*Agrobacterium* mid-log phase (O.D A600 = 0.6) cells were harvested by centrifugation at 4,000 rpm at 4 ◦C for 10 min. *A. tumefaciens* was resuspended in half-strength MS liquid media and cell density was adjusted to an O.D A600 of 1.0 for plant co-culture. The leaf explant from 3 weeks old seedling was wounded by using a scalpel, then immersed in *A. tumefaciens* GV3101 suspended liquid medium for 15–20 min. The leaf explants were then incubated on MS media supplemented with 2.0 mg/L of BAP and 0.1 mg/L of NAA without antibiotics for two days. The infected leaf explant was transferred to the plant regeneration media containing MS supplement with 2.0 mg/L of BAP, 0.1 mg/L of NAA, and antibiotics (50 mg/L of kanamycin + 500/250 mg/L of cefotaxime) for one week. The single shoot emerged from the wounded site was separated from the leaf explant and transferred to the rooting media containing 1 2 MS media supplemented with antibiotics (50 mg/L of kanamycin + 250 mg/L of cefotaxime). After 2 weeks of rooting, a single whole plant was transplanted to the pot and acclimatized for two weeks before transferring to the growth chamber.

#### *2.9. PCR Analysis of the Transgenic Lines*

Plant genomic DNA for polymerase chain reaction (PCR) was extracted by the Genomic DNA mini kit (Geneaid Biotech Ltd., New Taipei City, Taiwan), following the manufacturer's instructions. The leaves of transformation (AtPAP1) lines and control (GUS and GV3101) plants were ground into a fine powder (50–100 mg fresh weight) using a mortar and pestle. Extracted genomic DNA was subjected to PCR analysis using gene-specific primers provided in Table 1.

**Table 1.** Comparison of phenylpropanoid biosynthetic genes of *S. nigrum* with most orthologous genes.

The PCR conditions consisted of denaturation at 95 ◦C for 30 s, annealing at 58 ◦C for 30 s and extension at 72 ◦C for 30 s, followed by 35 cycles and the final extension at 72 ◦C for 5 min. The PCR products were analyzed on a 1% agarose gel stained with ethidium bromide and visualized under UV light.

#### *2.10. Transgenic Plant Seed Segregation Assay*

The transgenic plants were grown in a growth chamber for four months and the mature seeds of T0 primary transgenic plants were harvested. Twelve AtPAP1 overexpression transgenic seeds (T1) were harvested from seeds germinated on half-strength MS media with 50 mg/L Kanamycin selection marker. Then five shoots of kanamycin-resistant in each line were grown on the pot (T2) for three months. The transgenic plants were grown in a growth chamber for four months and the mature seeds of T0 primary transgenic plants were harvested. Following generations (i.e., T1 and T2) screened on 50 mg/L kanamycin media were subjected to phenotype and anthocyanin analysis.

#### *2.11. Total RNA Extraction and cDNA Synthesis*

Total RNA was isolated from *S. nigrum* at fruit maturity stage by using RNA Mini Kit (Geneaid) according to the manufacturer's protocol. The quantity and quality of total RNA were examined on a 1.0% agarose gel and using NanoVue Plus Spectrophotometer (GE Healthcare Life Sciences, Pittsburgh, PA, USA), respectively.

#### *2.12. cDNA Synthesis and Quantitative Real-time PCR*

The cDNA was synthesized from 1 μg of DNA free total RNA using ReverTra Ace-α Kit with oligo (dT) 20 primer (Toyobo, Osaka, Japan). The resulting cDNA products were used as templates for real time-PCR analysis. For all target genes, primers were designed using Primer Design software (GeneRunner.exe) and using an online program (https://www.genscript.com/ssl-bin/app/primer). Quantitative real-time PCR was performed in a BIO-RAD CFX96 Real-time PCR system (Bio-Rad Laboratories, Hercules, CA, USA) with 2X QuantiTect SYBR Green RT-PCR Master Mix, (QIAGEN, Hilden, Germany) under the condition as follows: reverse transcription at 50 ◦C for 30 min, PCR initial at 95 ◦C for 15 min, denaturation at 94 ◦C for 15 s, annealing at 60 ◦C for 30 s, extension at 72 ◦C for 30 s and reaction cycle was repeated for 39 cycles at extension followed by a final extension at 72 ◦C for 1 min. All reactions were from three biological replicates.

#### *2.13. Statistical Analysis*

Statistical analyses were performed using GraphPad InStat software (Graphpad Instat Software, San Diego, CA, USA). One-way ANOVA was applied for comparisons with the Turkey test. All data are expressed as mean ± SEM. The significance threshold was at a level of 5% (\* *P* < 0.05; \*\* *P* < 0.01, \*\*\* *P* < 0.001). All the experiments were biological triplicates.

#### **3. Results**

#### *3.1. Identification of Phenylpropanoid Biosynthesis Genes in Black Nightshade*

The sequences of genes involved in the phenylpropanoid biosynthesis pathway were identified from the RNA-seq data of the black nightshade. The retrieved sequences were subject to a BLAST search to confirm and compare with the orthologs of other *Solanaceae* plants. The genes identified in black nightshade associated with general phenylpropanoid pathways were designated as *SnPAL* (accession MT032183, 712 amino acids), *SnC4H* (accession MT032184, 505 amino acids), *Sn4CL* (accession MT032185, 545 amino acid), *SnC3H* (accession MT032186, 510 amino acids), and *SnCHI* (accession MT032187, 217 amino acids). In addition, a biosynthesis gene involved in the anthocyanin pathway (e.g., anthocyanin-5-*o*-glucosyl transferase) was designated as *SnUGT75C1* (accession MT032188, 473 amino acids), and genes more closely linked to the lignin synthesis (e.g., caffeate *O*-methyltransferase

(*COMT*)) and hydroxycinnamoyl-CoA shikimate (*HCT*)) were designated as *SnCOMT* (accession MT032189, 363 amino acids), and *SnHCT* (accession MT032190, 435 amino acids) (Table 1). To add perspective to the identified sequence of black nightshade, orthologs of several other *Solanaceae* plants that showed most similarity to the black nightshade genes were also compared. Except for *SnCHS*, all the deduced amino acid sequences from various *Solanaceae* plants had sequence identity from 90 to 99 percentile between orthologous genes in *Solanaceae* plants.

#### *3.2. Induction of Anthocyanin by Overexpression of AtPAP1 in Black Nightshade*

We used a gain-of-function approach to examine the effect of overexpression of AtPAP1 on black nightshade. To this end, we generated the transgenic black nightshade plants that harbor AtPAP1 cDNA and GUS (control) both driven by CaMV 35S (Figure 1A). The presence of the transgenes was confirmed by PCR analysis of AtPAP1 transgenic plants using Kan primers (Neo). Expected sizes of amplicons (630 bp) were observed in the corresponding *S. nigrum* plant lines (Figure 1B). In addition, the expression of *AtPAP1* transgene was verified by quantitative RT-PCR (Figure 1B).

More than 15 individual transgenic lines were obtained and maintained in a growth chamber (Figure 2A–F), for which the transgene was also confirmed by PCR using Kan primers (Neo) (Figure 2G). Overexpression of *Arabidopsis* AtAPAP1 resulted in enhanced color changes all over the plant including leaves and flowers compared with the control plant (Figure 3A). The appearance of purple color was especially notable in leaves and floral organs such as petals and anthers (Figure 3B,C). Thus, our data demonstrate that overexpression of a single *Arabidopsis* regulatory gene, *AtPAP1*, might have increased the content of anthocyanin in the heterologous system of black nightshade.

**Figure 2.** Schematic diagram of AtPAP1 overexpression transgenic lines of *S. nigrum* by Agrobacterium-mediated transformation. (**A**) Shoot regeneration for 30 days after AtPAP1 transformation. (**B**) Shoot regeneration after sub-culture for 30 days of AtPAP1 transformation. (**C**)&(**D**) Development of the whole plant. (**E**) Transgenic plant rooting. (**F**) Transgenic plant after transfer to the pot for 40 days. (**G**) Confirmation of transgene using kanamycin (Neo) primer in diverse transgenic lines.

**Figure 3.** Phenotype comparison of AtPAP1 overexpression transgenic lines of *S. nigrum*. (**A**) Whole plants of AtPAP1 transgenic lines and the control line at fruit development stage in a growth chamber. (**B**) Leaf and (**C**) flower phenotype comparison between AtPAP1 overexpression lines and control (GFP-GUS (Control) and GV3101 (Mock)) lines.

#### *3.3. Expression of Anthocyanin Biosynthesis Genes in the AtPAP1-overexpressed Black Nightshade Plants*

To understand the mechanism underlying the increased content of anthocyanin in the transgenic black nightshade, we examined gene expression of anthocyanin biosynthesis that might have been affected by overexpression of AtPAP1. As expected, expression of AtPAP1 transgene was up-regulated in the transgenic black nightshade plants (PAP1 #4 and #7) compared with the control (GFP-GUS) and Mock (GV3101) lines (Figure 4). When we examined the genes identified in this study, associated with the phenylpropanoid biosynthesis pathway, most of the genes were highly induced in the AtPAP1 overexpression lines (Figure 5). Of the genes identified in the current study, genes known to be associated with general phenylpropanoid pathways (EBGs), such as *SnPAL* (accession MT032183)*, SnC4H* (accession MT032184)*, Sn4CL* (accession MT032185)*,* and *SnCHI* (accession MT032187) were all highly induced in the AtPAP1 overexpressed plants. Expression of other biosynthesis genes associated with anthocyanin (*SnUGT75C1*, accession MT032188) and lignin (*SnHCT*, accession MT032190) was largely unaltered. This suggests that black nightshade genes involved in the general phenylpropanoid biosynthesis pathway may have been regulated by the AtPAP1 transcription factor that rendered the purple color in *AtPAP1*-overexpressing *S. nigrum* plants, which ultimately led to an increase in the levels of anthocyanin in vegetative tissue and flower parts.

**Figure 4.** Expression of AtPAP1 gene in AtPAP1 overexpressing *S. nigrum* transgenic lines and control lines. All data are expressed as mean and SEM. The significance threshold was at a level of 5% (\* *P* < 0.05; \*\* *P* < 0.01).

**Figure 5.** Expression of flavonoid biosynthetic genes in AtPAP1 overexpressing *S. nigrum* transgenic lines and control lines. The AtPAP1 transgenic lines were harvested and proceeded to qRT-PCR analysis. SnPAL, phenylalanine ammonia-lyase; SnC4H, cinnamate-4-hydroxylase; Sn4CL, 4-coumarate: CoA ligase; SnCHI, chalcone isomerase; SnUGT75C1, UDP-glycosyltransferase 75C1; SnHCT, quinate hydroxycinnamoyl transferase. All data are presented as mean and SEM. The significance threshold was at a level of 5% (\* *P* < 0.05; \*\* *P* < 0.01,). All the experiments were biological triplicates.

#### *3.4. Identification of Anthocyanins in the AtPAP1-overexpressed Black Nightshade Plants*

To understand the types of anthocyanins that might have accumulated resulting from the overexpression of AtPAP1, we investigated the metabolites that correspond to anthocyanins using HPLC. From the first and second generation of AtPAP1-overexpressed transgenic black nightshade lines, we have detected several peaks associated with the various anthocyanins in black nightshade transgenic lines, while no detectable peaks have been observed in the control line (Figure 6).

**Figure 6.** Identification of anthocyanins in AtPAP1 overexpressed *S. nigrum* transgenic lines by HPLC analysis. Above, AtPAP1 overexpression *S. nigrum* line. Detected eight peaks represented unknown anthocyanins at different retention time; Below, *S. nigrum* control line. No peak detected indicated the absence of anthocyanin.

Subsequent analysis of LC-MS/MS mass spectrometry has revealed that each peak of the HPLC data obtained demonstrated the same pattern of the mass ionization in both T0 and T1 transgenic lines, indicating that anthocyanins detected in two generations of transgenic plants were identical. The total ion chromatogram (TIC) was separated into 18 main peaks, from which only six peaks that represent anthocyanins were identified. The *m*/*z* ratio of each intact anthocyanin and its daughter fragments are listed in Table 2. Of these, three aglycones were determined as delphinidin aglycone (Dd, *m*/*z* 303), petunidin aglycone (Pt, *m*/*z* 317) and malvidin aglycone (Mv, *m*/*z* 331) [20]. In addition, three delphinids, two petunidin and one malvidin were detected in the AtPAP1-overexpressed black nightshade transgenic lines.

Specifically, the mass spectrum of peak 8 with a retention time at 16.41 min that had a molecular ion *m*/*z* of 773.2136 [M]+ and fragment *m*/*z* of 627.156 ([M-146]+) identified as delphinidin 3-O-rutinoside-5-O-glucoside with a loss of one molecule of rhamnose [21]. The peak 9 with retention time at 20.47 min that had a molecular ion *m*/*z* of 627.1557 and fragment *m*/*z* of 611.1610 was identified to be a 3,5-diglucoside glycone (m/z 303 [Dd]+ known as delphinidin aglycon), and this peak represented delphinidin-3,5-O-diglucoside. The peak 12 with a retention time at 25.42 min with *m*/*z* 611.1611[M]+ and fragmentation *m*/*z* of 464.1031 ([M-146]+) corresponds to a delphinidin aglycone that is derived from loss of one molecule of rhamnose with *m*/*z* 303 [Dp]+, was identified as an anthocyanin derivative (delphinidin 3-0-rutinoside) [21]. Peak 13 with a retention time at 28.46 min that showed a molecular ion *m*/*z* and fragment *m*/*z* of 933.2679 and 641.1721 [M-146-146], respectively, appeared as a petunidin aglycone with a loss of two molecules of coumaric acid (*m*/*z* 317([Pt]+) identified as petunidin-3-O-rutinoside (*cis*-*p*-coumaroyl)-5-O-glucoside or petunidin-3-O-rutinoside (*trans*-*p*-coumaroyl)-5-O-glucoside. The peak 15 with retention time at 32.68 min that had a molecular ion and fragment of 963.2803 and 317 [Pt]+ corresponded to a petunidin aglycone, which was tentatively identified as petunidin-3-(feruloyl)-rutinoside-5-glucoside [21–23]. Finally, the peak 17 with a retention time at 36.54 min was identified as malvidin-3-(feruloyl)-rutinoside-5-glucoside by the ion at *m*/*z* of 977.2945 [M]+ and the fragment at *m*/*z* of 801.2463 [M-176]+, produced by elimination of one molecule of ferulic acid (*m*/*z* 331[Mv]+) that is malvidin aglycone (Figure 7) [24].

**Table 2.** The molecular ionization characteristic of anthocyanin detected in AtPAP1 overexpression*S. nigrum*lines.

*Biomolecules* **2020**, *10*, 277

 Abbreviations: M, molecular; Dd, delphinidin; Pt, petunidin; Mv, malvidin. Glycone mass and tentative assignment: 331, malvidin; 317, petunidin; 303, delphinidin; 176, glucose/ ferulic acid;146,rhamnose/coumaricacid;611,3,5-diglucoside.

**Figure 7.** Mass spectrometric data of six anthocyanins detected in AtPAP1 overexpressed *S. nigrum* transgenic lines. (**A**) Delphinidin 3-O-rutinoside-5-O-glucoside, (**B**) Delphinidin-3,5-O-diglucoside, (**C**) Delphinidin-3-O-rutinoside, (**D**) Petunidin 3-O-(p-coumaroyl) rutinoside-5-O-glucoside, (**E**) Petunidin-3-(feruloyl)-rutinoside-5-glucoside, and (**F**) Malvidin-3-(feruloyl)-rutinoside-5-glucoside.

