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CHAPTER IV

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CHAPTER IV

RESULTS & DISCUSSION

4.1 EXTRACTION, FRACTIONATION AND ISOLATION

4.1.1 Extraction, fractionation and isolation of aqueous ethanol extract of Hericium erinaceus

The flow chart shows the extraction and fractionation procedures for H. erinaceus (Figure 4.1 and 4.2)

Figure 4.1: Aqueous ethanol extraction of Hericium erinaceus.

Fresh H. erinaceus (1.33 kg) yielded 200.00 g of dried and ground H.erinaceus.

The dried H. erinaceus sample (200.00 g) yielded 52.29 g of crude aqueous ethanol extract.

Fresh H. erinaceus (1.33 kg)

Dried and ground H. erinaceus (200.00 g)

Dried and ground to fine powder

Aqueous ethanol extract (52.29 g)

i. Extraction with 80 % ethanol (3 times) ii. Concentration under reduced pressure

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Figure 4.2: Fractionation of aqueous ethanol extract of Hericium erinaceus.

The aqueous ethanol extract was further extracted with hexane to give hexane- soluble fraction (3.85 g, 7.36 %) and hexane-insoluble residues. The hexane-insoluble residues were further partitioned between ethyl acetate-water (ratio 1:2) to give the ethyl acetate-soluble fraction (0.77 g, 1.47 %) and water fraction (44. 34 g, 84.80 %).

Aqueous ethanol extract

Hexane soluble fraction (3.85 g, 7.36 %)

Hexane insoluble fraction

Ethyl acetate fraction (0.77 g, 1.47 %)

Water fraction (44. 34 g, 84.80 %) i. Partition (v/v) between ethyl acetate and water (Ratio 1:2)

ii. Concentration under reduced pressure i. Extraction with hexane ii. Concentration under reduced

pressure

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The combined hexane and ethyl acetate fraction were subjected to flash column chromatography to yield 7 fractions, which were E1 (384.0 mg, 0.73 %), E2 (780.8 mg, 1.49 %), E3 (438.2 mg, 0.84 %), E4 (62.4 mg, 0.12 %), E5 (39.7 mg, 0.08 %), E6 (183.1 mg, 0.35 %), E7 (1068.2 mg, 2.04 %) (Figure 4.3). The percentage yields were calculated based on the crude aqueous ethanol extract.

Figure 4.3: Isolation of combined hexane and ethyl acetate extract of Hericium erinaceus obtained through flash column chromatography.

Combined fraction of hexane and ethyl acetate

Flash column chromatography (developing solvent: CHCl3 → CHCl3/Ac → CHCl3/MeOH →MeOH)

E1 (384.0mg, 0.73 %)

E2 (780.8mg, 1.49 %)

E3 (438.2mg, 0.84 %)

E4 (62.4mg 0.12 %)

E5 (39.7mg, 0.08 %)

E6 (183.1mg, 0.35 %)

E7 (1068.2mg, 2.04 %)

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Fraction E2 obtained from flash column chromatography were further subjected to preparative thin layer chromatography to yield subfraction sub4b (187.7 mg, 0.36 %) (Figure 4.4). Sub4b was then subjected to high performance liquid chromatography (HPLC) to give sub4b_4 (68.5 mg, 0.13 %) and sub4b_6 (38.5 mg, 0.07 %) (Figure 4.4).

Figure 4.4: Isolation of fraction E2 of Hericium erinaceus using preparative thin layer chromatography and high performance liquid chromatography.

E2

Sub4b (187.7 mg, 0.36 %)

Sub4b_4 (68.5 mg, 0.13 %)

Sub4b_6 (38.5 mg, 0.07 %) Preparative thin layer chromatography

High perfomance liquid chromatography (HPLC)

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4.2 NEURITE OUTGROWTH ACTIVITY

4.2.1 Effect of aqueous ethanol extract and fractions of Hericium erinaceus on the neural cell line NG108-15

Aqueous ethanol extract and fractions of H. erinaceus were screened for the in vitro neurite outgrowth activity on the neural hybrid cell line NG108-15 at various concentrations (µg/ml) (Figure 4.5; Table 4.1). Cells were observed under a phase contrast microscope for the neurite outgrowth and branching of neurites. The effect of the various extracts on the morphology and neurite extension of the NG108-15 cells are given in Figure 4.6 (crude aqueous ethanol extract), Figure 4.7 (hexane fraction), Figure 4.8 (ethyl acetate fraction) and Figure 4.9 (water fraction).

Figure 4.5: Percentage of neurite bearing cells incubated with varying concentrations of aqueous ethanol crude extract, hexane fraction, ethyl acetate fraction and water fraction of Hericium erinaceus (nerve growth factor, 20 ng/ml, used as positive control).

19.9 23.0 22.9 22.5 20.9 18.624.6 27.4 25.4 32.3 34.1 40.6

20.5 24.1 23.7 26.5 27.8 34.5

19.0 24.2 21.9 24.0 23.5 25.6

0 5 10 15 20 25 30 35 40 45 50

Negative control

NGF (20ng/ml)

10 25 50 100

Neurite- bearing cells (%)

Concentrations (µg/ml)

ethanol crude extract Hexane fraction Ethyl acetate fraction Water fraction

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Table 4.1: Stimulation of neurite outgrowth activity in the NG108-15 cells with varying concentrations of aqueous ethanol extract and fractions of Hericium erinaceus. NG108-15 cells without extract was negative control. 20 ng/ml of nerve growth factor (NGF) was used as positive control.

Treatment concentration

(µg/ml)

Ethanol crude extract Hexane fraction Ethyl acetate fraction Water fraction Neurite

bearing cells (%)

Increase compared to

control (%)

Neurite bearing cells

(%)

Increase compared to

control (%)

Neurite bearing cells

(%)

Increase compared to

control (%)

Neurite bearing cells

(%)

Increase compared to

control (%) Negative

control 19.9±1.5ab - 24.6±1.5a - 20.5±1.5a - 19.0±1.7a -

Positive

control (NGF) 23.0±1.8c 15.5 28.9±0.9bc 11.3 24.1±0.2b 17.8 24.2±0.2b 27.1

10 22.9±0.5c 15.0 25.4±1.3ab 3.3 23.7±0.8b 15.9 21.9±1.8ab 14.9

25 22.5±0.7bc 13.2 32.3±2.6bc 31.4 26.5±2.0bc 29.4 24.0±1.6b 26.3

50 20.9±0.9abc 4.8 34.1±0.1c 38.7 27.8±1.6c 35.4 23.5±1.9b 23.3

100 18.6±1.3a -6.4 40.6±2.5d 65.2 34.5±0.9d 68.5 25.6±1.3b 34.4

Note: Data are expressed as means ± standard deviation (n = 2). Means with different letters in the same column are significantly different (P < 0.05), one-way analysis of variance/ANOVA)

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Figure 4.6: The morphology of the NG108-15 cells treated with various concentrations of crude aqueous ethanol extract of Hericium erinaceus [24hr of incubation at 37 ºC in a 5 % CO2 humidified incubator. NG108-15 cells without extract or treated with NGF (20 ng/ml) was negative and positive control, respectively.]

A: negative control (cells without extract); B: positive control - NGF (20 ng/ml);

C: 10 µg/ml of crude aqueous ethanol extract;

D: 25 µg/ml of crude aqueous ethanol extract;

E: 50 µg/ml of crude aqueous ethanol extract;

F: 100µg/ml of crude aqueous ethanol extract

A B

C D

E F

neurite

neurite

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Figure 4.7: The morphology of the NG108-15 cells treated with various concentrations of hexane fraction of Hericium erinaceus [24 hr of incubation at 37 ºC in a 5 % CO2 humidified incubator. NG108-15 cells without extract or treated with NGF (20 ng/ml) was negative and positive control, respectively.]

