Euphorbia Diterpenes: An Update of Isolation, Structure, Pharmacological Activities and Structure–Activity Relationship

Euphorbia species have a rich history of ethnomedicinal use and ethnopharmacological applications in drug discovery. This is due to the presence of a wide range of diterpenes exhibiting great structural diversity and pharmacological activities. As a result, Euphorbia diterpenes have remained the focus of drug discovery investigations from natural products. The current review documents over 350 diterpenes, isolated from Euphorbia species, their structures, classification, biosynthetic pathways, and their structure–activity relationships for the period covering 2013–2020. Among the isolated diterpenes, over 20 skeletal structures were identified. Lathyrane, jatrophane, ingenane, ingenol, and ingol were identified as the major diterpenes in most Euphorbia species. Most of the isolated diterpenes were evaluated for their cytotoxicity activities, multidrug resistance abilities, and inhibitory activities in vitro, and reported good activities with significant half-inhibitory concentration (IC50) values ranging from 10–50 µM. The lathyranes, isopimaranes, and jatrophanes diterpenes were further found to show potent inhibition of P-glycoprotein, which is known to confer drug resistance abilities in cells leading to decreased cytotoxic effects. Structure–activity relationship (SAR) studies revealed the significance of a free hydroxyl group at position C-3 in enhancing the anticancer and anti-inflammatory activities and the negative effect it has in position C-2. Esterification of this functionality, in selected diterpenes, was found to enhance these activities. Thus, Euphorbia diterpenes offer a valuable source of lead compounds that could be investigated further as potential candidates for drug discovery.


Introduction
Euphorbia species have a rich history of ethnomedicinal use, as well as ethnopharmacological applications in drug discovery from ancient times to the present [1][2][3]. Plants of the Euphorbia genus are popular medicinal herbs applied in the prevention and treatment of diseases, like respiratory diseases, body/skin pain and irritations, indigestion disorders, inflammation, cancer, microbial infestations such as HIV, and gonorrhea, and eye disorders [2,4,5]. As early as the era before Christ (BC), Euphorbia species were utilized in the treatment of liver diseases, snake bites, sprains, convulsions, asthma, tumors, and rheumatisms in the Ayurvedic and Chinese medicine systems [1,2,4,6].
Reported evidence shows that medicinal usages of Euphorbia species are recorded worldwide and utilize the stems, stem barks, whole plant, latex, aerial part, seeds, leaves, and roots, with E. lathyris, E. maculata, and E. hirta as the most frequently used species [1,2,7]. Their classification, chemistry, and medicinal applications are ascribed to the presence of many phytochemical constituents, such as terpenes [6,[8][9][10]. Therefore, Euphorbia species remain a rich source of diverse natural products, with a wide range of pharmacological applications that can provide promising lead compounds for drugs and therapeutic agents' discoveries. The genus Euphorbia is amongst the largest genera in the Euphorbiaceae family of flowering plants, consisting of several sections and subgenera with over 2000 species [9,11,12]. Thus it is complex, and of immense research potential.
The genus Euphorbia is also known for the structural diversity of its isoprenoids, with most of them being macrocyclic diterpenes [9,13,14]. These diterpenes are the major chemical constituents in the genus and are known to occur in different types of core skeletal structures/frameworks, such as abietanes, tiglianes, ingenanes, daphnanes, lathyranes, jatrophane, myrsinols, and cembranes [10,14,15] amongst others. Jatrophanes, tiglianes, and lathyranes type diterpenoids are the main chemical constituents reported in the genus [9,13]. As a consequence, significant efforts have been made in the isolation and identification of these chemical constituents from the roots, aerial, stems, seeds, stem barks and whole plants of Euphorbia species.
In addition, most of the reported Euphorbia diterpenes exhibited a wide range of pharmacological activities such as cytotoxic, anti-inflammatory properties, anti-HIV, tumorpromoting abilities, and antibacterial properties [9,10,13,14]. As a successful example of drug development from naturally derived diterpenes, taxol and taxol derivatives are presently in wide use for cancer treatment [10]. Furthermore, the latest release of ingenol metabutate (picato) a diterpene isolated from E. peplus for treatment of actinic keratosis [5,16] has revitalized interest in the phytochemistry of Euphorbia species.
Diterpenes occurring in Euphorbia species are classified as either higher or lower diterpenes, and have diverse skeletal frameworks such as tigliane, ingenane, and daphnane [9,13]. Lower diterpenes are limited to the Euphorbiaceae and Thymeleaceae families. Euphorbia diterpenes can therefore offer better alternatives for the development of more selective and high potency prodrug derivatives based on their structure-activity relationships.
