A new diterpenoid from the leaves and twigs of Croton lachnocarpus Benth

Abstract A new clerodane diterpenoid, crotolanin A (1), along with three known clerodane diterpenoids, crotoeurin B (2), teucvidin (3) and teucvin (4), was isolated from the ethanol extract of the leaves and twigs of Croton lachnocarpus Benth. Their structures were identified by extensive NMR spectroscopic and HRESIMS analyses. The dopaminergic neuroprotective activity of compounds 1-4 was tested by using transgenic Caenorhabditis elegans pathological model. Compound 2 alleviated dopaminergic neuron degeneration of worms induced by 6-hydroxydopamine (6-OHDA) that represented a potential therapy for Parkinson’s disease (PD). Graphical Abstract


Introduction
The genus Croton belongs to the Euphorbiaceae family, containing probably 1300 species, and the majority of species are distributed in tropics and subtropics worldwide (Palmeira J uniora et al. 2006), with approximately 21 species in China (Flora of China Editorial Board of Chinese Academy of Sciences.1996a).Croton was characterised as a rich natural source of diterpenoids, which exhibited a diversity of biological activities (Xu et al. 2018), including anti-tumor (Sul et al. 2013), antimicrobial (Liu et al. 2014;Rajachan et al. 2021), anti-inflammatory (Kuo et al. 2013;Li et al. 2021), and NGF (nerve growth factor)-potentiating activities (Sun et al. 2014).Croton lachnocarpus Benth. is an evergreen shrub, 1-3 m in height with fruits, younger leaves and shoots densely stellate-pubescent while older branches subglabrous, mostly occurring in the south of China, such as Guangxi, Guangdong, Jiangxi, Hunan and Guizhou Province (Flora of China Editorial Board of Chinese Academy of Sciences.1996b).The roots or leaves of C. lachnocarpus have known in China as the folk medicine, 'Xiao-Ye-Shuang-Yan-Long', for the treatment of rheumatic diseases and arthralgia, snakebites, traumatic injuries, swelling and sore pain (Pan et al. 2014).The phytochemical study on the leaves and twigs of C. lachnocarpus was completed in order to search for more bioactive constituents, which led to the isolation and structural elucidation of four clerodane diterpenoids (1-4) (Figure 1), incorporating one new, crotolanin A (1), and three known ones (2-4).The structures of the isolated compounds were identified by extensive spectroscopic methods and HRESIMS.
Parkinson's disease (PD) is a chronic, severe and age-related neurodegenerative disease associated with movement problems including rigidity, resting tremor and bradykinesia, as well as the impairments of cognitive functions with dementia, anxiety, disturbances and depression (Cooper et al. 2015).PD is featured by dopaminergic neurons degeneration in the substantia nigra (SN).The mitochondrial neurotoxins such as 6-hydroxydopamine (6-OHDA), 1-methyl-4-phenylpyridinium (MPP þ ), and rotenone result in dopaminergic neurodegeneration, abnormal mitochondrial unfolded protein response (mito-UPR) and the death (Gonz alez-Hunt et al. 2014;Lehtonen et al. 2016).Caenorhabditis elegans has been frequently served as a model to test the activity of potential antiparkinsonian agents as a result of its highly conservation to the human genome (Kaletta and Hengartner 2006).In this study, a transgenic C. elegans strain (BZ555) induced by 6-OHDA, was employed as a pathological model of dopaminergic neuroinjury to explore the neuroprotective activity of compounds 1-4.The results showed that compound 2 attenuated dopaminergic neuron degeneration and represented the potential antiparkinsonian bioactivity.

