Biologically Active Diterpenoids in the Clerodendrum Genus—A Review

One of the key areas of interest in pharmacognosy is that of the diterpenoids; many studies have been performed to identify new sources, their optimal isolation and biological properties. An important source of abietane-, pimarane-, clerodane-type diterpenoids and their derivatives are the members of the genus Clerodendrum, of the Lamiaceae. Due to their diverse chemical nature, and the type of plant material, a range of extraction techniques are needed with various temperatures, solvent types and extraction times, as well as the use of an ultrasound bath. The diterpenoids isolated from Clerodendrum demonstrate a range of cytotoxic, anti-proliferative, antibacterial, anti-parasitic and anti-inflammatory activities. This review describes the various biological activities of the diterpenoids isolated so far from species of Clerodendrum with the indication of the most active ones, as well as those from other plant sources, taking into account their structure in terms of their activity, and summarises the methods for their extraction.


Introduction
The name Clerodendrum is derived from two Greek words: kleros (destiny or chance) and dendron (tree) [1]. It probably has a dual meaning: in ancient times, some plants were believed to have healing properties, while others were poisonous [1].
The genus Clerodendrum was first described by Linneus in 1753, and this was followed by the species C. infortunatum [2]. This unusual genus was originally classified into the Verbenaceae family by Liang [3] and Munir [4], among others, but is now included in the Lamiaceae family [2]. It currently includes about 500 species [5] growing in warm temperate and tropical regions of Africa, southern and eastern Asia, as well as America and northern Australia [6]. The genus includes a range of deciduous or evergreen shrubs, small trees, perennial herbs and woody vines [7]; in addition, some species are subshrubs or herbs. Young branchlets are usually four-angled with simple leaves, which are opposite or, rarely, whorled. Inflorescences are loosely cymose or capitate, arranged in terminal or, rarely, axillary paniculate thyrses. The calyx is campanulate or cup shaped. The corolla has a slender tube, five spreading lobes and four stamens. The style has two acute stigmatic lobes. A fruiting calyx is partly inflated. The fruit is a type of drupe and has four one-seeded pyrenes [3].
Diterpenes are natural plant-derived secondary metabolites with the general formula C 20 H 32 . They constitute a chemically diverse group of secondary metabolites which are biosynthesised in the flowering shoots, roots or rhizomes. Such plant diterpenoids most commonly occur in a cyclic form [22]. The diterpenoids are classified into abietane, caurane, caurene, clerodane and labdane types according to their main skeleton [23].
The diterpenes are believed to exert cytotoxic activities through a range of possible mechanisms. Fronza et al. (2012) suggest that they exert their cytotoxic activity by targetting the biological membrane, with its lipophilic character [31]. Other abietane diterpenes could exert their cytotoxicity effects by their alkylating [31] and protonophoric properties [32]. In addition, sphaeropsidin A, a pimarane diterpene isolated from a fungal pathogen, was found to significantly affect cellular homeostasis by modulating the ion-transporter activity of the Na-K-2Cl electrochemical cotransporter or the Cl − /HCO 3− anion exchanger, thus increasing cellular volume [33].
Due to their diverse and often strong biological activities, diterpenoids make an interesting class of natural compounds. There are also attempts to identify new plant sources. A range of reports indicate that the roots and aerial parts of the Clerodendrum genus are rich in abietane-, pimarane-and clerodane-type diterpenoids and their derivatives, including their glycoside forms ( Figure 1). triterpenoids, flavonoids and their glycosylated forms; in addition, various phenylethanoid glycosides, steroids and their glycoside derivatives, cyclohexylethanoids, anthraquinones and cyanogenic glycosides have been noted [12][13][14][15][16][17][18][19][20][21]. Diterpenes are natural plant-derived secondary metabolites with the general formula C20H32. They constitute a chemically diverse group of secondary metabolites which are biosynthesised in the flowering shoots, roots or rhizomes. Such plant diterpenoids most commonly occur in a cyclic form [22]. The diterpenoids are classified into abietane, caurane, caurene, clerodane and labdane types according to their main skeleton [23].