The different skeletal frameworks of Euphorbia diterpenes are derived from geranylgeranyl pyrophosphate (GGPP) and are subsequently categorized according to their biosynthetic pathways and cyclization patterns as acyclic; like phytanes, bicyclic; like labdanes, clerodanes and halimanes, tricyclic; including abietanes, rosanes, pimaranes, and cassanes, tetracyclic; like kauranes, atisanes and gibberellins and macrocyclic diterpenes including taxanes, daphnanes, cembranes, ingenanes and tiglianes [14,[17][18][19]. The detailed information about the biosynthetic pathway and classification of Euphorbia diterpenes is not dealt with within this review article and the reader can consult the references for detailed information.
That said, several review publications have summarized scientific reports about the phytochemical constituents of Euphorbia species. Most of the published reviews exclusively focused on partial studies about the chemical constituents, biological activities, and synthesis. For instance, Goel et al. [20] reviewed esters of phorbol highlighting the structural diversity, mode of action, toxic effects in animals, and how the compounds can be detoxified from the Jatropha species, of the Euphorbiaceae family. Shi et al. [13], reviewed the chemical and pharmacological activities of Euphorbia species chemical constituents covering the period 1998 to 2008. Previously undescribed diterpenes and common diterpenes isolated within the review period (1998)(1999)(2000)(2001)(2002)(2003)(2004)(2005)(2006)(2007)(2008) were discussed in the review. Vasas and Hohmann [9] reviewed the Euphorbia diterpenes isolated from Euphorbia species between 2008 and 2012, highlighting their structural diversity and pharmacological activities [9]. Different classes of Euphorbia diterpenes and biological activities reported within this period were reviewed. Wang et al. [21] reviewed the tigliane-type diterpenoids from the Euphorbiaceae and Thymelaeaceae families and their biological activities. Wongrakpanich et al. [22] reviewed the induction of apoptosis in cancer cells by chemical constituents isolated from Euphorbia species. Jin et al. [14] reported daphnane-type diterpenes of the Euphorbiaceae and Thymelaeaceae families and their pharmacological activities, while Salehi et al. [23] reported the essential oil constituents of Euphorbia species. In addition, in our previous study we reviewed the ethnomedicinal uses, triterpenoids of Euphorbia species, and their pharmacological activities [24].
In most of the reviewed publications, emphasis was given to a specific subclass of diterpenes isolated in Euphorbia species, their chemical structures, and reported biological activities within the review period (1998)(1999)(2000)(2001)(2002)(2003)(2004)(2005)(2006)(2007)(2008)(2009)(2010)(2011)(2012)(2013)(2014) with limited reference to the structureactivity relationship of these constituents, and that there is no comprehensive review of previously undescribed Euphorbia diterpenes covering the period from 2013-2020. Hence, to gain a more comprehensive insight on the latest information about the structural diversity of Euphorbia diterpenes, the current review reports the structures, occurrence, classification, and pharmacological activities of newly isolated Euphorbia diterpenes, their mechanisms of action, and the structure-activity relationships reported between June 2013 and October 2020. It is hoped that the review on the Euphorbia diterpenes will enrich the existing databases with the latest information about the structural diversity of Euphorbia diterpenes and their structure-activity relationships, which will help in identifying potential hit or lead compounds for drug discovery.

Literature Sources and Search Strategy
To gather all the relevant information about Euphorbia diterpenes, their pharmacological activities, and structure-activity relationships, the following online databases were used; Scifinder, Scopus, Springer Link, Science Direct, Wiley online, PubMed, Google Scholar, and Web of Science. The databases were systematically searched for articles published from 2013 until 2020. The syntax TITLE-ABSTR-KEY (title-abstract-keyword) was used in combination with keywords like 'Euphorbia', OR 'genus' OR 'Euphorbiaceae', OR 'diterpene compounds' OR 'Euphorbia diterpenes' OR 'macrocyclic diterpenes', OR 'tigliane' OR 'jatrophane' OR 'lathyrane', OR 'abietane' OR 'kaurane' OR 'atisane' OR 'biological studies' OR 'structure-activity relationship' OR 'anticancer activity' OR 'antibacterial', OR 'anti-inflammatory'. The search terms were run separately or as limited combinations depending on the database used. In addition, a plant-list database was used to ascertain the correct names of the species. The search strategy was limited to English-language publications and excluded research articles still under consideration for publication or not yet available in the databases. The search was restricted to between 2013 and 2020. The retrieved information was checked, critically read, and searched for descriptions of previously undescribed diterpenes, occurrence, structures, the biological activities, biosynthetic pathway, and the structure-activity relationships. Additional information was obtained by reviewing the listed references in the selected articles.