A: negative control (cells without extract); B: positive control - NGF (20 ng/ml);

C: 10 µg/ml of hexane fraction; D: 25 µg/ml of hexane fraction;

E: 50 µg/ml of hexane fraction; F: 100 µg/ml of hexane fraction

A B

C D

E F

neurite

neurite

neurite

neurite neurite

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Figure 4.8: The morphology of the NG108-15 cells treated with various concentrations of ethyl acetate fraction of Hericium erinaceus [24 hr of incubation at 37 ºC in a 5 % CO2 humidified incubator. NG108-15 cells without extract or treated with NGF (20 ng/ml) was negative and positive control, respectively.]

A: negative control (cells without extract); B: positive control - NGF (20 ng/ml);

C: 10 µg/ml of ethyl acetate fraction; D: 25 µg/ml of ethyl acetate fraction;

E: 50 µg/ml of ethyl acetate fraction; F: 100 µg/ml of ethyl acetate fraction

A B

C D

E F

neurite

neurite

neurite

neurite

neurite

neurite

neurite

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Figure 4.9: The morphology of the NG108-15 cells treated with various concentrations of water fraction of Hericium erinaceus [24hr of incubation at 37 ºC in a 5 % CO2

humidified incubator. NG108-15 cells without extract or treated with NGF (20 ng/ml) was negative and positive control, respectively.]

A: negative control (cells without extract); B: positive control - NGF (20 ng/ml);

C: 10 µg/ml of water fraction; D: 25 µg/ml of water fraction;

E: 50 µg/ml of water fraction; F: 100 µg/ml of water fraction

A B

C D

E F

neurite

neurite

neurite

neurite

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Table 4.1 and Figure 4.2 showed the effects of various concentrations of crude aqueous ethanol extract and fractions of H. erinaceus on neurite outgrowth of the NG108-15 cells after 24 hr of incubation. Aqueous ethanol extract at 10 µg/ml cause maximal stimulation of neurite outgrowth. The percentage of neurite bearing cells was significantly higher in extract concentration of 10 µg/ml compared to negative control.

Furthermore, increasing the concentration of the extract has a minimal effect on the number of neurite bearing cells. Extract concentration of 100 µg/ml showed 6.4 % decreased of neurite bearing cells when compared to negative control.

When the aqueous ethanol extract was further fractionated, three fractions were obtained (hexane, ethyl acetate and water fraction). Hexane fraction showed significant stimulation of neurite outgrowth at the concentration of 25 µg/ml. When the concentration was increased (10, 25, 50 and 100 µg/ml), there was an increase in the percentage of neurite bearing cells (25.4 %, 32.3 %, 34.1 % and 40.6 % respectively) for the hexane fraction. Hexane fraction showed 65.2 % increased in neurite bearing cells when compared to negative control at the highest tested concentration of 100 µg/ml.

Ethyl acetate fraction showed significant stimulation (p ˂ 0.05) of neurite outgrowth at the concentration as low as 10 µg/ml. There was an increase in the percentage of neurite bearing cells for the ethyl acetate fraction when the concentration is increased (23.7 %, 26.5 %, 27.8 %, 34.5 % for 10, 25, 50 and 100 µg/ml respectively).

Ethyl acetate fraction showed 68.5 % increased in neurite bearing cells compared to negative control at the highest tested concentration of 100 µg/ml.

At 25 µg/ml, water fractions caused significant stimulation (p ˂ 0.05) of neurite- bearing cells compared to negative control. However, water fraction did not show a significant difference of neurite bearing cells at concentration above 25 µg/ml.

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Upon comparison of the various extracts (crude ethanol, hexane, ethyl acetate and water), it was observed that the minimum concentration acquired by each extract to stimulate the neurite outgrowth were 10 µg/ml, 25 µg/ml, 10 µg/ml and 25 µg/ml respectively. In crude aqueous ethanol extract, increasing the concentration of the extract showed decreased on the number of neurite bearing cells. A similar situation was observed in the water fraction where further increase of concentration after 25 µg/ml did not give significant effect to the neurite outgrowth. Hexane and ethyl acetate fractions showed increase of the neurite outgrowth activity until the highest tested concentration, 100 µg/ml.

The maximum increase in neurite bearing cells compared to negative control were in the following descending order with respect to concentration: ethyl acetate fraction (68.5 % at 100 µg/ml) > hexane fraction (65.2 % at 100 µg/ml) > water fraction (34.4 % at 100 µg/ml) > aqueous ethanol extract (15.0 % at 10 µg/ml). These results were further supported by comparison of the cell morphology upon treatment with aqueous ethanol extract, hexane fraction, ethyl acetate fraction and water fraction. When the cells were treated with aqueous ethanol extract and water fraction, the cells exhibited short cellular extensions of neurites (Figure 4.6 and Figure 4.9). Obvious enhancement of neurite outgrowth was observed in cells treated with hexane and ethyl acetate fractions of H. erinaceus (Figure 4.7 and Figure 4.8). When the cells were treated with 100 µg/ml of hexane fraction and ethyl acetate fraction (Figure 4.7F and Figure 4.8F), cells exhibited neurite networks with an exuberant outgtrowth of long, diverse, beaded, multipolar and fine-meshed branching neurites.

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4.2.2 Effect of the fractions E1–E7 of Hericium erinaceus on the neural cell line NG108-15

Fractions E1-E7 of H. erinaceus were screened for the in vitro neurite outgrowth activity on the neural hybrid cell line NG108-15 at various concentrations (µg/ml). Cells were observed under a phase contrast microscope for the neurite outgrowth and branching of neurites after 24 hr of incubation. The results of effect on neurite extension and morphology of NG108-15 cells were shown in Table 4.2, 4.3; Figure 4.10 – 4.16.

Table 4.2 and Table 4.3 showed the effects of various concentrations of fraction E1-E7 on neurite outgrowth of the NG108-15 cells after 24 hr of incubation. An obvious enhancement of neurite stimulation was observed in the cells when treated with fraction E1 (Figure 4.10). Fraction E1 showed an increase in percentage of neurite bearing cells when the concentration is increased. The percentage of neurite bearing cells was significantly higher compared to negative control at 25 µg/ml which showed 66.2 % increase compared to negative control. At 50 µg/ml, 51.3 % of neurite bearing cells were observed in fraction E1 which showed an increase of 138.4 % compared to negative control. Further increment of the concentration after 50 µg/ml did not bring any significant difference to the neurite bearing cells.

Fraction E2 showed an increase in neurite bearing cells when the concentration of fraction E2 is increased from 10 µg/ml to 100 µg/ml. Fraction E2 showed good effect on the neurite stimulation of NG108-15 cells where long and diverse neurite were observed (Figure 4.11). The increment of the concentration (0 µg/ml, 10 µg/ml, 25 µg/ml, 50 µg/ml and 100 µg/ml) showed significant difference in the percentage of neurite bearing

cells (20.8 %, 28.1 %, 33.5 % 38.5 %, 51.7 %). At the highest tested concentration of 100 µg/ml, fraction E2 showed 149.1 % increase of neurite bearing cells when compared to negative control.

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Table 4.2: Stimulation of neurite outgrowth activity in the NG108-15 cells with varying concentrations of fractions (E1-E4) of Hericium erinaceus. NG108-15 cells without extract was negative control. 20 ng/ml of nerve growth factor (NGF) was used as positive control.