The diterpenes are believed to exert cytotoxic activities through a range of possible mechanisms. Fronza et al. (2012) suggest that they exert their cytotoxic activity by targetting the biological membrane, with its lipophilic character [31]. Other abietane diterpenes could exert their cytotoxicity effects by their alkylating [31] and protonophoric properties [32]. In addition, sphaeropsidin A, a pimarane diterpene isolated from a fungal pathogen, was found to significantly affect cellular homeostasis by modulating the ion-transporter activity of the Na-K-2Cl electrochemical cotransporter or the Cl − /HCO 3− anion exchanger, thus increasing cellular volume [33].
Due to their diverse and often strong biological activities, diterpenoids make an interesting class of natural compounds. There are also attempts to identify new plant sources. A range of reports indicate that the roots and aerial parts of the Clerodendrum genus are rich in abietane-, pimarane-and clerodane-type diterpenoids and their derivatives, including their glycoside forms ( Figure 1). Therefore, this review examines the members of the genus Clerodendrum and their diterpenoid presence, highlighting their biological potential in the area of the most often studied activities, such as cytotoxic, antibacterial, antifungal and others. The chemical structures of the constituents are shown in Figure 2. The present review encompasses the literature data describing the diterpenes present in Clerodendrum from 1981 to 2022. The main sources of literature data were Google Scholar, Google, PUBS ACS, ScienceDirect, Springer, Ebsco and others. Therefore, this review examines the members of the genus Clerodendrum and their diterpenoid presence, highlighting their biological potential in the area of the most often studied activities, such as cytotoxic, antibacterial, antifungal and others. The chemical structures of the constituents are shown in Figure 2. The present review encompasses the literature data describing the diterpenes present in Clerodendrum from 1981 to 2022. The main sources of literature data were Google Scholar, Google, PUBS ACS, ScienceDirect, Springer, Ebsco and others.

A Review of Diterpenoid Compounds Isolated from Clerodendrum Genus
Diterpenoids demonstrate various chemical properties, with a variety of polarities, affinity for the organic phase and solubility. Therefore, the solvent and extraction method must be chosen carefully to optimise the extraction process. The selection of solvent not only depends on the plant species, but also on the organ (overground, underground), and the amount of contaminants, including the presence of chlorophyll. The various solvents and methods used for diterpenoid extraction from Clerodendrum are given in Tables 1-13, together with the parts of the plants used for isolation.
The diterpenes sugiol (18), uncinatone (9) and cyrtophyllone B (21), also isolated from C. cyrtophyllum, have also been identified in Aegiphila lhotzkyan roots. These phytocompounds were tested for antiproliferative activity against leukaemia (CEM and HL-60), breast (MCF-7), colon (HCT-8) and skin (B-16) cancer cell lines in three independent experiments [43]. Of these, only cyrtophyllone B (21) is able to inhibit the proliferation of all tested tumour cell lines; however, it did not demonstrate strong inhibition (IC 50 values above 1 µg mL −1 ) [43]. In addition, diterpenoids isolated from Caryopteris mongolica roots were found to inhibit acethyl-and butyrylcholineesterase (AChE and BChE) [44]. The extraction method used for the phytochemical analyses of this plant species is shown in Table 2.
