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Induction of NGF synthesis in astrocytes by onjisaponins of Polygala tenuifolia, constituents of kampo (Japanese herbal) medicine, Ninjin-Yoei-To.

Summary

The effect of a kampo medicine, Ninjin-yoei-to (NYT; Ren-shen-yang-rong-tang in Chinese) on nerve growth factor (NGF) secretion from the cultured rat astrocytes was examined in vitro. When rat embryo astrocytes were cultured in the presence of NYT for 24 h, the amount of NGF in the medium was significantly increased in a dose dependent manner. Among 14 kinds of component herbs in NYT, the roots of Polygala tenuifolia and roots of Panax ginseng extracts increased NGF levels from the astrocytes. Saponin fraction from the roots of P. tenuifolia enhanced the production of NGF, however phenolic glycoside fraction showed no effect. Onjisaponins A, B, E, F and G as major saponins of the root of P. tenuifolia strongly increased the NGF level, whereas ginsenosides [Rb.sub.1] and [Rg.sub.1] did not affect the NGF level. Onjisaponin F also induced ChAT mRNA level in rat basal forebrain cells. These results indicate the possibility that NYT and/or onjisaponins in P. tenuifolia may have potential therapeutic effects for t he treatment of Alzheimer disease patients.

Key words: Polygala tenuifolia Willdenow, Polygalaceae, triterpene glycosides, onjisaponins, Ninjin-yoei-to, nerve growth factor

Introduction

Cholinergic neurons in basal forebrain innervate to the cortex and hippocampus, and may closely concern with the cognitive function and memory. It has been reported that Alzheimer disease (AD) and aging are associated with decreased levels of choline acetyltransferase (ChAT: EC2.3.1.6) activity and degeneration of cholinergic neurons in the central nervous system (Davis and Maloney,1976; Whitehouse et al., 1992; Bartus et al., 1982). Neurotrophins, such as nerve growth factor (NGF) and brain derived neurotrophic factor (BDNF), are responsible for the development and maintenance of basal forebrain cholinergic neurons (Thoenen, 1991). NGF has been shown to enhance the ChAT activity both in vitro and in vivo (Honegger and Lenoir, 1982; Seiler and Schwab, 1984; Gnahn et al., 1983) and improves some of the biological abnormalities that occur in disorders such as AD (Olson et al., 1992). However, there is a problem that peripheral administration of NGF has no effect in the central nervous system neuron, because NGF is a high molecular weight protein that can not pass through the blood-brain barrier. Therefore, the stimulating agents, which can pass through the blood brain barrier, may have a possibility of becoming a therapeutic agent for dementia.

One of kampo (Japanese herbal) medicines, Ninjin-Yoei-To (NYT) has been used traditionally for the treatment of patients with insomnia, anemia, amnesis, palpitation, and neurosis. Although NYT is suggested to play as an effective agent for the cholinergic nervous system (Egashira et al., 1996), action mechanism of NYT on central nervous system has not yet been studied.

In the present study, we examined the effect of NYT on NGF secretion from the cultured astrocytes in order to explore the possibility of NYT as a therapeutic agent for AD.

Materials and Methods

Materials

NGF 2.5S was purchased from Biomedical Technologies Inc. (USA). NYT extract preparation (TJ-108) was kindly supplied from Tsumura & Co. (Japan) as a spray dried powder. Medicinal plants used for preparation of kampo medicines were purchased from Uchida WakanYaku Co. Ltd. (Japan) and Tsumura & Co. Ginsenosides [Rb.sub1] and [Rg.sub.1] were purchased from Wako Chemical Co. Ltd. (Japan). Kami-untan-to (KUT: Jia-weiwen-dan-tang in Chinese) was prepared as previously described (Yabe and Yamada., 1996/1997). A voucher specimens of these plants were deposited at Oriental Medicine Research Center, the Kitasato Institute in Tokyo, Japan.

Preparation of extracts of component herbs in Ninjin-Yoei-to (NYT)

NYT was composed of the root of Panax ginseng Meyer (3 g), the root of Angelica acutiloba Kitagawa (4 g), the root of Rehmannia glutinosa Liboschitz var. purpurea Makino (4 g), the rhizome of Atraclylodes japonica Koizumi ex Kitamura (4 g), the scierotium of Poria cocos Wolf (4 g), the bark of the tank of Cinnamomum cassia Blume (2.5 g), the root of Polygala tenuifolia Willdenow (2 g), the root of Paeonia lactiflora Pallas (2 g), the pericarp of the ripe fruit of Citrus unshiu Markovich (2 g), the root of Astragalus membrancaeus Bunge (1.5 g), the root and stolon of Glycyrrhiza uralensis Fisher (1 g) and the fruit of Schisandra chinensis Baillon (1 g). The component herbs (5 g) in NYT were each decocted with 300 ml of water until the volume is reduced to half volume. Then the decoction was centrifuged at 7,000 rpm for 30 min, and the supernatant was lyophilised. Each lyophilised extract was dissolved in distilled water, and the solution was sterilized through the membrane filter (0.20 [micro]m) before using f or the assay. 3-dimensional HPLC analysis was carried out to know broad chemical profile of NYT (Fig. 1).