Treatment concentration

(µg/ml)

Fraction E1 Fraction E2 Fraction E3 Fraction E4

Neurite bearing cells

(%)

Increase compared to

control (%)

Neurite bearing cells

(%)

Increase compared to

control (%)

Neurite bearing cells

(%)

Increase compared to

control (%)

Neurite bearing cells

(%)

Increase compared to

control (%) Negative

control

21.5±2.0a - 20.8±1.5a - 18.7±0.9a - 18.0±0.4a -

Positive control (NGF)

26.8±0.8b 24.5 25.2±1.3b 21.4 22.7±0.2b 21.7 21.5±0.9b 19.1

10 25.1±1.1ab 16.6 28.1±1.3b 35.4 24.4±0.3b 30.9 17.9±0.7a -0.7

25 35.8±0.7c 66.2 33.5±2.2c 61.1 29.5±1.8c 57.9 26.4±1.8c 46.7

50 51.3±2.8d 138.4 38.5±2.7d 85.3 32.2±0.8d 72.3 27.3±1.2c 51.8

100 56.0±3.2d 160.6 51.7±0.3e 149.1 - - - -

Note: Data are expressed as means ± standard deviation (n = 2). Means with different letters in the same column are significantly different (P < 0.05), one-way analysis of variance/ANOVA).

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Table 4.3: Stimulation of neurite outgrowth activity in the NG108-15 cells with varying concentrations of fractions (E5-E7) of Hericium erinaceus. NG108-15 cells without extract was negative control. 20 ng/ml of nerve growth factor (NGF) was used as positive control.

Treatment concentration (µg/ml)

Fraction E5 Fraction E6 Fraction E7

Neurite bearing cells

(%)

Increase compared to

control (%)

Neurite bearing cells

(%)

Increase compared to

control (%)

Neurite bearing cells

(%)

Increase compared to

control (%)

Negative control 20.7±0.8a - 18.7±0.5a - 18.2±1.5a -

Positive control (NGF) 25.5±0.6b 23.0 22.7±1.0b 21.2 22.2±1.7b 22.2

10 22.3±0.8a 7.5 19.9±0.7a 6.1 16.1±0.6a -11.6

25 28.9±0.5c 39.6 19.7±0.9a 4.9 17.0±1.1a -6.6

50 34.1±0.2d 64.6 19.9±1.5a 6.1 16.6±1.0a -8.4

100 - - 19.3±0.4a 3.1 16.8±0.8a -7.3

Note: Data are expressed as means ± standard deviation (n = 2). Means with different letters in the same column are significantly different (P < 0.05), one-way analysis of variance/ANOVA).

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Figure 4.10: The morphology of the NG108-15 cells treated with various concentrations of fraction E1 of Hericium erinaceus [24 hr of incubation at 37 ºC in a 5 % CO2 humidified incubator. NG108-15 cells without extract or treated with NGF (20ng/ml) was negative and positive control, respectively.]

A: negative control (cells without extract); B: positive control - NGF (20 ng/ml);

C: 10 µg/ml of fraction E1; D: 25 µg/ml of fraction E1;

E: 50 µg/ml of fraction E1; F: 100 µg/ml of fraction E1

A B

C D

E F

neurite

neurite

neurite

neurite

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Figure 4.11: The morphology of the NG108-15 cells treated with various concentrations of fraction E2 of Hericium erinaceus [24 hr of incubation at 37 ºC in a 5 % CO2 humidified incubator. NG108-15 cells without extract or treated with NGF (20 ng/ml) was negative and positive control, respectively.]

A: negative control (cells without extract); B: positive control - NGF (20 ng/ml);

C: 10 µg/ml of fraction E2; D: 25 µg/ml of fraction E2;

E: 50 µg/ml of fraction E2; F: 100 µg/ml of fraction E2

A B

C D

E F

neurite

neurite

neurite

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Fraction E3 showed significant difference in percentage of neurite bearing cells (24.4 %) at the concentration as low as 10 µg/ml. Further increment of the concentration to 25 µg/ml and 50 µg/ml also showed an increment in the percentage of neurite bearing cells (29.5 % neurite bearing cells and 32.3 % neurite bearing cells respectively).

However, the cells showed toxicity effect when treated with 100 µg/ml of fraction E3.

The cells did not attach to the well surface due to the cell damage which make the cells cannot be counted (Figure 4.12F).

For fraction E4, the concentration of 10 µg/ml was too low to show significant effect on the cells. 25 µg/ml of fraction E4 showed significant difference effect on the cells with 26.4 % neurite bearing cells which equal to 46.7 % increase in neurite bearing cells when compared to negative control. Further increment of the concentration to 50 µg/ml did not give significant diffence effect on the cells if compared to 25 µg/ml. At

the concentration of 100 µg/ml, the fraction showed toxicity effect on the cells and the cells did not attach onto the well surface (Figure 4.13F).

The percentage of neurite bearing cells was increased when the concentration of fraction E5 was increased. However, 10 µg/ml of fraction E5 was too low to show significant effect on the neurite outgrowth of the cells. At 25 µg/ml, 28.9 % of neurite bearing cells were observed and showed 39.6 % increase compared to negative control.

50 µg/ml of fraction E5 also showed significant effect on the neurite outgrowth of the cells when compared to 25 µg/ml. At the highest tested concentration of 100 µg/ml, fraction E5 was toxic towards the cells which damage the cells and prevent the cells from attach to the well surface (Figure 4.14F).

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Figure 4.12: The morphology of the NG108-15 cells treated with various concentrations of fraction E3 of Hericium erinaceus [24 hr of incubation at 37 ºC in a 5 % CO2 humidified incubator. NG108-15 cells without extract or treated with NGF (20 ng/ml) was negative and positive control, respectively.]

A: negative control (cells without extract); B: positive control - NGF (20 ng/ml);

C: 10 µg/ml of fraction E3; D: 25 µg/ml of fraction E3;

E: 50 µg/ml of fraction E3; F: 100 µg/ml of fraction E3

A B

C D

E F

neurite

neurite

neurite

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Figure 4.13: The morphology of the NG108-15 cells treated with various concentrations of fraction E4 of Hericium erinaceus [24 hr of incubation at 37 ºC in a 5 % CO2 humidified incubator. NG108-15 cells without extract or treated with NGF (20ng/ml) was negative and positive control, respectively.]

A: negative control (cells without extract); B: positive control - NGF (20 ng/ml);

C: 10 µg/ml of fraction E4; D: 25 µg/ml of fraction E4;

E: 50 µg/ml of fraction e4; F: 100 µg/ml of fraction E4

A B

C D

E F

neurite

neurite

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Figure 4.14: The morphology of the NG108-15 cells treated with various concentrations of fraction E5 of Hericium erinaceus [24 hr of incubation at 37 ºC in a 5 % CO2 humidified incubator. NG108-15 cells without extract or treated with NGF (20 ng/ml) was negative and positive control, respectively.]

A: negative control (cells without extract); B: positive control - NGF (20 ng/ml);

C: 10 µg/ml of fraction E5; D: 25 µg/ml of fraction E5;

E: 50 µg/ml of fraction E5; F: 100 µg/ml of fraction E5

A

C D

E

B

F

neurite

neurite

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Fraction E6 did not give any significant effect on the neurite outgrowth of NG108-15 cells at all tested concentration (10, 25, 50 and 100 µg/ml). Fraction E7 showed inhibition effect of the neurite outgrowth at all tested concentration. However, this inhibition effect did not show significant difference if compared to negative control.

From the cells morphology, the NG108-15 cells exhibited short extension when treated with fraction E6 and E7 (Figure 4.15 and Figure 4.16 respectively).

