Diterpenoids with Potent Anti-Psoriasis Activity from Euphorbia helioscopia L.

Psoriasis, an immune-mediated inflammatory skin disorder, seriously affects the quality of life of nearly four percent of the world population. Euphorbia helioscopia L. is the monarch constituent of Chinese ZeQi powder preparation for psoriasis, so it is necessary to illustrate its active ingredients. Thus, twenty-three diterpenoids, including seven new ones, were isolated from the whole herb of E. helioscopia L. Compounds 1 and 2, each featuring a 2,3-dicarboxylic functionality, are the first examples in the ent-2,3-sceo-atisane or the ent-2,3-sceo-abietane family. Extensive spectroscopic analysis (1D, 2D NMR, and HRMS data) and computational methods were used to confirm their structures and absolute configurations. According to the previous study and NMR data from the jatropha diterpenes obtained in this study, some efficient 1H NMR spectroscopic rules for assigning the relative configurations of 3α-benzyloxy-jatroph-11E-ene and 7,8-seco-3α-benzyloxy-jatropha-11E-ene were summarized. Moreover, the hyperproliferation of T cells and keratinocytes is considered a key pathophysiology of psoriasis. Anti-proliferative activities against induced T/B lymphocytes and HaCaT cells were tested, and IC50 values of some compounds ranged from 6.7 to 31.5 μM. Compounds 7 and 11 reduced the secretions of IFN-γ and IL-2 significantly. Further immunofluorescence experiments and a docking study with NF-κB P65 showed that compound 13 interfered with the proliferation of HaCaT cells by inhibiting the NF-κB P65 phosphorylation at the protein level.


