Diterpenoids from the Aerial Parts of Isodon serra with Selective Cytotoxic Activity

Four new diterpenoids, isodosins A–D (1–4), together with nine known compounds (5–13) were isolated and identified from the aerial parts of Isodon serra (Maxim.) Hara. The structures of the new diterpenoids were elucidated based on the analysis of HR-ESI-MS data, 1D/2D-NMR-spectroscopic data, and electronic circular dichroism (ECD) calculations. Cytotoxicities of compounds 2, 3, 5, 6, and 9 against the HepG2 and H1975 cell lines were evaluated with the MTT assay. As a result, compounds 2, 3, and 6 revealed higher levels of cytotoxicity against HepG2 cells than against H1975 cells. Moreover, compund 6 demonstrated the most efficacy in inhibiting the proliferation of HepG2 cells, with an IC50 value of 41.13 ± 3.49 μM. This effect was achieved by inducing apoptosis in a dose-dependent manner. Furthermore, the relationships between the structures and activities of these compounds are briefly discussed.


Introduction
Hepatocellular carcinoma (HCC), which is the most common primary tumor of the liver, is ranked as the third most common cause of cancer death in 46 countries, thus remaining a global health challenge [1,2].The number of new cases and deaths from HCC is predicted to rise by more than 55% from 2020 to 2040, resulting in 1.3 million fatalities in 2040 [1].The risk of developing HCC has been associated with multiple factors, including chronic viral infections [3], alcohol consumption [4], and exposure to various chemicals [5].The development of chemotherapy has positively influenced the treatment of HCC and offers a substantial improvement in patient survival [6] from drugs such as Sorafenib, 5-Fluorouracil, and Cisplatin [7].However, the main limitation of the chemotherapeutic modality is their non-specificity and their inability to identify and target cancerous cells directly [8,9].The administration of anti-HCC chemotherapeutics often necessitates the utilization of delivery systems to ensure targeted drug delivery, thereby minimizing systemic side effects [7,10].Consequently, medicinal chemists have been making efforts to find potential chemotherapeutic agents with high selectivity and low toxicity effects over the past several decades [11].
Natural products (NPs) from medicinal plants play a crucial role in the exploration of new drugs for cancer therapy [12].Among the different types of NPs, diterpenoids have attracted considerable interest on account of their promising anti-cancer effects and modifiable skeleton, including NPs such as Taxol and Ingenol 3-angelate [13][14][15][16][17]. Recently, researchers have discovered a series of diterpenoids from medicinal plants with selective anti-cancer activities and have proved that those diterpenoids could induce apoptosis, autophagy, and metastasis suppression in cancer cells via the inhibition of Akt [18][19][20].These findings suggest that the isolation of more diterpenoids with selective cytotoxic activities from medicinal plants could make a substantial contribution to the progress of anti-cancer drugs [21].
Isodon serra (Maxim.)Hara, a perennial plant belonging to the family of Labiatae, is commonly used as a folk medicine for the treatment of acute jaundice, hepatitis, and acute cholecystitis [22][23][24].Previous phytochemical studies on I. serra have led to the isolation and identification of over 90 ent-kaurane diterpenoids, which are characterized by a perhydrophenanthrene subunit (A, B, and C rings) and a cyclopentane ring (D ring) [25].Pharmacological investigations have been conducted to highlight the therapeutic potential of ent-kaurane compounds isolated from I. serra against various cancers such as liver [22,26], colon [27,28], lung [29], muscle-invasive bladder [30], and human nasopharyngeal carcinoma [31] cancer.In an effort to search for diterpenoids with anti-liver cancer bioactivity from I. serra, the petroleum ether fraction of this plant was chemically investigated.
autophagy, and metastasis suppression in cancer cells via the inhibition of Akt [18][19][20].These findings suggest that the isolation of more diterpenoids with selective cytotoxic activities from medicinal plants could make a substantial contribution to the progress of anti-cancer drugs [21].
Isodon serra (Maxim.)Hara, a perennial plant belonging to the family of Labiatae, is commonly used as a folk medicine for the treatment of acute jaundice, hepatitis, and acute cholecystitis [22][23][24].Previous phytochemical studies on I. serra have led to the isolation and identification of over 90 ent-kaurane diterpenoids, which are characterized by a perhydrophenanthrene subunit (A, B, and C rings) and a cyclopentane ring (D ring) [25].Pharmacological investigations have been conducted to highlight the therapeutic potential of ent-kaurane compounds isolated from I. serra against various cancers such as liver [22,26], colon [27,28], lung [29], muscle-invasive bladder [30], and human nasopharyngeal carcinoma [31] cancer.In an effort to search for diterpenoids with anti-liver cancer bioactivity from I. serra, the petroleum ether fraction of this plant was chemically investigated.
In this study, four new diterpenoids (1-4) and nine known ones (5-13) were isolated and characterized (Figure 1).Compounds 2, 3, and 6 demonstrate high selectivity towards HepG2 cells over H1975 cells, which could be attributed to the presence of a double bond at C-2 and C-3 or the hydroxy groups at C-6 and C-15.Furthermore, compound 6 exhibited a dose-dependent induction of apoptosis in HepG2 cells.