A comparison of fraction E1-E7 showed that fraction E1 and fraction E2 possessed the same trend of activity where the percentage of neurite bearing cells increase when the concentration of the fraction increased. Fraction E3, E4 and E5 showed the same trend of activity where the percentage of neurite bearing cells showed maximum increment at 50 µg/ml and the further increment of the concentration of fraction to 100 µg/ml showed damage effect towards the cells which might be due to toxicity effect. Fractions E6 and E7 did not give significant effect on the neurite outgrowth of neural hybrid cells of NG108-15.

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Figure 4.15: The morphology of the NG108-15 cells treated with various concentrations of fraction E6 of Hericium erinaceus [24 hr of incubation at 37 ºC in a 5 % CO2 humidified incubator. NG108-15 cells without extract or treated with NGF (20 ng/ml) was negative and positive control, respectively.]

A: negative control (cells without extract); B: positive control - NGF (20 ng/ml);

C: 10 µg/ml of fraction E6; D: 25 µg/ml of fraction E6;

E: 50 µg/ml of fraction E6; F: 100 µg/ml of fraction E6

A B

C D

E F

neurite

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Figure 4.16: The morphology of the NG108-15 cells treated with various concentrations of fraction E7 of Hericium erinaceus [24 hr of incubation at 37 ºC in a 5 % CO2 humidified incubator. NG108-15 cells without extract or treated with NGF (20 ng/ml) was negative and positive control, respectively.]

A: negative control (cells without extract); B: positive control - NGF (20 ng/ml);

C: 10 µg/ml of fraction E7; D: 25 µg/ml of fraction E7;

E: 50 µg/ml of fraction E7; F: 100 µg/ml of fraction E7

A B

C D

E F

neurite

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4.2.3 Effect of subfraction sub4b_4 and sub4b_6 of Hericium erinaceus on the neural cell line NG108-15

Subfractions sub4b_4 and sub4b_6 of H. erinaceus were screened for the in vitro neurite outgrowth activity on the neural hybrid cell line NG108-15 at various concentrations (µg/ml) (Figure 4.17; Table 4.4). Cells were observed under a phase contrast microscope for the neurite outgrowth and branching of neurites. The effect of the subfractions sub4b_4 and sub4b_6 on the morphology and neurite extension of the NG108-15 cells are given in Figure 4.18 and Figure 4.19 respectively.

Figure 4.17: Percentage of neurite bearing cells incubated with varying concentrations of subfractions sub4b_4 and sub4b_6 of Hericium erinaceus (nerve growth factor, 20 ng/ml, used as positive control).

17.0 20.6 20.5 24.7 29.6 48.8

17.2 21.6 19.8 23.4 18.4

0 10 20 30 40 50 60

Negative control

NGF (20ng/ml)

10 25 50 100

Neurite-bearing cells (%)

Concentrations (µg/ml)

sub4b_4 sub4b_6

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Table 4.4: Stimulation of neurite outgrowth activity of the NG108-15 cells with varying concentrations of subfractions sub4b_4 and sub4b_6 of Hericium erinaceus. NG108-15 cells without extract was negative control. 20 ng/ml of nerve growth factor (NGF) was used as positive control.

Treatment concentration (µg/ml)

Sub4b_4 Sub4b_6

Neurite bearing cells (%) Increase compared to control (%)

Neurite bearing cells (%) Increase compared to control (%)

Negative control 17.0±1.3a - 17.2±1.4a -

Positive control (NGF) 20.6±0.6b 21.4 21.6±1.1bc 25.1

10 20.5±1.9b 20.3 19.8±1.7abc 15.1

25 24.7±1.3c 45.2 23.4±1.4c 35.8

50 29.6±0.5d 73.8 18.4±1.8ab 6.7

100 48.8±1.6e 187.1 - -

Note: Data are expressed as means ± standard deviation (n = 2). Means with different letters in the same column are significantly different (P < 0.05), one-way analysis of variance/ANOVA).

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Figure 4.18: The morphology of the NG108-15 cells treated with various concentrations of subfraction sub4b_4 of Hericium erinaceus [24 hr of incubation at 37 ºC in a 5 % CO2 humidified incubator. NG108-15 cells without extract or treated with NGF (20 ng/ml) was negative and positive control, respectively.]

A: negative control (cells without extract); B: positive control - NGF (20 ng/ml);

C: 10 µg/ml of subfraction sub4b_4; D: 25 µg/ml of subfraction sub4b_4;

E: 50 µg/ml of subfraction sub4b_4; F: 100 µg/ml of subfraction sub4b_4

A B

C D

E F

neurite

neurite

neurite

neurite neurite

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Figure 4.19: The morphology of the NG108-15 cells treated with various concentrations of subfraction sub4b_6 of Hericium erinaceus [24 hr of incubation at 37 ºC in a 5 % CO2 humidified incubator. NG108-15 cells without extract or treated with NGF (20 ng/ml) was negative and positive control, respectively.]

A: negative control (cells without extract); B: positive control - NGF (20 ng/ml);

C: 10 µg/ml of subfraction sub4b_6; D: 25 µg/ml of subfraction sub4b_6;

E: 50 µg/ml of subfraction sub4b_6; F: 100 µg/ml of subfraction sub4b_6

A B

C D

E F

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Table 4.4 and Figure 4.10 showed the effects of various concentrations of subfractions sub4b_4 and sub4b_6 on neurite outgrowth of the NG108-15 cells after 24 hr of incubation. Subfraction sub4b_4 showed significant increase of neurite bearing cells at the concentration as low as 10 µg/ml. Subfraction sub4b_4 showed an increase in neurite bearing cells when the dose was increased exponentially (10, 25, 50, 100 µg/ml). At the highest tested concentration of 100 µg/ml, an increase of 187.1 % of

neurite bearing cells was observed when compared to negative control.

At 25 µg/ml, subfraction sub4b_6 showed a maximal and significant increase of neurite bearing cells (23.4 % of neurite bearing cells and 35.8 % of increase in neurite bearing cells if compared to negative control). Other tested concentrations did not show significant difference in neurite bearing cells if compared to negative control. Further increase of the concentration after 25 µg/ml showed decrease in neurite bearing cells. At 100 µg/ml, the cells did not attach to the well surface due to the cell damage or cell death caused by the toxic effect of the extract.

Subfraction sub4b_4 (Figure 4.18) showed obvious stimulation of neurite outgrowth compared to subfraction sub4b_6 (Figure 4.19). Subfraction sub4b_4 showed maximal stimulation of 48.8 % neurite bearing cells at 100 µg/ml while subfraction sub4b_6 only showed maximal stimulation of 23.4 % neurite bearing cells at 25µg/ml.

Further increment of the concentration after 25 µg/ml for subfraction sub4b_6 showed negative effect on the cells. 20 ng/ml of nerve growth factor (NGF) was served as positive control in subfraction sub4b_4 and sub4b_6. In both subfraction, NGF showed significant effect on neurite bearing cells compared to the negative control.

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4.3 OVERALL COMPARISON OF AQUEOUS ETHANOL EXTRACT, HEXANE FRACTION, ETHYL ACETATE FRACTION, WATER FRACTION, FRACTION E1-E7, SUBFRACTION SUB4B_4 AND SUBFRACTION SUB4B_6.

After the overall comparison of all the extract, fractions and subfractions, the low neurite stimulation activity of crude aqueous ethanol extract might be due to the concentration of the active compounds in the crude extract form was too low to induce the neurite outgrowth activity. The crude extract contains a lot of compounds which were in the mixture form and the mixture of all compounds might show synergistic effects.

