Enzyme-assisted Extraction and Enrichment of Galanthamine from Lycoris aurea

Tian CL et al. Chinese Herbal Medicines, 2016, 8(2): 182-188

182

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Original article 

Enzyme-assisted Extraction and Enrichment of Galanthamine from Lycoris aurea Chun-lian Tian1*, Peng Wang1, Ji-xin Qin2, Xiao-pan Liu1, Ke Song1, Zhuo-bing Xiao1 1. Key Laboratory of Forest Products and Chemical Industry Engineering, Jishou University, Zhangjiajie 427000, China 2. The First High School of Zhangjiajie, Zhangjiajie 427000, China

ARTICLE INFO 

  ABSTRACT   

Article history

Objective

Received: November 21, 2015 Revised: January 5, 2016 Accepted: February 11, 2016 Available online: April 8, 2016

and separation effect of cation exchange resin on galanthamine. Methods Cellulase and pectinase solution was used as the extraction solvent. The effects of

pH value of enzyme, amount of complex enzyme, dissociation time, and enzymatic hydrolysis temperature on the extraction results were investigated. Results

10.1016/S1674-6384(16)60028-X

The

optimal conditions were obtained as follows: ratio of solid to liquid (g: mL) 1:10, pH

value 4.5, amount of complex enzymes 4%, enzymatic hydrolysis temperature 50 o

DOI:

To explore the optimum condition for complex enzyme-assisted

extraction of galanthamine from Lycoris aurea by L 9 (3 4) orthogonal array design

C, and reaction time 2.0 h. Under these conditions, the extraction yield of

galanthamine was 0.0294%. In addition, D-001 cation exchange resin was selected

for separation of galanthamine. The separation conditions were that adsorption flow rate was 3 BV/h with reagent of pH 2 and the desorption flow rate was 3 BV/h with 70% ethanol solution containing 1.5 mol/L ammonia. After separation, the content of galanthamine was increased to 12.31%. Conclusion a reference for industrial production of galanthamine.

The results provide

Key words

cation exchange resin; enzyme-assisted extraction; galanthamine; Lycoris aurea; separation

1.    Introduction  Lycoris aurea (L'Hér.) Herb., also called golden spider lily, is a perennial plant in the Amaryllidaceae family and found in eastern Asia, mainly Korea, eastern and southern China, and Japan (Kang et al, 2012). Most plants in Amaryllidaceae are known to produce unique alkaloids with diverse bioactivities, such as acetylcholinesterase inhibitory, analgesic, antibacterial, antifungal, antimalarial, antitumor, antiviral activities, and so on (Wu et al, 2014). Especially, *

Corresponding author: Tian CL

© 2016 published by TIPR Press. All rights reserved.

galanthamine exhibited the significant benefits in cognition behavior in rats, mice, and rabbits with cognitive deficits, which has been proven for the symptomatic treatment of Alzheimer’s disease (Tsvetkova et al, 2014; Marco et al, 2006). Some extraction methods have been developed to obtain alkaloids from the plants of Amaryllidaceae family, such as solvent extraction, ultrasonic extraction, microwave extraction, supercritical fluid extraction, and so on (Li et al, 2008; Fan et al, 2006; Wang et al, 2013; Xiao et al, 2005; Du et al, 2007). However, due to these methods also existed some

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Funds: Hunan Provincial Science and Technology Department (2012TP4002-3); Zhangjiajie Science and Technology Bureau (2014YB17)

Tian CL et al. Chinese Herbal Medicines, 2016, 8(2): 182-188 disadvantages like massive organic solvent consumption, high cost, while low yield, in recent years, enzyme-assisted extraction has attracted more attention relying on its advantages of efficiency, mild-condition, and pollution-free. According to Puri et al (2012), carbohydrate- hydrolyzing enzymes like cellulases, hemicellulases, and pectinases disrupt cell wall with the hydrolysis of its components are leading to a major permeability and allowing an easier release of the metabolites from plants. Until now, there is no related report on the utilization of carbohydrate-hydrolyzing enzymes in the extraction of galanthamine from L. aurea. We had also tried to utilize cation exchange resin for enhancing the separation efficiency of galanthamine. However, this technique has never been applied to galanthamine isolation. In the present study, in order to improve the extraction rate of galanthamine and develop a greener extraction method, the enzyme-assisted extraction method was investigated for reaction and extraction variables, such as enzyme types, enzyme ratio, reaction temperature, pH value, time, and the solid to liquid ratio. In addition, the separation of galanthamine by cation exchange resin was also studied.