Introduction
Psoriasis is a common autoimmune skin disease characterized by T cell-mediated hyperproliferation of keratinocytes.This disease has certain distinct but overlapping clinical phenotypes, including chronic plaque lesions (psoriasis vulgaris), acute and usually selflimiting guttate-type eruptions, seborrhoeic psoriasis, pustular lesions, and at least 10% of these patients develop arthritis [1,2].The global prevalence of the disease is approximately 2%, with regional variations [2,3].Biological agents, immunosuppressants derived from natural products, and traditional Chinese medicine formulas are the familiar drugs for treating psoriasis.However, the emergence of drug resistance necessitates the discovery of more potent agents for psoriasis treatment.
Euphorbia, the largest genus of Euphorbiaceae, contains over 2000 species.Its members inhabit worldwide, some of which are used as folk medicines in China to treat skin diseases (such as psoriasis), edema, tuberculosis, and constipation.The chemical constituents of Euphorbia are terpenes, flavonoids, tannins, and phenolic compounds.The characteristic components are diterpenoids, more than 150 diterpenoids with various skeletons and significant bioactivities, such as antitumor, anti-inflammatory, and anti-HIV activities, having been isolated from this genus [4,5].Diterpenoids from Euphorbia peplus (pepluacetal and pepluanols A-B), E. helioscopia (helioscopids A and O, euphohelioscoids A), and E. fischeriana inhibit Kv1.3 ion channel, a validated target for the treatment of autoimmune diseases, such as multiple sclerosis, type-1 diabetes, asthma, and psoriasis [6][7][8][9].Additionally, it was reported that the methanol extract of E. kansui radix alleviated the symptoms of psoriasis through the inhibition of Th17 differentiation and activation of dendritic cells.These effects are expected to be beneficial in treating and preventing psoriasis [10].
Euphorbia helioscopia L., commonly referred as Zeqi in traditional Chinese medicine, is a representative herb of the Euphorbia genus.In recent years, the chemical analysis of E. helioscopia L. has yielded novel diterpenoids with diverse biological activities, such as secoheliosphane B with activity against HSV-1, heliojatrone C with anti-inflammatory activity, euphohelioscopoids A-C against paclitaxel-resistant A549 human lung cancer cell line, and helioscopids A/O/euphohelioscoids A with Kv1.3 inhibitory activity [7,8,[11][12][13], which, in turn, sustains extensive attention from phytochemists.Additionally, multiple clinical studies have found that Chinese ZeQi powder preparation can treat psoriasis without significant adverse reactions [5,14], and E. helioscopia L. is the monarch constituent.To find more diterpenoids with promising activities for psoriasis, E. helioscopia L. was selected to be investigated in this study, resulting in the isolation of 23 diterpenoids.Among them, compounds 1 and 2 each feature an unusual 2,3-seco ent-2,3-dicarboxyl-atisan-2,3-dioic acid or 2,3-seco ent-abietan-2,3-dioic acid and compounds 3-7 are new jatropha diterpenes.In establishing the structures of new and known jatropha diterpenes, we summarized and examined the correlations between 1 H NMR signals (chemical shifts and coupling constants) and the relative configurations of C-2, C-13, and C-14 in the (7,8-seco)/3α-benzyloxyjatropha-11E-ene systems.Here, the isolation and structural elucidation of compounds were reported.Moreover, the anti-psoriatic potential of the isolated compounds was evaluated by assessing their immunosuppressive activities and anti-proliferation effects on keratinocytes.The possible mechanism of active compounds was explored through immunofluorescence experiments and docking studies.
OR PEER REVIEW 4 of 21     Based on the HRESIMS result and 13 C NMR spectrum, the molecular formula of 3 was determined to be the same as the co-isolate secoheliosphane B (21) [11], C 31 H 40 O 8 .These 1D NMR data presented a set of rare but typical signals for a seco-jatropha triester: four methyl singlets, two methyl doublets, two acetoxys, two double bonds, one benzyloxy, one ketone, and a ketal (Tables 2 and 3).Data from 3 were almost identical to those of 21, implying that the planer structure of compound 3 is the same as that of compound 21, which was supported by 1 H-1 H COSY and HMBC correlations (Figures S18 and S19, Supplementary Materials).The geometries of ∆ 5 and ∆ 11 double bonds were easily assigned as E for the large coupling constant ( 3 J H-11 , H-12 = 15.6 Hz) and the ROESY correlations (for ∆ 5 : H-4/H 3 -17, H-5/H-7; for ∆ 11 H-11/H-13, H 3 -18/H-12) (Figure S20, Supplementary Materials).For all jatropha diterpenes, the orientations of the substitutions are usually fixed at C-3 (an α-oriented benzoate substituent), C-4 (a β-oriented hydrogen), and C-15 (an α-oriented hydroxy or acetoxy), but orientations of CH 3 -16 at