This situation was proved when the crude aqueous ethanol extract was further fractionated into hexane, ethyl acetate and water fraction. The neurite stimulation ability of all the fractions was higher if compared to the crude extract and it was due to the active compounds were further separated and concentrated after the fractionation. It was further proved when the combined fraction of hexane and ethyl acetate was further separated into 7 fractions (fraction E1-E7). The two most active fractions, fraction E1 and E2, which showed the maximum increase of neurite bearing cells of 160.6 % and 149.1 % at 100 µg/ml when compared to the hexane and ethyl acetate fraction (65.2 % and 68.5 % respectively). It showed that most of the active compounds in hexane and ethyl acetate fraction were separated and concentrated into fraction E1 and E2. Besides that, H. erinaceus might contain some compounds that might toxic to the cells at high concentration which separated and concentrated into fraction E3-E5. At 100 µg/ml, fraction E3-E5 showed toxicity effect to the cells which damage the cells and prevent it from attach to the well surface.

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compared to fraction E2 (149.1 %) at 100 µg/ml. It showed that the active compounds that stimulate the neurite outgrowth might present in subfraction sub4b_4. The relatively low activity of neurite outgrowth in subfraction sub4b_4 when compared to subfraction sub4b_4 and fraction E2 indicated that the active compounds that present in subfraction sub4b_6 were nil.

Nerve growth factor (NGF) was served as positive control in the entire neurite outgrowth assay. The percentage of neurite bearing cells that induced by the NGF were significantly difference when compared to negative control in all the assay.

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4.4 IDENTIFICATION OF CHEMICAL CONSTITUENTS

4.4.1 Identification of chemical constituents of fraction E1

Identification of the chemical constituents of fraction E1 was carried out using GC-MS. Components in fraction E1 of H. erinaceus is presented in Table 4.5. The constituents were identified by matching their mass spectral data with those in the accompanying mass spectral database (NIST 05 – Mass Spectral Library, USA).

Table 4.5: Identified constituents of fraction E1 of Hericium erinaceus.

No.

Identified components

Area

percentage (%)

Molecular weight

Molecular formula

Method of identification 1 ethyl palmitate 29.81 284 C18H36O2 MS

2 ethyl stearate 2.26 312 C20H40O2 MS

3 ethyl oleate 18.56 310 C20H38O2 MS

4 ethyl linoleate 29.85 308 C20H36O2 MS

Total 80.48

Note: Percentages were calculated on the basis of results obtained from the total ion chromatogram.

MS = Mass Fragmentation

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The chemical components of fraction E1 of H. erinaceus was analyzed by GC- MS. Four compounds comprising about 80.48 % of the total volatile compounds in fraction E1 were identified. The major compound was ethyl linoleate (29.85 %), followed by ethyl palmitate (29.81 %), ethyl oleate (18.56 %) and ethyl stearate (2.26

%). The other remaining unidentified compounds comprised of 19.52 % of the total volatile compounds in fraction E1.

There were no previous reported studies on the presence of ethyl linoleate, ethyl palmitate, ethyl oleate and ethyl stearate in H. erinaceus. However, Miyazawa et al., 2008 reported the presence of methyl palmitate and methyl linoleate in H. erinaceus which were not detected in fraction E1. The two major fatty esters, ethyl linoleate and ethyl palmitate were the esterified products of the reported fatty acids isolated from the H. erinaceus; linoleic acid (Miyazawa et al., 2008) and palmitic acid (Wang et al., 2005;

Miyazawa et al., 2008).

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4.4.2 Identification of chemical constituents of the subfraction sub4b_4

Sub4b_4 was obtained as white crystals and the components in this fraction were identified by NMR spectroscopy and LC/MS/MS. Two compounds and their isomers were identified as (I) and its isomer and (II) and its isomer (Figure 4.20). The NMR spectral data (Table 4.6) can be assigned to hericenone C and 4-(3’,7’-dimethyl-5’-oxo- 2’,6’-octadienyl)-2-formyl-3-hydroxy-5-methoxylbenzyl oleate.

The peaks at retention time 7.66 and 8.72 in the LC/MS/MS chromatogram were identified as I, hericenone C and its isomer (Appendix 24 and Appendix 26). The 1H- NMR and 13C NMR spectrum (Table 4.6) of compound I was identical to that reported by Kawagishi et al. (1991) for hericenone C. The main structure of compound I was a penta-substituted phenyl moiety [δH=6.52 (s, 6CH); δC=138.75 (C-1), 112.96 (C-2), 162.99 (C-3), 117.38 (C-4), 163.55 (C-5) and 105.62 (C-6)], a hydroxyl group [δH=12.37 (s, OH)], a formyl group [δH = 10.10 (s, CHO); δC=193.18 (CHO)], a methoxyl group [δH=3.90 (s, OCH3); δC=56.00 (OCH3)], a 10-carbon side chain with two olefinic bonds [δH=3.39 (d, J=7.32Hz, 1’CH2), 5.31 (m, overlapping proton of 2’CH and C1-CH2-O), 2.99(s, 4’CH2), 6.08 (s, 6’CH), 1.83 (s, 8’CH3), 1.77 (s, 3’-CH3) and 2.11 (s, 7’-CH3); δC=21.68 (C-1’), 126.32 (C-2’), 130.43 (C-3’), 16.48 (C-3’-CH3), 55.63 (C-4’), 199.63 (C-5’), 122.89 (C-6’), 155.54 (C-7’), 20.74 (C-7’-CH3) and 27.75 (C-8’)], a methylene group [δH=5.31 (s, C1-CH2-O); δC=62.98 (C-1-CH2-O)] to which is attached a palmitate side chain [δH=2.32 (t, J=7.32Hz, 2”CH2), 1.60 (m, 3”CH2), 0.87 (t, J=6.88Hz, 16”CH3) and 1.23 (hide underneath, 7”CH2-15CH2); δC=173.28 (C-1”), 34.31 (C-2”), 32.01, 29.74, 29.66, 29.52, 29.44, 29.30, 29.19, 24.95, 22.77 (C-3” to C- 15”) and 14.20 (C-16”)]. Compound I in subfraction sub 4b_4 exhibited [M+H]+ ion at

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to the loss of the palmitate side chain. The base peak at m/z 176.9 is consistent with loss

of CH

O

+ H

from the fragment ion at m/z 315.0. However, there are observed identical nominal mass for 571.3 at different retention time (7.66 and 8.72 min) with similar fragmentation pattern. This might be due to potentially stereoisomeric compounds which cannot be determined using 1H-NMR and 13C NMR when present in a mixture. From the NMR and LC/MS/MS results, it can be concluded that the nmr data is consistent with 4-(3’,7’-dimethyl-5’-oxo-2’,6’-octadienyl)-2-formyl-3-hydroxy-5- methoxylbenzyl palmitate which also known as hericenone C.

Compound II and its isomer in sub4b_4 were identified using LC/MS/MS.

Compound II and its isomer showed identical nominal mass for m/z 597.3 at different retention time (7.92 and 9.01 min); both peaks sharing similar mass spectral fragmentation pattern. A comparison of the mass spectrum of compound II and its isomer with those of compound I and its isomer revealed that both had the same fragmentation pattern, the only difference was the molecular ion (m/z 571.3 for compound I and m/z 597.3 for compound II). Compound I and compound II only differ in the component of the fatty ester side chain which was the oleate in compound II instead of palmitate. The NMR data for compound II should be similar to hericenone C except for the presence of the olefinic protons at C-9”. It is predicted that these two protons would resonate at δ = 5.31 superimposing that of 2’CH and C1-CH2_O protons.

