New diterpenoid alkaloids from Delphinium pachycentrum Hemsl

Abstract Six diterpenoid alkaloids, namely, pachycentine (1), deacetylswinanine A (2), siwanine A (3), tatsiensine (4), deacetyltatsiensine (5), and 6-deoxydeltamine (6), were isolated from a China-specific Delphinium plant (family Ranunculaceae), Delphinium pachycentrum Hemsl. Their structures were established via detailed spectroscopic analyses, including IR, HR-ESI-MS, 1D and 2D NMR techniques. Pachycentine (1) is a previously undescribed hetisine-type C20-diterpenoid alkaloid, and compounds 5 and 6 were synthetic intermediates newly identified as natural products. In addition, compounds 2–4 were isolated from this species for the first time. The chemotaxonomic significance of all the isolates was summarized. Moreover, the new compound was evaluated for its potential anti-inflammatory effect using LPS-stimulated RAW 264.7 macrophages. Graphical Abstract


Introduction
Delphinium is a species-rich genus in the family Ranunculaceae, which comprises more than 400 species of hardy annual, biennial or perennial herbs distributed across the North temperate zone (Wang and Michael 2001).Among them, at least 232 species (200 endemic) have been found in China, which are mainly distributed in the high mountain areas in northern Yunnan, eastern Tibet and western Sichuan (He et al. 2019;Wang 2019).Globally, Delphinium plants have been widely used as horticultural and medicinal plants.Certain species of Delphinium, such as D. grandiflorum, D. elatum, Delphinium Â belladonna (a hybrid between D. elatum Â D. grandiflorum ex Bergmans), feature showy flowers with various colors, including white, pink, blue, light blue, violet, purple, and lavender, which are particularly popular worldwide and have been widely cultivated as gardens, potted plants, or cut flowers (Ichimura et al. 2009).On the other hand, in many countries and regions, such as China and India, Delphinium plants are commonly used as herbal medicines for treating various diseases, including analgesia, enteritis, rheumatism, spasm migraine, tooth ache, neuralgia, and various kinds of pain (Chen et al. 2017;Yin et al. 2020;Qasem et al. 2022).The multiple therapeutic effects of Delphinium-derived herbs could be attributed to their abundance of various active ingredients, including diterpenoid alkaloids (DAs), flavonoids, phenolic acids, and volatile oils (Marin 2011).In particular, DAs, which have been acknowledged as the characteristic ingredients for the genus Delphinium, have exhibited broad pharmacological activities, including anti-inflammatory, analgesic, antiarrhythmic, anticancer, antibacterial, antioxidant, and neuroprotective activities (Thawabteh et al. 2021;Yin et al. 2021;Liu et al. 2022).The discovery of novel metabolites from Delphinium plants supported and broadened their application as herbal medicines (Yin et al. 2021).Hence, in the present study, a China-specific Delphinium plant, D. pachycentrum Hemsl, was phytochemically investigated.This plant is restricted to western Sichuan and southeastern Qinghai in China and usually grows on stony grassy slopes at altitudes of 3600-4700 m.As a result, six DAs were isolated and identified via extensive spectroscopic analyses, including a new hetisine-type C 20 -DA pachycentine (1) and five lycaconitine-type C 19 -DAs (2-6) (Figure 1).The chemotaxonomic significance of all the isolates was summarized.Moreover, according to the medicinal use of this plant, the isolated new compound was evaluated for potential anti-inflammatory effects by the inhibition of NO production in lipopolysaccharide (LPS)-induced RAW 264.7 macrophages.Herein, the isolation, structural elucidation, and biological activities of these compounds are described.

