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. 2010 Oct 9;62(5):461–472. doi: 10.1007/s10616-010-9300-2

Enhancement of pheochromocytoma nerve cell growth by consecutive fractionization of Angelica gigas Nakai extracts

Myoung Hoon Jeong 1,2, Ji Seon Kim 1,2, Yunyun Zou 1,2, Chang Soon Yoon 3, Hye Won Lim 3, Juhee Ahn 1,2, Hyeon Yong Lee 1,2,✉
PMCID: PMC2993867  PMID: 20936343

Abstract

This work was to investigate the effect of flavonoids from Angelica gigas Nakai on the proliferation and differentiation of PC12 cells. Several solvents including hexane, chloroform, ethyl acetate, butanol and water consecutively partitioned. We determined the ethanol crude extract of Angelica gigas Nakai. The hexane fraction was shown to contain the highest number of flavonoids as follows; 21.48 mg/g and the composition of the flavonoids was as follows: 12.24 mg/g of quercetin, 4.39 mg/g of myricetin and 2.58 mg/g of catechin. In addition, this hexane fraction greatly increased both cell growth and outgrowth of the neurite, and whose effects were three times higher than those of the other fractions. The length of the neurites was measured as ca. 110 μm in adding 50 μg/mL of the hexane fraction, which was about the same as the case of adding 50 ng/mL of NGF as a positive control. This result indicates that the differentiation of PC12 cells by the addition of the hexane fraction was comparable to the case of adding NGF. The hexane fraction was also determined to prevent apoptosis of PC12 cells by suppressing DNA fragmentation. It is interesting that the mixture of three major flavonoids, quercetin, myricetin and catechin showed stronger activity on, both PC12 cell growth and neuritis outgrowth, than when adding each flavonoid alone. We believe this was due to the synergistic effects of the three flavonoids. The activities of these flavonoids from Angelica gigas Nakai are reported for the first time in this study.

Keywords: Angelica gigas Nakai, PC12 cell, Neurite outgrowth, Flavonoids

Introduction

Several efforts have been made to prevent nerve cell related diseases, such as Alzheimer’s and Parkinson’s. The prevalence of these diseases has recently greatly increased and are difficult to treat due to a lack of knowledge on the regeneration and/or outgrowth of nerve cells in vitro and in vivo (Selkoe 1991; Halliwell 1992; Smith et al. 1996; Fahn and Cohen 1992). Pheochromocytoma (PC12) cells have often been used as a model cell line to understand how to deal with nerve systems; this is because this sympathetic nerve cell secretes catecholamine, dopamine and norepinephrine, and its growth mechanisms are relatively well established (Oh et al. 2003). Thus, the effect of neurotrophic factors, such as the nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF), on the expression levels of neurotransmitters and/or the differentiation and outgrowth of nerve cells has been examined (Kazua et al. 1995; Martha 1999, 2000). These factors have shown to increase the concentration of dopamine by six times (Mudumba et al. 2002) and also induce the expression of 5-HT3 receptor, as well as the outgrowth of neurite’s and synaptic vesicles (Furukowa et al. 1992). In particular, BDNF improved the differentiation and proliferation of PC12 cells as well as promoted the outgrowth of neurite’s by activating tyrosinase kinase, which can activate neurotransmitting proteins and p21 Ras and MAP (Mitogen-activated protein) kinase (Vantini and Skaper 1992; Montalcini 1987). There are several neurotrophic factors that have structures similar to BDNF, includingthe nerve growth factor (NGF), neurotrophin-3 (NT-3) and neurotrophin-4/5 (NT-4/5). However, these neurotrophic factors are relatively expensive and require several steps for purification from mammalian cell culture due to low levels of secretion and heterogeneous culture systems (Miller 1959; Lindsay et al. 1994). Because of these limitations, recent trends in treating or protecting nerve related diseases have been aimed at discovering new naturally occurring compounds that could have new and/or improved efficacy.

It has been reported that the medicinal herb Angelica gigas Nakai might possess the ability to prevent ischemia and reperfusion injury (Cai et al. 2001) and the effect of pulmonary hypertension by hypoxia (Xi et al. 1998). However, there have not yet been many relevant studies that examined how and why this plant prevents nerve related diseases. To date, the only work that has been conducted to examine the potential of using this natural herb to prevent ischemia was just on crude extracts, and the effects of purified components from the crude extracts are considered to be further examined (Kim et al. 2002). Therefore, it is necessary to investigate which compounds in the Angelica gigas Nakai extract could potentially improve nerve cell growth, and to assess the potential of using them to eventually replace or assist the functions of relatively expensive neurotrophic factors in treating and/or preventing neuro-degrading disorders. To accomplish this, several consecutive solvents were used to partition the crude extracts of Angelica gigas, and the effect of the more purified extracts on improving both proliferation and differentiation of PC12 cells was examined.