Compounds Isolated from I. serra Were Selectively Toxic toward HepG2 over H1975 Cells
The global prevalence of HCC is increasing, posing a significant challenge to public health [46].Chemotherapy has emerged as a well-established therapeutic approach in cancer treatment, offering potential relief from the disease burden.The superior efficacy and safety profile of targeted therapeutic drugs, in comparison to traditional chemotherapy, has established them as the prevailing modality for cancer treatment [8].I. serra is a proven source of diterpenoid-containing natural products that often have anti-cancer activity [28].
In consideration of the anti-cancer activity of ent-kaurane diterpenoids, the main compounds 2, 3, 5, 6, and 9 were evaluated for their cytotoxic activities against two human cancer cell lines (HepG2 and H1975) by an MTT method.As shown in Figure 4, it is noteworthy that compounds 2, 3, and 6 exhibited significant inhibitory effects against HepG2 cells while displaying limited cytotoxic activity against H1975 cells.Compound 6 exhibited strong inhibition effects (IC 50 = 41.13 ± 3.49 µM) on HepG2 cells, whereas compounds 2 and 3 demonstrated potent cytotoxic activity, displaying IC 50 values of 121.33 ± 17.54 µM and 96.44 ± 9.52 µM, respectively.In contrast, compounds 5 and 9 exhibited relatively weak cytotoxic activity against HepG2 cells.Importantly, the tested compounds exhibited limited cytotoxic activity against H1975 cells (Figure 5).These results suggest a compelling structure-activity relationship (SAR) involving the cyclopentanone core.In the present study, the weak inhibition effect of compound 9 suggested that the cleavage of the C-6-C-7 could decrease its cytotoxicity.Antitumor activity is enhanced by a hydroxy group at C-6 and C-15, which was supported by the fact that compound 2 was more effective than compound 5 [32].Moreover, the antitumor activity is enhanced by the presence of a double bond at C-2 and C-3, as was demonstrated by the superior efficacy of compound 6 compared to compound 5.The presence of substantial evidence suggests that modifying or altering its conformation, such as introducing a fluorine atom, can significantly enhance anti-HCC activity [47].The compounds with the highest potency, or those exhibiting moderate efficacy possess multiple sites available for modification, and the development of their derivatives could potentially enhance anti-HCC activity.

Compound 6 Selectively Induced Apoptosis in Two Human Cancer Cell Lines
Apoptosis is essential for maintaining a delicate equilibrium between cell death and proliferation [48].Evasion of apoptosis results in uncontrolled cell multiplication, thereby contributing to various diseases such as cancer [49].The cytotoxic results have demonstrated that compound 6, the primary compound in the PE part, exhibited the highest efficacy in suppressing the proliferation of HepG2 cells.PI staining was conducted to assess whether its cytotoxicity was caused by apoptosis.As shown in Figure 6a,b, HepG2 cells treated with compound 6 displayed a significant dose-dependent increase in the percentage of apoptotic cells compared to that in the control group.Meanwhile, compound 6 induced a higher level of apoptosis in HepG2 cells than in H1975 cells, indicating that the effect was not cell-type dependent (Figure 6c,d).

Compound 6 Selectively Induced Apoptosis in Two Human Cancer Cell Lines
Apoptosis is essential for maintaining a delicate equilibrium between cell death and proliferation [48].Evasion of apoptosis results in uncontrolled cell multiplication, thereby contributing to various diseases such as cancer [49].The cytotoxic results have demonstrated that compound 6, the primary compound in the PE part, exhibited the highest efficacy in suppressing the proliferation of HepG2 cells.PI staining was conducted to assess whether its cytotoxicity was caused by apoptosis.As shown in Figure 6a,b, HepG2 cells treated with compound 6 displayed a significant dose-dependent increase in the percentage of apoptotic cells compared to that in the control group.Meanwhile, compound 6 induced a higher level of apoptosis in HepG2 cells than in H1975 cells, indicating that the effect was not cell-type dependent (Figure 6c,d).
Targeting the apoptotic pathway presents a compelling strategy for the discovery of novel anti-HCC therapies, as evading apoptosis is a hallmark of cancer [50].The present study conducted an unprecedented investigation into the cytotoxicity of compound 6 in inducing apoptotic cell death in HepG2 and H1975 cells.Moreover, this data indicated the potential of compound 6 as a promising targeted-anti-liver-cancer candidate by selectively inducing cell death in liver cancer cells.Targeting the apoptotic pathway presents a compelling strategy for the discovery of novel anti-HCC therapies, as evading apoptosis is a hallmark of cancer [50].The present study conducted an unprecedented investigation into the cytotoxicity of compound 6 in inducing apoptotic cell death in HepG2 and H1975 cells.Moreover, this data indicated the potential of compound 6 as a promising targeted-anti-liver-cancer candidate by selectively inducing cell death in liver cancer cells.

