Anti-Inflammatory and Cytotoxic Activities of Clerodane-Type Diterpenes

The secondary metabolites of clerodane diterpenoids have been found in several plant species from various families and in other organisms. In this review, we included articles on clerodanes and neo-clerodanes with cytotoxic or anti-inflammatory activity from 2015 to February 2023. A search was conducted in the following databases: PubMed, Google Scholar and Science Direct, using the keywords clerodanes or neo-clerodanes with cytotoxicity or anti-inflammatory activity. In this work, we present studies on these diterpenes with anti-inflammatory effects from 18 species belonging to 7 families and those with cytotoxic activity from 25 species belonging to 9 families. These plants are mostly from the Lamiaceae, Salicaceae, Menispermaceae and Euphorbiaceae families. In summary, clerodane diterpenes have activity against different cell cancer lines. Specific antiproliferative mechanisms related to the wide range of clerodanes known today have been described, since many of these compounds have been identified, some of which we barely know their properties. It is very possible that there are even more compounds than those described today, in such a way that makes it an open field to discover. Furthermore, some diterpenes presented in this review have already-known therapeutic targets, and therefore, their potential adverse effects can be predicted in some way.


Introduction
Diterpenes are metabolites that come from isoprene units; these compounds can be classified according to their structure [1]. One type of diterpene is clerodanes, which are found in a wide range of plant species, especially those from the Labiatae, Euphorbiaceae and Verbenaceae families [2,3]; they have also been found in bacteria, fungi and marine sponges. This type of diterpene has been extensively studied due to many of them having biological activity [1][2][3][4]. For example, clerodin has anthelminthic activity [5]; salvinorin A is an agonist of κ-opioid receptor-serotonin-2A [6] with potential for use as a treatment in neuropsychiatric disorders [7]; tinosinenosides A-C show cytotoxicity effects against HeLa [8]; and columbin has anti-inflammatory and anticancer efficacy [4].
Clerodanes are secondary metabolites; when these compounds are obtained from plants, they are biosynthesized in the chloroplasts from geranylgeranyl pyrophosphate, producing a labdane-type precursor skeleton, which can be transformed to a halimane-type intermediate, and then converted to either cisor transclerodanes [3] (Figure 1a).
Clerodanes are bicyclic diterpenoids with a fused ring of decalin structure (C 1 -C 10 ) and a side chain of six carbons at C 9 . They are classified according to the configuration at the ring fusion and the substituents in C 8 and C 9 into four types: trans-cis, trans-trans, cis-cis and cis-trans (Figure 1b). About 25% have a cis ring fusion, and 75% have 5:10 trans ring fusion [9]. the ring fusion and the substituents in C8 and C9 into four types: trans-cis, trans-trans, ciscis and cis-trans (Figure 1b). About 25% have a cis ring fusion, and 75% have 5:10 trans ring fusion [9]. In this review, we have included clerodanes and neo-clerodanes and their enantiomers ent-neo-clerodanes ( Figure 2). Additionally, carbons 12 to 16 are usually oxidized to diene, furan, lactone or hydrofurofuran, which give structural characteristics to clerodane [10]. Cancer is a global health problem and is currently one of the main causes contributing to premature death worldwide [11]. At the present time, even with the great advances in medicine in our understanding and treatment of cancer with multimodal therapies including immunotherapy, gene-targeted therapy, chemotherapy, hormonal therapy and cancer vaccines [12] against specific cell targets, there are needs that have not been covered. These include more effective therapies, with fewer adverse effects, but also therapies at a more affordable cost. Thus, there is still a need to investigate more effective and In this review, we have included clerodanes and neo-clerodanes and their enantiomers ent-neo-clerodanes ( Figure 2). Additionally, carbons 12 to 16 are usually oxidized to diene, furan, lactone or hydrofurofuran, which give structural characteristics to clerodane [10].
Molecules 2023, 28, x FOR PEER REVIEW 2 of 38 the ring fusion and the substituents in C8 and C9 into four types: trans-cis, trans-trans, ciscis and cis-trans (Figure 1b). About 25% have a cis ring fusion, and 75% have 5:10 trans ring fusion [9]. In this review, we have included clerodanes and neo-clerodanes and their enantiomers ent-neo-clerodanes ( Figure 2). Additionally, carbons 12 to 16 are usually oxidized to diene, furan, lactone or hydrofurofuran, which give structural characteristics to clerodane [10]. Cancer is a global health problem and is currently one of the main causes contributing to premature death worldwide [11]. At the present time, even with the great advances in medicine in our understanding and treatment of cancer with multimodal therapies including immunotherapy, gene-targeted therapy, chemotherapy, hormonal therapy and cancer vaccines [12] against specific cell targets, there are needs that have not been covered. These include more effective therapies, with fewer adverse effects, but also therapies at a more affordable cost. Thus, there is still a need to investigate more effective and Cancer is a global health problem and is currently one of the main causes contributing to premature death worldwide [11]. At the present time, even with the great advances in medicine in our understanding and treatment of cancer with multimodal therapies including immunotherapy, gene-targeted therapy, chemotherapy, hormonal therapy and cancer vaccines [12] against specific cell targets, there are needs that have not been covered. These include more effective therapies, with fewer adverse effects, but also therapies at a more affordable cost. Thus, there is still a need to investigate more effective and less toxic compounds. Most of the chemotherapeutic drugs (nearly 65%) that are used in current cancer treatment regimens were originally isolated from natural products or their derivatives such as plants or microorganisms [13]. For instance, paclitaxel, a diterpene isolated from Taxus brevifolia (yew trees), classified as a taxane, is used in the therapy of various types of cancers [14]. Other examples include anthracyclines derived from Streptomyces strains, among them being doxorubicin, bleomycin and many others [13]. The cytotoxic activity of several clerodanes in different cancer cell lines has been described [1].
On the other hand, inflammation is an immune response to different stimuli, such as pathogens such as viruses and bacteria, traumas and chemical irritants [15]; that is to say, inflammation is a protective response of the body against harmful stimuli. Additionally, long-term inflammation could lead to several symptoms, such as pain, fatigue, insomnia, depression and gastrointestinal problems [16]. Chronic inflammation is associated with diseases such as cancer, diabetes and arthritis [17]. The inflammatory response leads to the production of pro-inflammatory mediators, such as cytokines, serotonin, leukotrienes and histamine [18]. These mediators promote vascular permeability, leukocyte migration, blood vessel dilatation and pain. The anti-inflammatory activity of terpenes, such as carvacrol, some carotenes and diterpenes, such as clerodanes, and triterpenes, has been studied [2,19].