Another abietane-type diterpenoid is sugiol (18), isolated from Clerodendrum eriophyllum roots. This compound has an oxygen atom connected to the B ring and an aromatic C ring. This unusual aromatic diterpene demonstrates various antioxidant, antibacterial, antiviral, anticancer, anti-tumour and anti-inflammatory activities [55]. Its antioxidant activity is similar to those of α-tocopherol and ascorbic acid based on DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging assay (84% and 82%, respectively) [56]. Sugiol (18) also demonstrates a concentration-dependent inhibitory effect (72.4%) against NO (nitric oxide), at a concentration of 100 µg mL −1 ; it also demonstrated similar superoxide radical scavenging activity at a concentration of 250 µg mL −1 , to ascorbic acid and α-tocopherol activities (73% for sugiol compared to 73% and 74.5%, respectively) [56]. Sugiol (18) is also active against various foodborne pathogenic bacteria but neutralises Gram-positive bacteria more effectively than Gram-negative bacteria. When isolated from Metasequoia glyptostroboides cones, the compound was also found to demonstrate stronger antibacterial action against Gram-positive bacteria than the streptomycin used as a control [57]. Sugiol (18) has also been found to exhibit antiviral activity against the H1N1 virus in infected Madin-Darby canine kidney (MDCK) cells: no cytopathic changes were observed following 72 h of exposure following treatment with 500 µg mL −1 sugiol (18). Hence, sugiol (18) could be a potential antiviral compound that can prevent H1N1-mediated cytopathy in MDCK cells [58].
The diterpenoid sugiol (18) also demonstrated cytotoxic activity against tumour cell lines, inhibiting the growth of three prostate tumour cell lines (LNCap, PC3 and DU145) and a non-tumorigenic cell line (MCF10A) [55]. Similarly, sugiol (18) treatment was found to reduce tumour weight and volume by as much as 75% in mice subcutaneously injected with DU145 cells in comparison with the control group. However, sugiol (18) did not affect the body weight of the mouse [55].
The abietane diterpenoid 6-hydroxysalvinolone (27), containing oxygen and hydroxyl groups in the B ring, demonstrates strong cytotoxicity against carcinoma cell lines. Following isolation from Salvia chorassanica roots, the compound exhibited strong cytotoxic activity against HL-60 and K562 cell lines with IC 50 values of 36.3 and 33.3 µM, respectively. It appeared to demonstrate a substantially less cytotoxic effect on non-cancerous human cell lines. When administered at concentrations of 2.5 and 5.0 µM for 48 h, it also enhanced the expression of the proapoptotic protein Bax, and cleaved caspase-3 and PARP [63]. It also was found to exhibit moderate cytotoxic activity against monkey kidney fibroblasts (VERO) with an IC 50 level of 4.5 µg mL −1 [47]. Similarly to taxodione (24), 6-hydroxysalvinolone (27) also demonstrated antifungal activity, especially against Candida neoformans with an IC 50 value of 0.96 µg mL −1 . In the same assay, the IC 50 of taxodione (24) was found to be 0.58 µg mL −1 , which is comparable with that of standard amphotericin B (IC 50 = 0.44 µg mL −1 ) [47].

Clerodendrum inerme
Studies on the aerial parts of Clerodendrum inerme resulted in the isolation of cleroinermin (32) a neo-clerodane diterpenoid [66] consisting of a bicyclic ring decalin moiety and a six-carbon side chain including a furane skeleton. The compound, first isolated from Heteroplexis micocephala, showed neuroprotective activity against MPP+ induced PC12syn cell damage, with a relative cell proliferation rate of 104.32% [67]. Elsewhere, the neo-clerodane diterpenoids clerodendrin B (33), 3-epicaryoptin (34), clerodendrin C (35), 2-acetoxyclerodendrin B (36) and 15-hydroxyepicaryoptin (37) have since been isolated [68]. The extraction methods used for the phytochemical analyses of this plant species are given in Table 5. These compounds have been found to be effective antifeedants against Earias vitella at 10 µg cm −3 of diet (30 µg g −1 ) and against Spodoptera litura at 10 µg cm −2 of leaf mass [68]. C. inerme has become an interesting subject of research for diterpenoid isolation. The aerial parts are a source of the neo-clerodane-type diterpenoids: clerodermic acid (38), inermes A (39) and B (40), as well as 14,15-dihydro-15β-methoxy-3-epicaryoptin (41) [69,71]. Among these compounds, clerodermic acid (38) deserves special attention due to its strong biological activity. The compound, isolated from the dichloromethane extract of the aerial part of Salvia nemorosa, was found to reduce the viability of A549 cells in a concentration-dependent manner, with an IC 50 of 35 µg mL −1 at 48 h, based on the MTT assay [72]. Furthermore, clerodermic acid treatment resulted in various morphological changes, including diminished cell density, membrane blebbing and an increased number of floating cells, all of them being a manifestation of cell death (38). DNA ladder, DAPI staining, cell cycle analysis, and annexin V/PI testing indicated that clerodermic acid demonstrates strong geno-and cytotoxicity and is able to induce apoptosis in A549 cells, as evidenced also by DNA fragmentation and chromatin condensation [72].

