c] 2008 Elsevier GmbH. All rights reserved.

Keywords: Nelumbo nucifera; Bisbenzylisoquinoline alkaloids; Neferine; Sedation; Behavior; Central nervous system

Introduction

"Lian zi xin", embryo loti (embryo of the seeds of Nelumbo nucifera Gaertner, Nymphaeaceae), has been used in Chinese traditional medicine as a sedative, antipyretic and hemostat agent (Chiang Su New Medical College, 1978), indicating that it may possess central effects. A previous study reported that bisbenzylisoquinoline alkaloids, such as neferine and liensinine, were isolated from this plant (Furukawa, 1966); however, the central effects of the embryo of the seeds of Nelumbo nucifera have not been characterized. Furthermore, it has not been clarified whether the alkaloid involved in this plant contributes to the central effects.

Out of interest in the sedative activity of this herbal medicine, we reexamined the chemical constituents of the embryo of the seeds of Nelumbo nucifera Gaertner and examined the locomotor activity effects of various extracts of embryo loti in mice. Furthermore, to clarify the central effects, we examined the effects of neferine, which is the main alkaloid of CH[Cl.sub.3] extracts, on locomotor activity, rectal temperature, thiopental-induced sleeping, muscle coordination and drug-induced seizures. Furthermore, we examined the effects of nerefine in the elevated-plus maze test to evaluate antianxiety effects.

Material and methods

General experimental procedures

Optical rotations were measured on a JASCO DIP-370 digital polarimeter (Japan). UV spectra were recorded on a Shimadzu UV-2500PC spectrophotometer (Shimadzu, Japan) and TR spectra on a Shimadzu FTTR-8200 spectrophotometer (Japan). [.sup.1]H (500 MHz) and [.sup.13]C (125 MHz) NMR spectra were recorded on a Varian VXR-500 spectrometer (USA) with TMS as an internal standard. Mass spectrometry was obtained with a Hitachi M-4100 mass spectrometer (Japan). Glycerol was used as the matrix for SIMS. TLC was performed on precoated Kieselgel [60F.sub.254] plates (Merck, Germany).

Plant materials

Embryos of the seeds of Nelumbo nucifera Gaertner were purchased from Longhua Hospital Shanghai University of Traditional Chinese Medicine in Shanghai, China. A voucher specimen was deposited in the laboratory of Kobe Pharmaceutical University.

Extraction and isolation

Embryos of the seeds of Nelumbo nucifera (1200 g) were extracted with hot MeOH. The extracts were evaporated in vacuo and the resulting residue (232.6 g) was resuspended in H2O and extracted successively with n-hexane, [CHCl.sub.3] and n-BuOH. The extraction diagram is shown in Fig. 1.

[FIGURE 1 OMITTED]

The residue from the CH[Cl.sub.3] layers (8.0 g) was purified by a combination of silica gel CC (CH[Cl.sub.3]-MeOH) and preparative TLC (CH[Cl.sub.3]-MeOH, 19:1, 9:1; CH[Cl.sub.3]-MeOH-[NH.sub.4]OH, 95:4.5:0.5, 90:9:1; [C.sub.6][H.sub.6]-AcOEt-[Et.sub.2]NH, 7:2:1) to afford pronuciferine (33.7 mg), thalifoline (6.7 mg), neferine (2.99 g, Fig. 2), liensinine (57.4 mg) and isoliensinine (20.3 mg). The isolated compounds were identified by comparison of their spectral data with those described in the literature.

[FIGURE 2 OMITTED]

Animals

Male ICR mice weighting 25-30 g were purchased from SLC Japan (Japan). Mice had free access to food and water, and were maintained on a 12h dark/light cycle in a room with controlled temperature (23 [+ or -] 1 [degrees] C) and humidity (55 [+ or -] 5%). Experiments were performed in accordance with the Guiding Principles for Care and Use of Laboratory Animals approved by The Japanese Pharmacological Society.

Treatment of extracts and neferine

Extracts of embryos of the seeds of Nelumbo nucifera were suspended in 1% carboxylmethyl cellulose-Na and administered p.o. Neferine HCl was dissolved in Saline and given i.p. or p.o.

Drugs and treatment

Diazepam and thiopental Na (Wako. Japan) were dissolved in saline and injected i.p. Strychnine nitrate (Wako, Japan) or picrotoxin (Wako, Japan) was dissolved in saline and given s.c.