Docking Analysis In silico evaluation
Compound 74 docked into the active sites of MDM2, TNF-α, FAK and IL-6 Compound 77 docked into the active sites of MDM2, TNF-α, TGF-β and FAK

Discussion
This review discusses research from the last 8 years on clerodane and neo-clerodane diterpenes that exhibit cytotoxic and anti-inflammatory activities. It presents studies on these diterpenes with anti-inflammatory effects from 18 species belonging to 7 families and those with cytotoxic activity from 25 species belong to 9 families. These plants mostly belong to the Lamiaceae, Salicaceae, Menispermaceae and Euphorbiaceae families. They Figure 19. Compounds of different species of Teucrium fructicans, Tinospora crispa and Tinospora sagittata with anti-inflammatory activity.

Discussion
This review discusses research from the last 8 years on clerodane and neo-clerodane diterpenes that exhibit cytotoxic and anti-inflammatory activities. It presents studies on these diterpenes with anti-inflammatory effects from 18 species belonging to 7 families and those with cytotoxic activity from 25 species belong to 9 families. These plants mostly belong to the Lamiaceae, Salicaceae, Menispermaceae and Euphorbiaceae families. They include 228 clerodanes and neo-clerodanes, of which, 140 have cytotoxic activity, 88 have antiinflammatory activity and crassifolin Q-U (49-53), compounds 74-77 and (-)-hardwickiic acid (91) have both activities. Compound 75 and 77 were alone in including acute toxicity, but they did not indicate LD 50 .