C-2, CH 3 -20 at C-13, and OH(OAc)-14 at C-14 are usually changeable.Carefully analysis of 1 H and 13 C NMR data for compounds 3 and 21 both measured in CDCl 3 revealed the relative configurations at C-2 and C-14 may be opposite for the differences observed in chemical shifts of relative carbons [3: 2 and 3).The relative configurations of C-2 and C-14 in compound 3 were confirmed by the analysis below.A key ROESY correlation (Figure S20, Supplementary Materials) of H-2 (δ H 2.15, m) and H-4 (δ H 3.61, dd, J = 9.4, 4.0 Hz) indicated that the orientation of CH 3 -16 is α, leading to the obvious changes for J values of H-3 [3: δ H 5.65 (t, J = 4.0 Hz), 21: δ H 4.69 (t, J = 9.0 Hz)], and for chemical shifts of C-1, C-2, C-3, and C-16 (3: δ C 46.8 C-1, 80.7 C-3; 21: δ C 40.8 C-1, 82.5 C-3).Then, a large coupling constant between H-13 and H-14 (J = 9.5 Hz) indicated a trans-position for H-13 and H-14.Furthermore, the relative configuration of CH 3 -20 was deduced to be β-oriented for chemical shifts of H-11 (δ H 5.38) larger than H-12 (δ H 5.27) [12,13].Therefore, the structure of 3 was defined as shown in Figure 1 and named secoheliosphane C.  (21).A detailed analysis of 1D and 2D NMR data (Tables 2 and 3) showed that the planner structure of compound 4 is also a seco-jatropha ester.The only difference between 4 and 21 is that OAc-15 in 21 is changed into OH-15 in 4, which was supported by 1 H-1 H COSY and HMBC correlations (Figures S25 and S26, Supplementary Materials).Subsequently, the relative configuration of 4 was determined by multiple methods, including ROESY (Figure S27, Supplementary Materials), J-based configuration analysis (JBCA), and a comparison of chemical shifts.First, the geometries of ∆ 5 and ∆ 11 double bonds were E based on the ROESY correlations and J values.Subsequently, JBCA of H-3 [δ H 5.20 (dd, J = 8.5, 5.7 Hz)] together with a chemical shift of CH 3 -16 (δ C 18.5), suggested a 16β-CH 3 ; a small coupling constant between H-13 and H-14 (J = 3.1 Hz) indicated that CH 3 -20 and OAc-14 located co-facial.Moreover, compared with secoheliosphane A (δ H-11 5.41, δ H-12 5.46), secoheliosphane B (δ H-11 5.46, δ H-12 5.37) and compound 3 (δ H-11 5.38, δ H-12 5.27), for compound 4, the chemical shift of H-11 (δ H 5.58), larger than that of H-12 (δ H 5.64) suggests a 20α-CH 3 .Finally, the structure of 4 was defined as shown in Figure 1 and given a trivial name, secoheliosphane D. Based on the known jatropha-skeleton diterpenoids with acetoxy groups from E. helioscopia [5], 1D NMR data of compounds 5-7 (Tables 2 and 3) displayed a group of characteristic signals for jatropha ester framework corresponding to one benzyloxy group, one ketone, two methyl doublets, three methyl singlets, two double bonds, and one to three acetoxy groups.In addition, the remaining two degrees of unsaturation for compounds 5-7 revealed that they all are dicyclic jatropha esters.
The molecular formula of heliosco-jatrophane C (7), C29H38O7, implied one acetoxyl group in 5 was changed into a hydroxy in 7.In the 13 C NMR spectrum (Table 3), an obvious difference was the chemical shift of a quaternary carbon (δC 83.6 in 7; δC-15 90.2 in 5), indicating that the hydroxy at C-15 was reserved in 7. Similarly, the orientations of CH3-16, OAc-14, and CH3-20 were speculated to be β, α and β by analysis of ROESY spectrum (Figure S48, SI), JBCA [δH-3 5.17 The structural elucidation of natural products consistently challenges chemists despite the ongoing development of new methods, including applying derivatization with chiral reagents, using JBCA, examining the intensity of the nuclear Overhauser effect, and relying on quantum-chemical calculations or X-ray crystallographic analysis [12,13,18,19].Among these, the NMR technique is a convenient and useful procedure for determining the relative configurations of organic molecules.For all jatropha diterpenes, the relative configurations of 3α-, 7α-, and 15α-oxygenated functionalities, as well as H-4β, are stable, while configurations at C-2, C-13, and C-14 positions vary.However, outcomes from nuclear Overhauser effect spectroscopy (NOESY) and ROESY experiments are often inconclusive because of the high flexibility of the 12-membered ring.Previously, Su et al. summarized empirical rules for deducing the relative configurations of C-2, C-13, and C-14 based on the chemical shifts of specific proximal carbons (CH3-16, C-4, C-13) [12].When establishing the relative configurations of C-2, C-13, and C-14 for compounds 3-21, the chemical shifts of CH3-16 and C-13 are indeed correlated to the orientations of CH3-16 and H-13.However, once C-7 was oxygenated, leading to the chemical shift of C-4 exceeding 45 ppm, the orientation of OAc-14 does not always align with the empirical rules, such as euphoscopoid E (δC-4 45.0), epieuphoscopin B (δC-4 45.3), and euphornin H (δC-4 46.2) [13].Therefore, searching for additional methods dealing with the stereochemical questions of jatropha diterpenoids remains an urgent priority.We summarized some efficient 1 H NMR A-Type The molecular formula of heliosco-jatrophane B ( 6) is C 33 H 42 O 9 , implying that it may be an acetylated derivative of 5.Then, HMBC correlation from H-7 [δ H 5.03 (dd, J = 8.1, 1.7 Hz)] to a carbonyl (δ C 170.2) suggested that OH-7 in 5 was acetylated in 6 (Figure S40, Supplementary Materials).The relative configuration of 6 is the same as 5 for similar ROESY correlations (Figure S41, Supplementary Materials), coupling constants, and chemical shifts.Consequently, the structure of 6 was established, as shown in Figure 1.