2.    Materials and methods   

2.1    Materials and instruments  The bulbs of L. aurea were collected from Huaihua City, Hunan province, China. The botanical authentication was carried out by researcher Bo-ru Liao, plant taxonomist at Jishou University. Galanthamine was obtained from National Institute for Food and Drug Control with purity > 98%. The cellulase (2 × 104 U/g) was purchased from Baimai Green Bio-Energy Co., Ltd. (Huaian City, China). The hemicellulase (1 × 104 U/g) and pectinase (3 × 104 U/g) were from Ruji Biotech. Co., Ltd. (Shanghai, China). Cation exchange resins 732 (001 × 7), HD−8, HZ−008, D155, D001, and D152 were obtained from Zhengzhou Diligent Technology Limited (Zhengzhou, China). All organic solvents were of analytical grade, except for HPLC (Tedia, America). HPLC (Agilent 1260, America) is used for the determination of galanthamine. Vacuum Freeze Dryer FD5-3 (SIM, America) is applied for drying.

2.2    Methods    2.2.1    Selection of enzyme types  The bulbs of L. aurea were washed, dried, and ground. For enzyme-assisted extraction, bulb powders were suspended in water (pH 4.5) with the solid to liquid ratio at 1:10. Later on, 3% of enzymatic preparations, such as cellulase, hemicellulase, pectinase, mixture of cellulase and hemicellulase, mixture of cellulase and pectinase, mixture of hemicellulase and pectinase, and mixture of these three enzymes were added to the bulb powders respectively. Then the samples were cultured at 50 oC for 2 h. Afterward, the cultured solution was centrifuged at 5000 r/min for 20 min.

183

The supernatant was transferred into 50 mL volumetric flask and diluted to scale with water (pH 4.5). Obtained solutions were analyzed for their galanthamine content with HPLC after filtered with membrane (0.45 μm). All processes were repeated for three times. 2.2.2    Single factor experiment  The mixed enzymes of cellulase and pectinase were investigated. Reaction variables of enzyme to substrate ratio from 0−5%, reaction solution pH from 1.5 to 7.5, reaction temperature from 20 to 80 oC, and reaction time from 0.5 to 2.5 h were selected for single factor test, in addition, the solid to liquid ratio from 1:5 to 1:25 was also studied. All the determinations were made by triplicate and the means were reported. 2.2.3    Orthogonal test  The orthogonal test was carried out based on single factor experiment. Four variables of enzyme to substrate ratio, reaction pH, temperature, and time were investigated together as per L9 (34) orthogonal arrays. 2.2.4    HPLC analysis on galanthamine in L. aurea  HPLC analysis on samples before and after the enzymatic treatment was performed to identify the galanthamine releasing during the enzyme-assisted extraction from L. aurea. The sample (20 μL) were injected into HPLC equipped with Kromasil ODS column (250 mm × 4.6 mm, 5 μm), in a mobile phase of 17% acetonitrile and 83% phosphate buffer, and monitored at 290 nm under 35 oC. Phosphate buffer was prepared by dissolving 2.72 g of monopotassium phosphate into 100 mL water added with 1.4 mL of triethylamine, diluting to a volume of 1 L with water. The pH value of phosphate buffer was 7.02. 2.2.5    Establishment of galanthamine standard curve  A galanthamine reference solution with the concentration of 1.0 mg/mL was prepared. Then 0.05, 0.1, 0.2, 0.5, 1.0, 2.0, and 5.0 mL of this reference solution were transferred into 10 mL volumetric flask, respectively to obtain a series of diluted reference solutions with concentration of 0.005, 0.01, 0.02, 0.05, 0.1, 0.2, and 0.5 mg/mL. After filtrated (0.45 μm), these solutions were injected into HPLC for galanthamine content analysis. The regression curve was plotted with content of galanthamine (x) against peak area (y) and the regression equation was y = 1038x + 39 348, r = 0.9996. 2.2.6    Calculation of galanthamine extraction rate  The extraction rate of galanthamine was calculated using the formula y = cv / m0, Wherein c was the concentration of galanthamine. v was the final volume of extraction solution. m0 was the weight of bulb powders. 2.2.7    Selection of cation exchange resins and optimization  of chromatographic condition  The separation effect of two types of cation exchange resin, including strong acid cation exchange resins [732 (001 × 7), HD-8, HZ-008, and D001] and weak acid cation