Cytotoxic Activity
All clerodanes included in this review are oxygenated; 58% of them have at least one acetate group, 47% a hydroxyl group, 49% a ring of lactone and 22% a ring of furan as substituents. Additionally, it was found that three diterpenes isolated from Sheareria nana (125-127) have -OSO 3 H.
We found that 82 compounds out of 140 were evaluated using the MTT assay, which is broadly used to measure the cytotoxic effects of drugs on cancer cell lines, and it is considered a quantitative cytotoxicity analysis; the assay is used more often because in itself, it is relatively straightforward and provides benefits due to the ease of its utility.
Compared to standard cancer therapies, in vitro studies have shown the cytotoxic and antiproliferative properties of different clerodane compounds. The mechanisms involved include growth inhibition, apoptosis, interference with DNA synthesis and driving DNA fragmentation in many cancer cell lines of mesenchymal, epithelial and hematopoietic origin [1,3].
Some clerodane compounds inhibit growth in cancer cell lines. Anacolosins A-F (3-8) and corymbulosins X and Y (9-10) isolated from Anacolosa clarkii exhibit cytotoxic properties in four paediatric cancer types [21]. Caseakurzin B (29) and caseakurzin J (34) from Casearia kurzii were investigated in a lung epithelial carcinoma cell line; the former arrested the cell cycle at the G2/M phase and the second at the S phase. Obtained from the same plant, corymbulosin M (25), caseamembrin B (26) and caseamembrin U (27) were also cytotoxic in three types of cancer cell lines. Of note, corymbulosin M (25) was the most potent of them and apparently even more active than etoposide, and it was shown that it affects the cell cycle at the G0/G1 stage [28]. Kurzipene D (38), also obtained from C. kurzii, has a potent antiproliferative effect compared to other kurzipenes and affects proliferation at the S stage. Further, one in vivo study used a xenograft tumor model in zebra fish embryos; this compound suppressed tumor proliferation and migration comparable to etoposide [26]. Crassifolins Q-U (49-53) from Croton crassifolius inhibited angiogenesis in HUVECs, and crassifolin U (53) had the strongest activity in this model [32]. Notably, the antitumor properties of casearins have been shown using in vivo and ex vivo methods [30]. Epoxy clerodane diterpene (139) isolated from Tinospora cordifolia had cytotoxic activity, inhibiting MCF7 growth by regulating the expression of the functional genes Rb1 and Mdm2 [55].
Several specific antiproliferative mechanisms related to the wide range of clerodanes known today have been described, since many of these compounds have been identified, some which we barely know their properties. It is very possible that there are even more compounds than those described today, in such a way that makes it an open field to discover. However, it is important to mention that clinical studies are required to demonstrate their efficacy in the therapy of the current cancer pandemic, and demonstrating their safety is also of great importance.

Anti-Inflammatory Activity
A total of 45% of the clerodanes with anti-inflammatory activity have at least one hydroxyl, 69% compounds contain a ring of lactones, 50% a ring of furans and 26% an acetate group as substituents.
We found that 63 compounds reported to have anti-inflammatory activity were evaluated for nitric oxide inhibition with the Griess assay on RAW264.7 macrophages or BV-2-cell-stimulated- LPS, and the clerodanes 157, 158, 185, 186 and 207 showed the best activity in this test with IC 50 values of less than 2 µM. In this review, we found that in vivo studies have only been performed for hautriwaic acid (196) and nepetolide (204).
The anti-inflammatory activity of clerodane diterpenoids mediated by different mechanisms has been demonstrated in in vitro and in vivo animal models. Compounds 154, 155, 157 and 158 from Callicarpa arborea showed potent inhibitory effects against the NLRP3 inflammasome by inhibiting Casp-1 activation and IL-1β in reticulum cell sarcoma cells [59].
Clerodane 74-77 and 206 from extracts of Polyalthia longifolia seeds inhibit inflammation, blocking the synthesis of prostaglandins and leukotrienes through highly selective binding to cyclooxygenases (COX) 1 and 2 and 5-lipooxygenase (5-LOX), respectively, compared to the nonsteroidal anti-inflammatory drugs diclofenac and indomethacin [71]. In 2008, clerodane 206 was associated with the suppression of neutrophil respiratory burst and degranulation, and it is thought that it is mediated at least in part by the inhibition of calcium mobilization, AKT (protein kinase B) and p38 mitogen-activated protein kinase pathways [77]. Hautriwaic acid (196) from Dodonaea viscosa leaves, used for rheumatism, exhibited anti-inflammatory activity in a mouse ear edema model [66]. Clerodane compounds 164-175 from Callicarpa hypoleucophylla suppress superoxide anion generation and elastase release, inhibiting the function of human neutrophils [61]. Trans-crotonin inhibits dextran-and histamine-induced oedema [2].

Conclusions
In summary, clerodane diterpenes have activity against different cell cancer lines. Furthermore, some of the diterpenes presented in this review have already-known therapeutic targets, and therefore, their potential adverse effects can be predicted in some way, but the discovery of new compounds and new mechanisms remains to be seen. Anyway, the study of possible new therapies for inflammation continues to be important in order to expand the options for the treatment of inflammatory diseases that afflict the world.
More than 50% of clerodanes included in this review with cytotoxic activity contain acetate groups; on the other hand, 69% of the compounds with anti-inflammatory effects have a ring of lactone.