The structural elucidation of natural products consistently challenges chemists despite the ongoing development of new methods, including applying derivatization with chiral reagents, using JBCA, examining the intensity of the nuclear Overhauser effect, and relying on quantum-chemical calculations or X-ray crystallographic analysis [12,13,18,19].Among these, the NMR technique is a convenient and useful procedure for determining the relative configurations of organic molecules.For all jatropha diterpenes, the relative configurations of 3α-, 7α-, and 15α-oxygenated functionalities, as well as H-4β, are stable, while configurations at C-2, C-13, and C-14 positions vary.However, outcomes from nuclear Overhauser effect spectroscopy (NOESY) and ROESY experiments are often inconclusive because of the high flexibility of the 12-membered ring.Previously, Su et al. summarized empirical rules for deducing the relative configurations of C-2, C-13, and C-14 based on the chemical shifts of specific proximal carbons (CH 3 -16, C-4, C-13) [12].When establishing the relative configurations of C-2, C-13, and C-14 for compounds 3-21, the chemical shifts of CH 3 -16 and C-13 are indeed correlated to the orientations of CH 3 -16 and H-13.However, once C-7 was oxygenated, leading to the chemical shift of C-4 exceeding 45 ppm, the orientation of OAc-14 does not always align with the empirical rules, such as euphoscopoid E (δ C-4 45.0), epieuphoscopin B (δ C-4 45.3), and euphornin H (δ C-4 46.2) [13].Therefore, searching for additional methods dealing with the stereochemical questions of jatropha diterpenoids remains an urgent priority.We summarized some efficient 1 H NMR spectroscopic rules for assigning the relative configurations of C-2, C-13, and C-14 in (7,8-seco)/3α-benzyloxyjatropha-11E-ene derivatives, providing a valuable complement to 13 C NMR spectroscopic rules [12].Based on the previous 13 C NMR rules and literature research, the configurations of compounds 3-21 containing a (7,8-seco)/3α-benzyloxy-jatropha-11E-ene were successfully established.The relative configurations of C-2, C-13, and C-14 can be fully and readily assigned by analyzing 1 H NMR information, including coupling constants and chemical shifts of H-11 and H-12 (Figure 4, Tables 4 and 5).First of all, coupling constants of H-2/H-3 and H-13/H-14 determine whether the orientations of H-2/H-3 and CH 3 -20/14-OAc are the same or not.For instance, the signal of H-3 appeared as t-like (J ≈ 3.0-5.0Hz), indicating a 16α-CH 3 , while the signal of H-3 appeared as a doublet of doublets (J ≈ 8.0, 3.0-5.0Hz) indicating a 16β-CH 3 .Similarly, if the 3 J H-13 , H-14 values range from 1.0 to 4.0 Hz, the orientation of CH 3 -20 and 14-OAc/OH are the same, whereas they should adopt a different orientation.Moreover, if the chemical shift of H-11 was smaller than that of H-12, the CH 3 -20 group was α oriented; otherwise, the CH 3 -20 should be β oriented (Tables 4 and 5).Theoretically, these correlations are attributable to steric interactions within the molecules.For all compounds, 3 J values indicative of the dihedral angle between adjacent protons serve as a practical means to infer their orientation relationship.In these analogs with a 3α-benzyloxy-jatropha-11E-ene system, the C-13 position includes a methyl substituent (CH 3 -20), and the C-12 position contains olefinic hydrogen: When CH 3 -20 is α-oriented, the gauche relationship between 20α-CH 3 and H-11 induces a significant upfield shift in H-11, attributable to the γ-gauche effect (Figure 4); conversely, when CH 3 -20 is β-oriented, the increased distance between 20β-CH 3 and H-11 does not result in a steric interaction between these groups.The above correlations align with NMR data from the reported jatropha diterpenes, whose structures have been confirmed by X-ray analysis [11][12][13][19][20][21][22][23].These empirical 1 H NMR spectrum rules are also suitable for 7,8-seco-jatrophanes with similar signals (Figure 4).Therefore, according to the empirical rules summarized in this and previous research [12], determining the relative configurations of C-2, C-13, and C-14 in jatropha diterpenoids can be efficiently achieved by analyzing their 1 H and 13 C NMR spectra.

