The alkaloids with neuroprotective effect from the root bark of Ailanthus altissima

Abstract The chemical constituent investigation on the root bark of Ailanthus altissima leads to the isolation of a new β-carboline alkaloid, 14(S),15-dihydroxy-6-methoxy-β-carboline (1), along with nine known alkaloids. The structure of new compound was elucidated on basis of extensive spectroscopic analysis, especially two-dimensional (2D) NMR techniques and the absolute configuration of C-14 was determined by ECD calculation. The neuroprotective effect of the isolated compounds on PC12 cells against the serum deprivation injury was evaluated by MTT method. As a result, compound 7 revealed protective effect on PC12 cells and the cell survival rate was significantly increased. Graphical Abstract


Introduction
The plant of Ailanthus altissima (Mill.)Swingle is a deciduous tree which belongs to the genus Ailanthus of the family Simaroubaceae (Tan et al. 2020).Ailanthi Cortex was documented as the root bark of Ailanthus altissima in 2020 edition of Chinese Pharmacopoeia which was used as a traditional Chinese medicine with the effect of heat-clearing, deprive the evil wetness, astringent, antidiarrheal, hemostasis, and disinfection (Ni et al. 2019;Du et al. 2021;Li et al. 2021).Alkaloids are a large class of plant secondary metabolites, with a wide range of plant distribution, rich chemical structure types, and prominent pharmacological activities which have drawn a lot of attentions (Eguchi et al. 2019;Heinrich et al. 2021).Over the past decades, lots of researches have investigated the chemical composition of genus Ailanthus, quassinoids and alkaloids especially carbolines and canthin-6-ones have been found as the main active ingredients (Ohmoto et al. 1976;1981;Ohmoto and Koike 1984a;Kim et al. 2015;2016;Jeong et al. 2018).So far, more than 20 alkaloids have been isolated from the root bark of Ailanthus altissima, most of which have shown a wide range of bioactivities such as anti-tumour, antiproliferative, anti-bacterial, anti-inflammatory and so on (Anderson et al. 1983;Souleles and Kokkalou 1989;De Feo et al. 2005;Cho et al. 2018).
Central nervous system disorders including autoimmune inflammatory, hypoxic, and degenerative diseases such as Multiple Sclerosis, ischemic stroke, and Alzheimer's disease which have caused a high social and health cost (Linnerbauer and Rothhammer 2020).Alzheimer's disease (AD) is recognized as a leading neurodegenerative condition which might be caused by the hyperphosphorylation of tau and Ab peptide, metal ions, the choLinergic pathway or oxidative stress and neuroinflammation (Noori et al. 2021).However, at present, there were no efficient therapeutics.In recent years, the natural products especially alkaloids have exhibited therapeutic prospect which have shown anti-inflammatory, antioxidant, anti-amyloidogenic, and anti-choLinesterase properties, such as huperzine A, galantamine, berberine, betaine, caffeine, and so on.Whereas, the nerve cell protect action of the alkaloid in this genus was rarely reported (Sasaki et al. 2016).Therefore, this article focuses on the chemical composition and the neuroprotective effect of the alkaloids from the root bark of Ailanthus altissima.As a result, 10 alkaloids were separated and identified including six b-carboline type alkaloids and four canthin-6-one type alkaloids (Figure 1).The MTT method was used to evaluate the effect of the isolated alkaloids on the survival rate of serum limited PC12 cells.The result showed that compared with the serum-depleted model group, compound 7 increased the cell survival rate to 77.5 ± 2.3% (p < 0.05) which indicated a good neuronal protection effect.
14(S),15-dihydroxy-6-methoxy-b-carboline (1): white amorphous powder, was positive to Dragendorff's reagent chemical reaction, which indicated it might be an alkaloid (Raal et al. 2020;Tanzey et al. 2020).The molecular formula was determined as C 14 H 14 N 2 O 3 on the basis of the ion peak of HR-ESI-MS at m/z 259.1078 Thus, the degree of unsaturation of compound 1 was 9.
The comprehensive analysis of the 1 H-NMR (DMSO-d 6 , 800 MHz), 13   117.0, 119.7, 134.4, 139.3, 139.7, and 150.5.Therefore, compound 1 was proposed as a b-carboline type alkaloid by deducing from the characteristic proton and carbon signals above (Ma et al. 2020).And the assignments of NMR signals of 1 were derived from HSQC, 1 H-1 H COSY, and HMBC experiments (Table S1).As manifested by the 1 H-1 H COSY spectrum (Figure S1), the correlations of d H 7.20 (H-10) to d H 8.16 (H-9) and d H 7.48 (H-11), and d H 7.48 (H-11) to d H 7.66 (H-12), proved the connection from C-9 to C-12.The correlations of d H 5.01 (H-14) to d H 3.77 (H-15b) and 3.81 (H-15a) indicated the connection of C-14 to C-15.In the HMBC spectrum (Figure S1), the correlations of H-12 to C-8, H-9 to C-13, and both of H-10 and H-11 to C-8 and C-13, indicated that C-8 was connected to C-9, and C-12 was connected to C-13.From the chemical shift of C-13 (d C 139.7), it can be deduced that C-13 was connected to a nitrogen atom.Moreover, H-9 was correlated with C-7 in HMBC spectrum which suggested that C-8 was connected to C-7.The HMBC correlations of H-15 to C-3, H-14 to C-3 and C-2 showed that C-14 was connected to C-3 and C-2 sequentially.From the chemical shift of C-2 (d C 134.4), it can be learned that C-2 was connected to a nitrogen atom.This connection could be confirmed by the correlations of H-1 to C-2, C-7, C-8, and C-13 in the HMBC spectrum.The chemical shift of C-3 (d C 139.3) indicated that it was connected to the other nitrogen atom of the b-carboline alkaloid skeleton.The HMBC correlation of d H 4.09 (3H, s) to d C 150.5, indicated that the methoxy was connected to C-6.The stereochemistry at C-14 was established by ECD calculation.The experimental CD spectrum of 1 was consistent with the calculated ECD spectrum of 14S, while opposite to that of 14 R (Figure S2).To sum up, the structure of compound 1 was elucidated as 14(S),15-dihydroxy-6-methoxy-b-carboline.
The protective effect of compounds 1-10 on PC12 cells against the damage induced by serum deprivation was tested by MTT method in vitro.The results showed that compound 7 was the most effective one which exhibited significant protective effect on PC12 cells compared with the serum deprivation group, and the cell survival rate was increased to 77.5 ± 2.3% (Table S2).Unexpected, none of the b-carboline type alkaloids (1-6) showed any protective effect on PC12 cells.The canthin-6-one type alkaloids were much more effective than the b-carboline type alkaloids.Furthermore, the results of 7-9 suggested that the polarity plays a pivotal role on the protective effect of the compounds on PC12 cells.Whereas, the sample size of this experiment wasn't big enough to afford a certain conclusion, and the speculation above needs further data to confirm.