Fractionation of the roots of Polygala tenuifolia

The roots of P tenuifolia (50 g) was refluxed with MeOH (500 ml), and the resulting MeOH extract was partitioned between water (160 ml) and [Et.sub.2]O (80 ml). The [Et.sub.2]O layer was obtained as xanthon fraction. The water layer was then partitioned with BuOH (80 ml). The BuOH soluble fraction was further fractionated on Silica gel column (5 x 18 cm), and phenolic glycoside fraction and saponin fraction were obtained by eluting with EtOAc-MeOH (4:1) (1.5 1) and MeOH (2 1), respectively.

Preparation of onjisaponins

Onjisaponins were purified in according to the procedure of Sakuma et al. (198 La and b). Each onjisaponins were identified by structural analysis of presenegenin skeleton, component sugar analysis and comparison with authentic samples by HPLC on Senshu Pak [C.sub.6.H.sub.5] 3252-N (35% [CH.sub.3]CN containing 0.01% AcOH). In order to identify the presenegenin skeleton in onjisaponins, the purified saponins were saponified with 1M KOH, and tenuifolin was purified by silica gel chromatography using [CHCI.sub.3]-[MeOH-H.sub.2]O (7:3:1) (Sakuma et al., 1981a). Tenuifolin was identified by comparison of MS and NMR spectra with those of literature (Sakuma et al., 1981a). Sugar composition of each onjisaponin was analysed by GC using conventional TMS methylglycoside method (Park et al., 1986). To evaluate cell toxicity of onjisaponins, we analysed the effect of onjisaponins on haemolytic activity. All onjisaponins (A, B, E, F and G) did not show hemolytic activity up to 100 [micro]g/ml (Nagai et al., 2001).

Preparation of rat astrocytes and basal forebrain cells

Astrocytes and basal forebrain cells were cultured as previously described (Yabe and Yamada, 1996/1997). Astrocytes were used for assay after confluence was reached.

Determination of choline acetyltransferase (ChAT) activity

Rat embryo basal forebrain cells were washed with PBS and then solubilised in 210 [micro]1 of 50 mM Tris buffer (pH 6.8) containing 1% Triton X-100. The solubilisate was taken for the determination of ChAT activity according to the method of Fonnum (1975).

Determination of NGF

NGF content in culture medium of astroglial cells was determined by ELISA using affinity purified anti NGF polyclonal antibody (Yabe and Yamada. 1996/1997).

RT-PCR

RT-PCR analysis was performed as previously described (Yabe et al., 1997). The actual sequences of specific primers are as follows:
NGF (sense) 5'-GTAATGTCCATGTTG
 TTCTACACTCTG-3'

NGF (antisense) 5'-GGCAAGTCAGCCTCT
 TCTTGTTAGCCTTCC-3'
ChAT (sense) 5'-GGAGCCACCTGAGAT
 GTTCATGGAT-3'
ChAT (antisense) 5'-CACAGACGAGGCTCT
 TTGGCAGCT-3'
cyclophilin (sense) 5'-GGTCAACCCCACCGT
 GTCTTCGA-3'
cyclophilin (antisense) 5'-AACGGTTAGGTCGGT
 AAGTCAGAAC-3'


Statistics

Statistical significance of difference of means was performed ANO VA followed by Dunnett's post hoc procedure.

Results

Effect of NYT on NGF secretion and NGF mRNA expression in rat cultured astrocytes

Rat embryo astrocyte cultures were incubated over 24 h in the presence of NYT (25-100 [micro]g/ml), and the content of NGF in the medium was measured by the ELISA method. Fig. 2 shows that NYT (25-100 [micro]g/ml) significantly increased the amount of the NGF in the medium in a dose dependent manner as like the extract (200 [micro]g/ml) of another kampo medicine, Kami-untan-to (KUT), which has been reported as a potent activator of astroglial NGF secretion in vitro (Yabe and Yamada. 1996/1997). However, addition of NYT did not enhance the proliferation of astrocytes (data not shown). Furthermore, the level of NGF mRNA in the astrocytes was also increased by addition of NYT (Figs. 3A and B).

