Study on Biological Active Components of Eurycoma Longifolia

Nik Nur Shamiha N. D

1,3*

, Siti Shukriyah S.

1,2

, Ryuichiro S.

, Yoshiaki S.

, Jiyauddin K.

1,3

, Ibrahim

1,3

*, Mohd Nizam A. G.

1,3

, Mohd Fadli A.

1,3

and Eddy Y.

School of Pharmacy, Management and Science University, Selangor Darul Ehsan, Malaysia

Faculty of Pharmaceutical Sciences, Josai University, 1-1 Keyakidai, Sakado, Saitama 350-0295, Japan

ICHLAS, Management & Science University, Selangor, Malaysia

Keywords: Eurycoma longifolia, Anti-glycation activity, % of inhibition, Tongkat Ali, AGEs formation inhibition in

vitro.

Abstract: Background and aims: Constant hyperglycemia in diabetic patient may lead to excess glycation and thus is

believed to cause diabetic complications. Eurycoma longifolia (Simaroubaceae) is tested for inhibitory

activity of advanced glycation end-products (AGEs) formation in vitro. Materials and methods: Three

concentration of methanolic extract were tested together with bovine serum albumin in anti-CML antibody.

HRP- conjugated anti-mouse IgG antibodies were introduced and sample were reacted with phenyldiamine

dihydrochloride. Absorbance were read by using micro-ELISA and percentage of inhibition were

calculated. Results: The calculated percentage of AGEs formation inhibition by E. longifolia root are -3.62

% (0.1 mg/mL), 58.38 % (1 mg/mL) and 92.28 % (10 mg/mL) as compared to aminoguanidine 5.55 % (0.1

mg/mL), 39.32 % (1 mg/mL), 72.92 % (10 mg/mL) as referring to the concentration. Since the biological

activity was tested on the whole methanolic extract, the activity is suggested to be due to synergistic activity

of the extract. Conclusion: New biological activity of E. longifolia methanolic extract which is inhibition of

AGEs formation in vitro is seen. However, isolation of Fr.8-2, m/z:381 does not lead to any compound

isolated related to the plant.

1 INTRODUCTION

This study focus on the antiglycation activity of

Eurycoma longifolia (Simaroubaceae). To date,

there is no known activity on inhibition of advanced

glycation end products (AGEs) formation by E.

longifolia. The plant is widely known for anti-tumor

promoting activities, antischistosomal,

plasmodicidal activities (Jiwajinda, S. et al., 2002),

potent antiulcer activity (Tada, H. et al., 1991), helps

to improves stress hormone profile and certain mood

state parameters (Talbott, S. M., Talbott, J. A.,

George, A., & Pugh, M., 2013), cytotoxic activity

(Kuo, P.C., Damu, A.G., Lee, K.H., & Wu, T.S.,

2003), antibacterial action (Farouk, A., & Benafri,

A., 2007) as well as antimalarial activity (Ang, H.,

Chan, K., & Mak, J., 1995). Chronic use of E.

longifolia is said to increase the testosterone level in

men.

AGEs formation lead to kaput protein. AGEs

formation in normal healthy people may not be

detrimental to their health compared to diabetic

patient. It is believed that major complication of

diabetes; nephropathy, neuropathy and retinopathy

are augmented by AGEs formation as well as

constant hyperglycemia. This study utilizes the

method of evaluation based on a previous study

conducted by Okada, Y., Ishimaru, A., Suzuki, R.

and Okuyama, T. in 2004. Inhibition of AGEs

formation is based on the carboxymethyl lysine level

inhibition.

By determining the biological activity, it may

leads to isolation of compounds responsible for the

said activity. This study suggest for further progress

in formulating therapeutic agents to counter diabetic

complications and thus improve the patients’ quality

of life and reduce the mortality rate among diabetic

due to complications.

Shamiha, N., Shukriyah, S., S., R., S., Y., Khan, J., A., I., Nizam, M., Asmani, M. and Yusuf, E.

Study on Biological Active Components of Eurycoma Longifolia.

DOI: 10.5220/0009846400002406

In Proceedings of BROMO Conference (BROMO 2018) - Symposium on Natural Product and Biodiversity, page 1

ISBN: 978-989-758-347-6

2 MATERIALS AND METHODS

2.1 Materials

E. longifolia sliced, dried roots were procured from

Malaysia, 99.5 % Methanol (Wako Pure Chemical

Industries, LTD, Lot: DSH3947), 99.9 % Methanol

(Wako Pure Chemical Industries, LTD, Lot:

DSJ0787), 99.7 % Methanol (Wako Pure Chemical

Industries, LTD, Lot: DSF3711), ethyl acetate

(Kanto Chemical Co. Inc. Lot: 51B1137), 99.0 % 1-

butanol (Wako Pure Chemical Industries, LTD, Lot:

DSR6754), 99.0 % Chloroform (Wako Pure

Chemical Industries, LTD, Lot: DSF3237), 99.8 %

Chloroform D + Silver Foil (Cambridge Isotope

Laboratories, Inc., Lot: PR-27572/04086CL1),

dimethyl sulfoxide, bovine serum albumin,

phosphate buffer solution, glucose, PBS containing

0.05% Tween 20, 0.5% gelatin in 100 mL coating

buffer, HRP-conjugated anti-mouse IgG antibodies,

1,2-phenyldiamine dihydrochloride, 100 µL of 1 M

sulfuric acid.