Materials and methods

Materials

Angelica gigas Nakai was cultivated in Inje, Korea and harvested in November 2009. The collected samples were dried, finely chopped, and then stored in a dry and cold place. The three HPLC grade flavonol standards (quercetin, myricetin, catechin) were purchased from Sigma Chemicals Co. (St. Louis, MO, USA) for quantitative analysis. All other chemical including acetonitrile, methanol, and hydrochloric acid used in this study were from Merck (Darmstadt, Germany), unless stated otherwise.

Extraction and fractionation of Angelica gigas Nakai

For extraction, 2 kg of Angelica gigas Nakai were extracted with 20 L of 70% (v/v) ethanol at 80 °C for 8 h. Following filtration, the extracts were concentrated using a rotary vacuum evaporator (Eyela, Tokyo, Japan). The sample was then lyophilized in a freeze-dryer, as a dry powder, and stored at −4 °C before use. Next, the dried powder was dissolved in distilled water, and fractionized sequentially at room temperature using several extraction solvents including hexane, chloroform, ethyl acetate and butanol. The detailed fractionation process is presented in Fig. 1. After each fraction was filtered, the extracts were concentrated using a vacuum evaporator, and the sample was lyophilized in a freeze-dryer. Finally, the dried powder from each fraction was weighed to estimate the extraction yield, and stored at −4 °C before use.

Fig. 1.

Fig. 1

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Flow diagram of the fractionation of the Angelica gigas Nakai extract by consecutive solvent extracts

Measurement of cytotoxicity

Human Embryonic Lung cells (HEL299, ATCC, USA) and rat Pheochromocytoma nerve cells (PC12, ATCC, Rockville, USA) were grown in a DMEM/F12 basal medium containing 365 mg/L of l-glutamine (GIBCO, Grand Island, USA) and supplemented with 10% FBS (GIBCO, Grand Island, USA) in a 37 °C CO2 incubator with a supply of 5% CO2. Cytotoxicity of the sample was measured using a modified MTT assay. Briefly, 5 × 104 viable cells/mL were seeded in each well of a 96-well plate containing 100 μL of growth medium. Cells were incubated for 24 h, and then treated with each sample including the 70% ethanol extract and various partitioned fractions at concentrations ranging from 10 to 50 μg/mL. NGF was used as a positive control at concentrations, ranging from 10 to 50 ng/mL. Next, 20 μL of 5 mg/mL MTT in a phosphate buffered saline (PBS) was added to each well and the plate was incubated for an additional 4 h. The medium was then discarded and formazanblue was dissolved in 100 μL dimethyl sulphoxide (DMSO) was added. After incubation at 37 °C for 10 min, the absorbance of the dissolved solution at 490 nm was measured using a microplate ELISA reader (Thermo Labsystems). Then, the cytotoxicity was expressed as the ratio of the growth of PC12 cell or HEL299 cell cultivated in the presence of the samples to the growth in absence of the samples as a control (Plumb and Milroy 1989). Cytotoxicity of the samples against HEL299 cells are shown in Figs. 2 and 3 presents the cytotoxicity of the samples against PC12 cells.

Fig. 2.

Fig. 2

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Cytotoxicity of the ethanol extract and the fractions from Angelica gigas Nakai on the normal cell line, HEL299, at different concentrations. Means with different letters (A–E) within fractions are significantly different at p < 0.05 and means with different letters (a–d) within a concentration are significantly different at p < 0.05

Fig. 3.