Lacomotor activity

The lacomotor activity of animals during the testing period was counted by an activity sensor (NS-AS01, Neuroscience Inc., Japan). Experiments were performed 60 min after administration of extracts or drugs.

Rectal temperature

Rectal temperature was measured by a thermometer (BAT-12, Sensortek, USA). The thermister probe was inserted 2 cm into the rectum.

Thiopental Na-induced sleep

Thiopental Na at 60 mg/kg was injected i.p. 15 min after the administration of neferine or diazepam. The onset time and duration of loss of the righting reflex were recorded.

Elevated plus maze test

The elevated plus maze (method of according to Pellow et al., 1985; Lister, 1987) consisted of two closed arms (10 x 50 x 40 cm) and two open arms (10 x 50 cm) emanating from a common central platform (10 x 10 cm). The closed and open arms were arranged opposite to each other. Thirty minutes after the injection of neferine or diazepam, the mouse was placed at the central platform of the maze with its facing an open arm and allowed to explore the maze for 5 min. Entry into each arm was defined as placement of four paws into an arm. Number of entries into each type of arm, the percentage of time spent and the percent of arm entries in open arms were recorded.

Rota-rod test

The integrity of motor coordination was assessed with a rota-rod apparatus (Ugo Basile, Italy) at a rotating speed of 8 rpm, by counting the number of falls from the rod in 3 min.

Strychnine-and picrotoxin-induced convulsion 

Mice were treated with neferine or diazepam before the administration of strychnine (2mg/kg, s.c.) or picrotoxin (5mg/kg, s.c.). The death time and latency of first episode of clonic seizure were recorded. The cutoff time was set as 60 min after the convulsant administration.

Statistics

Dose-related effects were evaluated by one-way analysis of variance (ANOVA) followed by Dunnett's test. Significance between two groups was analyzed by Student's t-test. Analysis of data on rota-rod test was evaluated by [[chi].sup.2] test.

Results

Locomotor activity

As shown in Fig. 3, MeOH extracts of embryos of Nelumbo nucifera seeds significantly inhibited locomotor activity. Fig. 4 shows the effects of CH[Cl.sub.3], n-hexane, n-BuOH and water extracts on locomotor activity in mice. Only CH[Cl.sub.3] extracts showed apparent inhibition of locomotor activity.

[FIGURE 3 OMITTED]

[FIGURE 4 OMITTED]

Fig. 5 demonstrates the effect of neferine and diazepam on locomotor activity in mice. Neferine above the dosage of 50 mg/kg inhibited locomotor activity. Diazepam at 5 mg/kg decreased locomotor activity.

[FIGURE 5 OMITTED]

Rectal temperature

As shown in Fig. 6, neferine significantly decreased rectal temperature in mice. Diazepam also induced significant hypothermia in mice (Fig. 6).

[FIGURE 6 OMITTED]

Thiopental-induced sleeping

As shown in Fig. 7, neferine shortened the onset time of thiopental-elicited sleeping dose-dependently. Pretreatment with neferine remarkably prolonged the duration of sleeping induced by thiopental. Diazepam at 1 mg/kg also enhanced thiopental-induced sleeping.

[FIGURE 7 OMITTED]

Elevated plus maze test

As shown in Fig. 8, neferine and diazepam increased both the percent of open arm entry or time spent in open arms.

[FIGURE 8 OMITTED]

Motor coordination

In the rota-rod test, neferine (25-100 mg/kg) did not affect motor coordination, whereas diazepam impaired motor coordination in mice (Table 1).

Table 1. Effects of neferine and diazepam on motor coordination
evaluated by rots-rod test

Group              Motor incoordination

Saline                  0/6
Naferine 50mg/kg        0/6
Neferine 50mg/kg        0/6
Naferine 100mg/kg       0/6
Diazepam 5 mg/kg        5/6 **

Rota-rod test was performed 15 min after the injection of neferine or
diazepam. ** p < 0.01

Strychnine- and picrotoxin-induced convulsion in mice

Diazepam prolonged the latency of convulsion induced by strychnine and picrotoxin. Diazepam protected against death induced by picrotoxin and prolonged duration until death induced by strychnine (Table 2). Neferine at 50 and 100 mg/kg did not modify strychnine- and picrotoxin-induced convulsions (Table 2).