#### *3.5. Total Anthocyanin Contents induced by Overexpression of AtPAP1 in Black Nightshade*

In addition to six anthocyanins identified in the AtPAP1-overexpressed transgenic black nightshade plants, we measured the total anthocyanin content in the leaves of AtPAP1-overexpressed black nightshade transgenic plants using spectrophotometry. Compared with control and mock (GV3101) lines, both first and second generation plants of the AtPAP1-overexpression lines had higher levels of total anthocyanins (Figure 8). Our data collectively demonstrate that ectopic expression of a single regulatory gene, AtPAP1, can induce total anthocyanins in black nightshade plants, likely through regulation of the conserved biosynthesis pathway of anthocyanin in black nightshade plants.

**Figure 8.** Total anthocyanin contents in AtPAP1 overexpressed *S. nigrum* transgenic lines. Total anthocyanin was measured by spectrophotometry at A530 nm and A650 nm. All data are expressed as mean and SEM. All AtPAP1 overexpressed *S. nigrum* transgenic lines from first (T1) and second generation (T2) were compared with control and mock lines. The significance threshold was at a level of 5% (\*\*\* *P* < 0.001). All the experiments were biological triplicates.

#### **4. Discussion**

In the current study, we have demonstrated the accumulation of anthocyanins by overexpressing an AtPAP1 in the black nightshade plant. Expression analysis of the genes associated with the biosynthesis of general phenylpropanoid and flavonoid pathways indicates that not all the regulatory genes of anthocyanins may not be transcriptional targets of AtPAP1, and may require unidentified species-specific mechanisms. Nonetheless, a single *Arabidopsis* MYB transcription factor, AtPAP1, was sufficient to enhance the accumulation of anthocyanins in a heterologous system of black nightshade.

In *Arabidopsis*, it has been shown that the biosynthesis of anthocyanins and proanthocyanidins is predominantly regulated by the MBW transcriptional complex that consists of MYB, bHLH, and WD-40 [25]. Alternatively, an *Arabidopsis* MYB transcription factor, AtPAP1, has been shown to be sufficient to promote accumulation of anthocyanin in a heterologous plant system such as dandelion (*Taraxacum brevicorniculatum*) [26]. Novel MYB transcription factors from the American plum (*Prunus americana*) have been also demonstrated to be effective in the accumulation of anthocyanins in tobacco and citrus without requiring any further transcriptional co-regulators except for *PamMYBA3*, which appears to be dependent upon an interacting protein (bHLH) to form a functional transcriptional regulatory unit [27]. Moreover, overexpression of MYB transcription factor, EsMybA1, from *Herba epimedii* (*Epimedium sagittatum)* was sufficient to induce high levels of anthocyanins in the transgenic tobacco and *Arabidopsis*, via up-regulation of major flavonoid biosynthetic pathway genes [28]. These reports indicate that some MYB transcription factors physically associate with the co-regulators such as bHLH, while other MYB transcriptional factors appear to be sufficient to induce the accumulation of anthocyanins in diverse plant species [29].

From our study, ectopic expression of an *Arabidopsis* AtPAP1 alone was sufficient to induce anthocyanin production in black nightshade, resulting in the purple-colored phenotype in both vegetative and reproductive tissues. It is noteworthy that *Gerbera* MYB transcription factor GMYB10, an *Arabidopsis* ortholog of AtPAP1, activates some of EBGs of general phenylpropanoid pathway genes (*PAL, C4H, CHI,* and *F3H*) and accumulate anthocyanins [30]. It would be interesting to know if the genes identified as EBGs of black nightshade are also the transcriptional targets of AtPAP1, which confers the pigmentation of anthocyanins in the transgenic lines.

Although numerous studies about anthocyanins in berries (e.g., cherry, blueberry, blackcurrant) have been reported to date, studies of anthocyanins in black nightshade are still scarce. Nonetheless, a recent study by Wang et al. [20] has shown that eight anthocyanins with four anthocyanin aglycones such as cyanidin (Cy), malvidin (Mv), petunidin (Pt) and delphinidin (Dp) were detected in the peel and flesh of black nightshade fruit [20]. In our study, we have identified six anthocyanins that included three delphinids, two petunidin and one malvidin from leaf tissue of AtPAP1-overexpressed transgenic black nightshade plants. Among the anthocyanins identified in this study, delphinidin 3-O-rutinoside-5-O-glucoside, delphinidin-3,5-O-diglucoside, delphinidin-3-O-rutinoside, and malvidin-3-(feruloyl)-rutinoside-5-glucoside were also detected in the fruit of black nightshade [20]. In addition to the above four anthocyanins, two petunidin, petunidin-3-O-rutinoside (*cis*-*p*-coumaroyl)-5-O-glucoside and petunidin-3-(feruloyl)-rutinoside-5-glucoside, were identified only in our study. Thus the latter two appear to be leaf-specific anthocyanins that are regulated by AtPAP1 in black nightshade.

In addition to developmental regulation, biosynthesis of anthocyanins is affected by various abiotic factors (e.g., light, sucrose, nitrogen) and phytohormones [5]. Stress tolerance against drought, high salinity, light and cold are often correlated with anthocyanin accumulation in plants [31]. These various abiotic stress conditions are known to induce a distinct set of anthocyanins, indicating that anthocyanins have different biological functions, or that coloration patterns of each anthocyanin may play a unique role in response to a certain stress factor [32]. It would be interesting to examine whether enhancing anthocyanin production in a black nightshade would help mediate stress tolerance against stress conditions. Of interest in this regard was that anthocyanin pigmentation of transgenic black nightshade plants has disappeared when grown in the greenhouse (data not are shown), which suggests that biosynthesis of anthocyanins in black nightshade governed by AtPAP1 is under the control of light and likely temperature conditions. How diverse stress conditions that would affect the accumulation of anthocyanins in black nightshade remain to be investigated.

#### **5. Conclusions**

In this present study, we have shown that an *Arabidopsis* MYB transcription factor, AtPAP1, is sufficient to induce the accumulation of anthocyanins in black nightshade that may provide the platform for the basic analysis of phytochemical biosynthetic genes and their final compounds. Furthermore, a black nightshade can be a model system to study AtPAP1-induced anthocyanin in response to various stress conditions. Further identification of detailed mechanisms that are associated with an AtPAP1-mediated and environment-modulated accumulation of anthocyanins remains to be investigated. Taken together, our data lay a foundation to study the molecular mechanisms of biosynthesis of flavonoid and anthocyanin pathways to improve the phytochemical properties in this species.

**Author Contributions:** S.C. is the first author. J.J. is the contributing author. S.C. and S.U.P. conceived the study. Experimental data were generated and analyzed by S.C., J.J., J.K., S.U.P. All authors reviewed and wrote the manuscript. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was supported by the Bio & Medical Technology Development Program of the National Research Foundation (NRF) funded by the Ministry of Science, ICT & Future Planning (2016M3A9A5919548) (S.U.P.) and by the National Research Foundation of Korea (NRF) grant funded by the Korean government (Ministry of Science and ICT) (NRF-2017R1A2B4010356) (J.K.).

**Conflicts of Interest:** The authors declare no competing financial interest.

#### **References**

© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

*Article*

### **Asiatic Acid, Extracted from** *Centella asiatica* **and Induces Apoptosis Pathway through the Phosphorylation p38 Mitogen-Activated Protein Kinase in Cisplatin-Resistant Nasopharyngeal Carcinoma Cells**

**Yen-Tze Liu 1,2,3,4, Yi-Ching Chuang 4, Yu-Sheng Lo 4, Chia-Chieh Lin 4, Yi-Ting Hsi 4, Ming-Ju Hsieh 1,3,4,5,\* and Mu-Kuan Chen 6,\***

Received: 4 December 2019; Accepted: 23 January 2020; Published: 25 January 2020

**Abstract:** Nasopharyngeal carcinoma (NPC) is an important issue in Asia because of its unique geographical and ethnic distribution. Cisplatin-based regimens are commonly the first-line used chemotherapy, but resistance and toxicities remain a problem. Therefore, the use of anticancer agents derived from natural products may be a solution. Asiatic acid (AA), extracted from *Centella asiatica*, was found to have anticancer activity in various cancers. The aim of this study is to examine the cytotoxic effect and mediated mechanism of AA in cisplatin-resistant NPC cells. The results shows that AA significantly reduce the cell viability of cisplatin-resistant NPC cell lines (cis NPC-039 and cis NPC-BM) in dose and time dependent manners caused by apoptosis through the both intrinsic and extrinsic apoptotic pathways, including altered mitochondrial membrane potential, activated death receptors, increased Bax expression, and upregulated caspase 3, 8, and 9. The Western blot analysis of AA-treated cell lines reveals that the phosphorylation of MAPK pathway proteins is involved. Further, the results of adding inhibitors of these proteins indicates that the phosphorylation of p38 are the key mediators in AA-induced apoptosis in cisplatin-resistant human NPC cells. This is the first study to demonstrate the AA-induced apoptotic pathway through the phosphorylation p38 in human cisplatin-resistant nasopharyngeal carcinoma. AA is expected to be another therapeutic option for cisplatin-resistant NPC because of the promising anti-cancer effect and fewer toxic properties.

**Keywords:** Asiatic acid; nasopharyngeal cancer; apoptosis; cisplatin resistance; MAPK pathway

#### **1. Introduction**

Nasopharyngeal carcinoma (NPC) is an important and widely studied issue in Asia, especially in Eastern Asia and South-Eastern Asia, because of its unique geographical distribution. According to the GLOBOCAN estimates in 2018, the estimated number of new cases is 129,079 worldwide, of

which 109,221 are in Asia, accounting for 84.6% [1]. Etiological factors for NPC include Epstein–Barr virus (EBV) infection, human papillomavirus (HPV) infection, genetic susceptibility, and salted fish consumption [2]. Different from other head and neck cancer, the primary and only curative treatment for NPC is radiotherapy instead of surgery for early-stage disease. Chemotherapeutic agents are used as concurrent chemoradiotherapy (CCRT), adjuvant chemotherapy, or induction chemotherapy in locoregionally advanced disease and metastatic disease [3]. Cisplatin-based regimen is commonly the first-line used. Paiar el al. proposed that the overall survival (OS) and disease-free survival by CCRT had statistically significant improvement with five-year OS of about 70% in non-metastatic stage III and IV disease, but the efficacy of chemotherapy varied markedly among different trials [4]. Otherwise, chemoradiotherapy leads to various side effects including hematological acute toxicity, mucositis, xerostomia, loss of taste, dysphagia, skin damage, skull bone damage, sensorineural hearing loss, and osteoradionecrosis. Therefore, new effective treatments with fewer toxicities need to be identified.

Finding anti-cancer agents from natural compound is a burgeoning research filed recent years due to its fewer toxic characteristics. Asiatic acid (AA) is a pentacyclic triterpenoid, one of the subclasses of phytochemicals, and derived from *Centella asiatica* which is a kind of tropical plant used as a diet and medicine [5]. AA has been demonstrated to have antioxidant and anti-inflammatory activity, and to have been involved in numerous molecular mechanisms and cell signaling pathway which related to its cardio-protective, neuro-protective, anti-diabetic, anti-obesity, and anti-cancer effect [5]. Previous studies have reported many mechanisms observed in carcinogenesis and metastatic process. Hsu el al. discovered the anticancer effect of AA in breast cancer through cell cycle arrest and mitochondrial apoptotic pathway [6]. Another research proposed that AA inhibit transforming growth factor-beta1 (TGF-beta1)-induced epithelial-mesenchymal transition in lung cancer [7]. However, the effect of AA on nasopharyngeal cancer cells is still unclear.

As described above, radiotherapy and chemotherapy are effective in early-stage NPC. In contrast, the median overall survival in distant metastases is only about 12 to 15 months after platinum-based chemotherapy [8]. Multidrug combinations can improve the therapeutic effects, but accompanied with significant hematological and mucosal toxicities, even septic deaths [9]. Even patients who initially respond to cisplatin will recur, and it is often refractory to further platinum therapy [10]. Whether it is innate or acquired resistance, to discover effective and less-toxic agents for anti-cancer or chemosensitizers is important. This study aims to examine the cytotoxic effect and mechanisms of AA on cisplatin-resistance NPC cells.

#### **2. Materials and Methods**

#### *2.1. Chemicals*

Asiatic acid is a triterpenoid isolated from *Centella asiatica* (≥98% purity), purchased from Chemfaces. MolecularWeight: 488.7, Formula: C30H48O5, dissolved in dimethyl sulfoxide (DMSO), and stored at −20 ◦C. All experiment treatments were consistently less than 0.1% (*v*/*v*) DMSO concentration. Other chemicals were obtained from the following companies: MTT (3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyltetrazolium bromide) and DAPI dye (Sigma Aldrich, St Louis, MO, USA), cisplatin (Enzo Life Sciences), specific inhibitors (Santa Cruz Biotechnology, Santa Cruz, CA, USA).

#### *2.2. Cell Culture*

The cisplatin-resistant nasopharyngeal carcinoma cell lines (cis NPC-BM and cis NPC-039) were established by human nasopharyngeal carcinoma (NPC) cell lines (NPC-BM and NPC-039), a gift from Dr. Jen-Tsun Lin, Hematology & Oncology, Changhua Christian Hospital, continuous culture with increase concentrations of cisplatin 1–60 nM for 6 months. The culture medium contains 60 nM cisplatin to maintain drug resistance. All cells were cultured in RPMI 1640 medium (with 10% (*v*/*v*) fetal bovine serum (FBS), 1 mM glutamine, 1% (*v*/*v*) penicillin/streptomycin, 1.5 g/l sodium bicarbonate, and 1 mM sodium pyruvate). All cells were cultured at 37 ◦C in a 5% CO2 culture room.

#### *2.3. Cell Cytotoxicity Assay*

The effect of Asiatic Acid on cell growth was assayed by the MTT (3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyl tetrazolium bromide) method. Briefly, cells were cultured in 96-well plates (1 × 104/well) and treated with various concentrations of AA (0, 25, 50, 75 μM). After the medium was removed, MTT reagent was added to each well (0.5 mg/mL final concentration) with a further incubation for 4 h at 37 ◦C in 5% (*v*/*v*) CO2. After removing the supernatant, DMSO was added to dissolve the formed blue formazan crystals. The absorbance of the converted dye was measured at 595 nm by the ELISA plate reader. Each condition was performed in triplicate and data were obtained from at least 3 separate experiments.

#### *2.4. Cell Cycle Analysis*

Cells were seeded (5 × 105/well) on 6-well plates overnight and incubated with various concentrations of AA for 24 h. After wards, cells were fixed 70% (*v*/*v*) ice-cold ethanol overnight, and then stained with PI buffer (4 mg/mL PI, 1% (*v*/*v*) Triton X-100, 0.5 mg/mL RNase A in PBS) for 30 min in the dark at room temperature and then filtered through a 40-mm nylon filter (Falcon, Billerica, MA, USA). The cell cycle distribution was analyzed by flow cytometry.

#### *2.5. DAPI Staining*

Observation of morphology by DAPI staining cis NPC-BM and cis NPC-039 cells treated with different concentrations of AA (0, 25, 50, and 75 μM) were incubated for 24 h. The cells were fixed with 4% (*v*/*v*) formaldehyde for 30 min at room temperature and permeabilized with 0.1% (*v*/*v*) Triton X-100. Washed twice in PBS, cells were stained with DAPI (50 μg/mL) for 15 min in the dark, then after being washed in PBS, morphological changes were photographed using fluorescence microscopy (Lecia, Bensheim, Germany). Quantitatively, the percentage of apoptotic cells by number of dots was scored on at least 500 cells.

#### *2.6. Annexin V*/*PI Double Staining Assay*

Cells were seeded on 6-well overnight and then treated with different concentrations of AA for 24 h. Cells were harvested and suspended in PBS (2% (*v*/*v*) BSA), and then incubated with Muse™ Annexin V & Dead Cell reagent (EMD Millipore, Billerica, MA, USA) for 20 min at room temperature in dark. Samples were analyzed by Muse Cell Analyzer flow cytometry (EMD Millipore, Billerica, MA, USA) and by MUSE 1.4 Analysis software (EMD Millipore).