Fig. 3

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Cytotoxicity of the ethanol extract and the fractions from Angelica gigas Nakai on PC12 nerve cells at different concentrations. Means with different letters (A–E) within fractions are significantly different at p < 0.05 and means with different letters (a–d) within a concentration are significantly different at p < 0.05

Measurement of neuritis outgrowth and numbers of neuritis bearing cells

The growth of PC12 cells was estimated daily using the trypan blue dye exclusion method (Frehney 1983) after the addition of various concentrations of the 70% ethanol extract (I) and 5% extract fractions (hexane (II), chloroform (III), ethyl acetate (IV), butanol (V), water (VI)) of Angelica gigas Nakai. 50 ng/mL of NGF (Genzyme, Cambridge, USA) was used as a positive control. The neurite’s outgrowth was estimated by measuring the average length of the neurites from five different spots on a graduated 75 T-flask under an inverted microscope every day (Rukenstein and Greene 1983). The ratio of neurite bearing cells to total cells was also calculated by counting the average number of the total and neurite bearing cells at three different spots (Park et al. 2002).

Measurement of apoptosis of PC12 cells

The effect of the hexane fraction on the apoptosis of PC12 cells was determined by measuring DNA fragmentations of the non-treated PC12 cells (lane 1 in Fig. 7), which was used as a control, cells treated with 50 μg/mL of the hexane fractions (lane 2) and 10 ng/mL of etoposide (Josep et al. 1997), cells treated with only 50 μg/mL of hexane fraction, which was used as a negative control (lane 3), and 50 ng/mL of NGF, which was used as a positive control (lane 4). After treating the samples for 24 h, 1 × 106 cells/mL were suspended in 500 μL cell-lysis buffer (500 mM Tris–HCl (pH 7.6), 100 mM NaCl, 20 mM EDTA, 1% SDS) containing 1 mg/mL proteinase K and incubated overnight at 37 °C. Cell lysates were extracted with TE-saturated phenol and the DNA was precipitated with ethanol. The DNA pellet was washed twice with 90% ethanol and dissolved in a Tris–EDTA buffer. The DNA samples (1 μg) were then treated with RNase and electrophoresed on a 2% agarose gel. The gel was stained with ethidium bromide and photographed under UV light using a gel image analyzer (Type AE-6910, Atto Co., Tokyo, Japan) to measure the degree of DNA fragmentation by comparing DNA fragments to the non-treated control cells (lane 1) (Nariyoshi and Miho 2003).

Fig. 7.

Fig. 7

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Effect of the hexane fraction of the Angelica gigas Nakai extract on DNA fragmentation in PC12 cells. The DNA pellet was washed twice with 90% ethanol and dissolved in a Tris–EDTA buffer. The DNA samples (1 μg) were then treated with RNase and electrophoresed on a 2% agarose gel. The gel was stained with ethidium bromide and photographed under UV light with a gel image analyzer Lane 1, untreated cells; lane 2, cells treated with 50 μg/mL of hexane fraction; lane 3, cells treated with 10 μg/mL of etoposide + 50 μg/mL of hexane fraction; lane 4, cells treated with 50 ng/mL of NGF after 24 h of the cultivation

Measurement of flavonoid contents in the extracts

The total flavonoid contents were determined using the Dowd method modified by Arvouet-Grand et al. (1994) as follows: 5 mL of 2% aluminium trichloride (AlCl3) (Labosi, Paris, France) in methanol (Fluka Chemie, Switerland) was mixed with the same volume of extract and fraction solutions (0.01 or 0.02 mg/mL). Absorption readings at 415 nm (CECIL CE 2041 spectrophotometer 2,000 series) were taken after 10 min against a blank sample consisting of a 5 mL of extract and fraction solutions with 5 mL methanol without AlCl3. The total flavonoid content was determined with a standard curve using quercetin (Simga–Aldrich Chemie, Steinheim, Germany) (0–50 mg/L) as standard. The mean of three readings was used and expressed as mg of quercetin equivalents (QE)/kg of extract and fractions. The concentrations of the three main flavonoids, quercetin, myricetin and catechin, were also analyzed by High-Performance Liquid Chromatography (HPLC) (model LC-10A, Shimadzu, Kyoto, Japan) equipped with two LC-10 AS pumps, a SCL-10A column oven, and a SPD-10A UV–vis detector. Twenty μL of the filtered samples was injected into an analytical Supelco (Supelco Inc., Bellefonte, USA) ODS reverse phase (C18) column (250 × 4.6 mm; 5 μm particle size). Two solvent systems were applied as follows: Solvent A containing 3% trifluoroacetic acid and 97% methanol and Solvent B containing acetonitrile and methanol (80:20 v/v). The chromatograph separation was performed by isocratic elution of the mobile phase (mixture of solvent A and B (50:50 v/v) filtered under a vacuum through a 0.45 μm membrane before use) at a flow rate of 0.1 mL/min at 30 °C. Compounds were detected at a wavelength of 360 nm. The flavonoid contents in the fractions were estimated by comparing their retention times with those of authentic standards (Sigma Chemicals Co., St. Louis, MO, USA) in the calibration curves (Bushra and Farooq 2008).