Effect of individual component herbs in NYT on NGF secretion of cultured astrocytes

In order to clarify the active ingredient in NYT, the effects of individual component herbs in NYT on NGF secretion were examined. The astrocytes were cultured in the presence of NYT (50 [micro]g/ml) or the individual extract of component herbs (50 [micro]g/ml) for 24 h at 37 [degrees]C. As shown in Fig. 4, the induced NGF secretion was observed in NYT, P tenuifolia extract (Pt) or P. ginseng extract (Pg) -treated cells. Especially, markedly induction of NGF synthesis was observed in Pt-treated cells. These results suggest that Pt and Pg extracts contain the active ingredients that induce NGF synthesis in astrocytes.

Effects of saponins on NGF secretion in astrocyte cultures

To identify the active ingredients in P. tenuifolia, the roots of P. tenuifolia was fractionated from the MeOH extract (see Materials and Methods). When xanthon fraction, phenolic glycoside fraction and saponin fraction from the root of P. tenuifolia were tested for NGF secretion from the astrocytes, only saponin fraction enhanced the NGF secretion from the cells (data not shown). Because onjisaponins were major saponins in the saponin fraction of P. tenuifolia (Fig. 5; Sakuma et al., 1981a and b), the effects of onjisaponins A, B, E, F and G on NGF secretion were tested. As shown in Fig. 6, onjisaponins A, B, E, F and G significantly increased NGF secretion from the cultured astrocytes. Especially, onjisaponins F and G showed the potent activity among the tested samples as similar as a positive control, KUT. In contrast to onjisaponins, ginsenosides [Rb.sub.1] and [Rg.sub.1], which are well known saponins from P. ginseng, had no effect on NGF secretion from the cultured astocytes (data not shown).

Effect of onjisaponin F on ChAT mRNA level in rat embryo basal forebrain cells

We have previously reported that another kampo formula, KUT, which contains roots of P. tenuifolia as a component herb, induced ChAT activity and ChAT mRNA expression in the rat embryo basal forebrain cells (Yabe and Yamada. 1996/1997). To confirm whether P. tenuifolia-containing kampo formulas have ChAT-inducing activity, we tested the effect of NYT on the ChAT activity in the basal forebrain cells. When the cells were cultured in the presence of NYT (100 or 200 [micro]g/ml) for 3 days at 37 [degrees]C, the ChAT activity of the cultured cells was significantly increased at 200 [micro]g/ml of NYT (Fig. 7A). When the cells were cultured in the presence of onjisaponin F (1 or 10 [micro]g/ml) for 6 h, ChAT mRNA was also increased in the treated cells as similar as a positive control, NGF (Fig. 7B). Similar results were obtained from other onjisaponins-treated cells but ginsenosides [Rb.sub.1] and [Rg.sub.1] showed no effect on ChAT activity (data not shown).

* Discussion

In the central nervous system (CNS), astrocytes play an important role during the development and maintenance of neuronal function (Shao and McCathy. 1994). Several researchers have reported the production of members of the neurotrophin family such as NGF, BDNF, and neurotrophin-3 in the cultured astroglial cell lines (Moretto et al., 1994; Oderfeld-Nowak et al., 1992). We have reported that a Kampo medicine, kamiuntan-to (KUT) and one of its component herbs, the root of P. tenuifolia increased the NGF secretion from the cultured astrocytes and NGF level in aged rat brain (Yabe et al., 1997). Oral administration of KUT improved memory and learning related behaviour in several dementia model animals (Yabe and Yamada, 1996/1997; Yabe et al., 1996), and induced ChAT activity in cortical cortex of aged rats (Yabe et al., 1996). KUT also increased number of ChAT-immunopositive cells of basal forebain in aged mice (Wang et al., 2000). Recently, Suzuki et al., reported that AD patients who took KUT for about one yea r improved their memory disturbance (Suzuki et al., 2001). These observations suggest the possibility that KUT and/or P. tenuifolia may have potential therapeutic effects for the treatment of AD patients. To explore this hypothesis, we studied the effect of other P. tenuifolia-containing kampo medicine, NYT, on NGF secretion from the cultured astrocytes. Because of no suitable clinically useful positive control is available, present study used KUT as a positive control for NGF secretion. As we expected, the enhanced NGF secretion and mRNA expression were observed in NYT and P tenuifolia-treated cells.

Studies on the chemical components of P. tenuifolia have proved the presence of polygalitol (Takiura et al., 1964), N-acetyl-D-glucosamine (Takiura et al., 1964), onjisaponins A-G (Sakuma et al., 1981a and b), various xanthones (Ito et al., 1977; Ikeya et al., 1991), tenuifolioses A-P (Miyase et al., 1991), tenuifoliosides A-D (Ikeya et al., 1991) and sucrose delivatives (Miyase et al., 1991). Although the pharmacological activities of these compounds have not been well studied, onjisaponins have been reported to show some pharmacological activities (Wang et al., 1994; Nagai et al., 2001). Present results demonstrated that onjisaponins, especially onjisaponins F and G potentlly induced NGF secretion from the cultured astrocytes, and that ChAT mRNA expression of the basal forebrain cells was increased in onjisaponin F treated cells.