Table 2. Effects of neferine and diazepam on strychnine- and
picrotoxin-induced convulsion in mice

Group                    Latency (min)           Death (min)

Strychnine
  Saline               2.2 [+ or -]0.13       2.8 [+ or -] 0.18
  Neferine 50mg/kg     2.6 [+ or -]0.20       3.0 [+ or -] 0.21
  Neferine 100 mg/kg   2.8 [+ or -]0.22       3.3 [+ or -] 0.23
  Diazepam 1 mg/kg     3.2 [+ or -]0.24 *     5.4 [+ or -] 0.33 **

Picrotoxin
  Saline               9.3 [+ or -] 0.42     22.8 [+ or -] 1.41
  Neferine 50 mg/kg    9.6 [+ or -] 0.59     18.4 [+ or -] 2.09
  Neferine 100 mg/kg  10.5 [+ or -] 0.67     29.8 [+ or -] 2.09
  Diazepam 1 mg/kg    18.4 [+ or -] 2.02 **       > 60 **

Results are shown as mean [+ or -]S.E.M. (N = 5-9). Neferine and
diazepam were given i.p. 15 min before strycnine or pictrotoxin
Strychnine at 2 mg/kg and picrotoxin at 5 mg/kg were given s.c.
* p < 0.05 and ** p < 0.01

Discussion

In Chinese traditional medicine, embryos of the seeds of Nelumbo nucifera have been recognized to have depressant effects on the central nervous system, such as sedative activity, or to be effective against fever in humans; however now, detailed pharmacological examination of this plant and its effects on the central nervous system have not been performed. The present study system have not been performed. The present study demonstrates that plant extracts and neferine, the major alkaloid in the embryos of the seeds of Nelumbo nucifera, have apparent central effects.

MeOH extracts of embryos of the seeds of Nelumbo nucifera significantly inhibited locomotor activity. Furthermore, the effects of the partitioned extracts of MeOH, that is CH[Cl.sub.3), n-hexane, n-BuOH and [H.sub.3] extracts apparently reduced locomotor activity, whereas other extracts did not affect locomotion. This indicates that sedative elements may be present in CH[Cl.sub.3] extracts.

As a result of investigating alkaloids in CH[Cl.sub.3] extracts, several alkaloids were identified, including pronuciferine (Bernauer, 1963), thalifoline (Krane and Shamma, 1982), neferine (Furukawa, 1965), liensinine (Pan et al., 1962) and isoliensinine (Tomota et al., 1965) in this study. Thalifoline was isolated for the first time from this plant species. A previous report demonstrated that bisbenzylisoquinoline alkaloids, such as neferine and liensinine, were isolated from this plant (Furukawa, 1966), which is in agreement with the results of this study. Since neferine is a major alkaloid, the effects of neferine on locomotor activity in mice were studied.

Administration of neferine dose-dependently inhibited locomotor activity in mice. Neferine above 50 mg/kg induced significant effects and administration of 100 mg/kg elicited potent effects. Since embryos of the seeds of Nelumbo nucifera may be effective against fever, the effects of neferine on rectal temperature were studied in mice. Neferine induced significant hypothermia in mice. These results demonstrate for the first time that neferine actually induces sedation and hypothermia in mice, suggesting that neferine may contribute to the sedative and anti-fever effects of embryos of the seeds of Nelumbo nucifera.

An anxiolytic, diazepam, the most widely used benzodizaepine derivative, causes sedation and antianxiety effects by activation of benzodiazepine receptors. In the present study, we also found that diazepam injbited locomotor activity and induced hypothermia, similar to neferine. Neferine may therefore show pharmacological activity in the central nervous system, similar to diazepam; therefore we further studied the pharmacological effects of neferine on the central nervous system compared to those of diazepam.

Both neferine and diazepam prolonged thiopental-induced sleep in mice. The potentiation of neferine on thiopental-induced sleep further supports that neferine and diazepam showed anti-anxiety effects evaluated by the elevated plus maze test. Although diazepam displays sedative effects mediated by the benzodiazepine receptor it is well known to cause typical side effects and impairment of muscle coordination. Our results also demonstrate that in the rota-rod test, diazepam impairs motor coordination in mice; however, neferine did not affect motor coordination in this test. In addition, diazepam antagonized strychnine-and picrotoxin-induced convulsion, whereas neferine had on effect; therefore, the pharmacological profile of neferine is not the same as that of diazepam. The machanism of neferine may not relate to benzodiazepine receptors but to other neurotransmitter systems.