2.2 Apparatus

Evaporating flask, mantel heater, rotary evaporator

(EYELA NVC, No: 038006204), desiccator with

silica gel, TLC ODS plate, TLC silica gel plate. UV

light transmitter, microtube, micropipette, 96-well

plate, micro-ELISA plate reader, HPLC-RI detector

(Waters 600 Pump, Waters 600 Controller, Shodex

RI-201H Refractive Index Detector), MPLC Micro

pump KPW-20 (Kusano, Kagakukikai Co.),

Advantec Fraction Collector (CHF122SC), H-NMR

Varian (Agilent, 400 Hz), EI-MS (JEOL), HPLC

ODS-4151-N column (Senshu Pak, 10 x 150 mm,

No: 1110201H), MPLC column (MERCK,

LiChroprep Si 60 (40-63 µm), No: 540087666).

2.3 Preparation of Methanolic Extract

Methanolic extract of the roots of E. Longifolia (200

g) was obtained by extraction with MeOH (5.4 L)

three times under reflux for 3 hours. The solvent was

evaporated in vacuo to give MeOH extract (6.74 g).

2.4 Isolation of Components from

Methanolic Extract

The methanolic extract was suspended in water, then

extracted with EtOAc and n-BuOH, sequentially.

Each soluble portion was evaporated in vacuo to

give EtOAc (1.07 g) and n-BuOH (1.79 g) fractions,

respectively. The EtOAc fraction was

chromatographed on a prepacked silica gel column

(LiChroprep Si60 (40-63 µm) Merck Co. serial

number: 540087666, 140987) eluting with CHCl

give 15 fractions. Fr.8, Fr.9, and Fr.10 was further

purified with HPLC-RI (Detector: RI-201H,

SHODEX, Column: ODS-4151-N; size: 10 x 150

mm; number: 1110201H, Senshu Scientific Co. Ltd.)

detector. Fr.8 (0.0123 g) was further purified with

HPLC-RI detector using MeOH to provide four

fraction Fr.8-1 (0.0001 g) Fr.8-2 (0.0006 g) Fr.8-3

(0.0001 g) and Fr.8-4 (0.0001 g). Fr.9 (0.0148 g)

was further purified with MeOH-H

O mixture

(MeOH : H

O = 10 : 1) to gives four fraction Fr.9-1

(0.0014 g) Fr.9-2 (0.0010 g) Fr.9-3 (0.0014 g) and

Fr.9-4 (0.0007 g). Fr.10 (0.0074 g) was further

purified with MeOH-H

O mixture (MeOH : H

O =

10 : 1) to gives three fraction Fr.10-1 (0.0002 g)

Fr.10-2 (0.0003 g) Fr.10-3 (0.0001 g).

2.5 Inhibition Test on AGE Formation

in Vitro

BSA was incubated with 200 mmol/L glucose in

both presence and absence of test compound for 7

days in 0.1 M of phosphate buffer (pH 7.4) at 37 °C.

After incubation, coating buffer, blocking buffer and

anti-CML antibody were introduced to the cell.

HRP-conjugated anti-mouse IgG antibodies was

treated to the cells. 1 M sulfuric acid was used to

stop the reaction. The level of inhibition is measured

by calculating the level of CML measured by CML-

specific micro-ELISA plate reader at 492 nm

(SpectraMax PLUS 190PC ROM v1.23). Percentage

of inhibition was calculated as in following

equation:

Inhibition (%) = [1-(A

– A

)/(A

– A

)] x 100,

where A

is the CML level in the incubated mixture

with sample, A

is the CML level in the incubated

mixture without sample, and A

is the CML level in

the incubates mixture without sample and glucose

that served as blank control.

3 RESULTS

Methanolic extraction of E.longifolia root (200 g)

yielded 6.74 g of dried extract. Repeated extraction

under reflux ensures complete extraction from the

root. 20 mg of methanolic extract were subjected to

inhibition of AGEs formation in vitro by measuring

CML level using microELISA at 492 nm. Three

reading were recorded for each concentration. The

average reading is tabulated in Table 1.

BROMO 2018 - Bromo Conference, Symposium on Natural Products and Biodiversity

Table 1: Average reading of absorbance of AGEs

inhibition by microELISA.