Statistical analysis

An analysis of variance (ANOVA) was used to evaluate the treatment as a fixed effect. Significant mean differences among treatment were estimated by Fisher’s Least Significant Difference (LSD) at p < 0.05 (Norman and Smith 1981).

Results

Extraction yield and flavonoid contents in each fraction

Table 1 displays the results of estimating the extraction yields of each fraction obtained from the sequential fractionation process outlined in Fig. 1. This table reveals that the water fraction had the highest extraction yield (39.17%) followed by the hexane fraction (21.35%), butanol fraction (15.17%), 70% ethanol extract (13.53%), ethyl acetate fraction (7.94%) and chloroform fraction (2.84%), respectively. Table 2 presents the total flavonoid contents in each fraction and the 70% ethanol crude extract. We determined hexane fraction was shown to contain the highest number of flavonoids at 21.48 mg/g and the chloroform fraction had the lowest flavonoid content at 3.64 mg/g. To determine the flavonoid profile in the hexane fraction that contained the highest amounts of the flavonoids, the concentrations of the three main flavonoids in Angelica gigas Nakai, quercetin, myricetin and catechin, were determined as shown in Table 3. The concentration of quercetin was the highest (12.24 mg/g), which was followed by myricetin and catechin with 4.39 mg/g and 2.58 mg/g, respectively. The total content of the three flavonoids was 19.21 mg/g, which was somewhat lower than 21.48 mg/g presented in Table 3. We believe this occurred because there were other minor flavonoids present in the fraction of them. For all remaining experiments, 50 μg/mL of the hexane fraction were used, which corresponded to 612 ng/mL of quercetin, 219.5 ng/ml of myricetin and 129 ng/mL of catechin, and these concentrations were compared to the results of adding 50 ng/mL of NGF. The weight fraction of the mixture of the three flavonoids in the hexane fraction was calculated to bet 63.4/22.8/13.4 (w/w). Therefore, any potential synergistic effects of the three flavonoids in the hexane fraction on PC12 cell growth and neurites outgrowth were also examined (Table 4).

Table 1.

The extraction yields of the crude extracts and the fractions of Angelica gigas Nakai

Sample Extraction yieldsa (%, w/w)
70% Ethanol crude extract (I) 13.53 ± 0.18A
Hexane fraction (II) 21.35 ± 0.42B
Chloroform fraction (III) 2.84 ± 0.36C
Ethyl acetate fraction (IV) 7.94 ± 0.84D
Butanol fraction (V) 15.17 ± 0.46E
Water fraction (VI) 39.17 ± 0.35F
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aMeans with different letters (A–F) within column are significantly different at p < 0.05

Table 2.

Estimation of total flavonoid contents in several extracts of Angelica gigas Nakai

Sample Flavonoid (mg/g)a
70% ethanol extract (I) 15.36 ± 0.07Ab
Hexane fraction (II) 21.48 ± 0.05B
Chloroform fraction (III) 3.64 ± 0.71C
Ethyl acetate fraction (IV) 6.47 ± 0.84D
Butanol fraction (V) 5.21 ± 0.57D
Water fraction (VI) 9.47 ± 0.47E
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aThe total flavonoid content was determined by comparison with a standard curve generated with quercetin

bMeans with different letters (A–E) within column are significantly different at p < 0.05

Table 3.

Estimation of flavonol contents of quercetin, myricetin, catechin in hexane fraction of Angelica gigas Nakai

Quercetina(mg/g) Myricetin (mg/g) Catechin (mg/g) Total flavonoids (mg/g)
Hexane fraction 12.24 ± 0.21a 4.39 ± 0.18b 2.58 ± 0.41c 21.48 ± 0.05d
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aMean with different letters (a–d) within a row are significantly different at p < 0.05

Table 4.