C. infortunatum Aerial parts Leaves
The shade was dried, the crushed aerial parts were exhaustively extracted with n-hexane (3×). The extract was then  The purified diterpenoids, together with their extracts and fractions, also demonstrated insecticidal activity against the highly polyphagous cotton bollworm (Helicoverpa armigera) [77]. The antifeedant activity of the isolated diterpenoids was tested using choice and no-choice tests with 24-and 48-h observation intervals. In the no-choice test conditions, clerodin (45) and 15-methoxy-14,15-dihydroclerodin (46) demonstrated significantly higher antifeedant activity compared to high concentration azadirachtin, the key ingredient in many commercial pesticides [77], with the second diterpenoid demonstrating similar antifeedant activity to that of azadirachtin. In the choice test conditions, all isolated and identified compounds, as well as azadirachtin, demonstrated 100% antifeedant activity at the highest concentration. Furthermore, clerodin (45) has also been found to demonstrate antifeedant activity against Earias vitella and Spodoptera litura [68]. The antifeedant index (AI 50 ) values for clerodin (45), 15-methoxy-14,15-dihydroclerodin (46) and 15-hydroxy-14,15-dihyroclerodin (47) were found to be 6, 6, and 8 ppm in the choice tests, and 8, 9, and 11 ppm in the no-choice tests, respectively.
The antifeedant activity of clerodanes has been attributed to the presence of a perhydrofuranofuran moiety and the degree of its unsaturation; a significant role may also be played by the presence of a trans-decalin ring system bearing an epoxide, together with ac-etate groups [78,79]. These results suggest that the diterpenoids isolated from Clerodendrum infortunatum leaf extract offer promise as biopesticides and require further studies [77].
In addition, 11,14-dihydroxy-8,11,13-abietatrien-7-one (64), an abietane diterpenoid found in Clerodendrum kiangsiense aerial parts, exhibits some interesting biological activities. Costa-Lotufo et al. (2004) found it to demonstrate moderate cytotoxic activity against tumour cell lines, together with as well as carnasol, isolated from Hyptis martiusii roots [88]. Zadali et al. (2020) also reported it to be present in the aerial parts and roots of Zhumeria majdae and to show promising antiprotozoal activity; the IC 50 value was found to be 8.65 µM, with a selectivity index (SI) of 4.6 [89]. Additionally, it has also been found to demonstrate greater binding affinity at the active site of AChE in comparison to donepezil [90].