Results and discussion
Compound 1 was obtained as yellow oil and established a molecular formula of C 26 H 36 O 6 on the basis of the HRESIMS ion peak at m/z 445.2580S11), containing nine degrees of unsaturation.Its IR spectrum (Figure S12) showed the presence of ester carbonyl (1727, 1749, 1779 cm À1 ) and double bond (1637 cm À1 ) functionalities.The 1 H NMR spectrum (Figure S5) revealed   The relative stereochemistry of 1 was determined by the NOESY analysis (Figures S2  and S10).In the NOESY experiment, the key correlations of H 3 -20/H 2 -17 and H 3 -20/H-1a, indicated that H 3 -20, H 2 -17 and H-1a were cofacial and assigned as a-oriented arbitrarily.The cross-peaks of H-1b/H-3b and H-3b/H 3 -19 revealed the coplanarity of these protons to be b-oriented.On the basis of the relative configuration of 1, the absolute configuration of 1 was established by ECD calculations.The results suggested that the calculated weighted ECD spectrum of (4 R,8R,9S)-1 agreed well with the experimental ECD spectrum of 1, leading to the assignment of the absolute configurations at the stereogenic centers (Figure S3).Based on the above data, the structure of compound 1 was established and named as crotolanin A.
The dopaminergic neuroprotective bioactivity of compounds 1-4 was evaluated by the transgenic C. elegans strain (BZ555).n-Butylidenephthalide has been proved to have the antiparkinsonian property for effectively alleviating dopaminergic neuron degeneration induced by 6-OHDA (Fu et al. 2014), hence n-butylidenephthalide was presented as a positive control.In this work, the 6-OHDA-exposed animals exhibited selective degeneration of dopaminergic neurons.To analyse the dopaminergic neuroprotective bioactivity of all compounds, the loss of the expression of a GFP (green fluorescent protein) reporter gene in 6-OHDA-treated animals was measured.The fluorescence intensity of dopamine neurons of nematodes treated with 60 lM n-butylidenephthalide increased significantly.The mean fluorescence intensity remarkably raised by about 71% (p＜0.0001)compared to those 6-OHDA-treated C. elegans.Among all the 60 lM tested compounds, compounds 1, 3 and 4 had no effect on restoring the level of dopamine in 6-OHDA-treated C. elegans.While compound 2 attenuated 6-OHDA-induced degeneration of dopaminergic neurons, the mean fluorescence intensity raised by about 28% (p＜0.0001)compared to the 6-OHDA-treated C. elegans (Figure S4).

General experimental procedures
Optical rotations were obtained on a Rudolph Autopol IV automatic polarimeter.IR spectra were measured with a Nicolet NEXUS 670 FT-IR spectrometer in KBr. 1 D and 2 D NMR spectra were acquired on a Bruker Avance NEO-600 (600 MHz for 1 H NMR and 150 MHz for 13 C NMR) spectrometers with TMS as an internal standard.HRESIMS data were recorded on a Thermo LTQ Orbitrap Elite mass spectrometer.Sephadex LH-20 was supplied by GE Healthcare Bio-Sciences AB.MCI GEL CHP 20P (75-150 lm) was purchased from Mitsubishi Chemical Holdings in Japan.ODS (octadecyl silane, 50 lm) were purchased from the YMC CO., Ltd in Japan.Silica gel (200-300 mesh) used for column chromatography (CC) and silica gel GF 254 (10-40 lm) used for thin layer chromatography (TLC) were purchased from the Qingdao Marine Chemical Factory in China.n-Butylidenephthalide was purchased from Thermofisher Ltd in China.Spots were visualised on TLC by heating silica gel plates after spraying with 5% H 2 SO 4 in C 2 H 5 OH (v/v).

Plant material
The leaves and twigs of C. lachnocarpus were collected in September 2019 from Jinxiu County, Laibin City, Guangxi Province, P. R. China, and were identified by Dr. Jian-Yin Li (School of Pharmacy, Lanzhou University).A voucher specimen (No. 201909CLA) has been deposited at School of Pharmacy, Lanzhou University, P. R. China.

Dopaminergic neuroprotective bioactivity assay
The transgenic C. elegans strain BZ555 was purchased from Caenorhabditis Genetics Center (CGC).Worms were cultured on nematode growth medium (NGM) with the Escherichia coli OP50 as a food sources for 30 h at 22 C, and after washing by M9 buffer three times, about 80-100 eggs were transferred to OP50/S-medium which was added 50 mM 6-OHDA and 20 mM ascorbic acid, containing 60 lM tested compound in 0.1% DMSO.Meanwhile, 0.1% DMSO solvent was treated as a negative control and n-butylidenephthalide was used as a positive control.Subsequently, the worms were incubated for 2 h at 20 C and mixed lightly every 10 min. 2 h later, took them into the OP50/NGM plates with the tested compound, when the animals grew into L3 larvae, they were washed with M9 buffer three times again, and next transferred to OP50/ NGM plates for another 72 h.BZ555 animals were collected and washed with M9 buffer twice in order to remove the residual thalli, 10 lL worms were mounted onto a glass slide using 20 mM sodium azide till they got paralysis.Imaging of immobilised and paralysed worms were obtained under a fluorescence microscope (Nikon, Japan).The fluorescence intensity was indicated using ImageJ software.

Conclusion
In summary, a phytochemical investigation on the leaves and twigs of C. lachnocarpus led to the isolation and structural determination of four compounds, including a new clerodane diterpenoid, crotolanin A (1), along with three known compounds (2-4).
Their structures were ultimately elucidated by extensive spectroscopic methods and HRESIMS.All compounds were tested for their dopaminergic neuroprotective activity by a transgenic 6-OHDA-induced C. elegans.And compound 2 alleviated the dopaminergic neuron degeneration in the transgenic worms, which exhibited potential antiparkinsonian activity.