The cell growth and neurite’s extension in adding quercetin, myricetin, catechin and hexane fraction

Sample Concentration (ng/mL) Neuronal differentiation activitiesa
Viable cell density (×104 cell/mL) The length of neurite (μm)
Quercetin 100 3.31 ± 0.12A 27 ± 1A
300 5.15 ± 0.27B 51 ± 2B
500 6.47 ± 0.08C 92 ± 1C
Myricetin 100 3.13 ± 0.19A 18 ± 1D
300 4.01 ± 0.30D 35 ± 3E
500 4.94 ± 0.04E 41 ± 2F
Catechin 100 4.11 ± 0.14D 31 ± 3E
300 6.06 ± 0.05F 57 ± 4G
500 8.23 ± 0.06G 96 ± 2H
Sample Concentration (μg/mL) Viable cell density (×104 cell/mL) The length of neurite (μm)
Hexane fraction 10 6.39 ± 0.14G 61 ± 2G
30 10.42 ± 0.21H 138 ± 4H
50 16.24 ± 0.10I 230 ± 3I
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aMeans with different letters (A–Y) within column are significantly different at p < 0.05

Cytotoxicity of the samples

The cytotoxicity of the 70% ethanol extract and several fractions of Angelica gigas Nakai against two normal cell lines—the human lung cell, HEL299, and rat pheochromocytoma nerve cell, PC12 are shown in Figs. 2 and 3. In general, the cytotoxicity of the samples against both cell lines was less than 25% at the highest concentration of 50 μg/mL, and for both cases the water fraction showed the lowest cytotoxicity (approximately 10–15%) at the highest treatment concentration. All of the samples were found to have similar cytotoxicity against both cells in the ranges of 10–20%, which was not significantly different from other plant extracts (Cha et al. 2006; Wang et al. 2006). However, it is worth noting that the hexane and chloroform extracts showed the highest cytotoxicity against HEL299 cells while the butanol extract showed the highest cytotoxicity against PC12 cells. This result suggests that the hexane fraction could be used to promote the growth of PC12 cells.

Nerve cell growth and differentiation

As presented in Fig. 4, neurites outgrowth of PC12 cells were observed by adding 50 μg/mL of the crude extract, hexane fraction and water fraction, and 50 ng/mL of NGF as the positive control. Cell growth as a function of cultivation time was also examined because the water fraction had less cytotoxicity and relatively higher flavonoids than the other fractions. In previous studies, NGF was shown to have the ability to increase, both outgrowth of neurites and cell growth at a concentration of 10–50 ng/mL (Kaberi et al. 2004; Nariyoshi and Miho 2003). Based on this result, 50 ng/mL of NGF was used as a positive control in this work. The highest neurite’s extension was measured to be 235 μm after treatment with 50 μg/mL of the hexane fraction, which was approximately 1.8 times longer than the control 124 μm after 4 days of cultivation. However, the length of the neurite’s extension started to decrease at longer cultivation times. When the cells were treated with NGF, the length of the neurite’s extensions was approximately 100 μm after 4 days of cultivation (as described in Table 3). The highest cell growth was determined to be 1.6 × 105 viable cells/mL after treatment with the hexane fraction, which was approximately three times higher than the control growth of 5.2 × 104 viable cells/mL. The ethanol crude extract showed stronger cell growth than the other fractions, which implies that flavonoids must play a role in improving PC12 cell growth since only the hexane fraction and crude extract contained a significant amount of flavonoids when compared to the other fractions. These results clearly indicate that the hexane fraction could favorably promote PC12 cell growth and neurites outgrowth compared to the other fractions even though cell growth gradually decreased after 4 days of cultivation. It is also important to point out that, both, cell growth and neurites outgrowth remained relatively constant with 1.0 × 105 viable cells/mL and about 100 μm, respectively in adding 50 ng/mL of NGF after 7 days of cultivation. This was better than for the cases of adding the partitioned fractions because NGF could more effectively induce both cell growth and neurites outgrowth (Kaberi et al. 2004). In addition, comparing the length of the neurites in the presence of 50 μg/mL of the 70% ethanol extract (b), hexane fraction (c), ethyl acetate fraction (d), water fraction (e) and the control (a) after 4 days of cultivation (Fig. 6), it is quite obvious that the hexane fraction improves neuronal differentiation. The hexane fraction resulted in that the neurites were approximately 100 μm longer than those for the control (A). Very little neurites outgrowth was observed for the ethyl acetate (D) and water (E) fractions, compared to the hexane fraction, possibly because of the relatively low flavonoid concentrations in these fractions (as discussed in Table 2). Figure 5 also shows the changes of the ratio of neurite-bearing cell numbers to total viable cell numbers for different samples in function of cultivation time. The hexane fraction had the highest percentage of neurite-bearing cells at 81.21% after 4 days of cultivation, compared to 40.11% for the control. When adding 50 ng/mL of NGF, the ratio of neurite-bearing cells was estimated to be 58.11% after 4 days of cultivation, which was higher than the water fraction. However, after the 4th day of cultivation, the neurite-bearing cells started to slightly decrease. This was a somewhat different pattern than that observed for cell growth and neurites outgrowth when adding NGF.