Occurrence of Euphorbia Diterpenes
The isoprenoid constituents of Euphorbia species are very diverse. Over the last decade, phytochemical investigations of the roots, stems, stem barks, aerial, seeds, and whole plant extracts of Euphorbia species led to the isolation and structural identification of a wide range of diterpenoids. Within this time frame, over 350 (1-382) newly isolated diterpenoids, possessing different skeletal frameworks, were reported. At the same time, the compounds showed significant pharmacological activities. Over thirty Euphorbia species presented in this review were reported to contain these diterpenes. Furthermore, diterpenoids bearing new skeletal structures were described, and their biosynthesis was proposed. The newly reported diterpenoids structures led to new information about their biological activities and their biogenesis in plants. Plants newly investigated within this time frame were E. kopetdaghi [25], E. sanctae-catharinae [26], E. gaditana [27], E. saudiarabica [28], and E. glomerulans [29].
Euphorbia diterpenoids described for the first time possessed the parent skeletal structures, only differing by the type of substituent attached to the parent framework. Other diterpenoids, previously not described and classified in the genus, were isolated, such as meroterpenoids [30]. The structural diversity of the isolated diterpenoids and their analogs further enabled the studies of the structure-activity relationship to be conducted. From the findings, it was established that the existence of the hydroxyl group in some diterpenoids was essential for the activity, as it had both positive and negative effects. Esterification of the hydroxyl functionality in some of the diterpenes analogs was found to enhance their efficacy activities. These studies are important as they give vital information on pharmacophoric elements of diterpenes as promising lead compounds for drug discovery. It is also noteworthy that most of the diterpenoids were isolated from methanol and ethanol extracts of the roots, stems, aerial, stem barks, seeds, and in some cases the whole plant materials, of less than fifty species of the over 2000 species of Euphorbia species.

Higher Diterpenes
Euphorbia diterpenes are classified as higher diterpenes and lower diterpenes [9,13]. Higher diterpenes are not specific to the Euphorbiacaeae family, as they occur in many other plant families as well [76]. The skeletal structures of these diterpenes involve the cyclization of the precursor to yield different cyclized diterpenes including bicyclic labdanes, clerodanes, tricyclic abietanes and tetracyclic kauranes, atisanes, and bayeranes [14,[17][18][19]. In this review we have collected information about the occurrence, isolation, structure and biological activities of Euphorbia diterpenes between the years 2013 and 2020, as summarized in Table 2
Diterpenoids possessing rare or unusual skeletal structures were reported in Euphorbia species. For instance, chemical analysis of ethanol extracts of E. ebracteolata resulted in the isolation of ebraphenol A-D (332-335) [48] alongside ebralactone A (336), which were found to have a rosane skeletal structure with an uncommon aromatic C ring [48]. The previous unreported diterpenoids, considered as 18 (or 19)-norditerpenoids of the entisopimaranes skeleton, were isolated for the first time from E. neriifolia [73]. Furthermore, an unusual tetracyclic diterpenoid named eupholathone (343) [66] was isolated from E. lathyris and described for the first time in the genus. Besides, previously undescribed euphnerin A (337) and euphnerin B (338) [71] isolated from the stems of E. neriifolia were found to possess a spirocyclic carbon skeleton rarely found among rosane diterpenoids. This was proposed to be occasioned by rearrangement reactions. Interestingly, euphominoid E (73) [71], isolated from the same species, was found to co-occur with euphnerin A (337) [71] and euphnerin B (338) [71] as they had the same B/C ring systems. Based on the observations, euphominoid E (73) was postulated to be the precursor for the biosynthesis of euphnerin A (337) and euphnerin B (338) [71].
Within the wider Euphorbiacaeae family, other genera such as Excoecaria, Sapium, Isodon, Xylopia, and Spiracea are important sources of these diterpenes [89]. It is noted that the oxidation patterns observed of the isolated ent-atisanes varied distinctively with producing genus. This suggests the non-uniform expression of enzymes responsible for their biogenetic pathway across the genera. For instance, all the ent-atisane diterpenes derived from Elaeoselium and Isodon genera have oxidation patterns at C-16 and C-17. In contrast, Xyopia ent-atisanes diterpenes possess a C-16 tert hydroxyl groups while Euphorbia and Sapium ent-atisanes have unsaturated C-12. Besides, all reported 3,4-seco atisanes were isolated from Euphorbia, Excoecaria, Croton, and Sapium genera of the Euphorbiacaeae family [89]. Furthermore, daphane diterpenes were reported in Euphorbiacaeae and Thymelaeceae families [14]. Ent-kauranes and labdane diterpenes were reported in Rabdosia (Lamiaceae) [96], while abietanes were isolated from Toxodium (Toxodiaceae) species [97].