Since UV absorbance of onjisaponins is relatively low, onjisaponins were not detected in present 3D-HPLC analysis (Fig. 1). However significant amount of onjisaponins were detected in the hot water extract of P. tenuifolia by HPLC analysis, and when sensitivity of photodiode detector was set up, onjisaponins were also detected in NYT (unpublished results). When the roots of P. tenuifolia was eliminated from the preparation of NYT or KUT, P. tenuifolia-eliminated NYT or KUT formula did not enhance NGF secretion from cultured astrocytes (Yabe et al., 1997 and unpublished results). Furthermore, when xanthon fraction, phenolic glycoside fraction and saponin fraction from the root of P. tenuifolia were tested for NGF secretion from the astrocytes, only saponin fraction enhanced the NGF secretion from the cells (data not shown). These observations suggest that onjisaponins are one group of active ingredients in P. tenuifolia-containing kampo medicines such as NYT and KUT. However further studies are needed to estim ate the significance of onjisaponins in NYT or KUT-induced NGF synthesis.

Among the component herbs of NYT, P. ginseng extract also induced NGF secretion from the cultured astrocytes (Fig. 4). It has been reported that P. ginseng extract increases the survival rate of nerve cells and the content of monoamines in the brain. For example, Rudakewich et al., reported that ginsenosides [Rb.sub.1] and [Rg.sub.1] have neurotrophic and neuroprotective actions against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)- or [beta]-amyloid-induced neuronal cell death. (Rudakewich et al., 2001). In contrary to our expectation, ginsenosides [Rb.sub.1] and [Rg.sub.1] had no effect against NGF secretion from the astrocytes and ChAT activity in the basal forebrain neurons. However we cannot exclude the possibility that other ingredients in P. ginseng can induce NGF synthesis. Further studies are needed to find active compounds in P. ginseng.

AD has emerged in the past decade as a major problem in public health. The etiology of the disorder has progressed, however, at present there is no established therapeutic modality for AD. Nabeshima and co-authors (Nabeshima et al., 1994; Nitta et al., 1994) have reported that oral administration of substances, which stimulate NGF synthesis in cultured astrocytes, recovers reduced brain NGF content in aged rats and cognitive dysfunction in basal forebrain lesioned rats. As well as these observations, we have reported that KUT, a strong NGF stimulator, improves cognitive function in aged rats (Yabe et al., 1996). Furthermore, Olson et al. (1992) reported that intraventricular infusion of NGF to AD patient resulted in a marked transient increase in uptake and binding of C-nicotine (11) in frontal and temporal cortex, a persistent increase in cortical blood flow and improvement of verbal episodic memory. These observations suggest that the use of the stimulators for the NGF synthesis may provide a therapeutic ap proach against cholinergic dysfunction in AD patients. The results demonstrated here, taken together with others in the literature, indicate the possibility that NYT and/or onjisaponins in P. tenuifolia may have potential therapeutic effects for the treatment of AD patients.

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Acknowledgements

We thank Dr. Kazuo Toriizuka for helpful discussions. We also thank Ms. Naoko Hattori and Dr. Iwao Sakakibara for 3D HPLC work. Part of the present work was supported by a Grant-in Aid for Scientific Reseach from the Ministry of Education, Science, Sports and Culture of Japan (Grant No. 600966691) and research funds from Uehara Memorial foundation.

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T. Yabe, (1,2) H. Tuchida, (1,3) H. Kiyohara, (1,2) T. Takeda, (3) and H. Yamada (1,2)

(1.) Oriental Medicine Research Center, The Kitasato Institute, Tokyo, Japan

(2.) Kitasato Institute for Life Sciences, Kitasato University, Tokyo, Japan

(3.) Kyoritsu College of Pharmacy Tokyo, Japan

* Address

H. Yamada, Kitasato Institute for Life Sciences, Kitasato University, 5-9-1, Shirokane, Minato-ku, Tokyo, 108-8641, Japan

Te.: ++81-3-5991-6364; Fax: ++81-3-3445-1351; e-mail: yamada@lisci.kitasato-u.ac.jp
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Title Annotation:nerve growth factor
Author:Yabe, T.; Tuchida, H.; Kiyohara, H.; Takeda, T.; Yamada, H.
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Geographic Code:1USA
Date:Mar 1, 2003
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