#### *2.7. Mitochondrial Membrane Potential Measurement*

Cells were seeded on 6-well plates and incubated with different dose of AA for 24 h. The cells were harvested, washed and suspended in Muse MitoPotential working solution at 37 ◦C for 20 min. Add 5 μL of 7-AAD and incubate at room temperature for 5 min. Samples were monitored by a Muse Cell Analyzer flow cytometry (EMD Millipore) The data were analyzed by a Muse Cell Analyzer (Millipore).

#### *2.8. Western Blotting Analysis*

The cells are washed twice with cold PBS and lysed with RIPA buffer containing protease inhibitor cocktail and phosphatase inhibitor cocktail. The protein quantification of supernatants was determined using BCA protein assay (Pierce) and all samples are separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and the separated proteins were transferred electrophoretic ally from the gel to the surface of PVDF membrane (Millipore, Bedford, MA, USA). Membranes were blocked with 5% non-fat milk in TBST for 1 h. The analysis used primary antibodies as described by the manufacturers of the antibodies. Antibodies were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). Anti-ERK 1/2 (#4695), Anti-JNK 1/2 (#9252), Anti-p38 (#8690), Anti-Akt (#4685), Anti-phospho-ERK 1/2 (#4370), Anti-phospho-JNK 1/2 (#4668), Anti-phospho-p38

(#4511), Anti-phospho-Akt (#4060), Anti-cleaved caspase 3 (#9664), Anti-cleaved caspase 8(#9496), Anti-cleaved caspase 9 (#9505), Anti-cleaved PARP(#5625), Anti-Bax (#2772), Anti-Bak (#3814), Anti-Fas (#4233), Anti-TNF-R1(#3736), Anti-DCR2 (#8049), Anti-DR5 (#8074) or isotype-control rabbit mAbs (Cell Signaling Technology, Beverly, MA). After the final rinsing with TBST, the membrane was incubated with secondary HRP-linked anti-rabbit IgG or anti-mouse IgG for 1 h. After washing, the immunoblots were visualized using chemiluminescence (ECL) HRP substrate (Millipore).

#### *2.9. Statistical Analysis*

Values represent the means ± standard deviation and the experiments were repeated at least three times. Data were analyzed using Student's t-test when two groups were compared. A *p* value of <0.05 was considered statistically significant. Statistical analysis using one-way analysis of variance (ANOVA). Analysis of more than three groups using Dunnett's test.

#### **3. Results**

#### *3.1. Cytotoxicity of Asiatic acid on Cisplatin-Resistance Human NPC Cell Lines*

Figure 1a reveals the chemical structure of Asiatic acid, a pentacyclic triterpene, titrated extract from *Centella asiatica*. We assessed the cell viability of two kinds of cisplatin-resistant human NPC cell lines, cis NPC-039 and cis NPC-BM, by MTT assay. These cell lines were treated with an increasing concentration of AA (0, 25, 50, and 75 μM) with or without cisplatin for 24, 48, and 72 h. The results in Figure 1b,c show that the cell viability of both cis NPC-039 and cis NPC-BM was inhibited in a dose- and time-dependent manner regardless of whether with or without cisplatin. Cell viability was significantly reduced in concentration of 50 and 75 μM compared to control (0 μM).

**Figure 1.** Cytotoxic effects of Asiatic acid on cisplatin-resistant human NPC cell lines (NPC-039 and NPC-BM) (**a**) The chemical structure of Asiatic acid. (**b**) The cell viability significantly decreased in AA-treated cis NPC-BM cell and cis NPC-039 cell with cisplatin and (**c**) without cisplatin in a dose and time dependent manner under MTT assay. \* *p* < 0.05 as compared with control group.

#### *3.2. Asiatic Acid Induced Cell Apoptosis and Cell Cycle Arrest in Cisplatin-Resistance Human NPC Cell Lines*

PI staining and flow cytometry were applied to determine the cell cycle distribution of cisplatinresistance human NPC cell lines, cis NPC-039 and cis NPC-BM, and to examine the effect of AA on cell growth suppression. Figure 2 shows that the proportion in the sub-G1 phase increased in a concentration-dependent manner, especially in 75 μM group. Figure 3a illustrates the more condensed nuclei, suggesting the typical characteristic morphology of DNA fragmentation in apoptotic cells, in the higher concentration group of AA-treated NPC cell lines with the fluorescence microscopy after DAPI staining. The difference was statistically significant in 50 and 75 μM groups of both two cell lines (Figure 3b). Furthermore, we performed the Annexin-V and PI double staining and followed by flow cytometric analysis. The results in Figure 3c,d indicated that AA increased apoptotic cells in early stage and late stage of apoptosis, and significantly in 75 μM -treated cell lines.

**Figure 2.** Asiatic acid cause cell cycle arrest in cis NPC-039 and cis NPC-BM cell lines. (**a**) The cisplatinresistant human NPC cell lines were treated with Asiatic acid (0, 25, 50, and 75 μM). Cell cycle phase distribution (Sub-G1, G0/G1, S, and G2/M) were estimated by flow cytometry. (**b**) The rate of different phase of cell cycle in cis NPC-039 and cis NPC-BM cell lines. \* *p* < 0.05 as compared with control group.

**Figure 3.** Effect of Asiatic acid on apoptosis induction in cis NPC-039 and cis NPC-BM cell lines. (**a**) Nuclear counterstain level was detected by DAPI staining. Characteristic 'blebbing' morphology of cell nucleus was observed by fluorescence microscopy. (**b**) The DNA condensation fold compared with control. (**c**) and (**d**) Cell apoptosis was analyzed by flow cytometric with Annexin-V/PI Staining. \* *p* < 0.05 as compared with control group.

#### *3.3. Asiatic Acid Induced Cell Apoptosis in Cisplatin-Resistance Human NPC Cell Lines through the Mitochondrial Pathway and Death Receptor Related Pathway*

The pathway involved in the AA-induced apoptosis in cis NPC-039 and cis NPC-BM cell lines have to be verified. We performed the Muse MitoPotential Kit and Muse Cell Analyzer assays. The results in Figure 4a,b display that the mitochondrial membrane potential was changed and the depolarized cells was significantly increased with AA at 75 μM. On the other hand, the expressions of Fas, DCR2, and DR5 were all increased in AA-treated cisplatin-resistant NPC cell lines by Western blot analysis (Figure 4c,d).

**Figure 4.** Asiatic acid cause cis NPC-039 and cis NPC-BM cell apoptosis thought the intrinsic pathway and the extrinsic pathway. (**a**) Effect of Asiatic acid on mitochondrial membrane potential was analyzed by flow cytometric (**b**) The related mitochondrial membrane depolarized levels compared with control. (**c**) The expression levels of Fas, TNF-R1, DcR2, and DR5 were detected by Western blot. (**d**) The relative band density was normalized to GAPDH as a densitometer. \* *p* < 0.05 versus control group.

#### *3.4. Asiatic Acid Upregulates the Expression of Caspase-3, -8, -9, and Altered the Expression of Bax and Bak in Cisplatin-Resistance Human NPC-039 and NPC-BM Cell Lines*

We performed Western blot analysis of the cleaved forms of caspase-3, caspase-8, caspase-9, and Poly (ADP-ribose) polymerase (PARP) in AA-treated NPC cell lines to determine the caspase activation pathway in AA-induced apoptosis. As observed in Figure 5a,b, the expressions of caspase-3, -8, -9, and PARP increase significantly in cis NPC-039 cell lines with AA at 50 μM compared to control group. In cis NPC-BM cell clines, significantly increased expression of caspase-3, -8, and -9 was found in AA treatment of 75 μM for 24 h. In Figure 5c,d, the related expression level of Bax and Bak, two pro-apoptotic proteins, in cis NPC-BM cell line were significantly increased by 183% and 137% respectively. In another cis NPC-039 cell line, 24-h 50 μM and 75 μM AA treatment increased Bax expression, but decreased Bak expression.

**Figure 5.** Asiatic acid activates caspase 3, 8, and 9 and upregulated the expression of Bax and Bak. (**a**) The expression levels of cleaved caspase-3, -8, -9 and PARP were detected by Western blot. (**b**) The protein levels were quantified and normalized to GAPDH using a densitometer. (**c**) The expression levels of Bak and Bak were detected by Western blot. (**d**) The protein levels were quantified and normalized to GAPDH using a densitometer. \* *p* < 0.05 versus control group.

#### *3.5. Asiatic Acid Induced Cell Apoptosis via p38 Pathway in Cisplatin-Resistance Human NPC-039 and NPC-BM Cell Lines*

We applied the Western blot analysis to obtain the level of expression of ERK1/2, AKT, p38, and JNK1/2 to confirm the mechanisms related to AA-mediated apoptosis. The results in Figure 6a,b display the significantly increased expressions of phosphorylation of p38 and JNK1/2 with dose-dependent relationship in cis NPC-039 cell lines, whereas decreased the phosphorylation of AKT in 50- and 75 μM -treated groups and ERK1/2 in 50 μM-treated group. In cis BPC-BM cell lines, AA treatment of 50 and 75 μM for 24 h significantly increased the activation of phosphor-p38 by 298% and 348% respectively and significantly decreased the expression of phosphor-ERK1/2. Decreased expression of phosphor-AKT and increased of phosphor-JNK1/2 were found in 75 μM-treated cell group. To further examine the signal transduction pathways involved in apoptosis, we treated cis NPC-039 and cis NPC-BM cell lines with AA of 50 μM plus ERK1/2 inhibitor (U), AKT inhibitor (LY), p38 inhibitor (SB), and JNK1/2 inhibitor (SP). The related expression level of cleaved caspase 9 was increased when adding ERK1/2 inhibitor (U) and JNK1/2 inhibitor (SP) and decreased when adding AKT inhibitor (LY) and p38 inhibitor (SB) in both cell lines compared to AA-treated group. The expression of cleaved PARP was increased when adding JNK1/2 inhibitor (SP) and decreased when adding AKT inhibitor (LY), and p38 inhibitor (SB) in cis NPC-039. However, the expression of cleaved PARP was increased when adding ERK1/2 inhibitor (U) and decreased when adding AKT inhibitor (LY), p38 inhibitor (SB), and JNK1/2 inhibitor (SP) in cis NPC-BM. Therefore, we might propose that the phosphorylation of p38 plays a considerable role in the pathway of AA-related apoptosis.

#### **4. Discussion**

Chemotherapy is one of the mainstay options for the treatment against nasopharyngeal carcinoma, especially in the locoregionally advanced or metastatic stage. Cisplatin is most commonly used, but the therapeutic outcome may be restricted because of its toxicity and resistance. This research attempted to discover the promising natural compound against cisplatin-resistance NPC cells. Our results indicate that the Asiatic acid may induce apoptosis in cisplatin-resistance NPC-039 and NPC-BM cell lines via the p38 and ERK signaling transduction pathway.

It has been proposed that Asiatic acid has various pharmacological activities such as anti-inflammatory and antioxidant, as well as the potent anti-hypertensive, neuro and cardio-protective, antimicrobial, and antitumor activities [5].  Our study corresponds with these researches and revealed the anti-cancer effect of AA in both dose and time dependent manners in cisplatin-resistance NPC cell lines (Figure 1b). AA induced cell death by cell cycle arrest and apoptosis in this study (Figures 2 and 3). Previous study also revealed that AA induced cells undergo S-G2/M phase arrest and apoptosis in human breast cancer MCF-7 and MDA-MB-231 cell lines [6]. Siddique et al. suggested that AA induced apoptosis through decreasing the Bcl-2 and increasing the Bax, caspase-3 and -9 in a rat model of colon carcinogenesis [11].

Furthermore, we investigated the intrinsic and extrinsic pathway in apoptosis induced by AA. Figure 4 shows that both the mitochondrial pathway and the death receptor-initiated pathway are involved in AA-induced apoptosis, as well as the upregulated Bax and Bak in Figure 5. The research in 2017 revealed that AA-induced apoptosis in lung cancer was caused by altering the mitochondrial membrane potential and lead to activation of caspase and PARP signaling pathways [14]. Related researches mention that AA cause activation of mitochondrial pathway via regulating B cell CLL/lymphoma 2 (BCL2) family proteins and increasing the ratio of Bax to Bcl2 which lead to cancer cell apoptosis [19,20].

**Figure 6.** Effect of Asiatic acid on the MAPKs and AKT pathway in cis NPC-039 and cis NPC-BM cell lines (**a**) The expression levels of ERK1/2, AKT, p38, and JNK1/2 were detected by Western blot. (**b**) The protein levels were quantified and normalized to GAPDH using a densitometer. \* *p* < 0.05 versus control group. (**c**) After pretreated with ERK1/2, AKT, p38, and JNK1/2 inhibitors separately (U, LY, SB, SP), the expression levels of cleaved caspase-9 and PARP were detected by Western blot. (**d**) The protein levels were quantified and normalized to GAPDH using a densitometer. \* *p* < 0.05 versus control group. # *p* < 0.05, compared with Asiatic acid treated only.

Previous studies revealed that the AA-induced apoptosis of human cancer cell is mediated by the mitogen-activated protein kinase (MAPK) pathway with or without involving the activated caspases [5,6,21]. The inhibition of ERK and p38 phosphorylation in AA-treated cancer cell are also noted [15]. Our results in Figures 5 and 6 also indicate that caspase-3, -8, and -9 are upregulated in AA-induced apoptosis and that caspase-9 expression is mediated by the phosphorylation of p38 and ERK1/2 pathway in human NPC cell lines. Other research has indicated the anti-cancer activity of AA by Akt pathway [17,22], but our results didn't show it.

Based on previous research, the resistance of cisplatin comes from three mechanisms: altered DNA repair, cytosolic inactivation of drug, and altered cellular accumulation of drug, and the increased expression of excision repair cross complementation 1 (ERCC1) to alter DNA repair in NPC was related to cisplatin resistance [10]. Liu et al. proposed that the resistance might be sensitized by increasing Bax/Bcl-2 mRNA ratios caused by inhibited NFκB expression and downregulating ERCC1 and thymidine phosphorylase (TP) by inhibiting the phosphorylation of ERK, p38 and PI3K/AKT pathways, which all lead to apoptosis, in human hepatoma HepG2 cells [23]. Another research also revealed the decreased ERCC1 mRNA expression by downregulation of p38 MAPK pathway [21]. Consistent with these studies, our results revealed significantly increased expression of Bax and decreased phosphorylation of ERK in AA-treated cis-NPC cell lines. However, the phosphorylation of p38 was upregulated in our study. Hsieh et al. also proposed that another plant-derived triterpene, Celastrol, also induces cancer cell apoptosis of cisplatin-resistant nasopharyngeal carcinoma though increasing p38 MAPK signaling pathway, as well as ERK1/2 [24]. These inconsistent results of the regulatory signaling pathway and detailed mechanisms require further clarification.

#### **5. Conclusions**

In conclusion, this is the first study to demonstrate the AA-induced apoptotic pathway in human cisplatin-resistant nasopharyngeal carcinoma. The mechanism of both intrinsic and extrinsic pathways, including the phosphorylation p38 and Bax upregulation, is involved. Therefore, AA is expected to be another therapeutic option for cisplatin-resistant NPC because of the promising anti-cancer effect and fewer toxic properties.

#### **Data Availability Statement**

All the data that supports the findings of this study are available in this article.

**Author Contributions:** Conceptualization, M.-J.H. and M.-K.C.; methodology, Y.-C.C.; Y.-S.L.; C.-C.L. and Y.-T.H.; software, Y.-T.L.; writing—original draft preparation, Y.-T.L.; writing—review and editing, M.-J.H. and M.-K.C. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by grants from National Science Council, Taiwan (MOST 106-2314-B-371- 006-MY3) (MOST 106-2314-B-371-005-MY3) and Changhua Christian Hospital (108-CCH-IRP-041, 108-CCH-IRP-002, 108-CCH-ICO-145).