Lower Diterpenes
Lower diterpenes are macrocyclic diterpenes and their cyclized products. They are derived from a geranylgeranyl pyrophosphate precursor in a 'head-to-tail cyclization [19,98]. The different functionalization of these diterpenes proceeds via cyclization. Macrocyclic diterpenes are characteristic compounds of the Euphorbiaceae and Thymelaeaceae families and are used as chemotaxonomic markers. In this study both macrocyclic and polycyclic diterpenes (1-382, Table 2) were reported within the review period.

Ingenanes
Ingenane-type diterpenoids are characterized by a tetracyclic ring system of 5/7/7/3and having a ketone bridge between C-8 and C-10 in addition to β-hydroxylated at C-4. The rings A and B are usually trans-fused and have a double bond between C-1 and C-2 in ring A, and between C-6 and C-7 in ring B. The carbons; C-3, C-5, C-13, C-17, and C-20 positions, in most cases, are oxygenated or esterified [9].

Jatrophanes and Modified Jatrophanes
Jatrophane and modified diterpenes occur mainly as polyesters in the Euphorbiaceae family. These macrocyclic diterpenes are based on a bicycle [10.3.0] pentadecane skeleton and without a cyclopropane ring. The structural diversity of jatrophane diterpenes is based on the number and positions of the double bonds within the ring, nature, and the number of oxygen functionalities, and the structural configuration of the core skeletal framework. The oxygen functionalities vary from hydroxyl, epoxy, keto, ether, to ester groups [9,19]. As a result, they occur as modified jatrophanes and have shown interesting pharmacological activities.
Tiglianes (phorbol esters) are common to Euphorbiacaeae and Thymelaeceae families. Within the Euphorbiacaeae family, several genera such as Excoecaria, Croton, Jatropha, Ostedes, and Sapium were also reported to contain these diterpenes [89]. In the Thymelaeceae family, phorbol esters were reported in Pseudomyrmex and Danae genera [58].
Chemical analysis of the E. micractina roots extracts yielded a previously undescribed minor diterpenoid, named secoeuphoractin (379) [67], which had a new carbon skeleton framework [67]. From the same species, a new diterpenoid with a unique 6/5/7/3 fused-ring skeleton structure named, euphorbactin (380), was isolated and described. This skeletal structure had not been previously identified [104]. Phytochemical investigations of the aerial extracts of E. kopetdaghi yielded previously undescribed cyclomyrsinol diter-penoids, named kopetdaghinane A (381) and B (382) [25], which were found to possess an oxidation pattern of a new tetrahydrofuran pattern having a hemiacetal group. This was the first report of cyclomyrsinol diterpenes from this species. The above accounts show, the abundance and structural diversity of novel diterpenoids from Euphorbia species yet to be discovered that can provide a wide range of potential lead compounds that can be harnessed by pharmaceutical companies for drug discovery.

Pharmacological Activities and Structure-Activity Relationship (SAR)
Due to the ethnomedicinal usage of Euphorbia species in the prevention and treatment of various ailments, and the structural diversity of isolated compounds, different publications reported various biological studies. Analysis of the reported biological studies revealed that most of the publications explored cytotoxic effects and anti-inflammatory activities [10,13,108]. This was followed closely by the chemoreversal studies. Most of the studied species reported new bioactive diterpenes, particularly as anticancer agents.
Antibacterial and antimalarial biological activities were the least studied, while a significant number (5%) of isolated diterpenes were not evaluated ( Figure 16). Many of the reported biological studies used appropriate controls while few studies lacked information about the controls used. The structure-activity relationship (SAR) of these diterpenes revealed that acetylation and esterification of hydroxyl groups, particularly at C-3 and C-8, have a positive effect on these activities.