Sample

Reading

Average

reading

Blank

0.755

0.765

0.663

0.728

Control

2.592

2.280

2.287

2.386

AG 0.1

2.522

2.240

2.119

2.294

AG 1

1.657

1.758

1.787

1.734

AG 10

1.260

1.069

1.201

1.177

EL 0.1

3.146

2.220

1.972

2.446

EL1

1.344

1.683

1.226

1.418

EL 10

0.842

0.993

0.733

0.856

Upon calculating the average reading of each

sample, the percentage of AGEs formation inhibition

were determined by substituting the absorbance

obtained by Micro-ELISA plate reader in the

formula. -3.62 %, 58.38 % and 92.28 % of inhibition

were calculated in E. longifolia with concentration

of 0.1 mg/mL, 1 mg/mL and 10 mg/mL respectively.

The percentage of inhibition of AGEs by E.

longifolia compared to Aminoguanidine is as in

Figure 1.

Figure 1: Comparison between percentages of inhibition

of AGEs formation between aminoguanidine (positive

control) and E. longifolia root extract.

4 DISCUSSION

Three concentration of E. longifolia root were

tested; 0.1 mg/mL, 1 mg/mL, and 10 mg/mL. The

calculated percentage of AGEs formation inhibition

by E. longifolia root are -3.62 % (0.1 mg/mL), 58.38

% (1 mg/mL) and 92.28 % (10 mg/mL) as compared

to aminoguanidine 5.55 % (0.1 mg/mL), 39.32 % (1

mg/mL), 72.92 % (10 mg/mL) as referring to the

concentration. The activity of E. longifolia is

suggested to match the activity of aminoguanidine.

The percentage of inhibition is concentration

dependent. Since the biological activity was tested

on the whole methanolic extract, the activity is

suggested to be due to synergistic activity of the

extract. Due to the small amount of root, the isolated

fractions yield are very small. Thus, we are unable to

test the biological activity on each fraction. Based on

EI-MS analysis of Fr.8-2, the molecular weight is

suggested to be m/z: 381 as attached in Appendix.

However, based on this data alone, we are not able

to relate the finding to any compounds reported to be

having relationship with the plant E. longifolia.

5 CONCLUSIONS

Methanolic extract of E. longifolia is suggested to be

having the inhibitory activity of AGEs formation.

The antiglycation activity is believed to be

synergistic action between compounds in the extract.

Further isolation of fractions however does not lead

to identification of isolates. In the future study, it is

strongly suggested that large amount of plant sample

should be used and care attention to work procedure

must be applied to prevent possible impurity to the

components.

ACKNOWLEDGEMENTS

The authors would like to thank all members of

School of Pharmacy MSU and Josai university for

their support.

REFERENCES

Ang, H., Chan, K., & Mak, J. (1995). In Vitro

Antimalarial Activity of Quassinoids from

Eurycoma longifolia against Malaysian

Chloroquine-Resistant Plasmodium falciparum

Isolates. Planta Med Planta Medica, 61(02),

177-178. doi:10.1055/s-2006-958042

Farouk, A., & Benafri, A. (2007). Antibacterial

Activities of Eurycoma Longifolia A Malaysian

medicinal plant. Saudi Med J, 28(9), 1422-1424

-20

100

120

0.1 mg/mL 1 mg/mL 10 mg/mL

Percentage (%) of inhibition

Concentration

Aminoguanidine E. longifolia

Study on Biological Active Components of Eurycoma Longifolia

Jiwajinda, S., Santisopasri, V., Murakami, A.,

Kawanaka, M., Kawanaka, H., Gasquet,

M., Ohigashi, H. (2002). In vitro anti-tumor

promoting and anti-parasitic activities of the

quassinoids from Eurycoma longifolia, a

medicinal plant in Southeast Asia. Journal of

Ethnopharmacology, 82(1), 55-58.

doi:10.1016/s0378-8741(02)00160-5

Kuo, P.C.; Damu, A.G.; Lee, K.H.; Wu, T.S. (2003)

Cytotoxic and antimalarial constituents from the

roots of Eurycoma longifolia. Biorg. Med.

Chem.,12, 537–

Okada, Y., Ishimaru, A., Suzuki, R., & Okuyama, T.

(2004). A New Phloroglucinol Derivative from

the Brown Alga Eisenia bicyclis : Potential for

the Effective Treatment of Diabetic

Complications. Journal of Natural Products,

67(1), 103-105. doi:10.1021/np030323j

Tada, H., Yasuda, F., Otani, K., Doteuchi, M.,

Ishihara, Y,. Shiro, M., (1991). New antiulcer

quassinoids from Eurycoma longifolia. European

Journal of Med. Chem., 26(3): 345-349.

doi:10.1016/0223-5234(91)90069-Y

Talbott, S. M., Talbott, J. A., George, A., & Pugh,

M. (2013). Effect of Tongkat Ali on stress

hormones and psychological mood state in

moderately stressed subjects. J Int Soc Sports

Nutr Journal of the International Society of

Sports Nutrition, 10(1), 28. doi:10.1186/1550-

2783-10-28

BROMO 2018 - Bromo Conference, Symposium on Natural Products and Biodiversity

APPENDIX

Study on Biological Active Components of Eurycoma Longifolia