Fig. 4.

Fig. 4

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Cell growth (bar chart) and neurite’s extension (line chart) of PC12 nerve cells in the presence of 50 ng/mL of NGF and 50 μg/mL of several extracts from Angelica gigas Nakai. Means with different letters (A–E) within a fraction’s time are significantly different at p < 0.05 and means with different letters (a–f) within a cultivation time are significantly different at p < 0.05

Fig. 6.

Fig. 6

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The morphology of PC12 cells, not treated (a) or treated with 50 μg/mL of 70% ethanol extracts (b), 50 μg/mL of the hexane Fr. (c), 50 μg/mL of the ethyl acetate Fr. (d), 50 μg/mL of the water Fr. (e) after 4 days of cultivation

Fig. 5.

Fig. 5

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Comparison of the numbers of neurite-bearing cells in the presence of 50 ng/mL of NGF and several extracts (50 μg/mL). The ratio of neurite bearing cells to total cells was calculated by counting the total number of cells and neurite bearing cells using a haemacytometer. Means with different letters (A–E) within a sample are significantly different at p < 0.05 and means with different letters (a–g) within a cultivation time are significantly different at p < 0.05

The hexane fraction showed better activity in promoting PC12 cell growth than the other fractions, and was comparable to the cases of adding NGF. We believe that this was because of high amounts of the three flavonoids in the hexane fraction (Table 3) and these three flavonoids acted synergistically. Table 4 summarizes both cell growth and neuritis outgrowth in adding different concentrations of the three flavonoids and the hexane fraction, which can be compared with the results shown in Fig. 4. All samples enhanced the proliferation and differentiation of PC12 cells as the treatment concentration was increased. At the highest flavonoid concentration of 500 ng/mL, catechin showed the best effect on both cell growth and neurite’s extension with 6.57 × 104 viable cells/mL and 96 μm, respectively, followed by quercetin and myricetin. This result was consistent with other reports where catechin also showed stronger effects than the other flavonoids (Lacopini et al. 2008). In considering the effects of each flavonoid on PC12 cells, the hexane fraction dramatically improved cell growth and the outgrowth of neurites even though 50 μg/mL of the hexane fraction contained only 612 ng/mL of quercetin, 219 ng/mL of myricetin and 129 ng/mL of catechin, which were lower than the 300–500 ng/mL of each single flavonoid used in these experiments. These results strongly indicate that the efficacy of the hexane fraction was mainly influenced by synergistic effects of all three flavonoids, which resulted in enhancing PC12 cell growth and neuronal differentiation. This synergistic effect of a combination of different flavonoids has not been reported elsewhere.

Prevention of apoptosis in PC12 cells

Figure 7 illustrates the effect of the hexane fraction on the apoptosis of PC12 cell. DNA fragmentation was compared between PC12 cells treated with 50 μg/mL of the hexane fraction (lanes 2), 50 ng/mL of NGF (lane 4) and 10 ng/mL of etoposide, 50 μg/mL of the hexane fractions (lane 3) and non-treated cells (lane 1) using electrophoretic analysis. No significant apoptosis of PC12 cells was observed after treating with 50 ng/mL of NGF or 50 μg/mL of the hexane fraction, compared to the non-treated cells (control, lane 1) while significant DNA fragmentation was observed when treating with 10 ng/mL of etoposide (positive control). These data clearly indicate that the flavonoids in the hexane fraction of Angelica gigas Nakai prevented apoptosis of PC12 cells, which resulted in an enhancing cell growth as well as neurites outgrowth. Furthermore, these results are in good agreement with other previous studies that examined the effect of plant extracts on apoptosis of several human cell lines (Sun et al. 1999; Ma et al. 1998).