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **Abbreviations**

Nasopharyngeal carcinoma (NPC); Asiatic acid AA); Extracellular signal–regulated kinases (ERKs); c-Jun N-terminal kinases (JNKs); Mitogen-activated protein kinases (MAPK); Propidium iodide (PI)

#### **References**

© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

### *Review* **Can Medicinal Plants and Bioactive Compounds Combat Lipid Peroxidation Product 4-HNE-Induced Deleterious E**ff**ects?**

#### **Fei-Xuan Wang 1,\*, Hong-Yan Li 2, Yun-Qian Li 2 and Ling-Dong Kong 3,\***

Received: 1 December 2019; Accepted: 14 January 2020; Published: 16 January 2020

**Abstract:** The toxic reactive aldehyde 4-hydroxynonenal (4-HNE) belongs to the advanced lipid peroxidation end products. Accumulation of 4-HNE and formation of 4-HNE adducts induced by redox imbalance participate in several cytotoxic processes, which contribute to the pathogenesis and progression of oxidative stress-related human disorders. Medicinal plants and bioactive natural compounds are suggested to be attractive sources of potential agents to mitigate oxidative stress, but little is known about the therapeutic potentials especially on combating 4-HNE-induced deleterious effects. Of note, some investigations clarify the attenuation of medicinal plants and bioactive compounds on 4-HNE-induced disturbances, but strong evidence is needed that these plants and compounds serve as potent agents in the prevention and treatment of disorders driven by 4-HNE. Therefore, this review highlights the pharmacological basis of these medicinal plants and bioactive compounds to combat 4-HNE-induced deleterious effects in oxidative stress-related disorders, such as neurotoxicity and neurological disorder, eye damage, cardiovascular injury, liver injury, and energy metabolism disorder. In addition, this review briefly discusses with special attention to the strategies for developing potential therapies by future applications of these medicinal plants and bioactive compounds, which will help biological and pharmacological scientists to explore the new vistas of medicinal plants in combating 4-HNE-induced deleterious effects.

**Keywords:** medicinal plants; bioactive compounds; herbs; biological activity; 4-hydroxynonenal; deleterious effects

#### **1. Introduction**

The toxic reactive aldehyde 4-hydroxynonenal (4-HNE) belongs to the advanced lipid peroxidation end products [1]. As the second toxic messenger of free radicals, 4-HNE modifies specific amino acid residues on proteins to form highly cytotoxic reactive 4-HNE adducts via Michael addition or Schiff base reaction or reacts with DNA via epoxidation under oxidative stress conditions. Oxidative stress is one of the main pathogeneses for human disorders [2]. Accumulation of 4-HNE and formation of 4-HNE adducts are induced by mainly reactive oxygen species (ROS) and other free radicals; participate in several cytotoxic processes such as protein dysfunction, apoptosis, and inflammatory injury; and produce deleterious effects [2–8]. As a result, 4-HNE serves as a biomarker for both lipid peroxidation and oxidative stress [1,2]. Therefore, the inhibition, neutralization, and detoxification of

4-HNE and 4-HNE adducts has become one of the directions of developing potential therapies for oxidative stress-related disorders.

Medicinal plants have been widely used to prevent and treat various kinds of disorders for thousands of years. Their extracts and bioactive compounds have the ability to prevent the cells from oxidative stress injury [9–11]. Some of them have been suggested to be attractive sources of potential agents to mitigate oxidative stress and attenuate 4-HNE-induced toxicity. However, researchers pay little attention to reviewing these medicinal plants and natural bioactive compounds on combating 4-HNE-induced deleterious effects in neurotoxicity and neurological disorder, eye damage, cardiovascular injury, liver injury, and energy metabolism disorder [12–16].

Therefore, this review focuses on the therapeutic effects of medicinal plants in treating 4-HNE-related disorders under oxidative stress. An open-ended, English-restricted search of PubMed/MEDLINE database and Web of Science database has been conducted (up to 31 December 2019) using terms such as 4-HNE-induced or driven/induced by 4-HNE, and medicinal plant/herb/phytochemical/natural compounds compound/constituent. This review aims to highlight the pharmacological basis of medicinal plants and bioactive compounds in the attenuation of 4-HNE-induced deleterious effects, as well as the underlying molecular mechanisms. Additionally, this review briefly discusses the strategies for developing potential therapies in future applications of these medicinal plants and compounds, which will benefit biological and pharmacological scientists to explore the new vistas of medicinal plants in combating 4-HNE-induced deleterious effects.

#### **2. Neuroprotection**

Neurotoxicity exacerbated by oxidative stress is strongly associated with 4-HNE toxicity; its further deterioration cleaves nuclear enzyme poly (ADP-ribose) polymerase (PARP) to affect DNA repair and apoptosis in neurons, leading to neurological disorders [1,17]. Polyphenol extract from red wine offers health benefits and can help to avoid neurodegenerative diseases [18]. This extract inhibits 4-HNE-induced cleavage of PARP and protects against apoptosis in neuronal-like catecholaminergic cells (rat pheochromocytoma, PC12 cells) by reducing intracellular ROS [19]. Polyphenolic flavonoids quercetin and myricetin isolated from red wine extract or *Ginkgo biloba* extract have cytoprotective effects on 4-HNE-induced PC12 cell death [19], further demonstrating the benefits in neurodegeneration [20,21]. Other medicinal plant flavonoids luteolin and apigenin have antioxidant, anti-inflammatory, and neuroprotective effects. These two flavonoid compounds also attenuate 4-HNE-induced PARP-1 and caspase-3 activation as well as cell viability in PC12 cells [22].

The accumulation of 4-HNE up-regulates mitogen-activated protein kinase (MAPK) superfamily, especially C-Jun-N-terminal kinase (JNK), which plays a crucial role in oxidative stress-mediated cellular apoptosis [23,24]. Piceatannol, a bioactive stilbene derivative from medicinal plants *Passiflora edulis* or *Gnetum parvifolium*, restores 4-HNE-induced PARP cleavage and apoptosis regulator Bcl-2 expression, and down-regulates the phosphorylation of JNK (p-JNK) in PC12 cell death and nuclear condensation [25]. Recently, maternal supplementation with piceatannol has been demonstrated to have neuroprotection against neonate brain damage and reverse the sensorimotor deficit and cognitive impairment in rats [26]. Additionally, as a traditional medicinal plant in China and Korea, citri reticulatae viride pericarpium also restores 4-HNE-induced inflammatory injury in PC12 cells via inhibiting the activation of p-JNK and nuclear factor kappa-B (NF-κB) [27]. Mitogen-activated protein kinase kinase 4 (MKK4) as an upstream activator of JNK plays a critical role in response to cellular oxidative stress. Cocoa procyanidin fraction and its major antioxidant compound procyanidin B2 are reported to attenuate 4-HNE-induced nuclear condensation and increase sub-G1 fraction (markers of apoptotic cell death) as well as intracellular ROS accumulation in PC12 cells. This fraction and procyanidin B2 also protect against 4-HNE-induced PC12 cell apoptosis by blocking MKK4 activity [28]. Thus, the modulation of JNK and its upstream activator by medicinal plants and bioactive compounds may have potential therapeutic indications for neurodegenerative diseases.

Nuclear factor (erythroid-derived 2)-like-2 factor (Nrf2) is a ubiquitous master transcription factor that binds to the antioxidant response elements (ARE) and then enhances the expression levels of antioxidant enzymes and cytoprotective genes within DNA in response to oxidative stress [29]. Trans-resveratrol, a natural stilbene constituent isolated from medicinal plant grapevinele, is known to activate Nrf2 signaling pathway. Trans-resveratrol counters the cytotoxic response of 4-HNE consistently and reduces ROS generation and lipid peroxidation in PC-12 cells, showing its improvement of antioxidant defense system. It also restores 4-HNE-induced protein expression changes of mitochondria-mediated apoptosis markers (caspase-3, Bax, and B-cell lymphoma-2 (Bcl-2)) as well as oxidative damage, resulting in the alleviation of apoptotic neurodegeneration in PC12 cells [30]. More importantly, sulforaphane (an isothiocyanate mainly derived from crucifers such as broccoli, cabbages and olives) and carnosic acid (a plastidial catecholic diterpene mainly from *Rosmarinus o*ffi*cinalis*) are two phytochemicals Nrf2/ARE activators, which are found to attenuate 4-HNE-induced inhibition of mitochondrial respiration for complex I; the latter has been found to protect complex II in the isolated cortical mitochondria in young adult male CF-1 mice [14]. Furthermore, sulforaphane and carnosic acid decrease the amount of 4-HNE bound to mitochondrial proteins [14]. Microtubule-associated protein 1 light chain 3 alpha (LC3) is a marker of autophagosome, which is required for clearing dead cells. Study has shown that polyphenolic flavonoids luteolin and apigenin mitigate LC3 conversion and ROS production, activate Nrf2 signaling, and inhibit cytotoxicity in 4-HNE-exposed PC12 cells [22]. Therefore, the above phytochemicals have the capability of Nrf2 signaling induction to prevent the mitotoxicity of 4-HNE and provide neuroprotection.

Hyperactivity of NADPH oxidases (NOX) induced by 4-HNE has a crucial role in ROS overproduction and apoptosis; thus, NOX-derived ROS is a central mechanism in the development of neurodegeneration and neuroinflammation [31]. Kaempferol, a natural flavonoid presented in many medicinal plants, has been proved as a potential neuroprotective agent. It suppresses 4-HNE-mediated p-JNK and apoptosis in PC12 cells. Interestingly, kaempferol directly binds p47(phox), a cytosolic subunit of NOX, and effectively blocks 4-HNE-induced NOX activation in PC12 cells, exerting its potential neuroprotection against NOX-mediated neurodegeneration [13]. Scutellarin, a bioactive flavone isolated from *Scutellaria baicalensis*, has anti-inflammatory, anti-oxidative, anti-apoptotic, and anti-neurotoxic properties. More recently, a molecular docking study showed that scutellarin selectively binds to NOX2 with high affinity. Substantially, scutellarin down-regulates NOX2 expression and reduces 4-HNE and ROS levels in astrocytes subjected to ischemia/reperfusion and in rats with focal cerebral ischemia [32]. These results indicate that scutellarin is a neuroprotective flavone against ischemic injury.

Alzheimer's disease (AD) is a progressive and irreversible neurodegenerative disorder. High levels of 4-HNE are detected in the AD brain with the induction of neuronal apoptosis and oxidative stress, indicating that 4-HNE may play a role in AD subjects and/or animal models of age-related neurodegenerative disorders [33,34]. Acetylcholine esterase (AChE), cyclooxygenase-2 (COX-2), and matrix metalloproteinase-8 (MMP-8) are important target proteins implicated in AD pathogenesis. Phytocompounds albiziasaponin-A, iso-orientin, and salvadorin have antioxidant and anticholinesterase activities. The results from molecular docking studies show that there are strong interactions of albiziasaponin-A, iso-orientin, and salvadorin with AChE, COX2, and MMP8, respectively. Hence, these three bioactive compounds reduce serum 4-HNE levels in colchicine-induced AD model of rats, suggesting that albiziasaponin-A, iso-orientin, and salvadorin may be potential neuroprotective agents [35].

Glutathione *S*-transferase (GST) is a critical enzyme that participates in the detoxification of 4-HNE and has potential to prevent degenerative cellular processes [36]. Naringenin, a flavonoid found in grapefruit juice, is reported to improve learning and memory in a rat model of AD [37]. In an intracerebroventricular-streptozotocin-induced AD-type model of rats with cognitive impairment, naringenin increases GST, glutathione peroxidase (GSH-Px), glutathione reductase (GR), superoxide dismutase (SOD), and catalase (CAT) to detoxify 4-HNE in the hippocampus [38]. Collapsin response

mediator protein-2 (CRMP-2) is hyperphosphorylated in AD. More recently, using computational tools with structural and dynamic analyses, naringenin and naringenin-7-O-glucuronide have been found to selectively bind CRMP-2 and then reduce p-CRMP-2, being the therapeutic activity of novel CRMP-2 inhibitor [39,40].

In senescence-accelerated mouse prone 8 (SAMP8) model of mice with age-related cognitive decline and AD, rosemary extract as well as spearmint extract (containing carnosic acid and rosmarinic acid) are reported to reduce 4-HNE levels in brain cortex and protein carbonyls in brain hippocampus, resulting in the improvement of learning and memory [41]. More importantly, rosmarinic acid increases antioxidant enzyme activities (such as SOD, CAT, and GSH-Px) and glutathione levels and then reduces 4-HNE in amyloid beta 42-induced echoic memory decline in rats with oxidative stress and cholinergic impairment, possibly contributing to the improvement of neural network dynamics of auditory processes [42]. Dark tea prevents and treats age-related degenerative diseases. l-theanine, 3,3 -azanediylbis (4-hydroxybenzoic acid) and one of 8-C N-ethyl-2-pyrrolidinone substituted flavan-3-ols were recently isolated for in silico characterization of microbial metabolites extracted from dark tea. Oral administration of these three compounds indeed inhibits the formation of 4-HNE and ubiquitinated protein aggregates, regulates Aβ metabolic pathway, increases endogenous antioxidant capacity, and reduces neuronal apoptosis rate in SAMP8 mice, showing the protection of SAMP8 neurons [43].

Parkinson's disease (PD) is an age-related neurodegenerative disorder that severely affects quality of life. Chrysin, a flavone found in *Passiflora caerulea*, *P. incarnata*, and *Oroxylum indicum*, improves antioxidant defense system to protect against oxidative stress by increasing GSH to reduce 4-HNE. Consistently, chrysin improves behavioral and cognitive ability in a 6-hydroxydopamine-induced mouse model of PD [44]. Quercetin also markedly improves the motor balance and coordination in 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine-induced PD of mice. Moreover, quercetin significantly decreases 4-HNE immunoreactivity in striatum of mouse brains and increases GPx, SOD, AchE, and dopamine contents, showing the antiparkinsonian property [45].

#### **3. Prevention of Eye Damage**

The main visual ocular structures are the cornea and conjunctiva, lens, vitreum, and retina. Any structure dysfunction can cause visual problems and even blindness. Overproduction of ROS leads to the increased level of 4-HNE and induces lipid oxidative damage and inflammation in the cornea and conjunctiva [46,47], whereas 4-HNE accumulation causes the death of lens epithelia cells to induce lens opacification and cataract [48] as well as retinal pigment epithelium (RPE) cell death [49,50]. In the ocular surface of mouse model with dry eye disease, antioxidant extracts from *Schizonepeta tenuifolia*, *Angelica dahurica*, *Rehmannia glutinosa*, and *Cassia tora* reduce the number of 4-HNE-positive cells to protect from lipid peroxidation-related membrane damage. These antioxidant extracts also inhibit extracellular ROS production and decrease IL-1β, IL-6, tumor necrosis factor-α (TNF-α), and interferon-γ (IFN-γ) levels of the ocular surface, resulting in the improvement of clinical signs [51].

Aldose reductase (AR, an NADPH-dependent oxidoreductase) hyperactivity in the lens plays an important role in the pathogenesis of oxidative stress-mediated diabetic cataract [52]. Quercetin possesses therapeutic effect in the management and treatment of diabetic cataract. Quercetin and its glycoside derivative rutin, or *G. biloba* extract, remarkably inhibit AR activity, stimulate GSH production, and decrease the levels of 4-HNE, lipid peroxidation malondialdehyde (MDA), and advanced glycation end-products in the lenses of streptozotocin-induced diabetic cataract rats, delaying the progression of lens opacification [53]. Curcumin, a diketone constituent extracted from *Curcuma longa*, is a wonderful natural antioxidant and anti-cataract agent. This bioactive compound enhances the formation of GST isoenzymes to detoxify 4-HNE by aiding in the conjugation of 4-HNE to glutathione, showing its prevention against cataract [48].