Biological Activity Results
In China, E. helioscopia is one of the main herbs to treat psoriasis [5,7,8,14], a skin disease characterized by the excessive proliferation of keratinocytes and lymphocytes.Therefore, compounds 1-23 were evaluated for their immunosuppressive activities against the proliferation of T/B lymphocyte cells and HaCaT cells (Human immortalized keratinocytes) in vitro.Preliminary results (Table 6) indicated that nearly all diterpenoids moderately inhibited concanavalin A (ConA)-induced T lymphocytes and/or lipopolysaccharide (LPS)-induced B lymphocytes proliferation (c 20.0 µM), as well as HaCaT cell proliferation (c 40.0 µM).Moreover, compounds 5 and 7 displayed significant immunosuppressive activities, with IC 50 values of 17.6/10.2and 6.7/11.5 µM (Table 7) against induced T and B cells.Compound 13 exhibited stronger anti-proliferative activities on HaCaT cells than the positive control, MTX (Table 8).Since compounds 7 and 21 in different jatrophatype families showed considerable immunosuppressive activity, they were also evaluated for their inhibitory effect by EdU experiments.Compared with the model or control controls, EdU experiments demonstrated that compounds 7/21 and 9/13 significantly inhibited T/B and HaCaT cells, respectively (Figures 5 and 6) (Figure S54, Supplementary Materials).In addition, compounds 7 and 21 were also evaluated for their inhibitory effect on the secretion of cytokine interferon (IFN) γ, interleukin (IL) 2, and IL-17A of mouse splenocytes, as previously described [29].Lymphocytes were seeded in 96-well plates and treated with compounds 7 and 21 at concentrations of 20, 10, 5, 2.5, and 0 µM for 48 h.Cell viability was analyzed using a cell counting kit-8 (CCK-8) assay [30].Since there were no obvious differences in cell viability between 7/21 (c = 2.5 µM) and the control group, inhibitory activity against the secretion of cytokines was evaluated at a concentration of 2.5 µM and 1 µM (Figure 5A).As shown in Figure 5B, after being stimulated with ConA, the levels of IFN-γ/IL-2/IL-17A increased significantly in the model group via the control group (p < 0.0001); when dealt with compounds 7 and 21, the secretions of IFN-γ (p < 0.0001) and IL-2 (p < 0.01) were significantly inhibited.Although compounds 7 and 21 reduced the secretion of IL-17A, there is no significant difference compared with the model group.In addition, compounds 7 and 21 were also evaluated for their inhibitory effect on the secretion of cytokine interferon (IFN) γ, interleukin (IL) 2, and IL-17A of mouse splenocytes, as previously described [29].Lymphocytes were seeded in 96-well plates and treated with compounds 7 and 21 at concentrations of 20, 10, 5, 2.5, and 0 µM for 48 h.Cell viability was analyzed using a cell counting kit-8 (CCK-8) assay [30].Since there were no obvious differences in cell viability between 7/21 (c = 2.5 µM) and the control group, inhibitory activity against the secretion of cytokines was evaluated at a concentration of 2.5 µM and 1 µM (Figure 5A).As shown in Figure 5B, after being stimulated with ConA, the levels of IFN-γ/IL-2/IL-17A increased significantly in the model group via the control group (p < 0.0001); when dealt with compounds 7 and 21, the secretions of IFN-γ (p < 0.0001) and IL-2 (p < 0.01) were significantly inhibited.Although compounds 7 and 21 reduced the secretion of IL-17A, there is no significant difference compared with the model group.
Nuclear factor-κB (NF-κB) is a family of heterodimeric proteins, including proteolytic processing of the p50 subunit as well as the P65 subunit.NF-κB activation results in the expression of genes associated with cellular proliferation, differentiation, and survival [31].Therefore, the most promising compound 13 for psoriasis was chosen to evaluate its molecular mechanisms associated with the NF-κB pathway for the antiproliferative effect on HaCaT cells.As indicated in Figure 7, compound 13 attenuated the activation of NF-κB in a concentration-dependent manner (10 and 5 µM).Molecular docking analysis between 13 and P65 revealed that (i) the oxygen at C-3 and the carbonyl group of acetoxy groups at C-7 and C-14 formed hydrogen bonds with the residues THR164, ARG73, and GLN142, which make the structure of complex P65-13 stable; (ii) alkyl interaction was generated between 16-CH 3 and the residue PRO172 (Figure 7).The binding energy of 13 with P65 is −7.3 kcal/mol.These results suggested that compound 13 inhibits the phosphorylation of NF-κB p65 in HaCaT cells.