It is highly probable that compound II might be 4-(3’,7’-dimethyl-5’-oxo-2’,6’- octadienyl)-2-formyl-3-hydroxy-5-methoxylbenzyl oleate.

The remaining unidentified compound in subfraction sub4b_4 displayed [M+H]+ at m/z 148.8 with fragment ions at m/z 76.7, 94.7 and 104.7 (Appendix 23).

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(I) R =

O 1'' 2''

3'' 4''

5'' 6''

7'' 8''

9'' 10''

11'' 12''

13'' 14''

15'' 16''

Hericenone C and its isomer [mw = 570 (identified using NMR and LC/MS/MS)]

(II) R =

O 1'' 2''

3'' 4''

5'' 6''

7'' 8''

9'' 10'' 11''

12'' 13''

14'' 15''

16'' 17''

18''

4-(3’,7’-dimethyl-5’-oxo-2’,6’-octadienyl)-2-formyl-3-hydroxy-5-

methoxylbenzyl oleate and its isomer [mw = 597 (identified by using LC/MS/MS)]

Figure 4.20: Compounds (I and II) identified in subfraction sub4b_4.

O

H3CO

OH

CHO

1 OR

2 3 4

5 6 1'

2' 3' 4' 5' 6' 7' 8'

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Table 4.6: 1H- and 13C-NMR for subfraction sub4b_4 in CDCl3. Proton δH (multiplicity, Hz) Reference for

δH *

Carbon-13 δC Reference for δC *

6CH 6.52 (s) 6.53 (s) C-6 105.62 105.55

C1-CH2-OR 5.31 (s) 5.32 (s) C-1-CH2

C-1

62.98 138.75

62.90 138.68

CHO 10.10 (s) 10.11 (s) CHO

C-2

193.18 112.96

193.11 112.88

OH 12.37 (s) 12.38 (s) C-3 162.99 162.92

OCH3 3.90 (s) 3.91 (s) OCH3

C-5

56.00 163.55

55.93 163.47 1’-CH2 3.39 (d, 7.32) 3.40 (d, 7.33) C-1’

C-4

21.68 117.38

21.61 117.29 2’CH 5.31 (overlapping

with H-1-CH2 proton)

5.32 (t, 7.33) C-2’ 126.32 126.25

3’-CH3 1.77 (s) 1.78 (s) C-3’-CH3

C-3’

16.48 130.43

16.40 130.34

4’CH2 2.99 (s) 3.01 (s) C-4’

C-5’

55.63 199.63

55.56 199.53

6’-CH 6.08 (s) 6.09 (s) C-6’ 122.89 122.81

7’-CH3 2.11 (s) 2.12 (s) C-7’-CH3

C-7’

20.74 155.54

20.67 155.45

8’-CH3 1.83 (s) 1.84 (s) C-8’ 27.75 27.67

C-1” 173.28 173.19 2”CH2 2.32 (t, 7.72) 2.33 (t, 7.70) C-2” 34.31 34.23

3”-CH2 1.60 (m) 1.61 (m)

C-3” to C- 15”

32.01 29.74 29.66 29.52 29.44 29.30 29.19 24.95 22.77

31.93 29.70 29.68 29,66 29.65 29,44 29.36 29.23 29.12 24.88 22.70 7”CH2,

15”CH2

1.23 (hide underneath)

1.25 (m)

16”CH3 0.87 (t, 6.88) 0.88 (t, 6.96) C-16” 14.20 14.32

* Reference for δH and δC (Kawagishi et al., 1991)

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4.4.3 Identification of chemical constituents of the subfraction sub4b_6

Sub4b_6 was isolated as white crystals. The chemical components of sub4b_6 were analyzed by JEOL 400MHz NMR spectroscopy and LC/MS/MS. Three compounds identified in this subfraction were hericenone C (compound I), 4-(3’,7’- dimethyl-5’-oxo-2’,6’-octadienyl)-2-formyl-3-hydroxy-5-methoxylbenzyl oleate (compound II) and compound III (Figure 4.21).

The peak at retention time 9.11 minute (Appendix 30) cleaved a molecular ion peak at 571 (M+H)+ with mass spectral data m/z 315.0, 233.0, 217.0, 176.9 and 82.7.

The peak at m/z 315 is consistent with the fragment ion which arises from the following fragmentation due to loss of fatty acid side chain.

m/z=571.3

m/z=315

The 1H-NMR and 13C NMR spectrum (Table 4.7) could be assigned to those of O

H3CO

OH

CHO

1 OR

2 3 4

5 6 1'

2' 3' 4' 5' 6' 7' 8'

O

H3CO

OH

CHO

CH2

1 2 3 4

5 6 1'

2' 3' 4' 5' 6' 7' 8'

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(C-3), 117.38 (C-4), 163.55 (C-5) and 105.62 (C-6)], a hydroxyl group [δH=12.37 (s, OH)], a formyl group [δH = 10.10 (s, CHO); δC=193.18 (CHO)], a methoxyl group [δH=3.90 (s, OCH3); δC=56.00 (OCH3)], a 10-carbon side chain at C-4 with two olefinic bonds [δH=3.39 (d, J=7.32Hz, 1’CH2), 5.31 (m, overlapping proton of 2’CH and C1- CH2-O), 2.99(s, 4’CH2), 6.08 (s, 6’CH), 1.83 (s, 8’CH3), 1.77 (s, 3’-CH3) and 2.11 (s, 7’-CH3); δC=21.68 (C-1’), 126.32 (C-2’), 130.43 (C-3’), 16.48 (C-3’-CH3), 55.63 (C-4’), 199.63 (C-5’), 122.89 (C-6’), 155.54 (C-7’), 20.74 (C-7’-CH3) and 27.75 (C-8’)], a methylene group [δH=5.31 (s, C1-CH2-O); δC=62.98 (C-1-CH2-O)] to which is attached a palmitate side chain [δH=2.32 (t, J=7.32Hz, 2”CH2), 1.60 (m, 3”CH2), 0.87 (t, J=6.88Hz, 16”CH3) and 1.23 (7”CH2-15CH2); δC=173.28 (C-1”), 34.31 (C-2”), 32.01, 29.74, 29.66, 29.52, 29.44, 29.30, 29.19, 24.95, 22.77 (C-3” to C-15”) and 14.20 (C- 16”)]. Therefore, it can be concluded that the nmr data is consistent with 4-(3’,7’- dimethyl-5’-oxo-2’,6’-octadienyl)-2-formyl-3-hydroxy-5-methoxylbenzyl palmitate also called hericenone C.

As the NMR spectrum was determined for a mixture, only some peaks can be accounted for whilst others could not be assigned. The peak at retention time 9.31 min was identified as 4-(3’,7’-dimethyl-5’-oxo-2’,6’-octadienyl)-2-formyl-3-hydroxy-5- methoxylbenzyl oleate using LC/MS/MS. A comparison of the mass spectrum of compound II with those of hericenone C revealed that both had the same fragmentation pattern, the only difference was the molecular ion (m/z 571.3 for hericenone C and m/z 597.3 for compound II). Hericenone C and compound II only differ in the component of the fatty ester side chain which was the oleate in compound II instead of palmitate.

The peak at retention time 8.02 minute was identified as compound III using LC/MS/MS (Appendix 29). Compound III exhibited an [M+H]+ ion at m/z 569.3. In contrast to compound I which showed the base peak at m/z 176.9, compound III has a

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base peak at 178.9 and absence of peak at m/z 315. Exclusion of the 10-carbon side chain containing two olefins attached to the phenyl ring at C-4 of hericenone C, gave the base peak of m/z 178.9. The base peak at m/z 178.9 is therefore consistent with the following structure.