Proliferative diabetic retinopathy, a leading cause of blindness, is also promoted by 4-HNE. Berberine, an isoquinoline alkaloid from a medicinal plant rhizoma of *Coptidis chinesis*, has favorable

effects on glucose and lipid metabolism in animal experimental and human clinical studies [54]. In confluent human retinal Müller cells exposed to 4-HNE, cell death is partially attenuated by berberine pretreatment. Berberine also restores 4-HNE-induced autophagy in this cell model, showing its potential to prevent diabetic retinopathy [55].

In fact, 4-HNE can destabilize actin and induce cytoskeletal damage, resulting in retinopathy and age-related macular degeneration [56]. Cyanidin-3-glucoside is a major anthocyanin in mulberry or *Lonicera caerulea,* and its supplement has potential to prevent eye diseases. In human adult retinal pigmented epithelial (ARPE-19) cells, cyanidin-3-glucoside protects against 4-HNE-induced cell apoptosis, inflammatory damage, and angiogenesis [12,57]. Quercetin also increases viability and decreases inflammation and cytotoxicity in 4-HNE-exposed ARPE-19 cells [58]. Recently, solid dispersion of quercetin has been reported to decrease retinal pigment epithelium sediments and Bruch's membrane thickness in Nrf2 wild-type (WT) mice with dry age-related macular degeneration. This solid dispersion reduces ROS and MDA contents and increases SOD, GSH-PX, and CAT activities in serum and retinal tissues of Nrf2 WT mice. Solid dispersion of quercetin also up-regulates Nrf2 mRNA expression and enhances its nuclear translocation, as well as Nrf2 target gene hemeoxygenase-1(HO-1) in retinal tissues of Nrf2 WT mice [59]. These observations suggest that quercetin may alleviate oxidative injury to prevent dry age-related macular degeneration by enhancing Nrf2 activation. Medicinal plants marigold or grape seed (containing macular pigments lutein and zeaxanthin) are reported to have anti-oxidative activity and prevent 4-HNE adduct formation, actin solubility, and lipofuscin accumulation as well as age-related cone and rod photoreceptor dysfunction in β5(-/-) mice with cytoskeletal damage in aging RPE cells [56]. Of note, 4-HNE release and oxidative damage are also induced by irradiation exposure, causing retinopathy. Cyanidin-3-glucoside and quercetin decrease 4-HNE release in rod outer segments incubated with all-trans-retinal to generate bisretinoid under irradiation [60], whereas lutein and zeaxanthin isomers have recently been demonstrated to protect against light-induced retinopathy by reducing oxidative and endoplasmic reticulum stress in BALB/cJ mice [61]. Photodegradation of N-retinylidene-N-retinylethanolamine (A2E) is known to release reactive carbonyls. Medicinal plant compounds cyanidin-3-glucoside, quercetin, ferulic acid, and chlorogenic acid diminish cellular ROS and protect GSH from the reaction with photooxidized A2E in A2E accumulated-RPE cells irradiated with short-wavelength light [60].

This is especially important in exploring the possible molecular mechanisms by which bioactive compounds prevent 4-HNE-related retinopathy. NOD-like receptor protein 3 (NLRP3) inflammasome activation in the eyes is associated with the pathogenesis of age-related macular degeneration in RPE cells [62]. Cyanidin-3-glucoside has been found to inhibit NLRP3 inflammasome activation by reducing NLRP3, caspase-1, IL-1β, and IL-18 levels in 4-HNE-exposed ARPE-19 cells. This inhibitory mechanism may be mediated by regulating JNK-c-Jun/activator protein 1 (AP-1) pathway, further demonstrating the potential of cyanidin-3-glucoside to prevent retinal degenerative diseases [63,64]. Quercetin improves cell membrane integrity and mitochondrial function and decreases IL-6, IL-8, and monocyte chemotactic protein 1 (MCP-1) production, presumably by regulating MAPK/NF-κB signaling pathway in ARPE-19 cells stimulated by inflammatory cytokine [65].

#### **4. Protection against Cardiovascular Injury**

The normal redox status can be altered by 4-HNE adducts, which cause cardiomyocyte damage and vascular dysfunction. Thus, 4-HNE may play a key role in the progression of cardiovascular diseases [5,66,67]. *Thymbra capitata* is a Mediterranean culinary herb. Its essential oil prevents 4-HNE-induced cell death and avoids mitochondrial membrane potential loss and ROS generation in primary cultures of neonatal rat cardiomyocytes [15]. Olive (*Olea europaea*) leaf, a popular traditional herbal medicine, has a cardioprotective function. The ethanolic and methanolic extracts of *O. europaea* are reported to inhibit 4-HNE-induced apoptosis, ROS production, viability impairment, mitochondrial dysfunction, and pro-apoptotic activation in rat cardiomyocyte cell line (H9c2 cells). These extracts also reduce 4-HNE-induced phosphorylation of stress-activated transcription factors in

H9c2 cells [68]. Furthermore, phenolic compounds oleuropein, hydroxytyrosol, and quercetin derived from olive leaf are found to prevent against 4-HNE-induced cardiomyocyte carbonyl stress and toxicity and eventually regulate the cellular redox status [68]. Other medicinal plant constituents such as epigallocatechin-3-gallate, sulforaphane, and puerarin are reported to protect against cardiomyopathy. These compounds also reduce 4-HNE and lipid peroxidation to prevent oxidative stress in animal models of cardiovascular injury [69–71].

Aldehyde dehydrogenase 2 (ALDH2) mediates detoxification of toxic aldehydes, being necessary and sufficient to confer cardioprotection. Alpha-lipoic acid isolated from spinach and broccoli acts as a free radical scavenger. This bioactive compound activates myocardial ALDH2 and reduces 4-HNE and MDA levels in a Langendorff model of rat ischemia/reperfusion [72]. A recent report shows that α-lipoic acid prominently down-regulates the expression of 4-HNE and NOX subunit p67phox to inhibit oxidative stress in fistula-created deterioration of cardiac function rats [73], further demonstrating the cardioprotective effect. Flavonoid glycoside baicalin, extracted from *Scutellaria baicalensis* root, protects against hypoxia/reoxygenation-induced H9c2 cell injury. Baicalin also enhances ALDH activity to reduce of 4-HNE and MAD levels and increases SOD activity to suppress ROS production in H9c2 cells, showing its cardioprotection against hypoxia/reoxygenation-induced cardiomyocyte injury [74].

Atherosclerotic lesion is associated with the accumulation of reactive aldehydes derived from oxidized lipids. Accumulation of 4-HNE becomes an important risk factor that contributes to the atherogenicity of oxidized low-density lipoprotein (ox-LDL) and the development of atherosclerosis [75]. *G. biloba leaf* extract is reported to reduce ox-LDL and attenuate 4-HNE-induced production of matrix metalloproteinase-1 (MMP-1), probably through suppressing the activation of tyrosine-phosphorylated form of platelet-derived growth factor receptor beta in human coronary smooth muscle cells [76]. Medicinal plant *Opuntia cladodes* powder containing phenolic acid and flavonoids also reduces ox-LDL and 4-HNE toxicity in normal (Apc +/+) and preneoplastic (Apc min/+) immortalized epithelial colon cells [77]. Moreover, *O. cladodes* significantly reduces the formation of atherosclerotic lesions and the accumulation of 4-HNE adducts in the vascular wall of apoE-KO mice [78]. These observations demonstrate that *O. cladodes* may have therapeutic potential in 4-HNE-related cardiovascular diseases.

#### **5. Protection against Liver Injury**

There is evidence that the increased level of 4-HNE is correlated with the pathogenesis of liver injury [2,79]. Ginseng is a well-known medicinal plant. Ginseng extracts exert protection against liver injury via multiple pathways. For example, the fine root extract of ginseng with ginsenosides profiles has been found to attenuate 4-HNE-induced DNA damage in HepG2 cells. Methanolic extract of the main root also shows a decrease of DNA damage in this cell model [80]. Water extract of *P. ginseng*, rich in ginsenosides Rg1 and Rb1, significantly reduces total ROS generation and especially down-regulates 4-HNE signals in radiation-induced steatohepatitis of mice [81]. Red ginseng or black ginseng is a type of the repeated steam-processed *P. ginseng*. Red ginseng extract improves chronic alcohol-induced histopathological changes of liver in mice. This extract also inhibits oxidative stress and lipid peroxidation by reducing the formation of 4-HNE and the number of 4-HNE-positive cells and protects hepatocytes from inflammation and necrosis by activating AMP-activated protein kinase (AMPK)/Sirt1 pathway [82]. Recently, black ginseng extract has been reported to maintain the cellular redox status by restoring NOX and GSH level change and alleviate oxidative stress by reducing intracellular ROS production and lipid peroxide 4-HNE signals, resulting in the protection of hydrogen peroxide-induced oxidative damage in AML-12 cells [83]. American ginseng berry, the ripe fruit of *Panax quinquefolius*, contains a variety of protopanaxadiol ginsenosides and protopanaxatriol ginsenosides. This ripe fruit extract inhibits alanine aminotransferase and aspartate transaminase activity and mitigates oxidative stress and lipid peroxidation by reinforcing SOD activity, suppressing GSH depletion, and reducing 4-HNE formation in acetaminophen-induced liver injury of mice [84].

Several other bioactive compounds of medicinal plants also mitigate oxidative stress, improve antioxidant function, and reduce 4-HNE, protecting against liver injury. Tanshinone II-A is a diterpene quinone constituent of *Salvia miltiorrhiza*. This bioactive compound restores 4-HNE-induced hepatocyte damage in normal liver tissue NCTC 1469 cells. Tanshinone II-A also up-regulates peroxisome proliferator-activated receptor α (PPARα) expression and scavenges 4-HNE in this hepatocyte model [85]. Fisetin, one of the most popular polyphenols in fruits and vegetables, exhibits senotherapeutic activity in treating aged mice. It reduces liver oxidative stress damage by decreasing 4-HNE adducts and increasing the ratio of intracellular oxidized glutathione [86,87]. *p*-Coumaric acid, rich in fruits, vegetables, and plants, protects hepatocytes against oxidative stress and lipid peroxidation efficiently by reducing high levels of ROS and formation of 4-HNE protein adducts. Consistently, this active compound attenuates acetaminophen-induced hepatotoxicity, apoptosis, and inflammation in mice by suppressing the translocation of apoptosis-inducing factor and MAPK signaling and decreasing NF-κB activity [88]. Chlorogenic acid is an active compound in *Ligustrum lucidum* fruit that protects hepatocyte mitochondria from oxidative stress mediated by the activation of AMPK in carbon tetrachloride-induced acute liver injury of mice [89]. Water extract of *L. lucidum* inhibits ROS generation and increases intracellular GSH to reduce 4-HNE levels in this animal model, demonstrating that *L. lucidum* inhibits oxidative stress to prevent liver injury via AMPK activation [89].

Of note, high 4-HNE levels cause rapid cell death associated with the depletion of sulfhydryl groups and inhibition of key metabolic enzymes. Aldo-keto reductase family 7 member A2 (AKR7A2) is the most abundant anthracycline metabolizing enzyme, which has been found to protect against 4-HNE toxicity. AKR7A2 over-expression rescues the effect of Nrf2-knockdown on 4-HNE-induced cytotoxicity [90]. The active coumarin compound 7-Hydroxycoumain is presented in many medicinal plants, such as the seeds of *Clausena lansium*, *Parasenecio forrestii*, *Artemisia argyi*, and *Pharbitis purpurea*. This phytochemical acts as an AKR7A2 inducer and protects against 4-HNE-induced HepG2 cell damage via AKR7A2 induction. Thus, the inducible AKR7A2 has provided a new therapeutic target to treat oxidative stress-related chronic liver disease [90]. Additionally, the effects of bioactive compounds on AR activity have been reported. Plant triterpenoid saponins soyasaponin Ba (V), soyasaponin Bb, soyasaponin Bd (sandosaponin A), soyasaponin αg, 3-O-[R-L-rhamnopyranosyl (1→2)-α-D-glucopyranosyl (1→2)-α-D-glucuronopyranosyl]olean-12-en-22-oxo-3α,-24-diol, and soyasaponin βg isolated from methanolic extract of *Phaseolus vulgaris* seeds (Zolfino bean) have the ability to inhibit highly purified human recombinant aldose reductase (hAKR1B1), as well as hAKR1B1-dependent reduction of 4-HNE [91]. This work suggests that the reduction of 4-HNE by metabolic enzymes is an important determinant of the prevention of oxidative stress-driven liver injury.

#### **6. Improvement of Energy Metabolism Disorder**

Oxidative stress induced by 4-HNE also causes energy metabolism disorder that leads to tissue injury [92–94]. A harmful effect is exerted on adipocytes by 4-HNE, and 4-HNE also promotes insulin resistance [92,95,96]. In fact, 4-HNE induction and its lipolytic response are observed in insulin resistance patients with obesity and type 2 diabetes mellitus [92]. Insulin signaling pathway plays an important role in mediating energy metabolism. Tocotrienol-rich fraction in olive oil is reported to protect against oxidative stress, inflammation, and apoptosis, prevent the increased levels of 4-HNE and protein carbonyls from hyperglycemia-induced muscle damage, and effectively reduce insulin resistance in type 2 diabetic mice, via regulating the AMPK/Sirt1/peroxisome proliferator-activated receptor γ coactivator 1 (PGC1) pathway [97]. Carnosic acid decreases 4-HNE-mediated free fatty acid release and activates Tyr632 phosphorylation of insulin receptor substrate-1 (p-IRS-1Tyr632) and protein kinase B (p-Akt), inhibits as well as p-IRS-1Ser307 in insulin signaling pathway in 3T3-L1 adipocytes. This rosemary diterpene compound also suppresses 4-HNE-induced phosphorylation of protein kinase A (p-PKA) and hormone-sensitive lipase (HSL) and attenuates 4-HNE-induced down-regulation of p-AMPK and acetyl-CoA carboxylase, demonstrating the alleviation of insulin resistance driven by 4-HNE [16].

The improvement of antioxidant defense system in treating energy metabolism disorders is also important. GST can protect against 4-HNE-induced insulin resistance [36]. Carnosic acid up-regulates GST expression to reduce 4-HNE-conjugated proteins, being consistent with its attenuation of 4-HNE-induced lipolytic response and insulin resistance [16]. *Morus alba* (mulberry), a popular ornamental plant, has several well-documented beneficial effects to prevent and treat metabolic diseases [98]. The root bark extract of *M. alba*, rich in Diels–Alder type adducts cudraflavone B and other flavonoids, markedly protects against lipid peroxidation-induced pancreatic injury by increasing GSH and reducing 4-HNE in rats [99]. Mulberry leaf contains a variety of flavonoids to control obesity [100]. Recent study shows that mulberry leaf aqueous extract significantly reduces plasma levels of 4-HNE adducts and enhances the cellular antioxidant defense system to inhibit oxidative stress via normalizing Nrf2 activation in high fat diet-fed mice [101]. Mulberry fruits, a well-known source of resveratrol, prevent diabetic complications by reducing lipid peroxidation and 4-HNE accumulation in diabetic pregnancy [102]. Consistently, mulberry bioactive compound trans-resveratrol mitigates oxidative stress by reducing lipid peroxidation and inhibiting 4-HNE expression and DNA damage in streptozotocin-induced type 1 diabetes of rats [103]. In addition, the inhibition of NOX plays a key role in the mitigation of oxidative stress and prevention of insulin resistance, steatosis, and muscle damage [104,105]. NOX inhibitor apocynin, isolated from *Picrorhiza kurroa*, has been found to attenuate insulin resistance in skeletal muscle of mice with metabolic syndrome. Another phytochemical, (−)-epicatechin, found in green tea and cocoa, also down-regulates NOX1/NOX4, reduces 4-HNE adducts from ileum cells, and mitigates high fat diet-induced insulin resistance and steatosis in male C57BL/6J mice by preventing oxidative stress [106]. Cyanidin and delphinidin are the most common aglycon forms of anthocyanins and also have NOX inhibitory activity to attenuate high fat-induced steatosis and control the adverse effects by reducing 4-HNE-protein adducts in mice [107].

