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Biol. Pharm. Bull. 43(5): 767-773 (2020)

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Biol. Pharm. Bull. 43(5): 767-773 (2020) Vol. 43, No. 5 Biol. Pharm. Bull. 43, 767–773 (2020) 767 Regular Article Effects of Gentiana delavayi Flower Extract on APP Processing in APP/PS1 CHO Cells Min Yang, a Kai-yi Zhou,b Feng-feng Li,a Hui-y un Yang, a Ming Yin,b Li-hang Zhang,*,b and Fu-sheng Wang*,a a College of Pharmacy and Chemistry, Dali University; Dali, Yunnan 671000, China: and b School of Pharmacy, Shanghai Jiao Tong University; Shanghai 200240, China. Received August 26, 2019; accepted February 20, 2020 Ethnopharmacological relevance: Gentiana delavayi Franch. (Gentianaceae) as an ethnomedicinal plant contains a variety of effective active ingredients and exhibits diverse pharmacological actions, such as hepa- toprotective, anti-inflammatory and central nervous system effects. In this study we investigated the influ- ence of G. delavayi flower extract on amyloid precursor protein (APP) processing at molecular and cellular levels. APP/PS1 Chinese hamster ovary (CHO) cells were treated with chloroform extract of G. delavayi flow- er in different concentrations for 24 h. Concentrations of amyloid β (Aβ) 40 and Aβ42 in the cell supernatant and activity of β-site amyloid precursor protein cleaving enzyme 1 (BACE1), BACE2, and cathepsin D were determined. The expression of APP and neprilysin (NEP) within the cell were further determined. Compared with the control group, the levels of Aβ40 and Aβ42 declined notably and the activity of BACE1 was inhib- ited significantly in the APP/PS1 CHO cells after treatment with the chloroform extract of G. delavayi flower. Although the activities of BACE2 and cathepsin D were not changed, the expression of Aβ degrading enzyme NEP increased remarkably. Our experiments have clearly showed that the chloroform extract of G. delavayi flower inhibits the generation of β-amyloid by specifically inhibiting β-secretase and increases the expression of NEP which fastens the degradation of Aβ, exhibiting the effect of decreasing Aβ accumulation in APP/PS1 CHO cells. These results suggest that the active components from the chloroform extract of G. delavayi flower have a further prospect to be developed as potential anti-Aβ drug. Key words Alzheimer’s disease; Gentiana delavayi extract; ethnomedicinal plant; amyloid precursor protein; cathepsin D; neprilysin INTRODUCTION tors of Aβ production, BACE1, as a rate-limiting enzyme for Aβ generation, has received much attention.10) However, no Alzheimer’s disease (AD) is the primary dementia-causing BACE1 inhibitor has passed clinical trials.11) But the activity disease and approximately 50 million people worldwide has of BACE1 was still suggested as a new approach for anti-AD dementia in 2019.1) As the disease advances, symptoms in- drug discovery10) based on the fact that suppression of BACE1 clude disorientation, deepening confusion about events, loss activity efficiently improves AD symptoms.12) Beside the pro- of motivation, failure to perform self-care, and finally the loss duction of Aβ by the amyloidogenic processing of APP and of bodily functions leading to death. The main pathological β-secretase, Aβ levels are also determined by its degradation, changes of AD are large deposition of amyloid β (Aβ) pep- mediated mainly by neprilysin (NEP).13) tides and intracellular neurofibrillary tangles.2) Aβ are gener- All of the currently available cholinesterase inhibitors ated by the lysis of amyloid precursor protein (APP). APP (ChEIs: donepezil, galantamine, and rivastigmine) are indi- is proteolytically processed by α-, β-, and γ-Secretases. The cated for mild-to-moderate AD. They remain to be symptom- three proteases process APP in two different pathways, one of atic drugs with no success in preventing, treating or curing which is called amyloidogenic and another is anti-amyloido- the disease.14) Advances in research and development of new genic. In the amyloidogenic pathway APP is first cleaved by drugs for the prevention and treatment of AD is not very β-site amyloid precursor protein cleaving enzyme 1 (BACE1)3) fortunate, and a very high attrition rate was found, with an and generates soluble β fragment of amyloid precursor pro- overall success rate during the 2002 to 2012 period by 0.4%.15) tein (sAPPβ) and C-terminal fragment β (CTF-β). γ-Secretase Therefore, a multi-target-based strategy has gradually been further cleaves CTF-β to release APP intracellular domain accepted as a promising trend for the discovery of drug com- (AICD) and Aβ, which aggregates to form amyloid plaques.4) pounds against AD.16) Natural products have been recognized Among all Aβ isoforms, Aβ40 and Aβ42 are believed to be as sources of new lead compounds for the treatment of vari- the most important ones.5) In recent years, several studies ous diseases, including AD.17) Luckily, an extract of Gentiana have demonstrated that Aβ42 could form