H3C

H3CO

OH

CHO

CH2 1

2 3 4

5 6

m/z=178.9

The fatty ester side chain attached to the methylene group most probably contained 26 carbons with 3 double bonds. However, the chemical structure of the fatty ester side chain cannot be determined using the LC/MS/MS mass spectral data.

The remaining unidentified compound in subfraction sub4b_6 displayed [M+H]+ at m/z 162.9 with fragment ions at m/z 76.7, 94.7 and 104.7.

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(I) R =

O 1'' 2''

3'' 4''

5'' 6''

7'' 8''

9'' 10''

11'' 12''

13'' 14''

15'' 16''

Hericenone C and its isomer [mw = 570 (identified using NMR and LC/MS/MS)]

(II) R =

O 1'' 2''

3'' 4''

5'' 6''

7'' 8''

9'' 10'' 11''

12'' 13''

14'' 15''

16'' 17''

18''

4-(3’,7’-dimethyl-5’-oxo-2’,6’-octadienyl)-2-formyl-3-hydroxy-5-

methoxylbenzyl oleate and its isomer [mw = 597 (identified by using LC/MS/MS)]

OH

CHO

H3CO H3C

OR

(III) R = fatty ester side chain containing 26 carbons with 3 double bonds Figure 4.21: Compounds (I, II and III) identified in subfraction sub4b_6.

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Table 4.7: 1H- and 13C-NMR for subfraction sub4b_6 in CDCl3. Proton δH (multiplicity, Hz) Reference for

δH *

Carbon-13 δC Reference for δC *

6CH 6.52 (s) 6.53 (s) C-6 105.62 105.55

C1-CH2-OR 5.31 (s) 5.32 (s) C-1-CH2

C-1

62.98 138.75

62.90 138.68

CHO 10.10 (s) 10.11 (s) CHO

C-2

193.18 112.96

193.11 112.88

OH 12.37 (s) 12.38 (s) C-3 162.99 162.92

OCH3 3.90 (s) 3.91 (s) OCH3

C-5

56.00 163.55

55.93 163.47 1’-CH2 3.39 (d, 7.32) 3.40 (d, 7.33) C-1’

C-4

21.68 117.38

21.61 117.29 2’CH 5.31 (overlapping

with H-1-CH2 proton)

5.32 (t, 7.33) C-2’ 126.32 126.25

3’-CH3 1.77 (s) 1.78 (s) C-3’-CH3

C-3’

16.48 130.43

16.40 130.34

4’CH2 2.99 (s) 3.01 (s) C-4’

C-5’

55.63 199.63

55.56 199.53

6’-CH 6.08 (s) 6.09 (s) C-6’ 122.89 122.81

7’-CH3 2.11 (s) 2.12 (s) C-7’-CH3

C-7’

20.74 155.54

20.67 155.45

8’-CH3 1.83 (s) 1.84 (s) C-8’ 27.75 27.67

C-1” 173.28 173.19 2”CH2 2.32 (t, 7.72) 2.33 (t, 7.70) C-2” 34.31 34.23

3”-CH2 1.60 (m) 1.61 (m)

C-3” to C- 15”

32.01 29.74 29.66 29.52 29.44 29.30 29.19 24.95 22.77

31.93 29.70 29.68 29,66 29.65 29,44 29.36 29.23 29.12 24.88 22.70 7”CH2,

15”CH2

1.23 (hide underneath)

1.25 (m)

16”CH3 0.87 (t, 6.88) 0.88 (t, 6.96) C-16” 14.20 14.32

* Reference for δH and δC (Kawagishi et al., 1991)

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4.5 OVERALL COMPARISON OF THE IDENTIFIED COMPOUNDS AND THE NEURITE STIMULATION ACTIVITY IN FRACTION E1, FRACTION E2 (SUBFRACTION SUB4B_4 AND SUBFRACTION SUB4B_6) OF HERICIUM ERINACEUS

When the active fractions were evaluated by the neurite outgrowth assay, the components in the active fractions of H. erinaceus displayed some correlated results.

Four compounds were identified from the active fraction E1 namely ethyl linoleate, ethyl palmitate, ethyl oleate and ethyl stearate. In the fatty acid form, linoleic acid and oleic acid have been reported to play an important role in the neurological system.

Linoleic acid is an essential fatty acid which is the starting material for the biosynthesis of arachidonic acid. Arachidonic acid was shown to play important roles in the maintenance of the hippocampal neuron membrane fluidity in the brain (Fukaya et al., 2007), enhancement of the stimulation of neurite outgrowth by activating the plasma membrane protein, syntaxin 3 (Darios and Davletov, 2006) and protection against oxidative stress in the brain (Wang et al., 2006). Oleic acid showed decrease in the content of the saturated very long chain fatty acids level in vitro and in vivo (Rizzo et al., 1986; 1987). Therefore, it might be useful in the treatment of adrenoleukodystrophy, a neurological disease that leads to the damage of brain and adrenal gland due to the accumulation of the saturated very long chain fatty acids level.

Subfractions sub4b_4 and subfraction sub4b_6 were the isolated subfractions of fraction E2. Hericenone C was the compound present in both subfraction. Hericenone C was reported to have stimulating activity for the biosynthesis of NGF in vitro at the concentration of 33µg/ml (Kawagishi et al., 1991). The neurite stimulating activity was increased when the cells were treated with subfraction sub 4b_4 at all tested concentrations (10, 25, 50, 100 µg/ml) while the neurite stimulating activity of

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subfraction sub 4b_6 decreased after 25 µg/ml. From the neurite stimulating results, it revealed that hericenone C might not be the major compound responsible for neurite stimulation. This is in agreement with the results whereby hericenone C did not increase NGF mRNA expression in 1321N1 human astrocytoma cells and primary cultured rat astroglial cells (Mori et al., 2008). The relatively low activity of neurite stimulation in subfraction sub4b_6 also indicate that 4-(3’,7’-dimethyl-5’-oxo-2’,6’-octadienyl)-2- formyl-3-hydroxy-5-methoxylbenzyl oleate and compound III might not be the active compounds that stimulate the neurite outgrowth. The good ability of subfraction sub4b_4 in stimulating neurite outgrowth indicates the presence of active compounds in this subfraction. Isomer of hericenone C, isomer of 4-(3’,7’-dimethyl-5’-oxo-2’,6’- octadienyl)-2-formyl-3-hydroxy-5-methoxylbenzyl oleate and another unidentified compound with molecular weight of 148 were first reported present in H. erinaceus.

Therefore, the ability of these compounds to stimulate neurite outgrowth still remained unknown. It is highly probable that other compounds in the respective subfraction maybe responsible for the meurite stimulation activity.

All of the identified compounds in active fractions [ethyl linoleate, ethyl palmitate, ethyl oleate and ethyl stearate in fraction E1; hericenone C and its isomer, 4- (3’,7’-dimethyl-5’-oxo-2’,6’-octadienyl)-2-formyl-3-hydroxy-5-methoxylbenzyl oleate and its isomer, and another unidentified compound with molecular weight of 148 in subfraction sub 4b_4] are small and lipid soluble compounds. According to Banks (2009), lipid soluble compounds with molecular weight less than 600 Da are able to cross blood-brain barrier by transmembrane diffusion. Therefore, the identified compounds that cross the blood-brain barrier and stimulate NGF synthesis in brain might be responsible in stimulating the neurons to regrow. Due to the limitation of NGF

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molecular weight that elicit stimulatory activity on NGF synthesis are much more useful and practical for therapeutic purposes.