Structural elucidation
Compound 1 was obtained as a white amorphous powder.According to the pseudo ion peak at m/z 358.1628 [M þ H] þ in the HR-ESI-MS spectrum, the molecular formula of 1 was determined to be C 20 H 23 NO 5 with an unsaturation degree of ten.Its IR spectrum exhibited absorption bands for hydroxyl groups (3741 cm À1 ), ester carbonyl groups (1712 cm À1 ), and conjugated double bonds (1651 cm À1 ).The NMR spectrum revealed the presence of twenty carbons in compound 1, namely, an angular methyl (d H 1.16, s, 3H; d C 18.3 q); four methylenes, including three aliphatic methylenes (d C 26.3 t, 27.1 t, and 27.7 t) and a terminal olefinic methylene (d H 5.27, 5.37, each 1H; d C 116.7 t); nine methines, including three oxygenated methines (d C 65.5 d, 73.9 d, and 88.3 d); and six quaternary carbons, including three saturated quaternary carbons (d C 49.7 s, 49.9 s, and 61.5 s), two ketonic carbonyls (d C 206.5 s, 207.3 s), and an exocyclic double-bond quaternary carbon (d C 144.2 s).In addition, there were no signals corresponding to N-ethyl or N-methyl groups or methoxyl groups.The above-mentioned evidence, in conjunction with biogenetic consideration, suggested that 1 might be a hetisine-type C 20 -diterpenoid alkaloid with a heptacyclic skeleton (Lin et al. 2014;Zong et al. 2019).
Three oxygenated methines, corresponding to the molecular formula, suggested the existence of three hydroxyl groups in addition to two ketonic carbonyls.Two ketonic carbonyls could be positioned at C-11 and C-13 based on the HMBC correlations from H-9 (d H 2.71, brs) and H-12 (d H 2.97, s) to C-11 (d C 206.5 s) and from H-12, H-14 (d H 2.48, d), and H-20 (d H 3.74, brs) to C-13 (d C 207.3 s), respectively.Three hydroxyl groups were placed at C-3, C-15, and C-19 on the basis of the HMBC correlations from H-3 (d H 3.66, t, J ¼ 6.9 Hz, 1H) to C-1 (d C 26.3 t), C-4 (d C 49.9, s), and C-18 (d C 18.3 q), from H-15 (d H 4.81, s) S2).The location of OH-3 could also be confirmed by the 1 H-1 H COSY correlation between H-2 and H-3.Thus, the plane structure of compound 1 was established.The relative configuration of 1 was deduced from the NOESY experiment (He et al. 2019).The NOESY correction between H-3b and CH 3 -18 revealed the a-orientation of OH-3, while the NOESY correction between H-14 and H-15a established the b-orientation of OH-15.The NOESY correction between H-19 and H-6a revealed the b-orientation of OH-19.Therefore, the structure of 1 was determined.
DAs have been acknowledged as good chemical markers for the chemotaxonomy of Delphinium plants (Tursunkhodzhaeva et al. 2016;Wada et al. 2019;Yamashita et al. 2022).In this study, new compound 1 and compounds 5 and 6 have never been isolated from other Delphinium species and thus could be used as chemotaxonomical markers to differentiate D. pachycentrum from its easily confused species, which might be a challenging task by using classical taxonomy morphology due to their highly similar morphological characteristics.In addition, four of the isolated lycaconitine-type C 19 -DAs possesses a double bond between C-2 and C-3.Lycaconitine-type DAs with double bonds are not commonly encountered in nature (Liu et al. 2022).Compound 2 was only isolated from D. orthocentrum previously (Ding et al. 2000), while compound 3 was only found in three Delphinium species previously (Zhang et al. 1994;Ding et al. 2000;He Yiqing et al. 2009).Thus, compounds 2 and 3 could also be used as valuable chemotaxonomic markers for D. pachycentrum within the genus Delphinium in the future studies.
In addition, according to the medicinal use of Delphinium plants (Li et al. 2021), which are commonly used to treat inflammatory illnesses such as arthritis and enteritis, the new compound 1 was evaluated for its potential anti-inflammatory effect by the inhibition of NO production in lipopolysaccharide (LPS)-induced RAW 264.7 macrophages.However, the results indicated that compound 1 possesses no obvious in vitro anti-inflammatory activity.

Plant material
Whole herbs of D. pachycentrum were collected from Heishui County in Sichuan Province of China in November 2021.The voucher specimen (2021-dp-1) was identified by Zhang Jun from Kunming GenPHYTech Co., Ltd. is deposited in Zunyi Medical University, China.

Extraction and isolation process
Air-dried whole herbs of D. (5 kg) were extracted with 95% EtOH three times at room temperature, and each extraction lasted three days.Removal of solvent under reduced pressure afforded the ethanol extract, adjusted to pH 1 with HCl and extracted with ethyl acetate (3 Â 3 L).The acidic aqueous solution was adjusted to pH 10 with ammonia solution and then extracted with CHCl 3 to afford the crude alkaloid extract (240 g).

Conclusion
In conclusion, a new hetisine-type C 20 -DA pachycentine (1) along with five known lycaconitine-type C 19 -DAs (2-6) were isolated from the whole plant of D. pachycentrum.Their structures were established on the basis of extensive spectroscopic experiments.These isolates, such as compounds 1-3 and 5-6, could be useful markers for the chemotaxonomy of D. pachycentrum within the genus Delphinium in the future studies.

Disclosure statement
No potential conflict of interest was reported by the authors.