oligomers signifi- delavayi flower was discovered by us to inhibit BACE1 activ- cantly faster than Aβ40, and the oligomers show the greatest ity and increase the expression of NEP. level of neurotoxicity.6,7) The more generally accepted hypoth- Gentiana delavayi Franch. (Wei Zi Long Dan by Chinese esis of the pathogenesis about AD indicates that the effect of name) grows in southwest of China and is a well-known tra- Aβ oligomers on neurons and astrocyte plays an important ditional but pharmacologically less explored Chinese herbal role in the development of AD.8,9) In the discovery of inhibi- medicine.18) It is an ethnomedicinal plant used for choleretic, * To whom correspondence should be addressed. e-mail: [email protected]; [email protected] © 2020 The Pharmaceutical Society of Japan 768 Biol. Pharm. Bull. Vol. 43, No. 5 (2020) antihepatotoxic, antioxidative, and anti-inflammatory pur- were more than 98%. poses. However, little is known about its neuroactive potential HPLC Analysis of the Chloroform Extract of G. dela- on AD. In a latest study performed in our laboratory, we vayi The chloroform extract of G. delavayi was dissolved investigated the inhibitory effects of chloroform extract of G. in methanol. That solution was filtered using a Greenherbs® delavayi flower on acetyl cholinesterases (AChE). Our results NYLON 13 mm syringe filter (0.45 µm, Beijing, China), and showed that the chloroform extract of G. delavayi flower then filtrates were subjected to HPLC. The analytical column inhibits AChE activity significantly. And its inhibiting rate was a Agilent Eclipse XDB-C18 (Agilent Technologies, Santa at 100 µg/mL was 51.1% by comparison with huperine A at Clara, CA, U.S.A.) and was kept at 30°C during performance. 500 nM was 92.4%.19) The data were analyzed by Chemstation software (Agilent It seems that only inhibiting the cholinesterase is not ideal Technologies). The mobile phase conditions contained solution for the development of AD drugs. However, previous studies A 0.1% phosphoric acid in water and acetonitrile. The gra- showed that the ethnomedicinal plant was used as anti-inflam- dient flow was as follows; 0–8 min, 5–14% D1; 8–13 min, matory,19) and antioxidative agent.20) We speculate that the 14–20% D1; 13–27 min, 20–25% D1; 27–52 min, 25–36% D1; components within the extract may have some other positive 52–73 min, 36–42% D1; 73–75 min, 42–66% D1; 75–90 min, effects. Then it will show the property of multi-target action 66–100%. The injection volume was 10 µL, and the analysis and have a better potential for further research and develop- was carried out at a flow rate of 1.0 mL/min with detection at ment. Therefore, in the present study, we examined the effect 254 nm. of chloroform extract of G. delavayi flower on the Aβ40 and Cells and Cell Culture Conditions APP/PS1 CHO Aβ42 secreted in APP/PS1 Chinese hamster ovary (CHO) cells were maintained in Dulbecco’s modified eagle medium cells. We also analyzed whether the extract change the intra- (DMEM) (Thermo Fisher Scientific, U.S.A.) supplemented cellular APP, and the activity on BACE1 in vitro and NEP with 10% fetal bovine serum (Thermo Fisher Scientific), 100 U protein levels. penicillin–0.1 mg/mL streptomycin (Thermo Fisher Scientific) at 37°C with 5% CO2 in a tissue culture incubator (Thermo MATERIALS AND METHODS Fisher Scientific). The cells were passaged every 2 d when they reached 80% confluence. G. delavayi Plant Collection Gentiana delavayi Franch. Cell Viability Assay To explore the cytotoxic effect of (Gentianaceae) source plant material was collected in October G. delavayi chloroform extract on APP/PS1 CHO cells, cell 2013 from Er Yuan, Yunnan Province, China. The plant was proliferation was assessed using the 3-(4,5-dimethylthiazol- identified and authenticated by Dr. Baozhong Duan, a plant 2-yl)-2,5-diphenyltetrazolium bromide (MTT) Kit (JianCheng, taxonomist at Dali University, Yunnan, China. China). The cells were treated with increasing doses of the G. delavayi Characterization and Extract Preparation chloroform extract of G. delavayi (0.05, 0.5, 5, 50, 500 µg/L) G. delavayi source plant material was identified. A herbar- for 24 h. The MTT assay was performed after the treatments. ium voucher specimen has been kept at our research group The cells were incubated for 4 h at 37°C with 0.5 mg/mL of (W201310). An extract from dried ground G. delavayi flowers MTT dissolved in fresh complete medium. The extract was was prepared at Dali University. dissolved in dimethyl sulphoxide (DMSO) (Sigma-Aldrich, The plant material was dried in room temperature and U.S.A.), and the absorbance was measured on a microplate powdered. Three and half kilograms of the dry flower powder reader using a test wavelength of 570 nm. The data were ex- was extracted with 12 L of 95% (v/v) ethanol/water for 3 times pressed as the mean percentages of viable cells versus the (12 h each) in simply stirring the powder at room temperature.
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