Tian CL et al. Chinese Herbal Medicines, 2016, 8(2): 182-188

184

exchange resins (D155 and D152), on galanthamine from the bulbs of L. aurea were examined. The cation exchange resin was soaked in deionized water overnight and rinsed until the water became clear. 5 BV of 5% HCl solution, 5% NaOH solutions and 5% HCl solution were used to soak the resin for 12 h successively. Then the resin was eluted with deionized water until its pH value reached the range of 6 to 7, to obtain the H+ cation exchange resin. The bulb extraction solution of L. aurea was loaded to CC (H+ cation exchange resin) at a certain flow rate. An eluent of 70% ethanol containing 1.5 mol/L ammonia water was applied. Fractions with certain volume were collected and the content of galanthamine in them was analyzed by HPLC. Separation variables of the pH value, loading flow rate, eluent types, and elution flow rate of loading solution were investigated. 2.2.8    Calculation  for  adsorption  rate  and  desorption  rate  of cation exchange resin  ( c 0 − c 1) × v 2 w c2× v2 × 100 % rate (%) = ( c 0 − c 1) × v 1

Adsorption amount (mg / g) = Desorption

where c0, c1, and c2 respectively refer to the concentration (mg/mL) of extraction solution, extraction solution treated with cation exchange resin and solution eluted from the cation exchange resin. v1 and v2 are the volume of extraction solution and solution eluted from the cation exchange resin separately (mL). w are the quality of resin (g). Table 1

3.    Results and discussion   

3.1    Selection of enzyme types  According to Table 1, the mixed enzymes of cellulase and pectinase show the best extraction effect and its extraction rate can reach 0.0205%, followed by the mixture of cellulase and hemicellulase. Under single enzyme condition, galanthamine can not be extracted from the bulbs of L. aurea adequately. According to Ajay et al (2011), the pectinase enzyme can result in cell separation by pectin hydrolysis and cellulase can progressively degrade cellulose as well weaken the cell wall. The increase of extraction yield of galanthamine can be attributed to the synergistic effect of cellulase and pectinase generated under the extraction condition provided. Therefore, the mixed enzymes of cellulase and pectinase were chosen in the subsequent experiments.

  3.2    Single factor experiment results  3.2.1    The effects of the reaction pH value on the galanthamine extraction from the bulbs of L. aurea are shown in Figure 1A. The extraction yield of galanthamine increased sharply and reached a peak at the reaction solution of pH 4.5, and then sharply decreased to the pH 6.0, and dropped to the lowest at the pH 7.0, which may be attributed to the impact of reaction solution pH value on conformation of enzyme and on dissociation status of substrate (Ma et al, 2012).