Another risk factor of obesity and type 2 diabetes is increased following exposure to fine particular matter (PM2.5) [108–110]. Research has shown 4-Hydroxytyrosol to be beneficial for metabolic and cardiovascular disorders. It also reduces liver and serum 4-HNE levels and alleviates PM2.5-induced, adiposity, and insulin resistance in adult female C57BL/6j mice by mitigating oxidative stress as well as restraining NF-κB activation and gut microbiota [111]. Additionally, there is evidence that 4-hydroxytyrosol significantly reduces 4-HNE expression in red blood cells from hyperlipemic patients and protects red blood cells against 4-HNE toxicity [112].

#### **7. Amelioration of Other Disorders**

Medicinal plants and bioactive compounds also combat 4-HNE-induced deleterious effects and ameliorate the clinical signs in other disorders. *Aloe vera* is a medicinal herb that contains numerous bioactive components. Aqueous extract of *A. vera* has an antioxidant capacity on endogenous ROS production and 4-HNE-protein adducts induced by 4-HNE in human cervical cancer (HeLa), human microvascular endothelial cells (HMEC), human keratinocytes (HaCat), and human osteosarcoma (HOS) cell cultures [113]. Triterpene saponin aescin isolated from *Aesculus hippocastanum* restricts lipid peroxidation to reduce cytotoxic 4-HNE levels and keeps more neurons and myelin sheaths alive in the spinal cord injury of rats, which may be mediated by enhancing HO-1 expression and inhibiting NF-κB activation [114,115]. Natural anti-oxidation compounds capsaicin (chili peppers), curcumin (turmeric), and polyphenols as well as *G. biloba* and *Polypodium leucotomos* are reported to inhibit 4-HNE-induced oxidative stress in human melanocytes, resulting in the inhibition of cell apoptosis [116]. Rutin effectively decreases 4-HNE and increases GSH-Px and GSH; thus, it prevents against UV-induced skin fibroblast membrane disruption via the regulation of antioxidant system [117]. Ascorbic acid has a similar effect in reducing 4-HNE levels in human skin fibroblasts exposed to UV radiation and hydrogen peroxide [118].

Additionally, 4-HNE is reported to disrupt gap junction-mediated intercellular communication in the lateral wall structures of the cochlea [119]. Some medicinal plant constituents such as astragaloside IV, baicalein, catalpol, curcumin, kaempferol, luteolin, quercetin, resveratrol, and rosmarinic acid are reported to prevent hair cell death [120]. Astragaloside IV (the major constituent of *Astragalus membranaceus*) significantly down-regulates 4-HNE expression in guinea pig cochlea exposed to impulse noise, being consistent with the attenuation of impulse noise-induced trauma [121,122]. Rosmarinic acid enhances the endogenous antioxidant defense and decreases 4-HNE expression by the regulation of the Nrf2/HO-1 signaling pathway, resulting in the attenuation of noise-induced hearing loss and injury in rat cochlea [123]. Therefore, these natural compounds may be the effective therapy to ameliorate 4-HNE-driven hearing loss.

During food digestion, the absorption of highly toxic 4-HNE from the gastrointestinal tract into the blood system may contribute to the development of multiple oxidative stress-related disorders [124]. Polyphenol-rich beverages such as Japanese Sencha green tea and grape juice promote the stability of polyunsaturated fatty acids to oxidation and inhibit 4-HNE formation during the simulated digestion of linseed oil emulsions in the intestinal phase [125]. Algae (microalgae *Schizochytrium* sp.), the division of lower plants, also has pharmaceutical applications. Algae oil, rich in phenolic compounds β-carotene and tocopherols, is reported to prevent lipid peroxidation and inhibit 4-HNE formation in digestion during both in vitro gastric and duodenal digestions [126]. (−)-epicatechin also restores fat diet-induced increase of intestinal permeability, down-regulation of ileal tight junction proteins, as well as reduction of endotoxemia, being consistent with the improvement of insulin resistance. More importantly, (−)-epicatechin and apocynin are found to suppress NOX1/NOX4 overexpression, 4-HNE adducts and monolayer permeabilization by regulating redox sensitive signals in TNFα-exposed Caco-2 cells, further demonstrating the protection against the high fat diet-induced increased intestinal permeability. These results are consistent with the prevention of steatosis and insulin resistance under the treatment of (−)-epicatechin and apocynin [106]. The edible amaranth plants from around the world have strong antioxidant activity [127]. Clinical study shows that the supplementation of amaranth seed oil in addition to standard anti-helicobacter pylori treatment significantly decreases the accumulation of 4-HNE-histidine adducts in gastric mucosa and increases heart rate variability in duodenal peptic ulcer patients. Therefore, the standard treatment of duodenal peptic ulcer requires additional therapeutic approaches by using amaranth seed oil [128].

Energy metabolism disorders induced by 4-HNE may alter longevity by orchestrating the development of a biological phenotype. Plant fruits rich in high levels of antioxidants can promote longevity and health span. For example, the supplementation with 4% nectarine has been found to extend life and increase fecundity. Nectarine reduces 4-HNE-protein adduct in wild-type females fed with high-fat diet. Moreover, nectarine ameliorates aging-related death and reduces oxidative damage in female sod1-mutant flies [129]. These results demonstrate that the attenuation of 4-HNE-induced energy metabolism disorders by medicinal plants and bioactive compounds may promote longevity.

#### **8. The Strategy for Developing Potential Therapy**

As a toxic end-product of lipid peroxidation, 4-HNE is an important mediator in physiological adaptive reaction and signal transport, as well as in the pathogenesis of multiple oxidative stress-related disorders. Medicinal plants and bioactive compounds are found to enhance metabolism, detoxification or clearance of 4-HNE by regulating activities of endogenous enzymes (such as AR, GST, and AKRs), which may provide the effective strategies for combating 4-HNE-driven deleterious effects. Because of differential metabolisms of 4-HNE observed in liver, lung, and brain of rodents [130], further study is warranted to determine whether these medicinal plants and bioactive compounds have the capacity to mediate oxidative, reductive, and conjugative pathways to metabolize 4-HNE and 4-HNE adducts in oxidative stress-triggered injury of these tissues.

The Nrf2/ARE pathway is suggested to mediate the adaptive induction of antioxidant and detoxifying enzymes including GST and AKR1C1, resulting in the metabolism of 4-HNE [131]. The capability of the Nrf2/ARE pathway induction may constitute a pleiotropic cytoprotective defense to prevent 4-HNE toxicity. Ongoing studies will determine the therapeutic effects of Nrf2/ARE activators derived from medicinal plants and bioactive compounds to mitigate 4-HNE cytotoxicity-induced pathophysiology of disorders or diseases.

Of note, PPARα modulates several biological processes that are perturbed in energy homeostasis [132]. A recent report shows that PPARα is unable to block β-catenin transcriptional activity induced by a constitutively active mutant of lipoprotein receptor-related protein 6 (LRP6). In fact, 4-HNE-induced ROS production enhances LRP6 stability; this event is inhibited by PPARα overexpression [133]. It is known that anti-obesity hormone leptin signaling as well as insulin signaling mediate the regulation of PPARα. Insulin resistance is improved by 4-HNE [96], which also selectively inhibits leptin signaling, possibly promoting the pathogenesis of leptin resistance in obesity [134]. Some medicinal plants and natural compounds acting on PPARα, leptin, or insulin signaling may be responsible for the therapeutic effects on 4-HNE-induced energy metabolism disorders [85].

However, several important issues related to 4-HNE-driven molecular events in diseases, or the relevance of medicinal plants and bioactive compounds in redox homeostasis, still need more studies and new comprehensive approaches. In this regard, preclinical studies and clinical intervention trials are required, which should include the use of accurate analytical techniques, such as the determination of 4-HNE and 4-HNE adducts by immunohistochemistry and enzyme-linked immunosorbent assay, as well as matrix-assisted laser desorption/ionization–tandem time of flight (MALDI–TOF/TOF) mass spectrometry and liquid chromatography–tandem mass spectrometry (LC–MS/MS) [135–138].

#### **9. Conclusions and Future Perspectives**

As a biomarker of lipid peroxidation and oxidative stress, cytotoxic 4-HNE contributes to the progression of oxidative stress-related disorders. Some medicinal plants and bioactive compounds have been demonstrated to reduce 4-HNE levels, detoxify 4-HNE, and inhibit 4-HNE adduct formation through the up-regulation of the antioxidant enzymes and suppression of 4-HNE-mediated downstream signals. More experiments have proved that these medicinal plants and bioactive compounds combat 4-HNE-induced deleterious effects in oxidative stress-related neurological disorder, eye damage, cardiovascular injury, liver injury, energy metabolism disorder, and other disorders (Table 1). The understanding of the molecular basis for the function of medicinal plants and bioactive compounds would be useful to facilitate the selection of 4-HNE metabolism for future intervention investigation and health claim support and develop new treatment for 4-HNE-induced deleterious effects in these disorders. Moreover, the identification and analysis of 4-HNE and 4-HNE adducts are necessary to further support the association between medicinal plants and oxidative stress-related disorders in clinical trials.

**Table1.**Medicinalplantsandbioactivecompoundsintheattenuationof4-HNE-induceddeleterious

*Biomolecules* **2020**, *10*, 146

*Biomolecules* **2020**, *10*, 146

**Table1.***Cont.*

**Author Contributions:** Conceptualization, F.-X.W. and L.-D.K.; software, F.-X.W.; writing—original draft preparation, F.-X.W., H.-Y.L., and Y.-Q.L.; review and editing, F.-X.W. and H.-Y.L.; revising, L.-D.K. and F.-X.W. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Conflicts of Interest:** The authors declared no conflict of interest.

#### **Abbreviations**

#### **References**

© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

### **Mechanistic Pathways and Molecular Targets of Plant-Derived Anticancer** *ent***-Kaurane Diterpenes**

**Md. Shahid Sarwar 1,2, Yi-Xuan Xia 1, Zheng-Ming Liang 1, Siu Wai Tsang 1 and Hong-Jie Zhang 1,\***

Received: 18 December 2019; Accepted: 14 January 2020; Published: 16 January 2020

**Abstract:** Since the first discovery in 1961, more than 1300 *ent*-kaurane diterpenoids have been isolated and identified from different plant sources, mainly the genus *Isodon*. Chemically, they consist of a perhydrophenanthrene subunit and a cyclopentane ring. A large number of reports describe the anticancer potential and mechanism of action of *ent*-kaurane compounds in a series of cancer cell lines. Oridonin is one of the prime anticancer *ent*-kaurane diterpenoids that is currently in a phase-I clinical trial in China. In this review, we have extensively summarized the anticancer activities of *ent*-kaurane diterpenoids according to their plant sources, mechanistic pathways, and biological targets. Literature analysis found that anticancer effect of *ent*-kauranes are mainly mediated through regulation of apoptosis, cell cycle arrest, autophagy, and metastasis. Induction of apoptosis is associated with modulation of BCL-2, BAX, PARP, cytochrome c, and cleaved caspase-3, -8, and -9, while cell cycle arrest is controlled by cyclin D1, c-Myc, p21, p53, and CDK-2 and -4. The most common metastatic target proteins of *ent*-kauranes are MMP-2, MMP-9, VEGF, and VEGFR whereas LC-II and mTOR are key regulators to induce autophagy.

**Keywords:** *isodon* genus; *ent*-kaurane diterpenoids; cancer; natural compounds; pathways

#### **1. Introduction**

Cancer is a heterogeneous disease which caused 9.6 million deaths globally in 2018 and the incidence of cancer is projected to increase to 13.1 million deaths by 2030 [1,2]. In addition, a large number of cancer patients have forms of the disease that are resistant to the mainstay anticancer drugs. Currently used anticancer drugs are mainly obtained from natural sources (plants, microorganisms, and marine organisms) or from synthetic reactions [3]. Unfortunately, most of these drugs also kill normal cells generating severe side effects [4,5]. These factors indicate the urgent need for the development of new therapeutics and treatment strategies.

Medicinal plants are an important source of drug entities. Over the past few decades, plants became an indispensable source of anticancer agents which are commonly used to treat different types of cancers in clinical settings. The World Health Organization (WHO) estimates that 80% of the total world's population residing in rural areas rely on plant sources as a part of their primary health care system. In addition, more than one-quarter of the prescribed drugs in developed countries are prepared directly or indirectly from plants [6]. In developing countries, 75% of the population use plant-derived drugs for the treatment of various illnesses [7]. Since the early 1900s, thousands of medicinal plants have been thoroughly investigated to screen cytotoxic compounds, yielding numerous anticancer drugs and lead compounds [5]. The structure of the cytotoxic plant metabolite podophyllotoxin was

first described in 1932 [8], this was followed by the discovery of vinca alkaloids (vinblastine and vincristine) in 1958 [9], and later, the identification of paclitaxel in 1971 [10]. These plant-derived molecules have revolutionized cancer treatment, yet more lead structures continue to be needed.

*ent*-Kaurane diterpenoids have recently attracted the attention of scientists and doctors due to their promising anticancer effect and safety profile [11]. They are subclass of tetracyclic diterpenes which consist of a perhydrophenantrene unit (A, B, and C rings) and cyclopentane unit (D ring) connected by a bridge of two carbons between C-8 and C-13 (Figure 1). Diterpenes containing a kaurane scaffold with configurational inversion at all chiral centers are known as *ent*-kaurane diterpenes. In this case, the prefix "*ent*-" is added before the name of this class of compounds to indicate that they are enantiomers of kaurene diterpenes. Moreover, International Union of Pure and Applied Chemistry (IUPAC) has established structured-guidelines for the stereochemistry, nomenclature, and numbering of the *ent*-kaurane skeleton. With the exception of those compounds containing an alkene functionality between C-9 and C-11, most of *ent*-kauranes show characteristic negative values for the specific optical rotation [12]. This class of compounds are mainly available in *Isodon* genus which mainly consists of a group of flowering plants. Other plant species that also contain *ent*-kaurane compounds belong to different families such as Asteraceae, Lamiaceae, Euphorbiaceae, Jungermanniaceae, and Chrysobalanaceae [13]. Accumulating evidences described the anticancer activity and underlying mechanism of *ent*-kaurane compounds against numerous cancers such as lung, colon, breast, prostate, liver, and gastric cancer both in vitro and in vivo. [14,15]. In this article, we discuss the anticancer *ent*-kaurane diterpenoids reported in the literature along with their source and the biological targets regulated by this type of compound.

**Figure 1.** General structure and numbering system of *ent*-kaurane diterpenes.

#### *Methodology*

Literature search was performed through the commonly used databases such as PubMed, MEDLINE, Scopus, Embase, Google Scholar, ScienceDirect, and Wiley Online. The articles which described the anticancer activity, mechanism of action and protein targets of *ent*-kaurane diterpenes were included in the review whilst those papers which mentioned only the isolation and characterization without further exploration of their molecular pathways were excluded. The molecular structures of diterpenoids were generated using the ChemDraw software. The chemical formulae, molecular weight, and related information of the diterpenoids were comprehensively matched with SciFinder®, a well-known portal for chemical compounds.