The antiproliferative activities of previously undescribed tigliane diterpenoids isolated from E. fischeriana were evaluated in vitro against human gastric cancer cell lines (AGS) and human liver cancer cells (Hep-G2) using cell counting kit-8 (CCK-8) assay. These diterpenes exhibited potent activities against AGS cells. Among the diterpenoids, prostratin 20-O-(6acetate)-β-D-glucopyranoside (339) [63], and fischeroside A (340) [63] [63] Moreover, cytotoxic activities of fischernolides A-D (311-314) isolated from E. fischeriana, against five human cancer cell lines using the MTS method, showed that fischernolide B (312) [30] and fischernolide D (314) [30]  In related studies, previously isolated compounds; 12-deoxyphorbol esters, 12-deoxyphorbol-13-acetate (prostratin), 12-deoxyphorbol-13-hexadecanoate, and 12-deoxyphorbol-13-(9Z)-octadecanoate-20-acetate from E. fischeriana were evaluated for their cytotoxicity against Ramos B cells. The results showed that 12-deoxyphorbol 13hexadecanoate, having a long acyl chain at C-13 and a free hydroxy at C-20, exhibited promising cytotoxic activity against Ramos B cells with an IC 50 value of 0.0051 µg/mL. The findings suggested that the presence of saturated aliphatic acyl group at C-13, and a carbonyl at C-3 and free hydroxyl at C-20, were important to the cytotoxic activity against Ramos B cells of these compounds [58]. Furthermore, evaluation of the cytotoxic activities of 6α,7α-epoxy-5β-hydroxyphorbol ester isolated from Excoecaria acerifolia of Euphorbiaceae family against five cancer cell lines (HL-60, SMMC-7721, A-549, MCF-7, and SW480) showed significant IC 50 values in the range of 7.62−10.87 µg/mL [58]. From the findings, it was inferred that the type of aliphatic long-chain acyl group at C-12 or C-13, a trans-fused A/B ring system, a 6,7-olefinic group, and free C-20 hydroxyl, was important to the anticancer activities of these diterpenes. Furthermore, it was evident that the main active groups present in tigliane type diterpenes with promising anticancer activities were generally like those in related diterpenes displaying tumor-promoting activities [110]. The type and nature of the long-chain acyl groups are important to their anticancer activities. In general, the high activities of these diterpenes toward the cancer cells were attributed to the 6,7-olefinic, 3-carbonyl and the acyl groups attached to the skeleton [58].
Meng et al. [65] examined the antiproliferative activities of ingenane and jatrophane diterpenoids, isolated from E. kansui against human hepatoma cancer cells ( This further suggested that the 3,4-(methylenedioxy) cinnamyl group could be responsible for the bioactive activities [65]. These observations agreed with Kulig et al.'s [111] assertion that vicinal diol groups contribute significantly to the bioactivities of naturally occurring compounds possessing them. Other studies on the structure-activity relationship of jatrophane diterpenoids showed that substitution of a benzoate at C-8 and C-9 are favorable, while isobutanoyloxy group substitution at C-3 increased the observable effects on human lymphocytes deoxyribonucleic acid (DNA) [112].
Structural modification of these constituents by esterification of hydroxyl groups revealed that the 5-O-acetyl derivative presented triglyceride-lowering abilities with an EC 50 value of 0.61 µM. Structure-activity relationships showed that the trans-fused 5/7/6 ring system occurring in an angular shape was relevant to these activities [37]. In addition, the presence of a cyclopropane ring, an isopropyl substituent, and cyclobutane ring on these diterpenes did not have an effect. A nicotinoyl group at C-3 was also found not to be favourable, as derivatives with this functionality recorded poor activities. Equally, the availability of a free hydroxyl group at C-8 was found to be beneficial to the activity of these compounds, while acylation of 8-OH resulted in decreased activities. Tigliane diterpenoid, 12-O-benzoyl-13-O-[2-methylpropanoyl]-4,20-dideoxy-5-hydroxyphorbol, an acetylated derivative of phorbol exhibited promising lipid-lowering activity, with an EC 50 value of 0.32 µM and selectivity index of IC 50 /EC 50 > 312. The SAR studies showed that phorbol derivatives, bearing a trans-fused 5/7 ring system, presented significant activities compared to those possessing a cis-fused system, indicating that the trans-fused system of 5/7 ring was beneficial or had a positive effect on the activities of tigliane diterpenes [37].
In a related study, Zolfaghari et al. [113] evaluated the potential cytotoxic activities of previously described cyclomyrsinanes and premyrsinane against EJ-138 bladder carcinoma and Jurkat T-leukaemia cell lines in vitro. Most of the tested compounds showed promising activities against EJ-138 (A) and Jurkat T cells (B), with IC 50 values ranging from 33.31-15.3 µM against EJ-138 and 21.10-12.3 µM against Jurkat T cells, using doxorubicin as a positive control. The structure-activity relationship of the cyclomyrsinanes diterpenes revealed that their activities were modulated by the position of the substituents. In particular, substituents at C-8 had a positive influence and the activity increased with the length of the acyl chain (MeiPe > MeBu > iBu) increased [113].