Extraction results of galanthamine with different types of enzymes

Enzyme types

Extraction yield of galanthamine / %

Cellulase

0.0192

Hemicellulase

0.0189

Pectinase

0.0185

Mixed enzymes of cellulase and hemicellulase

0.0203

Mixed enzymes of cellulase and pectinase

0.0205

Mixed enzymes of hemicellulase and pectinase

0.0191

Mixed enzymes of cellulase, hemicellulase, and pectinase

0.0190

No enzyme added

0.0179

3.2.2    The effects of the enzyme to substrate ratio on the galanthamine extraction from the bulbs of L. aurea are shown in Figure 1B. When the ratio of enzyme to substrate is within the range of 0 — 3%, the extraction rate of galanthamine increased significantly, and reached a peak at the ratio of 3%. The enzymes play a role in disintegrating the cell wall matrix and facilitating the galanthamine extraction.

time of 1.5 h, the mass transfer resistance formed by pectin and cellulose in cell wall and intercellular substance is completely removed and the highest extraction yield of galanthamine is obtained. Since galanthamine is not stable and easy to be oxidized in the air, part of galanthamine may be degraded and the extraction yield decreases (Wang, 2010). Therefore, the reaction time should be controlled within 1.5 h.

3.2.3    The effects of the reaction time on the galanthamine extraction from the bulbs of L. aurea are shown in Figure 1C. The extraction yield of galanthamine increases along with the reaction time changing, and reaches a peak at the reaction time of 1.5 h, then decreases a little with time. At the beginning of reaction, galanthamine is released gradually along with the concentration of substrate and enzyme activity. At the reaction

3.2.4    The effects of the reaction temperature on the galanthamine extraction from the bulbs of L. aurea were investigated (Figure 1D). Extraction rate of galanthamine increased sharply along with the temperature, changing during 20−50 oC, and reaching the maximum at 50 oC which may be caused by the fact that high temperature has a significantly negative effect on the enzyme activity.

Tian CL et al. Chinese Herbal Medicines, 2016, 8(2): 182-188

viscosity of reaction solution and the galanthamine can not be infiltrated adequately. High ratio of solid to liquid can decrease the viscosity of reaction solution and accelerate the mass transfer processes. However, the reaction probability between enzymes and substrate can be also influenced accordingly. Therefore, the yield of galanthamine varies little when a most favorable solid to liquid ratio is obtained.

3.2.5    The effects of solid to liquid ratio on the galanthamine extraction from the bulbs of L. aurea are also examined, together with other variables and are shown in Figure 1E. Extraction rate of galanthamine increased dramatically along with ratio of solid to liquid and reached the peak when the ratio is 1:10. Low ratio of solid to liquid benefits the interaction between enzymes and substrate, but it also increases the A

0.0250

0.023

0.0245

0.022

0.0240 0.0235 0.0230 0.0225 0.0220 1

B

0.024

Yield of galarhamine/%

Yield of galarhamine/%

0.0255

2

3

4

5

6

7

0.021 0.020 0.019 0.018 0.017

8

0

1

pH

2

3

4

5

6

Addition amount of enzyme /% 0.028 0.027

C

0.026 Yield of galarhamine/%

Yield of galarhamine/%

0.027

185

0.025 0.024 0.023 0.022 0.021

D

0.026 0.025 0.024 0.023 0.022 0.021 0.020

0.0

0.5

1.0

1.5

2.0

2.5

3.0

20

40

50

60

70

80

90

o

enzymolysis time / h 0.028

30

enzymolysis temperature / C E

Yield of galarhamine/%

0.026 0.024 0.022 0.020 0.018 0.016 1:5

1:10

1:15

1:20

1:25

Liquid-solid ratio / (g:mL) Figure 1 Effect of reaction pH value (A), adding amount of enzyme (B), reaction time (C), reaction temperature (D), and liquid-solid ratio (E) on extraction yield of galanthamine

3.3    Results of orthogonal test  According to Table 2, the best extraction yield of galanthamine can be obtained through a combination of extraction variables A2B3C3D2. When the L. aurea bulb powder is extracted with aqueous solution of pH 4.5, added with amount of 4% mixed enzymes of cellulase and pectinase, under 50 oC for 2.0 h condition, the highest galanthamine extraction rate can be achieved. The results of orthogonal test show that

reaction temperature has the most impact, followed by enzyme to substrate ratio, reaction time, and reaction pH value. The variance analysis results are summarized in Table 3, the effect of reaction temperature has extremely remarkable difference (P < 0.01), meanwhile the influence of enzyme to substrate ration has remarkable difference (P < 0.1). The extraction yield of galanthamine can be 0.0294% when L. aurea bulb powder is extracted under the most favorable combination of extraction variables for two times.