Neurodegenerative diseases are always correlated with the neuronal cell death caused by the production of free radicals and reactive oxygen species (Halliwell et al., 1999; Lee et al., 2003). Thus, it is reasonable to suspect molecules which are able to attenuate the production or scavenge the free radicals and reactive oxygen species might reduce the risk of gaining the neurodegenerative diseases. Linoleic acid, the fatty acid form of ethyl linoleate, is an essential fatty acid required for biological processes.

According to Ismail et al. (2004) and Kirmizigul et al. (2007), a positive correlation between essential fatty acid and antioxidant was found. The identified phenols (hericenone C and its isomer, 4-(3’,7’-dimethyl-5’-oxo-2’,6’-octadienyl)-2-formyl-3- hydroxy-5-methoxylbenzyl oleate and its isomer) might have antioxidant activity as several studies which reported a highly positive relation between phenolic content and antioxidant activity in plants and fruits (Tabart et al., 2009; Sim et al., 2010; Mandana et al., 2012). Besides that, Li et al. (2012) proved that phenolic compounds present in H.

erinaceus exhibited strong antioxidant activity.

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CHAPTER V

GENERAL DISCUSSION & CONCLUSION

Neurite outgrowth, a process responsible for neuronal patterning and connections, is crucial for the development of the nervous system. The NG108-15 cell line is most widely used as an in vitro model of neuronal differentiation because of its high proliferative activity and rapid elaboration of neurites (Smalheiser, 1991). The advantage of this bioassay is that it uses a continuous cell line, thus avoiding the need for dissection. Experiment using this cell line is highly reproducible and this cell line is sensitive to sub-nanogram concentrations of NGF. Cells at plating were spherical and extended multiple filopodial processes were observed within the first 15-30 minutes.

Cell adherence to the surface occurred within 4 hr after plating. A cell was considered as positive for bearing neurites if it had at least one thin extension longer than one full diameter of its cell body. Short neurite-like extensions appeared within the first 4 hr, but less than 5 % of cells reached the criterion length of one cell body to be scored as a

‘neurite’.

After 24 hr of plating, cells expressing neurites were scored. In the presence of the extract, obvious enhancement of outgrowth was observed in cells. A cell was scored positive for bearing neurites if it had at least one short extension longer than one full diameter of its cell body (Smalheiser, 1991). Further, after 24 hr most cells remained as individual and there was minimal clumping which made scoring easier.

Nerve growth factor (NGF) was used as the positive control in the neurite outgrowth assay and showed significant effect for the stimulation of neurite outgrowth.

NGF is a protein molecule composed of 118 amino acid residues. It is known to be

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development. Since the discovery, NGF has been expected to be a potent anti-dementia drug, and many efforts have been made to confirm its therapeutic effects on the memory disorders. Intracerebroventricular and intranasal administrations of NGF improve the degeneration disease in the brain (Fischer et al., 1987; Kromer, 1987; Ricceri et al., 1996; Capsoni et al., 2002). However, neurotrophic factors are proteins, and unable to cross the blood brain barrier; they are also easily metabolized by peptidases. Therefore, their application as a medicine for the neurodegenerative disorders is assumed to be difficult. A useful strategy to overcome this problem is to develop drugs that could either cross the blood brain barrier or show NGF-like effect. Compounds which stimulate NGF synthesis in the brain will stimulate neurons to regrow.

In this study, poly-D-lysine was used to coat the 6 well plates. From the previous study, polylysine did exhibit attachment-promoting activity with a minimal of rapid-onset neurite formation (Smalheiser, 1991). Laminin, another coating agent, which have been used in most previous studies of serum deprivation-induced neurite outgrowth, induce rapid onset neurites to an unacceptable extent (12 % of cells bearing neurites within 4 hr post plating) (Smalheiser, 1991).

Freeze drying may be the best method for long term preservation of bioactive compounds in mushrooms that stimulate neurite outgrowth. It has been reported that extracts of oven dried fruit bodies of H. erinaceus stimulated short cellular extensions that were insufficiently elongated to be scored as neurites while freeze dried fruit bodies showed potent neurite stimulation ability. (Wong et al., 2007).

80% (v/v) aqueous ethanol was used to extract the fruiting bodies of H.

erinaceus because the present quantitative data revealed that aqueous and ethanol extracts showed good activity of stimulation of NGF. Erinacines A, B, C, E, F, G, stimulators of NGF synthesis, were extracted from the mycelium of H. erinaceus using

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ethanol (Kawagishi et al., 1994; 1996). However, from previous reported study, ethanol promotes the neuronal differentiation of NG108-15 resulting in reduction in the number of cell, whilst the cell size and incidence of neurite formation was increased (Harrison et al., 1997). Therefore, ethanol was used to extract the fruiting bodies of H. erinaceus must be completely evaporated to avoid the neurite outgrowth effect caused by ethanol.

DMSO was used to dissolve the extract and fractions of H. erinaceus. The final concentration of DMSO added to the culture was relatively low (less than 0.5 % v/v).

DMSO is recognized to have radical scavenging activity and may contribute to the protective effect against cell lines (Bishayee et al., 2000). Furthermore, high concentration of DMSO may cause toxicity to the cell. From the reported study, 0.5 % v/v of DMSO had no effect on cell viability in the hybrid cell NG108-15 (Mahakunakorn et al., 2003).

Tween 80 was added to the stock solution of subfraction sub4b_4 and subfraction sub4b_6 to prevent the precipitation problem that occurred during the serial dilution process with complete growth medium. Tween 80 also known as polysorbate 80 was widely used in food products and oral pharmaceutical. Tween 80 was used in the emulsion pharmaceutical preparation in order to improve the solubility of therapeutic agents and serve as the drug delivery system carrier (Weis & Liao, 2000; Malingre et al., 2001; Zhang et al., 2003). According to previous reported studies, Tween 80 did not show toxic and adverse effects when examined the growth, food utilization and metabolism, physiological behavior, post-mortem pathology in rats (Oser et al., 1956) and on the human colonic adenocarcinoma cell (Sullivan et al., 2004). It was approved as a pharmaceutical excepient for use in oral preparations by The United States Pharmacopeia–National Formulary (USP–NF).

Rujukan

DOKUMEN BERKAITAN

Table B.4: Percentage of neurite bearing cells in the cell line PC12 in response to treatments with aqueous and ethanol extracts of Lignosus rhinocerotis and

polyanthum methanol extract (ME), chloroform fraction (CF), water fraction (WF), and n-hexane fraction (SF-1), and squalene (SQ) on glucose uptake of abdominal muscle strips in

Crude methanol extract, n-hexane fraction, ethyl acetate fraction, n-butanol fraction as well as daucosterol were subjected to the neuraminidase inhibition assay (MUNANA

Figure 3.8 The effects of leaf ethyl acetate fraction of ethanol/water (1:1) extract of Piper betle (BLF) and leaf ethyl acetate fraction of ethanol extract of

The hexane fraction added with BHT has lowered the decreasing of peak at wavenumber of 1709 cm-1 rather than hexane fraction containing α-tocopherol, meaning that

Figure 3.8 The effects of leaf ethyl acetate fraction of ethanol/water (1:1) extract of Piper betle (BLF) and leaf ethyl acetate fraction of ethanol extract of

Proposed model of Phaleria macrocarpa ethyl acetate fraction (PMEAF) mechanism of action for apoptosis in human breast cancer MDA-MB-231 cell

(a) Extract the TPC and evaluate its antioxidant activity using five different extraction solvents (water, 50% aqueous methanol, methanol, ethyl acetate and hexane) from