Multidrug Resistance Activities
All diterpenoids isolated from E. royleana were evaluated for their chemoreversal activities against multidrug-resistant (MDR) liver cancer cells with doxorubicin (Hep-G2/DOX). All the compounds recorded weak cytotoxicity with IC 50 of less than 50.00 µM against liver cancer cell lines (Hep-G2) and Hep-G2/DOX cell lines, using verapamil (Vrp; 10.65 µM) and tariquidar (Tar; 2.31 µM) as positive controls [33]. With cognizance of the fact that expression of P-glycoprotein (P-gp) is the basis for multidrug mechanisms, exprimentatoin expressing P-gp in Hep-G2/DOX cells was further conducted. The results showed a significantly high expression of P-gp in the MDR cell line, compared to the parental cell line [33]. Hence, it was deduced that the multidrug (MDR) mechanisms of the lathyranes diterpenoids could be related to the modulation of the P-glycoproteins (P-gp) by down-regulation of protein expression or by blocking of their functions. It was also found that all the isolated diterpenoids inhibited the transport activities of P-glycoproteins (P-gp), rather than its expression, when tested for their effects on the expression of P-glycoproteins (P-gp) in cancer cells with doxorubicin (Hep-G2/DOX) [33].
Evaluation of multidrug resistance (MDR) reversal ability of isolated ingol diterpenoids from E. marginata against cancer cell line Hep-G2/ADR (Pgp-dependent) showed no significant cytotoxicity activities, with IC 50 values of less than 50 µM, compared to anticancer drug adriamycin (ADR) as the positive control. Euphornans A-N (142-155) [41] showed greater reversal activities compared to verapamil, the positive control. Euphornans, K (152), N (155), and R (159) [41], recorded better activities than tariquidar (IC 50 > 25 µM at 5 µM), using adriamycin as a control, and were further investigated for dose-effect relationships. The compounds (Euphornans; K (152), N (155), and R (159)), exhibited better dose-dependent activities and were found to reverse the sensitivity of adriamycin, the cancer drug, to 20-fold, at a concentration of 5.00 µM. In P-gp modulation-mechanism analysis, it was further established that the compounds reverse the sensitivities of multidrug (MDR) cancer cell lines by the inhibition of the P-glycoprotein (P-gp) [41].
Due to the various substitutions patterns in isolated ingol-type diterpenoids, structureactivity relationships were investigated. It was established that acetylation of the hydroxyl group at C-3 and C-8 improved the anticancer activities. In particular, the acylation of hydroxyl groups (OH-3 and OH-8) improved the activity, as shown in ingol-3,7,12-triacetate-8-benzoate (262) (IC 50 [33]. Molecular mechanisms of these diterpenoids and P-glycoprotein (P-gp) were further explored by in silico analysis. All the compounds were found to dock well in the transmembrane domain (TMD) of P-gp. Formations of three hydrogen bonds between 8-OBz and Gln-990, and Tyr310 and between 3-OAc and Tyr953 were observed. It was also found that their core structures formed hydrophobic forces between the aromatic moieties and hydrophobic residues of the transmembrane domain (TMD) pocket that favoured the binding. It is this binding that was used to explain the structure-activity relationships (SARs) of the isolated lathyranes diterpenoids [33]. Equally, molecular docking experiments of the lathyrane diterpenoids presented lower binding energies, compared to the positive controls, adriamycin, and verapamil. The data further established that the isolated lathyrane diterpenes could act as a substrate of high-affinity, P-glycoprotein (P-gp) which is effluxed with its monomer to reverse multidrug resistance (MDR). Hence, the MDR-reversal activities of these diterpenes were postulated to occur via two strategies. The main strategy was by maintaining the chemotherapeutic drug concentrations as high as possible, by suppressing overexpression of P-glycoprotein (P-gp) in the MDR cells. The second strategy involved reducing the efflux of P-glycoprotein (P-gp)-regulated drugs or chemotherapeutics. In this model, compounds (diterpenes) were found to replace the chemotherapeutic drugs as the P-glycoprotein (P-gp) efflux substrate. The findings showed that lathyrane diterpenoids are good (P-gp) efflux substrates with high affinity. Hence, they were praised for their ability to suppress the overproduction of (P-gp) in multidrug cell strains and could be potential candidates for cancer agents [106]. Chemical modification of the diterpenoids presents promising multidrug resistance modulators.