Discussion
The structural elucidation of natural products remains a significant challenge for chemists.Developing new methods using JBCA, examining the intensity of the nuclear overhauser effect, relying on quantum-chemical calculations or X-ray crystallographic analysis, and summarizing empirical rules of NMR data are effective procedures for determining the relative configurations of organic molecules [12,13,18,19].Recently, the relationships between the relative configurations of C-2/C-13/C-14 and 13 C NMR chemical shifts (C-16, C-4, C-13) were summarized in specific jatropha diterpenes [12].However, these rules did not encompass jatropha-11E-ene systems with (7,8-seco) 3α-benzyloxy (or hydroxy) derivatives or these hydroxy-substituted at C-3/C-14/C-15.Therefore, this work further analyzed the correlations between 3 J values/chemical shifts of the relevant protons/carbons and the relative configuration of C-2, C-13, and C-14 in A/B-type jatropha diterpenes based on the previous study (Tables 4 and 5) [12].
Psoriasis is a common immune-mediated skin disease with unclear cellular and mo-

Discussion
The structural elucidation of natural products remains a significant challenge for chemists.Developing new methods using JBCA, examining the intensity of the nuclear overhauser effect, relying on quantum-chemical calculations or X-ray crystallographic analysis, and summarizing empirical rules of NMR data are effective procedures for determining the relative configurations of organic molecules [12,13,18,19].Recently, the relationships between the relative configurations of C-2/C-13/C-14 and 13 C NMR chemical shifts (C-16, C-4, C-13) were summarized in specific jatropha diterpenes [12].However, these rules did not encompass jatropha-11E-ene systems with (7,8-seco) 3α-benzyloxy (or hydroxy) derivatives or these hydroxy-substituted at C-3/C-14/C-15.Therefore, this work further analyzed the correlations between 3 J values/chemical shifts of the relevant protons/carbons and the relative configuration of C-2, C-13, and C-14 in A/B-type jatropha diterpenes based on the previous study (Tables 4 and 5) [12].
Psoriasis is a common immune-mediated skin disease with unclear cellular and molecular mechanisms; however, T cell-mediated hyperproliferation of keratinocytes is the key feature of the pathophysiology of psoriasis.Immunosuppressants, vitamin A acid, methotrexate, and glucocorticoids are traditional and classical clinical medicines [2].Moreover, E. helioscopia is the predominant ingredient in the Chinese ZeQi Powder Preparation for treating psoriasis [5,14].Kv1.3 ion channel is predominant in activated effector memory T (T EM ) cells to further stimulate T EM cell proliferation and cytokine production [8].Although some studies have found helioscopids A/O/euphohelioscoids A from E. helioscopia inhibited Kv1.3 activity in human embryonic kidney cells 293 (HEK293) cells [7,8], this activity only indirectly suggests the immunosuppressive potential of these compounds.Thus, more relevant bioassay experiments are needed to elucidate how and why compounds from E. helioscopia contribute to psoriasis treatment.Additionally, natural products often vary due to their different origins.
Lymphocytes, which are the most important immune cells, can be divided into T and B lymphocytes.During an immune response, T and B lymphocytes secrete various inflammatory factors (such as IL-2, IFN-γ, IL-17A, and so on) or antibodies to maintain internal environmental stability jointly.However, the overactivation of T or B lymphocytes can lead to various autoimmune diseases, such as psoriasis [32].In vitro, the proliferation of T and B lymphocytes can be selectively stimulated by Con A and LPS, respectively.Meanwhile, the production level of cytokines and antibodies increases significantly when T and B lymphocytes are stimulated.Therefore, based on the specificity of these two mitogens and reference to the experimental model of splenic cell proliferation inhibition, this study evaluated the immunosuppressive activity of compounds from E. helioscopia using the previously established experimental model [33].As depicted in Table 7 and Figure 5, most compounds displayed anti-proliferative effects on induced T and B cells.In addition, compounds 7 and 21 significantly reduced the secretions of IFN-γ (p < 0.0001) and IL-2 (p < 0.01).IL-2 and IFN-γ are closely associated with T cell proliferation [34].Therefore, compounds 7 and 21 may exert anti-proliferative effects on T lymphocytes by reducing IFN-γ and IL-2 levels.
As previously reported, excessive proliferation and aberrant differentiation of keratinocytes are the main cellular events in psoriasis development [35].Since the HaCaT cell line exhibits good stability and infinite proliferation ability, similar to the characteristics of the rapid proliferation of epidermal cells in psoriasis pathophysiological changes, it has been widely used in psoriasis models in vitro [35].As shown in Table 8 and Figure 6, more than half of the compounds effectively inhibited HaCaT cells, particularly compound 13.NF-κB proteins are a family of transcription factors central to inflammation and immunity, and they also play crucial roles in development, cell growth, survival, proliferation, and various pathological conditions [36].The mechanism of 13 was then explored, focusing on NF-κB P65.Compound 13 may bind with NF-κB P65, inhibiting its phosphorylation of NF-κB P65 and thereby reducing the proliferation of HaCaT cells (Figure 7).