Plant Materials
The aerial parts of I. serra were collected from the Qingyuan City of Guangdong Province, People's Republic of China, in October 2021 and identified by one of the authors, Dr. Ji Yang.The voucher specimen (IS-2021-10) was stored at the School of Traditional Chinese Materia Medica Guangdong Pharmaceutical University.

ECD Calculations
The conformational analyses were performed using random searching in Spartan 16 (Wavenfunction, Irvine, CA, USA, 2016) employing the MMFF94 force field.Subsequently, the conformers were re-optimized using DFT at the B3LYP/6-31G(d,p) level and utilizing the polarizable conductor calculation model (SMD) provided by the Gaussian 09 program.The energies, oscillator strengths, and rotational strengths (velocity) of conformers with a Boltzmann distribution greater than 1% were calculated using TDDFT methodology at the B3LYP/6-311+g(d,p) level in MeOH.The ECD spectra were simulated by convolving Gaussian functions (half bandwidth at 1/e peak height, sigma ¼ 0.30 for all).The final spectra were obtained by averaging the simulated spectra of each conformer based on Boltzmann distribution theory and their relative Gibbs free energy (∆ G ) values.

Cell Lines and Cell Culture
Human hepatocellular carcinoma HepG2 and H1975 cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).HepG2 cells were cultured in DMEM medium supplemented with 10% FBS and 1% antibiotics (penicillin and streptomycin) in an incubator containing 5% CO 2 at 37 • C. H1975 cells were grown in RPMI 1640 medium containing 10% (v/v) FBS and 1% antibiotics in a cell incubator with 5% CO 2 at 37 • C.

Cytotoxicity Assay
Cytotoxicities of the main compounds (2, 3, 5, 6, and 9) against HepG2 and H1975 cell lines were evaluated with the MTT assay as described in the literature [32].Briefly, HepG2 or H1975 cells (5 × 10 3 cells/well) were cultured in 96-well plates.After seeding and incubating for 24 h, the cells were treated with the compounds at various concentrations (0, 25, 50, and 100 µM) for 24 h.After this treatment, MTT solution was added and the cells were incubated for 4 h at 37 • C in an atmosphere of 5% CO 2 .DMSO was added to dissolve the formazan after the supernatant medium was removed.Finally, absorbance was detected at 570 nm using a Multiskan SkyHigh Spectrum (Thermo Scientific™, Waltham, MA, USA).

Analysis of Apoptosis by PI Staining
HepG2 and H1975 cells (1.5 × 10 5 cells/well) were plated in 6-well plates and treated with compound 6 at 0, 20, 40, 60, 80, or 100 µM for 24 h.The cells were subsequently collected, re-suspended in 70% ethanol, and fixed at 4 • C for 30 min.Cells were centrifuged at 1000 rpm for 5 min to separate the supernatant.Each cell pellet was stained in 300 µL propidium iodide (PI) (50 µg/mL) staining solution at 37 • C for 30 min in the dark.Finally, the cells were washed twice with PBS and measured using flow cytometry (Beckman Coulter, CytoFLEX, Indianapolis, IN, USA).

Statistical Analysis
All values presented in the figures, tables, and text are presented as means ± SD.Statistical analysis was performed with a one-way analysis of variance (ANOVA) test using GraphPad Prism, Version 8.2.1 (GraphPad Software, Inc., San Diego, CA, USA).p < 0.05 was considered statistically significant.

Conclusions
In conclusion, a total of 13 diterpenoids (1-13) were isolated from the aerial parts of I. serra.Among them, compounds 1-3 were identified as new ent-kaurane diterpenoids, and compound 4 was identified as a new ent-abietane diterpenoid.Compounds 2, 3, and 6 demonstrate high selectivity towards HepG2 cells over H1975 cells.Moreover, compound 6 demonstrated a significant induction of apoptotic cell death specifically in HepG2 cells compared to H1975 cells.Our results suggest that compound 6 might be a highly promising lead candidate for targeted anti-liver cancer therapy by inducing apoptotic cell death.Overall, the findings of our study have significantly expanded the structural diversity of secondary metabolites derived from I. serra and provided evidence for the potential use of these diterpenoids in HCC treatment.

Figure 6 .
Figure 6.Flow cytometric analysis of apoptosis in HepG2 and H1975 cells (a,c).The apoptosis of HepG2 or H1975 cell lines was detected by flow cytometry based on PI staining after treatment with various concentrations of compound 6 (b,d).Histogram showing the proportions of apoptotic cells after 24 h of treatment with compound 6.The results are expressed as mean ± SD. (* p < 0.05, ** p < 0.01, *** p < 0.001 versus the control group).

Figure 6 .
Figure 6.Flow cytometric analysis of apoptosis in HepG2 and H1975 cells (a,c).The apoptosis of HepG2 or H1975 cell lines was detected by flow cytometry based on PI staining after treatment with various concentrations of compound 6 (b,d).Histogram showing the proportions of apoptotic cells after 24 h of treatment with compound 6.The results are expressed as mean ± SD. (* p < 0.05, ** p < 0.01, *** p < 0.001 versus the control group).