In summary, the bioactivities of jatrophane and lathyrane diterpenoids can be increased by acylation and esterification of the hydroxyls groups, which subsequently im-proves the hydrophobicity with the P-glycoprotein (P-gp) inhibitor. For instance, esterification of the hydroxyl group at C-3 and C-8 were vital for the activities of lathyranes diterpenoids, as the presence of hydroxyl groups was found to decrease activity due to the interference of hydrogen bonds. Likewise, diterpenoids having a benzoyl group at C-7 or C-8 displayed higher activities compared to those with tigloyl, angeloyl, and MeBu groups, as observed in some diterpenoids. This could be due to the interaction of the π electrons in the phenyl ring of the 8-OBz with the hydrophobic pockets favoring the binding [33]. Similar observations were made in ingol diterpenoids, isolated from E. marginata. Esterification of the C-OH by acylation was found to enhance the activities, as observed in euphornan B (143) and G (148), euphornans J (151), and O (156), euphornans K (152) and P (157) [41]. Acylation of the C-7 hydroxyl group was found to reduce activity, as observed in euphornan F (147) and B (143), as well in euphornan N (155) and J (151). In contrast, the substitution of OH-7 with benzoyl displayed better activity than when substituted with acetyl, as observed in euphornan K (152) and euphornan I (150) [41]. Yet, the replacement of nicotinoyl by benzoyl at the hydroxylated C-9 increased activity remarkably [41].
Likewise, evaluation of the multidrug resistance (MDR) activity of jatrophane diterpenoids, from E. esula, against cancer cell lines that are dependent on P-glycoprotein (Hep-G2/ADR), showed comparable activities to adriamycin (ADR), the positive control drug. Most compounds did not show obvious cytotoxicity in Hep-G2/ ADR cell lines, with IC 50 values less than 50.00 µM. However, euphoresulane H (212) [59] was the best multidrug resistance (MDR), modulator with IC 50 of 165.30 µM, compared to ADR (IC 50 of 284.50 µM), and was established to further enhance the anticancer activities of adriamycin by 33-fold at 5.00 µM. Hence, euphoresulane H (212) was further studied for a dose-effect dependence and ireported good dose-dependent activities, as it enhanced the activities of adriamycin (ADR) by 33-fold at 5 µM [59]. The cytotoxic evaluation of jatrophane diterpenoids isolated from the acetone extracts of E. glomerulans on multidrug-resistant breast cancer cells (MCF-7/ADR) was found to overexpress the P-glycoprotein (P-gp) with varying chemoreversal abilities and with reduced cytotoxicity activity. Euphoglomeruphane K (233) and L (234) showed better MDR reversal activity, with IC 50 values of 5.00 µM and 5.10 µM, respectively, compared to verapamil, the positive control, with IC 50 value of 4.70 µM [29].
The different substitutions patterns of the isolated jatrophane diterpenoids formed the basis for further evaluation of their structure-activity relationships. It was established that the existence of a keto carbonyl at C-9 in euphoresulane J-M (214-217) (IC 50 > 100 µM; ADR; IC 50 = 284.50 µM) [59] adversely affected their activities. It was noted that the existence of the acetoxy group at C-15 resulted in better activities in compounds bearing the acetoxy group than those with free hydroxyl at this position. It was also established that the acylated group at C-9 enhanced activity. Nonetheless, compounds with 9-OBz showed better activities than those with 9-OAc, as observed in euphoresulane F (210) (IC 50  in euphoresulane F (210) and euphoresulane G (211). Taken together, it was deduced that the acyloxy substitution at C-9 in jatrophane is essential to its activity, while the existence of C-OH enhances activity [59].
The biological evaluation of these jatrophanes and modified jatrophanes showed the significance of substitutions at C-3, C-6, and C-15, in addition to the configuration of the hydroxyl group. For instance, substitution at C-6 was found to affect the inhibitory activities in a way that was dependent on the position of the free hydroxyl group, while substitution of benzoyl and propyl at C-9 and C-3 reported positive inhibitory activities. Furthermore, jatrophanes possessing acetyl at C-8 and nicotinyl at C-9 reported significantly higher activities. These observations showed that jatrophanes and the modified jatrophanes possess common pharmacophoric elements that affect their activities as in Figure 17 [114,115].