Tian CL et al. Chinese Herbal Medicines, 2016, 8(2): 182-188

186

Table 2 Orthogonal test design and corresponding range analysis results

A No.

C

B

Reaction

Reaction time / min

o

temperature / C

D

Enzyme to substrate ratio / %

Yield of galanthamine / 103 %

Reaction pH

1

35

1.0

2

4.0

20.1

2

35

1.5

3

4.5

21.2

3

35

2.0

4

5.0

21.5

4

50

1.0

3

5.0

23.6

5

50

1.5

4

4.0

22.3

6

50

2.0

2

4.5

21.9

7

65

1.0

4

4.5

17.9

8

65

1.5

2

5.0

14.7

9

65

2.0

3

4.0

17.2

k1

21.300

20.533

18.900

19.867

k2

22.600

19.400

20.667

20.333

k3

16.600

20.567

20.933

20.300

R

6.000

1.167

2.033

0.466

Table 3

Results of variance analysis

Soruce of variation

Deviation square

Degree of freedom

Mean square

F value

P value

A

59.780

2

29.890

146.880

＜ 0.01

B

2.647

2

1.324

6.504

＞ 0.1

C

7.327

2

3.664

18.002

＜ 0.1

Deviation (D)

0.407

2

Sum

11.161

  3.4    Results and analyses for separation of  galanthamine by cation exchange resin    3.4.1    Selection of cation exchange resin types    Based on Table 4 which shows the static adsorptiondesorption rate of six kinds of cation exchange resins to galanthamine, the adsorption effect of strong acid cation exchange resins is better than that of weak acid cation exchange resins. The desorption rate of resin D001 is higher than any other resins, which is 93.76%, therefore, cation exchange resin D001 is chosen for the separation of galanthamine considering the influence of adsorption and desorption (Dong et al, 2014).   3.4.2    Adsorption effect of D001 to galanthamine  The separation effect of cation exchange resin to galanthamine can be influenced by pH value of loading solution, since pH value decides the ratio of alkaloids and alkaloid salts in loading solution. According to Figure 2A, when the pH value of loading solution is 2, the adsorption effect of D001 is the best. In addition, the influence of loading flow rate on adsorption effect of D001 to galanthamine is also investigated. As shown in Figure 2B, Table 4 Resin model Adsorption amount / (mg:g) Desorption rate / %

D001 exhibited the maximum adsorption capacity when the loading flow rate is 3 BV/h. The dynamic adsorption curve of cation exchange resin D001 (20 mL) to galanthamine is shown in Figure 2C. Based on Figure 2C, galanthamine reaches its leakage point when the volume of loading solution is 18 BV and reaches saturated after 71 BV of loading solution.

3.4.3    Elution  results  after  galanthamine  being  adsorbed onto D001 resin  According to Figure 3, when the concentration of ammonia water is 1.5 mol/L and the content of ethanol 70%, the galanthamine can be eluted from the column well. According to Figure 4, 3 BV/h is chosen as the best elution flow rate since under this flow rate, the amount of galanthamine eluted from column is the largest. The dynamic curve of galanthamine eluted from D001 column was shown in Figure 5. Galanthamine is almost totally eluted from the column when the volume of eluent reached 5 BV. All solution eluted from the column is combined and dried with Vacuum Freeze Dryer, and analyzed with HPLC, the content of galanthamine is 12.31%.