Plant materials
The root bark of Ailanthus altissima was collected from Xi'an, Shaanxi Province, China, in August 2016.The voucher sample (No.CC20160808) was deposited in the Department of Chinese Materia Medica and Natural Medicines, School of Pharmacy, Air Force Medical University, Xi'an, China.

Extraction and isolation
The air-dried root bark of Ailanthus altissima (20 kg) was refluxed with 85% ethanol for 3 times and 2 h for each time.The extracts were combined and concentrated under reduced pressure to obtain the crude extract.Then it was dispersed in water and adjusted to pH 1 $ 2 with 1% HCl which was followed by filtration.Subsequently, the filtrate was adjusted to pH 9 $ 10 with 1 mol/L NaOH.The solution was extracted with ethyl acetate for 4 times, and then the ethyl acetate layer was concentrated to obtain the total alkaloids.Ten fractions (Fr.1 to Fr.10) were obtained from the total alkaloids by silica gel column chromatography which was eluted with CHCl 3 /CH 3 OH/H 2 O (100:1:0 -6.5:3.5:1)gradually.Fr.3 was further purified and separated by Sephadex LH-20 gel column chromatography to give six sub-fractions .Fr.3-2 was subjected to semi-preparative HPLC eluting with CH 3 CN/H 2 O (85 : 15) to afford compounds 3 (10.7 mg, t R ¼ 15.3 min), 4 (15.2 mg, t R ¼ 17.2 min) and 5 (9.4 mg, t R ¼ 24.6 min) at a flow rate of 6 mL/min.Compounds 6 (12.1 mg, t R ¼ 33.3 min) and 10 (3.6 mg, t R ¼ 36.9 min) were isolated from Fr.3-3 by HPLC eluting with CH 3 CN/H 2 O (85 : 15) at a flow rate of 6 mL/min.And compound 9 (9.7 mg, t R ¼ 23.4 min) was isolated from Fr.3-5.Fr.4 was purified by a reversed-phase column chromatography, and then it was further separated by semi-preparative HPLC eluting with CH 3 CN/H 2 O (85 : 15) at a flow rate of 6 mL/min to afford compounds 7 (90.5 mg, t R ¼ 26.3 min) and 8 (7.4 mg, t R ¼ 34.7 min).Compounds 1 (4.0 mg) and 2 (4.0 mg) were obtained from Fr.8 which was separated and purified by reversed-phase column chromatography and semi-preparative HPLC sequentially.S1

The protective effect of the isolated compounds on the serum-free PC12 cells
The protective effect of the isolated compounds on the serum-free PC12 cells was evaluated by MTT method.The logarithmic phase cells were seeded on a 96-well plate at a concentration of 1 Â 10 4 cell/mL, and incubated for 12 h at 37 C with humidity atmosphere of 5% CO 2 .After that, the cells were divided into three groups, which were the normal group, the serum-free group and the test group.The normal group was given complete medium, the serum-free group was given DMEM medium without serum, and the test group was given serum-free medium and the isolated compounds at concentration of 100 lmol/L.Moreover, each group takes 5 parallel wells and incubated for 48 h.Then 20 lL of MTT solution was added to each well, and incubated for another 4 h.The water-insoluble dark blue formazan crystals formed during MTT cleavage in actively metabolizing cells which were dissolved in DMSO.The absorbance of each well was measured on the microplate reader at 490 nm.The cell survival rate: survival rate ¼ absorbance value of the tested drug group/absorbance value of the normal model group Â 100%.