Acetylcholinesterase inhibitory-active components of

Rhodiola rosea L.Hua Wang, Gaochao Zhou, Xiangdong Gao, Yidan Wang, Wenbing Yao *

School of Life Science and Technology, China Pharmaceutical University, 24 Tongjia xiang, Nanjing 210009, PR China

授課老師：詹于誼 教授報告者：田仲源、莫博欽

IntroductionRhodiola rosea ("golden root" or "rose root") : 景天科

(Crassulaceae) 紅景天屬 (Rhodiola sp.) is a popular plant in traditional medical systems in Eastern Europe and Asia.

西藏稱“索羅瑪寶 ”

Rhodiola rosea: in a variety of diverse areas of physiological function, including neurotransmitter levels, central nervous system activity, and cardiovascular function.

紅景天的多樣性

生理功能的主要有效成份為 : 紅景天苷 (salidroside ；C14H20O7) and 酪醇 (p-tyrosol ； C6H10O2)

Application :decreasing depression, enhancing work performance, eliminating fatigue, and preventing high altitude sickness…

Acetylcholine

Acetylcholinesterase

Acetylcholinesterase (AChE) inhibitors have been widely used in the treatment of Alzheimer’s disease (AD)

The medicinal effect of RR in the treatment of AD is probably mediated by the acetylcholinesterase inhibitory capability of this plant (Wu, Yao, Gao, & Wang, 2003). However, the specific compounds responsible for the anti-cholinesterase activity of RR remain unknown.

Alzheimer’s disease (AD)

1. 非正常的老化現象2. 喪失記憶和語言障礙3. 在美國，阿茲海默症高居成人死因第四位4. 神經纖維糾結與斑塊5. 膽鹼酯分解酵素抑制劑 Cognex 與 Aricept6. 維他命 E 、阿司匹靈和銀杏

BPSD, which includes symptoms such as depression, anxiety, delusions, and agitation, is currently treated with psychotropic medications including antipsychotics, antidepressants, and hypnotics.

Cholinesterase inhibitors may be useful in the management of behavioral and psychological symptoms of dementia (BPSD).

Materials and methodsChemicals

Acetylthiocholine iodide (ATCI) as substrate

AChE as enzyme

5,5’-dithio-bis-(2-nitrobenzoic acid) (DTNB)

Plant materials

the dried rhizome of Rhodiola rosea L.

HPLC mobile phases :Solvent A (water) and solvent B (acetonitrile)

detector : 280 nm

UV–visible spectrophotometric analysis The sample solution was scanned from 200 to 700 nm

Molecular weight estimation ionization energy was 70 eV; ion source temperature was 230

℃. The scan range of m/z was 40–800.

NMR spectroscopy

Extraction and isolation proceduresplant material (200 g)

boiled ethanol (1 l) for 1 h

filtered through a filter paper

boiled ethanol (1 l) for 1 h

filtered through a filter paper

Filtered extracts evaporated to dryness (46.4 g)

Filtered extracts evaporated to dryness (46.4 g)

Extract (40 g) in 400 ml of deionized water

centrifugation (3000 rpm, 5 min)

Supernatant (360 ml) was precipited with gelatine

centrifuged ( 3000 rpm ,10 min )to remove tannins

The supernatant was reprecipitatedwith ethanol

centrifuged ( 3000 rpm , 10 min ) remove the remnant gelatine

The supernatant was evaporated to dryness

supernatant(dryness)

H2Ochloroform

EtOAcn-butanol

column chromatography

chloroform/ethyl acetate (1:1)

100 fractions (A)

Fractions A36–A59

Sephadex LH-20 column

methanol/water (1:1)

100 fractions (B)

FractionsB29–B39compound 1purifier HPLC

system

Bioassay for anti-cholinesterase activitythe colorimetric method of Ellman

ATCIthiocholin

e

Compare the method of Ellman and Rhee Ellman’ s method Rhee’ s method(false-positive

effect)

First step substrate (1 ml) sodium phosphate buffer (0.1 mM, pH 7.4) 1 ml 0.1 ml of plant extract incubated at 37 for 5 min.℃

First step substrates (1 ml) sodium phosphate buffer (1 ml) enzyme (0.1 ml) Incubated at 37 for 5 min. ℃

Second step 0.1 ml of enzyme was added and the mixture was incubated at 37 for a ℃ further 8 min.

Second step Plant extract (0.1 ml) was added and the mixture was incubated at 37 for ℃ another 8 min.

Third step The reaction was terminated by adding 3% SDS (1 ml), then 0.1 ml of 0.2% DTNB was added to produce the yellow anion of 5-thio-2-nitrobenzoic acid.

Results