Static adsorption-desorption properties of six kinds of cation exchange resins 732 (001 × 7)

HD-8

HZ-008

D155

D001

D152

1.309

1.218

0.699

0.3826

1.211

0.0708

45.950

58.090

17.750

41.470

93.760

73.2800

A

0.7 0.6

Desorption rate / %

Adsorption amount for galanthamine /(mg·g-1)

Tian CL et al. Chinese Herbal Medicines, 2016, 8(2): 182-188

0.5 pH = 1

0.4

pH = 2

0.3

pH = 4

0.2

pH = 6

0.1

0.0

0.5

1.0

1.5

2.0

2.5

3.0

-1

0

0.6

5

10

15

20

Ammonia concentration / (mol·L )

25 100

0.5 0.4 0.3

90 85 80 85

0.2

40

70 1 BV/h 2 BV/h 3 BV/h

0.1 0.0

Figure 3

50 60 70 80 Ethanol concentration / %

90

Selection of ammonia concentration (A) and ethanol

concentration (B) for eluent 0

5

10

15

20

25

1.2

Eluation amount of galanthamine / mg

Bed volume C

1.0 0.8 0.6 0.4 0.2 0.0 0

20

40

60

80

55 50 45 40 35

1 BV/h

30

2 BV/h

Effect of adsorption conditions on loading solutions pH

20 0

The most favorable condition of galanthamine extraction from the bulds of L. aurea with enzyme treatment is determined to be the ratio of 4% for enzyme (mixed enzymes of cellulase and pectinase) to substrate, 50 oC, pH 4.5, and 2.0 h for hydrolysis reaction and extraction with water for two times in the present study. Under the optimal extraction condition, the extraction yield of galanthamine is 0.0294%. Among four reaction variables, reaction temperature shows the most influence on the extraction yield of galanthamine, ratio of enzyme to substrate is secondary, and then reaction time and pH value.

2

3

4

5

6

7

8

9

Eluent volume / BV

Galanthamine concentration /(mg·mL-1)

  4.    Conclusion 

1

Figure 4 Effect of elution flow rate on desorption of galanthamine

value (A), loading flow rate (B), and dynamic adsorption curve (C) of galanthamine

3 BV/h

25

100

Bed volume Figure 2

B

95

B Desorption rate / %

Galanthamine c / c0

A

0.0

t/h

Galanthamine c / c0

90 88 86 84 82 80 78 76 74 72 70

187

14 12 10 8 6 4 2 0 0

2

4

6

8

10

Elution volume/ BV Figure 5

Dynamic desorption curve

12

14

188

Tian CL et al. Chinese Herbal Medicines, 2016, 8(2): 182-188

The separation studies of galanthamine from the bulds of L. aurea with cation exchange resins demonstrate that resin D-001 is most suitable for isolation of galanthamine when its extraction solution is subjected to chromatographic column with the loading flow rate 3 BV/h until the resin becomes saturation, and eluted with 70% ethanol solution containing 1.5 mol/L ammonia under 3 BV/h elution rate. The content of galanthamine in dried eluent can reach 12.31% after separated under the best condition. We had also investigated the extraction efficiency of galanthamine by ultrasound-assisted extraction (Wang et al, 2013). Compared with our previous study, the extraction yield of galanthamine raised from 0.0253% to 0.0294%. According to our study, acidic aqueous water is used and the investment cost and environment pollution are reduced. In addition, considering the advantages of enzyme assisted-extraction method such as high extraction yield and simplified manipulation, enzyme-assisted extraction may be an effective and advisable method for the extract of alkaloids in L. aurea. References  Ajay P, Farhath K, 2011. Efficacy of xylanase purified from Aspergillus niger DFR-5 alone and in combination with pectinase and cellulose to improve yield and clarity of pineapple juice. Food Sci Technol 48(5): 562. Dong AW, Zhuo Qi, Bu XY, Zhu SH, Wang H, 2014. Adsorption properties for separation of apigenin from Viola yedoensis on LSA-10 resin. Chin Herb Med 6(1): 58-64. Du FY, Xiao XH, Li GK, 2007. Microwave-assisted extraction of alkaloids in Lycoris Radiata using ionic liquids solution. Chin J

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