#### **2. Anticancer** *ent***-Kauranes and Their Targets**

#### *2.1. Oridonin*

Oridonin (C20H28O6, MW: 364.44, Figure 2), was first isolated from *Isodon rubescens*in the 1960s and one of its derivatives l-alanine-(14-oridonin) ester trifluoroacetate (HAO472) (Figure 2) has progressed to phase-I clinical trials by Hengrui Medicine Co. Ltd., Lianyungang, China [16]. Numerous studies reported the anticancer activities of oridonin against diverse human cell lines such as HCT116 (colorectal carcinoma, IC50: 32.6 μM) [17], OCI-AML3 (acute myeloid leukemia, IC50: 3.27 μM) [18], and BxPC-3 (pancreatic carcinoma, IC50: 53.0 μM) [19] after 24 h treatment; KYSE-150 (esophageal carcinoma, IC50: 28.7 μM), EC9706 (esophageal carcinoma, IC50: 34.4 μM), KYS-30 cells (esophageal carcinoma, IC50: 32.3 μM) [20], Jurkat (T-cell leukemia, IC50: 0.73 μM) [21], MG-63 (osteosarcoma, IC50: 10.9 μM), HOS (osteosarcoma, IC50: 11.9 μM), Saos-2 (osteosarcoma, IC50: 17.3 μM), U-2OS cells (osteosarcoma, IC50: 17.7 μM) [22], EC109 (esophageal carcinoma, IC50: 19.7 μM), EC9706 (esophageal carcinoma, IC50: 31.3 μM), EC1 (esophageal carcinoma, IC50: 25.8 μM) [23], HUVECs (human umbilical vein endothelial cells, IC50: 420 μM) [24], CNE1 (nasopharyngeal carcinoma, IC50: 3.66 μM), and CNE2 (nasopharyngeal carcinoma, IC50: 5.93 μM) cells [25] after 48 h treatment; and EC109 (esophageal carcinoma, IC50: 67.1 μM), SHG-44 (glioblastoma, IC50: 53.5 μM), MCF-7 (breast carcinomas, IC50: 72.1 μM) [26], and SGC-7901 (gastric carcinoma, IC50: 22.7 μM) [27] after 72 h treatment. Kadioglu et al. (2018) tested the cytotoxicity of oridonin in spectrum of drug-resistant cancer cell lines including CCRF-CEM (leukemia, IC50: 1.65 μM), CEM/ADR5000 (leukemia, IC50: 8.53 μM), MDA-MB231 (breast carcinoma, IC50: 6.06 μM), MDA-MB231/BCRP (breast carcinoma, IC50: 9.74 μM), HCT116 (p53+/+) (IC50: 18.0 μM), HCT-116 (p53−/−) (IC50: 34.7 μM), U87MG (glioblastoma, IC50: 17.4 μM), HepG2 (liver carcinoma, IC50: 25.7 μM), and AML12 (liver normal cells, IC50: >109 μM) cells, but did not mention the incubation time with oridonin in these cytotoxic test experiments [28].

A wide variety of sources have reported the anticancer mechanism of oridonin in multiple cancer cell lines [17,24,27,29–32]. According to the study of Yao et al. (2017) oridonin induced autophagy by decreasing the protein levels of glucose transporter 1 (GLUT1) and monocarboxylate transporter 1 (MCT1) in SW480 human colorectal cancer cells, in the BALB/c xenograft model. Autophagy induction by oridonin was correlated to increased expression of light chain-I (LC-I) and LC-II, while decreasing the phosphorylation of adenosine monophosphate-activated protein kinase (p-AMPK) [17]. In human umbilical vein endothelial cells (HUVECs), oridonin suppressed the proliferation, tube formation, migration, and invasion, but induced apoptosis. Oridonin was also found to block the angiogenesis of zebrafish by decreasing the mRNA expression of vascular endothelial growth factor A (VEGFA), vascular endothelial growth factor receptor 2 (VEGFR2) and VEGFR3, while reducing the expression of metastatic proteins claudin-1, -4, and -7. Antimetastatic activity of oridonin was further confirmed in a xenograft zebrafish model tested at a dose of 8 mg/kg that showed significant effect (*p* < 0.01) in comparison to the vehicle control group [24]. Gao et al. (2016) reported that oridonin induced apoptosis by downmodulating the expression level of B-cell lymphoma 2 (BCL-2), but upmodulating the expression of BAX, thus reducing the BCL-2/BAX ratio in human gastric cancer SGC-7901 cells. Moreover, oridonin treatment activated caspase-3 by promoting the release of cytochrome c from mitochondria to the cytosol [27]. In another study, oridonin potentiated the anticancer activity of lentinan, a polysaccharide isolated from shiitake mushrooms, by upregulating the expression levels of caspase-3, -8, -9, BAX, p53, and p21 while downregulating the expression of BCL-2, B-cell lymphoma extra-large (BCL-XL) and epidermal growth factor (EGF) in SMMC-7721 human hepatoma cells [29].

In prostate cancer cells (PC-3 and DU-145), oridonin increased the expression levels of p53, p21, caspase-3, -9, and poly (ADP-ribose) polymerase (PARP), while it decreased cyclin-dependent kinase 1 (CDK1) levels [30]. Moreover, it inhibited the expression of phosphoinositide 3-kinase (PI3K) and blocked phosphorylation of protein kinase B (p-Akt). Sun et al. (2018) reported synergistic anticancer activity of oridonin in combination with lentinan by decreasing the expression of BCL-2 and nuclear factor kappa B (NF-κB) and increasing the expression of caspase-3, -9, p53, p21, NF-κB inhibitor-α (IκB-α) by increasing transcription of mRNA and translation of the mRNA into their respective protein products in human hepatoblastoma HepG2 cells [31]. In 4T1 human breast cancer cells, oridonin inhibited cellular proliferation, migration, and invasion via a negative modulation of notch1-4. Furthermore, the administration of oridonin (5 mg/kg) significantly (*p* < 0.01) reduced the weight (84%) and volume (72%) of 4T1 tumors compared with the negative control group in a xenograft nude mouse study [32].

#### *2.2. Eriocalyxin B*

Eriocalyxin B (C20H24O5, MW: 344.41, Figure 2) was isolated from the plant *Isodon eriocalyx* var. laxiflora [33]. The cytotoxicity of eriocalyxin B was tested in panel of human cell lines such as SMMC-7721 (hepatocarcinoma, IC50: 0.76 μM) [33], MCF-7 (IC50: 0.75 μM), MDA-MB-231 (IC50: 0.47 μM) [34] after 48 h treatment; PANC1 (pancreatic carcinoma, IC50: 1.79 μM), CAPAN1 (pancreatic carcinoma, IC50: 0.86 μM), CAPAN2 (pancreatic carcinoma, IC50: 0.73 μM), SW1990 (pancreatic carcinoma, IC50: 1.40 μM), WRL68 (normal human liver cells, IC50: >3.58 μM), PBMC (human peripheral blood mononuclear cells, IC50: >5.83 μM) [35], SU-DHL-4 (lymphoma, IC50: 1.00 μM), Namalwa (lymphoma, IC50: 1.50 μM), Raji (lymphoma, IC50: 2.00 μM), Jurkat (lymphoma, IC50: 2.00 μM), U266 (lymphoma, IC50: 5.60 μM) and HUT78 (lymphoma, IC50: 2.50 μM) cells [36] after 72 h treatment.

The molecular pathways of eriocalyxin B against different cancers have been intensively investigated [33,34,37–39]. In hepatocellular carcinoma SMMC-7721 cells, eriocalyxin B induced apoptosis by interfering with the binding of NF-κB with the response elements via targeting cysteine 62 moiety of p50 [33]. In a recent study, eriocalyxin B was reported to induce autophagy upon an upregulation of LC3B-II and beclin-1 in MCF-7 and MDA-MB-231 human breast cancer cells; however, the expression of p62 was downregulated. In addition to autophagy, eriocalyxin B also induced apoptosis by activating caspase-3 and PARP while decreasing BCL-2. In fact, the phosphorylation levels of Akt, mammalian target of rapamycin (mTOR), and p70S6K were decreased in a dose- and time-dependent manner post eriocalyxin B treatment. Therefore, the mechanism of action of eriocalyxin B is suggested to be associated with the Akt/mTOR/p70S6K signaling pathway [34].

Earlier studies demonstrated that eriocalyxin B induced G1 phase cell cycle arrest by decreasing the expression of cyclin D1, CDK4, and phosphorylated retinoblastoma (p-Rb). When applied to HUVECs at 50 and 100 nM, eriocalyxin B notably inhibited the VEGF-induced cell proliferation, tube formation, cell migration, and invasion [37]. Further to its modulation on the VEGF cascade, eriocalyxin B suppressed the VEGF-induced phosphorylation of VEGFR-2 via an interaction with various ATP-binding sites, thus leading to the repression of several VEGFR-2 downstream molecules such as VEGFR-1, focal adhesion kinase (FAK), Src, pSer473-Akt, extracellular signal-regulated kinase (ERK1/2), and pThr180/Tyr182-mitogen-activated protein kinase (p38-MAPK). When administered in vivo, eriocalyxin B restrained the formation of new blood vessels, vascularization, and growth of the 4T1 breast tumor xenografts. According to the study of Lu et al. (2016), eriocalyxin B inhibited the cell proliferation, migration, invasion, and angiogenesis of human colon cancer cells SW1116 [38]. Mechanistic studies found that it inhibited the phosphorylation of janus kinase 2 (JAK2) and signal transducer and activator of transcription-3 (STAT3) as well decreased the expression of VEGF, VEGFR-2, matrix metallopeptidase-2 (MMP-2), MMP-9, and proliferating cell nuclear antigen (PCNA). Yu et al., (2015) reported that eriocalyxin B inhibited both constitutive- and interleukin-6 (IL-6)-induced phosphorylation of STAT-3 in A549 lung cancer cells without affecting the upstream kinases such as JAK1, JAK2, and tyrosine kinase 2 (TYK2) [39].

#### *2.3. Excisanin A*

Excisanin A (C20H30O5, MW: 350.455, Figure 2) was isolated from *Isodon macrocalyxin* which exhibited cytotoxic potential in breast cancer MDA-MB-231 (IC50: 22.4 μM), SKBR3 (IC50: 27.3 μM) [40] and MDA-MB-453 (IC50: 10.3 μM) cells as well as in hepatoma Hep3B (IC50: 6.45 μM) cells [41] after 72 h treatment. At 10–40 μM concentration, excisanin A suppressed the expression of MMP-2, MMP-9, integrin β1, β-catenin, and reduced the phosphorylation of FAK and Src in breast cancer (MDA-MB-231 and SKBR3) cells. Moreover, the phosphorylation of PI3K, Akt and glycogen synthase kinase 3β (GSK3β) was also reduced after treatment with excisanin A [40]. In addition to its effect on breast cancer metastasis, excisanin A inhibited the proliferation of human hepatocellular carcinoma cell line Hep3B and breast cancer cell line MDA-MB-453 cells by inducing apoptosis. It significantly (*p* < 0.01) reduced the tumor weight (46.4%) as well as induced tumor cell apoptosis in Hep3B xenograft mice at a dose of

20 mg/kg/day. Mechanistic studies showed that excisanin A reduced the expression of p-GSK-3α/β, Thr308-Akt, Ser473-Akt, p-mTOR, and p-FKHR expression in both Hep3B and MDA-MB-453 cells [41]. Zhang et al. (2013) reported dose- and time-dependent induction of autophagy by increasing the expression of LC3-II and decreasing the expression of p62 in nasopharyngeal carcinoma (NPC) cell lines CNE1 and CNE2 cells after excisanin A treatment. Moreover, excisanin A also upregulated p-JNK (c-jun N-terminal kinase), p-c-Jun and sestrin 2 expression in NPC cells [42].

#### *2.4. Ponicidin*

Ponicidin (C20H26O6, MW: 362.42, Figure 2) was isolated from *Isodon rubescens* and *I. japonicas*, and showed cytotoxic activity against the human carcinoma cell lines K562 (leukemia), Bcap37 (breast), GC823 (gastric), BIU87 (bladder), HeLa (cervical) and PC-3 (prostate) cells [43]. Further studies also described the cytotoxicity of ponicidin in HeLa (IC50: 23.1 μM) [44], A549 (IC50: 38.0 μM) and GLC-82 (lung, IC50: 32.0 μM) cancer cell lines after 24 h; A549 cells (IC50: 31.0 μM) and GLC-82 (IC50: 26.0 μM) after 48 h; A549 cells (IC50: 15.0 μM) and GLC-82 cells (IC50: 13.0 μM) [45] after 72 h. It suppressed the growth of gastric carcinoma MKN28 cells in dose- and time-dependent manner by downregulating BCL-2, p-JAK2, and p-STAT3 expression, while upregulating BAX and cleaved caspase-3 expression [43]. An earlier study reported induction of apoptosis and disruption of mitochondrial membrane potential in ponicidin-treated lung cancer A549 and GLC-82 cells by the upregulating expression of cleaved caspase-3, -8, -9, and BAX, and downregulating the expression of BCL-2 and survivin [45]. Du et al., (2015) reported that ponicidin induced apoptosis and cell cycle arrest in colon cancer cells HCT-116 by increasing caspase-3, BAX, and p-p38 expression, while decreasing BCL-2, p-ERK, and p-Akt expression [46].

#### *2.5. Pharicins A and B*

Pharicins A (C24H34O7, MW: 434.53, Figure 2) and B (C24H34O8, MW: 450.53) were isolated from *Isodon pharicus*. The only structural difference between these two compounds is the position of hydroxyl group which is located at C-1 in pharicin A and at C-12 in pharicin B. Pharicin A induced mitotic arrest in leukemia cell line Jurkat and solid tumor-derived cell line raji by inhibiting auto phosphorylation activity of BubR1 [47]. Investigation of the effects of pharicin B in several acute myeloid leukemia (AML) cell lines and primary leukemia cells from AML patients such as NB-4, U937 and THP-1 cells showed cytotoxicity with IC50 values around 3.5 μM after 48 h of treatment. Further studies found that it can quickly stabilize the retinoic acid receptor-α (RAR-α) protein, even in the presence of all-trans-retinoic acid (ATRA) which generally induce the loss of RAR-α protein. Moreover, pharicin B also increased ATRA-dependent transcriptional activity of RAR-α protein in NB4 cells, a type of promyelocytic leukemia–RAR-α–positive APL cell line [48].

#### *2.6. Jaridonin*

Jaridonin (C22H32O5, MW: 376.49, Figure 2) was isolated from the Chinese herb *Isodon rubescens* which exhibited cytotoxicity in esophageal squamous cancer EC109 (IC50: 12.0 μM), EC9706 (IC50: 11.2 μM), and EC1 (IC50: 4.60 μM) cells [23] after 48 h as well as in human glioma cell line SHG-44 (IC50: 14.7 μM) and breast cancer MCF-7 (IC50: 16.7 μM) cell [26] after 72 h. A mechanistic study found that it induced apoptosis and G2/M phase cell cycle arrest in esophageal cancer EC109, EC9706 and EC1 cells. Jaridonin treatment caused remarkable reduction of mitochondrial membrane potential, release of cytochrome c into the cytosol, and increased expression of caspase-3 and -9, leading to activation of the mitochondria-mediated apoptosis. Moreover, jaridonin also increased the production of reactive oxygen species (ROS) and upregulated p53, p21waf1/Cip1, and BAX expression [23]. Another study confirmed the induction of G2/M phase arrest in esophageal squamous cancer EC9706 cells [49]. However, cell cycle arrest was related with increased phosphorylation of ataxia-telangiectasia mutated (ATM) (Ser1981) protein kinase, cell division control 2 (Cdc2) (Tyr15), Cdc25C, and H2A histone family member X (H2A.X)(Ser139) as well as increased expression of check point kinase 1(Chk1) and Chk2.