Plant Material
The whole plant, E. helioscopia L. (Euphorbiaceae), was collected from Kaifeng City, Henan Province, China, in May 2017 and identified by Prof. Cheng-Ming Dong (Henan University of Chinese Medicine), an expert in the taxonomic field of Chinese medicine.A voucher specimen (No.HZY201705) was deposited at the School of Pharmacy, Henan University of Chinese Medicine.

Biological Activity Assays
Immunosuppressive activities assay and antiproliferative activity against HaCaT cells were performed according to the reported methods [33].

Immunosuppressive Activities Assay
Female or male Balb/c mice (6-8 weeks old) were sacrificed, and their spleens were removed aseptically.A single-cell suspension was prepared by pressing the spleens against the bottom of the Petri dish with a 5 mL syringe plunger, and cell debris and clumps were removed.Erythrocytes were depleted with ammonium chloride buffer solution, and the mononuclear cell suspensions were maintained in RPMI 1640 media containing 10% fetal bovine serum, penicillin (100 U/mL), and streptomycin (100 µg/mL).Then the mononuclear cell suspensions were dispensed into 96-well plates (2 × 10 5 cells/well), in the absence or presence of compounds (c = 20.0 µM), were stimulated with ConA (5 µg/mL) or LPS (15 µg/mL) to induce T cell or B cell proliferative responses, respectively.Dexamethasone (Dex, c = 2.0 µM) was used as a positive control.In the 96-well plates, the wells cultured with cells and ConA/LPS were assigned as the modal group (Mod), the wells containing cells were described as the control group (Con), and the wells containing culture medium were described as the blank group.These 96-well plates were maintained under a humidified 5% CO 2 atmosphere at 37 • C for 48 h. 10 µL of CCK-8 was added to each well at the final 4-5 h of culture, and the absorbance (OD) values were measured with a microplate reader (SpectraMax iD3, Molecular Devices, LLC.) at 450 nm.Stimulation Index (SI) = (OD sample −OD blank )/(OD Con −OD blank ); Inhibition rate = (1−SI sample /SI Mod ) ×%.The IC 50 value for each compound was calculated using the Reed and Muench method.All experiments were performed in triplicate.

Cell Viability Assays of Lymphocytes
Similarly, the mononuclear cell suspensions of lymphocytes (2 × 10 5 cells/well) were cultured within 96-well plates in the absence or presence of compounds 7 or 21 (c = 20.0, 10, 5, 2.5, and 0 µM).In the 96-well plates, the wells containing cells were described as the control group (Con), and the wells containing culture medium were described as the blank group.These 96-well plates were maintained under a humidified 5% CO 2 atmosphere at 37 • C for 48 h. 10 µL of CCK-8 was added to each well at the final 5 h of culture, and the OD values were measured with a microplate reader at 450 nm.Cell vialibity (%) = (OD sample −OD blank )/(OD Con −OD blank ) × 100%.