In summary, we demonstrated that CH[Cl.sub.3] extracts of the embryos of the seeds of Nelumbo nucifera show sedative effects. The major alkaloid neferine induced appartent sedation and hypothermia or anxiolytic effects in mice. The effects of neferine are therefore different from those of diazepam. The anti-hypertensive activity of neferine was characterized (Nishibe et al., 1986), and recently the inhibitory effects of isoliensinine on pulmonary fibrosis were reported (Xiao et al., 2005). Further pharmacological studies of alkaloids, including neferine, in the embryos of the embryos of the seeds of Nelumbo nucifera are now under investigation.

Acknowlegements

We thank Dr. Sugiura (Kobe Pharmaceutical University) for the NMR spectra and Dr K. Saiki (Kobe Pharmaceutical University) for the Ms measurements.

References

Bernauer, K., 1963. Pronuciferin, ein Benzylisochinolin-alkaloid mit para-Cycolhexadienon-Gruppierung. Helv. Chim. Acta 46, 1783-1785.

Chainag Su New Medical Collage (ED.), 1978. Zhong-yao-dai-ci-dain (Dicitionary of Chinese Crude Druges). Shamghai Scientific Technologic publisher, Shan ghai, p. 1806.

Furukawa, H., 1965. On the alkaloids of Nelumbo nucifrea Gaertn. IX. Alkaloids of loti embryo. (2). Structure of neferine, a new biscoclaurine alkaloid. Yakygaku Zasshi 85, 335-338.

Furukawa, H., 1966. Studies on the alkaloids of Melumbo nucifera Gaertn. NMR spetcra of liensinie type alkaloids Yakgaku Zesshi b86,883-886.

Krane, B.R., Shamma, M., 1982. The isoqunokine alkalioids. J. Nat. Prod. 45,377-383.

Lister, R.G., 1987. The use of a plus-maze anxiety in the mouse Psychopharmacology 93, 180-185.

Nishibe, S., Tsukamoto, H., Kinoshits, H., Kitagawa, S., Nelumbo nucifera. J. Nat. Prod. 49, 47-548.

Pan, p.-c., Chou, Y.-L., Sun, T.-T., Kao Y.-S., 1962. Studies on the alkloids of embryo loti, Nelumbo nusifera Gaertn. II. Structure of liensinine. Sci. Sin. 11, 321-336.

Pellow, S., Chopin, File, S.E., Briley, M., 1985. Validation of open:closed arm entries in an elevated plus maze as a measure of amxiety in the rat. J. Neurosci. Methds 14, 149-167.

Tomota, M., Furukawa, H., Yang, T.-H., Lin, T.-J., 1965. On the alkaloids of Nelumbo nucofera Gaertn. VIII. Studies on the alkoids of loti embryo. (1) Structure of isoliensinine, a new biscoclaurine type alkakoid. Chem. Pharm, Bull. 13, 39-43.

Xiao, J.-H., Zhang, J.-H., Chen, H.-L., Feng, X.-L., 2005. Inhibitory effects of isoliensinine on bleomycin-induced pulmonary fibrosis in mice. Planta Med. 71, 225-230.

Yumi Sugimoto (a), *, Sachiko Furutani (b), Atsuko Itoh (c), Takao Tanahashi (c), Hiroshi Nakajima(d), Hideo Oshiro (e), Shujian Sun(e), Jun Yamada (a)

(a) Laboratory of Pharmacology, Department of Clinical Pharmacy, Yokohama College of Pharmacy, Matano-cho, Totsuka-ku, Yokohama 245-0066, Japan

(b) Department of Pharmacology, Kobe Pharmaceutical University, Motoyamakita-machi, Higashianda-ku, Kobe 658-8558, Japan

(c) Department of Organic Chemistry, Kobe Pharmaceutical University, Motoyamakita-machi, Higashianda-ku, Kobe 658-8558, Japan

(d) Osaka City University, Research Center for Industry Innovation, Sugimoto, Sumiyoshi-ku, 558-8585 Osaka, Japan

(e) Shanghai University of Traditional Chinese Medicine, Japan 1-7-20, Nishitenma Kita-ku, Osaka, Japan

* Corresponding author. Tel.: + 81 45859 1300; fax: + 81 45859 1301.

E-mail address: [e-mail] (Y. Sugimoto).

0944-7113/$-see front matter [C] 2008 Elsevier GmbH. All rights reserved.

doi:10.1016/j.phymed.2008.09.005