#### *2.7. Jungermannenones A and B*

Jungermannenones A (C20H28O2, MW: 300.44, Figure 2) and B (C20H28O3, MW: 316.44, Figure 2) were isolated from *Jungermannia fauriana*, and exhibited cancer cell killing activity in multiple cell lines [50]. The IC50 values of jungermannenone A were determined in PC3 (IC50: 1.34 μM), DU145 (prostate carcinoma, IC50: 5.01 μM), LNCaP (prostate carcinoma, IC50: 2.78 μM), A549 (IC50: 8.64 μM), MCF-7 (IC50: 18.3 μM), HepG2 (IC50: 5.29 μM) and RWPE1 (normal prostate epithelial, IC50: 5.09 μM) cell lines, while the IC50 values of jungermannenone B were measured in PC3 (4.93 μM), DU145 (5.50 μM), LNCaP (3.18 μM), A549 (5.26 μM), MCF-7 (14.2 μM), HepG2 (6.02 μM), and RWPE1 (18.2 μM). Both compounds induced apoptosis by stimulating ROS accumulation and inducing cell cycle arrest. Western blot analysis showed reduced expression of c-myc, cyclin D1, cyclin E, CDK4, and p-Cdc2, while increased expression of p21 and p-ERK. Furthermore, both jungermannenones A and B induced DNA damage by reducing the expression of DNA repair proteins Ku70/Ku80 and RDA51 [50].

#### *2.8. E*ff*usanin E*

Effusanin E (C20H28O6, MW: 364.44, Figure 2) was isolated from *Rabdosia serra* (*Rabdosia* is a synonym of *Isodon*), and is cytotoxic to nasopharyngeal carcinoma cell CNE2 (IC50: ~60 μM) but non-toxic up to 500 μM in normal nasopharyngeal epithelial cell line after 48 h. It inhibited cell proliferation and induced apoptosis in CNE2 cells by increasing the expression of cleaved PARP, caspase-3 and -9 proteins, as well as decreasing nuclear translocation of p65 NF-κB [51]. Moreover, the binding ability of NF-κB to the promoter region of cyclooxygenase-2 (COX-2) abolished after treating the cells with effusanin E, therefore, inhibiting the expression and promoter activity of COX-2. In vivo studies also found significant reduction of tumor growth (*p* < 0.05) at 30 mg/kg without any sign of toxicity in comparison to negative control group (DMSO) as well as decreased expression of p50 NF-κB and COX-2 in tumor tissue.

#### *2.9. Longikaurin A*

Longikaurin A (C20H28O5, MW: 348.44, Figure 2) was described from *Isodon ternifolius*, and displayed cytotoxic potential in human hepatocarcinoma SMMC-7721 (IC50: ~1.8 μM), HepG2 (IC50: ~2 μM), BEL7402 (IC50: ~6 μM), Huh7 (IC50: ~6 μM), and LO2 (IC50: ~9 μM) cells [52], and the nasopharyngeal carcinoma CNE1 (IC50: 1.26 μM) and CNE2 (IC50: 1.52 μM) cells [25] with 48 h incubation. This *ent*-kaurane compound induced apoptosis and G2/M phase cell cycle arrest in SMMC-7721 and HepG2 cells. In vivo studies found longikaurin A treatment (6 mg/kg) significantly suppressed tumor development (*p* < 0.01) in a SMMC-7721 xenograft mouse model study and the antitumor effect was comparable to the positive control 5-FU (fluorouracil) (10 mg/kg). A mechanistic study showed reduced expression of S-phase kinase-associated protein 2 (Skp2) after longikaurin A treatment, which was correlated with increased expression of p21 and p-Cdc2 (Try15) and decreased expression of cyclin B1 and Cdc2 proteins [52]. It also induced ROS production and phosphorylation of JNK.

In nasopharyngeal carcinoma (NPC) cell lines S18 and S26, longikaurin A exerted anticancer activity and suppressed the stemness of cells. Western blot analysis showed downregulation of c-myc and fibronectin in NPC cells [53]. At a low concentration (0.2 μM), longikaurin A induced S phase arrest while at a higher concentration (3.12 μM) it induced apoptosis in human NPC cell lines CNE1 and CNE2 cells [25]. Mechanistic studies found upregulation of cleaved caspase-3, cleaved PARP, and BAX, while downregulation of BCL-xL, p-Akt, and p-GSK-3β. In CNE2 xenograft model, longikaurin A significantly suppressed tumor growth without affecting the body weights of the mice [53].

#### *2.10. Glaucocalyxins A and B*

Glaucocalyxin A (C20H28O4, MW: 332.44, Figure 2) was isolated from *Isodon japonica*, and demonstrated strong anticancer effects in the human cell lines of HL-60 (leukemia, IC50: 6.15 μM) cells [54] after 24 h; Focus (hepatocarcinoma, IC50: 2.70 μM), SMMC-7721 (IC50: 5.58 μM), HepG2 (IC50: 8.22 μM), SK-HEP1 (hepatocarcinoma, IC50: 2.87 μM) [55], HOS (IC50: 7.02 μM), Saos-2 (IC50: 7.32 μM), U-2OS (IC50: 8.36 μM), MG-63 (IC50: 5.30 μM) [56], and UMUC3 (bladder carcinoma, IC50: 9.77 μM) cells [57] after 48 h; and MCF-7 (IC50: 1.00 μM) and Hs578T (breast carcinoma, IC50: 4.00 μM) [58] after 72 h.

Glaucocalyxin A dose- and concentration-dependently inhibited the growth of the liver cancer Focus and SMMC7721 cells. Mechanistic studies found induction of G2/M phase cell cycle arrest as well as increased expression of cleaved caspase-3 and PARP [55]. In MCF-7 and Hs578T breast cancer cells, glaucocalyxin A induced apoptosis, and G2/M phase arrest by increasing the expression of intrinsic apoptotic markers cleaved caspase-3, BAX, and p53, while decreasing BCL-2 expression. It also upregulated the expression of extrinsic apoptotic markers such as Fas and Fas ligand (FasL) at the mRNA and protein level. Moreover, the expression of p-ERK and p-JNK increased in a dose-dependent manner after glaucocalyxin A treatment [58]. Furthermore, it concentration-dependently suppressed the proliferation and induced apoptosis in human brain glioblastoma U87MG cells by activating caspase-3, while downregulating p-Akt, p-Bad, and X-linked inhibitor of apoptosis protein (XIAP) [59]. In another study, glaucocalyxin A induced a dose-dependent apoptosis in HL-60 cells by increasing the expression of caspase-3, -9, and BAX, while decreasing the expression of BCL-2. It caused loss of mitochondrial membrane potential and release of cytochrome c from mitochondria into the cytosol as well as elevated intracellular ROS generation [60].

Glaucocalyxin B (C22H30O5, MW: 374.48, Figure 2), which differs from glaucocalyxin A by an acetylation of the hydroxyl group at C14, exerted anticancer activity in leukemia cell line HL-60 (IC50: 5.86 μM) after 24 h [54], gastric cancer cell line SGC-7901 (IC50: 13.4 μM) [61] after 60 h, and cervical cancer cell lines HeLa (IC50: 4.61 μM) and SiHa (IC50: 3.11 μM) after 72 h [62]. The anticancer activity in HeLa and SiHa cells was related with induction of apoptosis and autophagy by increasing the expression of phosphatase and tensin homolog (PTEN) protein and cleaved PARP, as well as increasing the cleavage of LC3 II/I protein. Moreover, glaucocalyxin B also reduced the expression of p-Akt in HeLa and SiHa cells [62].

#### *2.11. Lasiodin*

Lasiodin (C22H30O7, MW: 406.48, Figure 2) was isolated from *Isodon serra*, and inhibited the proliferation and migration of human nasopharyngeal carcinoma cells CNE1 (IC50: ~6 μM) and CNE2 (IC50: ~5 μM) at 24 h incubation [63]. Mechanistic studies showed that lasiodin-induced apoptosis was related to increased expression of apoptotic protease activating factor 1 (Apaf-1), release of cytochrome c, and cleavages of PARP, caspase-3 and -9. Moreover, the expression of p-Akt, p-ERK1/2, p-p38, and p-JNK was also reduced after 24 h treatment with lasiodin. However, lasiodin-mediated inhibition of cell proliferation was blocked in the cells treated with Akt or MAPK inhibitors. Lasiodin treatment also downregulated the expression of COX-2, revoked NF-κB binding to the COX-2 promoter and stimulated the nuclear translocation of NF-κB. Therefore, lasiodin inhibited the proliferation of CNE1 and CNE2 cells by activating Apaf-1/caspase-dependent apoptotic pathways and suppressing Akt/MAPK and COX-2/NF-κB signaling cascades.

#### *2.12. Adenanthin*

Adenanthin (C26H34O9, MW: 490.55, Figure 2) was isolated from the leaves of *Isodon adenanthus*, and showed antiproliferative effects against hepatocellular carcinoma cells such as HepG2 (IC50: 2.31 μM), Bel-7402 (IC50: 6.67 μM), and SMMC-7721 (IC50: 8.13 μM) while exhibiting less toxicity in the two normal hepatic cell lines QSG-7701 (IC50: 19.6 μM) and HL-7702 cells (IC50: 20.4 μM) at 48 h exposure [64]. At 72 h treatment, the IC50 values of adenanthin in esophageal carcinoma cell line EC109, glioma cell line SHG-44, and breast cancer cell line MCF-7 were 6.50, 4.80, and 7.60 μM, respectively [26]. According to the study of Hou et al. (2014), adenanthin killed malignant liver cells by stimulating ROS generation and targeting peroxiredoxin (Prx) I and II proteins which are considered

essential for survival of HCC cells. In-vivo studies with SMMC-7721 cells xenograft mice showed significantly reduced tumor size at 10 mg/kg without any notable side effects. However, the body weight of the mice decreased after treatment with 20 mg/kg of adenanthin [64]. In a different study, the same research group also found that adenanthin stimulated acute promyelocytic leukemia (APL) cell differentiation by targeting Prx I and Prx II as well as blocking their peroxidase activities. Moreover, the level H2O2 increased after adenanthin treatment, which led to activation of p-ERK, p-c-Jun, and increased transcription of CCAAT-enhancer-binding protein β (C/EBPβ) [65].

#### *2.13. Kaurenic Acid*

Kaurenic acid or kaurenoic acid (KA) (C21H32O2, MW: 316.49, Figure 2), chemically known as *ent*-kaur-16-en-19-oic acid, was isolated from *Espeletia semiglobulata*, and exhibited antimelanoma effects with an IC50 value of 0.79 μM in B16F1 cells. An in vivo study found that KA (160 mg/kg) markedly (*p* < 0.001) reduced the tumor sizes (49.51%) compared with the vehicle control group (87.5%) in a C57BL/6 mice model. RT-PCR analysis showed reduced expression of BCL-xL at the mRNA level after KA treatment in C57BL/6 mice [66]. In another study, KA selectively reduced the cell viability of two breast cancer cells MCF7 (proficient P53) and SKBR3 (mutated p53), while KA treatment was resistant to HB4A cell line [67]. At 70 μM, KA caused 40% and 25% cell death of MCF7 and SKBR3 cells suggesting that p53 protein may play a vital role in anticancer activity of KA. A recent study reported significant genotoxicity, apoptosis, and cell cycle arrest in gastric cancer cells after KA treatment [68]. At 10 μg/mL concentration, KA downregulated the expression of c-myc, CCND1, BCL-2, and caspase-3, while it upregulated ATM, Chk2, and TP53 expression.

#### *2.14. Weisiensin B*

Weisiensin B (C20H28O5, MW: 348.44, Figure 2) was isolated from the traditional Chinese herb *Isodon weisiensis* and inhibited the growth and proliferation of human hepatoma cell lines BEL-7402 and HepG2, ovarian cancer HO-8910 cells, and gastric cancer SGC-7901 cells [69]. The IC50 values of the compound were 10.0, 3.24, 32, and 4.34 μM in BEL-7402, HepG2, HO-8910 cells and SGC-7901 cells, respectively, after 48 h. DNA fragmentation assay and Hoechst 33,258 staining showed that weisiensin B significantly induced apoptosis. Flow cytometry analysis with propidium iodide (PI) staining revealed induction of G2/M phase arrest after weisiensin B treatment. Another study reported weisinensis B-mediated induction of apoptosis, G2/M phase arrest, and significant ROS generation in human chronic myeloid leukemia K562 cells [70].

#### *2.15. Inflexinol*

Inflexinol (C24H34O8, MW: 450.53, Figure 2) was isolated from *Isodon excisus*, which is native to China, Korea, and Japan. It inhibited the growth of the colon cancer cell lines SW620 (IC50: 29.0 μM), HCT116 (p53+/+) (IC50: 30.0 μM) and HCT116 (p53−/−) (IC50: 34.0 μM) but did not show toxicity in the normal colon CCD-112 CoN cells up to 40 μM [71]. Antiproliferative effects of inflexinol were mediated through induction of apoptosis via downregulation of antiapoptotic markers BCL-2, XIAP, and cIAP1/2, with simultaneous upregulation of cleaved caspase-3, -9, and PARP. Inflexinol treatment also significantly increased the ratio of BAX/BCL-2 and suppressed the expression of cyclin D1 and BCL-2. Moreover, inhibitory effects of inflexinol on the growth of colon cancer cells were related to inactivation of NF-κB by modification of a cysteine residue in the p50 subunit. An in vivo study in a SW620 xenograft model showed dose-dependent reduction of tumor volumes (64.5% to 47.8%) and weights (75.9% to 58%) at 12 and 36 mg/kg, respectively, compared with the control group. Moreover, it also exhibited inhibition of DNA binding activity of NF-κB in tumor tissue and suppressed nuclear translocation of p65 and p50 as well as inhibited the phosphorylation of IκB in the cytosol [71].

#### *2.16. Xerophilusin B*

Xerophilusin B (C20H26O5, MW: 346.42, Figure 2) was isolated from *Isodon xerophilus* and showed dose-dependent growth inhibition of esophageal squamous cell carcinoma KYSE-140 (IC50: 2.80 μM), KYSE-150 (IC50: 1.20 μM), KYSE-450 (IC50: 1.70 μM), and KYSE-510 (IC50: 2.60 μM) cells when incubated for 72 h. Further studies found that xerophilusin B induced apoptosis and G2/M phase cell cycle arrest in KYSE-150 and KYSE-450 cells [72]. Treatment with xerophilusin B increased the release of mitochondrial cytochrome c and upregulated the expression of cleaved caspase-3 and -9, while downregulated caspase-7 and PARP levels. Moreover, the ratio of BCL-2/BAX decreased after xerophilusin B treatment. An in vivo study in BALB/c nude mice showed significant inhibition of tumor growth at 15 mg/kg without any major adverse effects.

#### *2.17. Henryin*

Henryin (C22H32O6, MW: 392.49, Figure 2) was isolated from *Isodon rubescens*, and inhibited the proliferation of colorectal cancer SW480 (IC50: 0.27 μM), HT-29 (IC50: 0.77 μM), and HCT-116 cells (IC50: 0.90 μM), and lung cancer A549 cells (IC50: 2.47 μM), but is slightly less toxic to normal colon cells CCD-841-CoN (IC50: 2.98 μM) and normal bronchus cells BEAS-2B (IC50: 3.55 μM). The inhibitory effects of henryin were correlated to reduced expression of cyclin D1 and c-myc. Moreover, it inhibited the binding of β-catenin to transcription factor 4 (TCF-4) [73].

#### *2.18. 11*α*, 12*α*-epoxyleukamenin E*

11α, 12α-epoxyleukamenin E (EPLE) (C22H30O6, MW: 390.48, Figure 2) was isolated from *Salvia cavaleriei*, and exhibited cytotoxic activity in colorectal cancer cell lines HCT-116 and SW480 by downregulating the markers of the Wnt-signaling pathway such as c-myc, axin2 and survivin as well as inhibiting β-catenin transcriptional activity [74]. It induced apoptosis by decreasing the expression of BCL-2 and Bcl-xL, while increasing the expression of Bim and caspase-3. Moreover, a combination of EPLE and 5-fluorouracil produced synergistic anticancer effects in colon cancer cells. An in vivo study in the xenograft model also showed significant reduction of tumor size after EPLE treatment [74].

#### *2.19. DEK*