Antiproliferative Activity against HaCaT Cells
The keratinocyte cell line was HaCaT (FH0186), bought from Fufeng Biology (Shanghai, China).HaCaT cells were cultured using the trypsin enzyme digesting technique and then passaged in vitro.The number of digested cells was counted by cell counter and were maintained in DMEM media containing 10% fetal bovine serum, penicillin (100 U/mL), and streptomycin (100 µg/mL) under a humidified 5% CO 2 atmosphere at 37 • C. In brief, each well of a 96-well cell culture plate was seeded with 100 µL of cells (1 ×10 5 cells/mL) and kept for 12 h for adherence and then added with test compounds (at a final concentration of 40 µM).The wells containing cells were described as the control group (Con), and the wells containing culture medium were described as the blank group.Methotrexate (MTX) was used as a positive control.After different concentrations of test compounds addition, each cell line was incubated for 48 h under a humidified 5% CO 2 atmosphere at 37 • C.After the incubation, each well was treated with CCK-8 (10 µL), and incubation continued for 4 h at 37 • C.Then, the 96-well cell culture plates were subjected to a measure of optical density at 450 nm with a 96-well microplate reader.The IC 50 value for each compound was calculated using the Reed and Muench method.All experiments were performed in triplicate.

EdU Assay
EdU fluorescence labeling for cell proliferation of induced T/B and HaCaT cells was performed as previously reported [37,38].EdU (the Click-iT™ EdU Flow Cytometry Assay Kit, APExBIO, Houston, TX, USA) was added 24 h (final concentration 50 µM, T/B cells) and 4 h (final concentration 10 µM, HaCaT cells) before harvesting the cells.For the Click reaction, cells were fixed with 100 µL of 4% paraformaldehyde for 15 min.Cells were washed again and incubated with 100 µL of saponin-based permeabilization buffer for 15 min.After additional washing, cells were incubated with 100 µL Click-iT reaction buffer for 1 h and washed again with 200 µL permeabilization buffer.All procedures were performed according to the manufacturer's instructions.

Statistics and Reproducibility
Data analyses were carried out using Prism 8.0, and One-way ANOVA was used to compare the differences between groups.LSD or Dunnett's T3 test was used for pair comparison according to standard deviation values.p < 0.05 or p < 0.01 were considered to be statistically significant.

Ethic Statement
The animal study (No.DWLL202003116) was reviewed and approved by the Animal Welfare Ethics Committee of the Henan University of Chinese Medicine.

Molecular Docking Studies
The crystal structure of the inducible nitric oxide synthase (P65) (PDB: 1NFI) was downloaded from RCSB PDB (http://www.rcsb.org/,accessed on 6 June 2024), the structure of 13 was drawn by ChemDraw 14.0, and the 3D structure file was transformed by Chem3D 14.0.Before docking, the water molecules on the receptors were removed, and polar hydrogen atoms, charge, and magnetic field were added.AutoDockTools 1.5.6 was used to process ligands and receptors, and AutoDock vina was used for molecular docking.The 3D and 2D diagrams of the best-scored binding pose were visualized by Discovery Studio Visualizer v20 (http://www.discoverystudio.net/accessed on 6 June 2024).

Conclusions
Phytochemical investigation of E. helioscopia resulted in isolating 23 diterpenoids, including four rare novel seco-diterpenoids (1-4) and three new jatropha diterpenes (5)(6)(7).Based on the NMR data obtained in this study and the previously summarized configuration rules, as well as those with crystallographic structures reported in the literature, the correlations between 3 J values/chemical shifts of the relevant protons and the relative configuration of C-2, C-13, and C-14 in jatropha diterpenes were considered.

Figure 2 .
Figure 2. Key 2D NMR correlations of compounds 1 and 2 (four rings numbered as A-D).Figure 2. Key 2D NMR correlations of compounds 1 and 2 (four rings numbered as A-D).

Figure 2 .
Figure 2. Key 2D NMR correlations of compounds 1 and 2 (four rings numbered as A-D).

Table 6 .
Inhibition rate of compounds 1-23 (c 20.0 µM) and Dex (c 2.0 µM) against the ConA-induced proliferation of T and/or LPS-induced proliferation of B cells.

Table 7 .
IC 50 values of compounds 5 and 7 against the induced proliferation of T cells (ConA) and B cells (LPS).

Table 8 .
IC 50 values of active compounds against the proliferation of HaCaT cells.