Discussion

In this study, we demonstrated that the neuronal protection activities of Angelica gigas Nakai can be increased by concentrating the biological active components or flavonoids in the partitioned fractions using several extraction solvents. Koh et al. (2003) reported that flavonoids such as epigallo-catechin showed positive effects on PC12 cells in terms of decreasing cytotoxicity, and also protecting oxidative damages due to its strong antioxidant abilities (Rice-Evans et al. 1996). However, it has been hypothesized that this classical hydrogen-donating antioxidant activity cannot be the reason for the bioactivity of flavonoids in vivo, particularly in the brain, where they are found at only very low concentrations (Spencer 2008). Rather, it has been postulated that their effects on the brain are mediated by their ability to protect vulnerable neurons, enhance existing neuronal function, stimulate neuronal regeneration and induce neurogenesis (Spencer 2008; Joseph et al. 2005). These hypotheses support our results; that is, that flavoniods directly improve nerve cell growth and differentiation. Our results also showed that treatment using a combination of flavonoids, enhanced the neuroprotective functions by reducing apoptosis of PC12 cells relative to treatment with individual flavonoids. This is in agreement with other reported data that flavonoids are able to exert neuroprotective actions (at low concentrations) via their interactions with critical neuronal intracellular signaling pathways pivotal to controlling neuronal survival and differentiation, long-term potentiation (LTP) and memory (Williams et al. 2004; Spencer 2007; Spencer 2009). Therefore, these results strongly suggest that flavonoids could improve memory, learning and neuro-cognitive performance.

To more clearly understand the effects of the flavonoids in the hexane fraction the effects of each flavonoid on PC12 cell growth were quantitatively examined along with the hexane fraction for the first time. For example, it was found that there were 612 ng/mL of quercetin, 219 ng/mL of myricetin and 129 ng/mL of catechin, respectively in 50 μg/mL of the hexane fraction. In these studies, 50 ng/mL of NGF was used as a positive control since it has been reported that about 10–50 ng/mL of NGF can protect PC12 cells. For instance, Kaberi et al. (2004) reported an extension of neurites of PC12 cells of 200 μm by adding 50 ng/mL of NGF after 7 days of cultivation. In addition, its length increased with increasing cultivation time. Another study also examined the effect of adding 1 mg/mL of Aplysia californica (Michael and David 1995) on neurite’s extenstion and found that it grew 50% longer than the control. In this study, the neurite length was 70% greater after addition of 50 μg/mL of the hexane fraction than for the control. The result was comparable to those obtained after addition of Aplysia californica although the cells were only treated with half of the concentration (Kuninori et al. 1996). In previous studies, 100 ng/mL of IGF-I (insulin-like growth factor-I) and E2 (estradiol) increased the length of the neurites by approximately 40% relative to treatment with the hexane fraction of Angelica gigas Nakai after cultivation (Ilir and Anne 2004). However, their effects cannot be extracted from the data to conclusively prove their effectiveness because these compounds are no natural hormones.

In general, the hexane fraction showed higher activity on PC12 cells than the cases of treating the individual flavonoids, which implies that these three key flavonoids display synergistic properties in the hexane fraction. To verify the effect of the hexane fraction, apoptosis of PC12 cells was also monitored by measuring DNA fragmentation formation, performed using 100 μM of DDT (dichlorodiphenyltrichloroethane) (Nariyoshi and Miho 2003). Less DNA fragments were observed when treating with the hexane fraction relative to the treatment with NGF. These results, in conjunction with the cytotoxicity data, imply that the hexane fraction would be safe at the cellular level. In PC12 cells, the butanol fraction showed the highest cytotoxicity (25.15%) while the water and hexane fractions had the lowest cytotoxicity (16–20%). These differences in the cytotoxicity of each fraction were also shown in other studies (Byun et al. 2005), probably because the kind and the amount of the active components in the extracts were different. Taken together, our results strongly suggest that the synergistic effects of the flavonoids in the hexane fraction have the potential to promote nerve cell growth and neurites extension, as well as increase the percentage of neurite’s bearing cells. It is worth noting that each of the three flavonoids such as quercetin, myrictin or catechin did not show better proliferation and differentiation activities relative to the hexane fraction. This implies for developing medicines from natural resources, that it would be efficient and/or economical to focus on mixtures of several active substances rather than focusing on purifying a single specific compound from plants because it could save time and cost and increase the efficacy of medicine (Harvey 1999, 2008). Moreover, this is the first report to examine the effects of complex flavonoids from Angelica gigas Nakai on PC12 cells by comparing each of the three major flavonoids on the cellular properties.

Acknowledgments

This work was supported by the Grant of the Korean Ministry of Education, Science and Technology (The Regional Core Research Program/Medical and Bio-Material Research Center)

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