The Latest Data
Woody branches and healthy stems of Clerodendrum bracteatum were the plant materials used for the extraction, isolation and purification to identify two new abietane diterpenes compounds, which are defined as: (10S,16S)-12,16-epoxy-17(15→16)-abeo-3,5,8,12abietatetraen-7,11,14-trione (103) and 11,14,16-trihydroxy-6,12-dimethoxy-17(15→16)-abeo-5,8,11,13-abietatetraen-3,7-dione (104) [97]. The extraction method used for the phytochemical analyses of this plant species is shown in Table 12. Both phytochemicals have an abeo-abietane structure, the first of which has p-quinone and p-benzoquinone moieties. In compound 104, four methyls, two ketones at C-3 and C-7, as well as the presence of a methine group at C-16 were detected. According to Li et al. (2021), two newly isolated structures (103 and 104) have the strongest antioxidant and cytotoxic activities against HL-60 and A-549 tumour cell lines among seven isolated diterpenes [97].  [97] Interesting new data on diterpenes were published in the work of Qi et al. in 2021 [98]. The authors successfully undertook the extraction of Clerodendrum chinense roots, which resulted in the isolation and identification of 6 new diterpenes: Clerodenoids A-F (105-110) ( Table 13). All of these compounds have an aromatised C ring. It is worth noting that structures 106-108 are the rearranged abietane diterpenoids sharing a 17(15→16)-abeoabietane skeleton, while compounds 109-110 are 17(15→16),18(4→3)-diabeo-abietane moieties. Furthermore, compound 110 has a persubstituted ∆3 double bond and methylhydroxyl function in the A ring. All six newly isolated diterpenes were examined towards antiproliferative activities against HL-60 and A-549 human tumour cell lines [98]. The most active diterpene was compound 110 demonstrating IC 50 values at 1.36 and 1.00 µM against HL-60 and A-549 cell lines, respectively [98]. Clerodendrum infortunatum aerial parts were used for extraction and isolation of terpenoid compounds [76]. Among various known compounds, two previously unknown diterpenes were isolated and identified as (5R,10S,16R)-11,16,19-trihydroxy-  [76]. The extraction method used for the phytochemicals analyses of this plant species is shown in Table 6. Inhibition of converting carbohydrates into monosaccharides is considered to be an adjunct to the treatment of type 2 diabetes. Therefore, it was justified by the authors to investigate this activity among isolated compounds. The isolated secondary metabolites were tested for their ability to inhibit α-amylase and α-glucosidase.  [76]. Additionally, the studied compounds were tested for acethyl-and buthyrylcholinesterase (AChE and BChE) inhibition, showing weak activity against AChE with an IC 50 of 191 and 139 µM, respectively [76].
The diterpenes present in C. inerme [69] were isolated from the dried roots of Clerodendrum bungei crolerodendrum A (43) and B (42) for the first time in this species [20,22].
Compound 42 is known for its antioxidant properties [70] and exhibits significant inhibition against the α-glucosidase enzyme with an IC 50 value of 17 µM [39].
The biological activities of the diterpenoids isolated from the Clerodendrum genus are summarised in Table 14.

Concluding Remarks
The members of the genus Clerodendrum, of the family Lamiaceae, are rich in diterpenoid secondary metabolites, both in the aerial parts and the roots. Due to their moderate, and in some cases strong, biological activities, these diterpenoids are interesting experimental objects. This is particularly true for in vivo pharmacological evaluation. Some of the diterpenes isolated from Clerodendrum spp. are structurally similar to the more highly active phytocompounds; however, they have not been tested for their potential biological activities. This is an important area for further study, as both infectious and civilization diseases, such as cancer, require the search for new therapeutically active structures. The new metabolites obtained from Clerodendrum spp. demonstrate high pharmacological potential, and could be an interesting object of further studies, particularly plant in vitro culture aimed at optimizing the cultivation conditions to increase biomass and secondary metabolite production, especially diterpenes. These biotechnological investigations should determine the effect of culture type (callus, shoot, modified root) and growth conditions such as basal medium and light wavelength, regardless of climatic conditions, season and environmental pollution.
Another interesting area of research concerning the diterpenes from Clerodendrum could be the chemical modification of the isolated phytocompounds. These would include the production of semisynthetic analogues with enhanced biological activities, and improved bioavailability or safety [99,100].
Another equally interesting area of research into these diterpenes is biotransformation [101]. Biotransformation is a very useful tool for the structural modification of natural products with complex chemical structures. Research into the biotransformation of metabolic pathways is essential to understand the potential toxicity and efficacy of new drug candidates and should be a mandatory part of preclinical studies [102].