Euphorin A (37) and euphorin B (38), from E. antiquorum, displayed inhibition of NO production in BV-2NO with IC 50 value of 35.80 and 41.40 µM compared to 2-methyl-2thiopseudourea, sulphate (SMT) (4.2 µM) [56], while diterpenes (39) and (40) [75]. Previously, evaluation of the water fraction of E. royleana latex displayed dose-dependent anti-arthritic and anti-inflammatory activities in acute and chronic test models in mice and rats. Further studies showed that it reduced the migration of leukocytes and had poor inhibitory effects on the granuloma formation induced by cotton pellets. The ethyl acetate fraction on the other hand showed dose-related peripheral analgesic effects [116]. These findings support the use of E. royleana as an analgesic in traditional medicine. These effects could be due to the presence of ent-isopimaranes diterpenoids. Ebraphenol A-D (332-335) and ebralactone A (336), from the root extracts of E. ebracteolate, showed high lipase-inhibitory activity, with IC 50 values of between 1.0 and 24 µM, compared to lovastatin positive control; IC 50 = 0.24 µM [48].
In another structure-activity relationship study by Wang et al. [31], the previously isolated lathyrane diterpenoids with anti-inflammatories named euphorbia factors L 2 (247), L 3 (248) [31], were found to reduce the formation of inflammatory factors and decreasing the expression of nuclear factor kappa B (NF-κB). They further investigated the influence of substituted benzoic acid, cinnamic acid, and other heterocyclic acids through esterification reactions on the anti-inflammatory efficacy of the analogs. The results showed that, when the hydroxyl group on C-7 of the euphorbia factors L 3 (248) was esterified, many of the yielded intermediates exhibited weaker inhibitory activities compared to the parent compound. This was an indication that the hydroxyl group on C-7 is essential in retaining the anti-inflammatory activities of euphorbia factors. However, when the hydroxyl was esterified using fatty acids like nicotinic acid and glycine, the yielded derivatives displayed better inhibition activities. While isonicotinic acid derivatives showed poor inhibition activities. This suggested that the anti-inflammatory activities of lathyranes diterpenoids could be increased by esterification [31].
This was also evident when the hydroxyl on C-5 was esterified. When the hydroxyl groups on C-3 and C-5 were esterified simultaneously, the observed activities were found to be higher. It was also established that compounds with aromatic groups exhibited high efficacy than those with aliphatic substituents [31]. Interestingly, when the substituents of the benzene were changed or when the ring was converted into a heterocyclic ring, the inhibition activities of these compounds were weakened. Also, the presence of an electron-donating group on the benzene ring was found to weaken anti-inflammatory activity more than when an electron-withdrawing moiety was attached [31]. It was further shown that lathyrane diterpenoids with an exocyclic ∆ 6(17) double bond presented higher inhibitory activities than those with a 5α, 6β-epoxy or ∆ 5(6) double bond. In addition, a substituted aromatic moiety at C-3 and nitrogen-containing aromatic substituent at C-7 were essential for retaining the inhibition of NO production [103]. Hence, it was concluded that Euphorbia lathyrane diterpenoids present good scaffolds for structure modification concerning drug discovery.
All The structure-activity relationships (SAR), revealed that most of the euphoesulatins A-L (184-193) [100] possessing a double bond exhibited stronger activities with IC 50 values of less than 10 µM, while some showed weaker activities with IC 50 values of >10 µM. Substitution of hydroxyl at C-15 with an acetoxy group was found to increase the activities in euphoesulatin A (184) registering IC 50 of 1.20 µM, compared to euphoesulatin B (185) with IC 50 of less than 10 µM, and for euphoesulatin H (191), with IC 50 value of 3.50 µM compared to euphoesulatin I (191), with IC 50 value of more than 10 µM [100]. It was observed that the presence of a hydroxyl group at C-5 destroyed these activities.
The antiosteoporotic activities of compounds having a double bond and a hydroxyl group at C-2, with identical structures other than the substituents at C-11 and C-12, showed increased activity. For instance, euphoesulatin E (188) compared to euphoesulatin N (197) ( [34], and euphoesulatin H (191) compared to euphoesulatin M (196) [34]. Replacement of the ∆ 11 (12) double bond with an epoxide resulted in increased activity. In contrast, euphoesulatins having an epoxy group and a 2-OH substituent recorded decreased or no activitys. For euphoesulatins, having a ∆ 11, 12 double bond in addition to a 2-OH functionality, either an 8-OH as in esulone B (202) or a 15-OH as in esulone A (204), resulted in no activity. This is an indication that higher numbers of hydroxyl groups does not translate to enhanced bioactivities. The SAR of the jatrophane diterpenoids supported the fact that a ∆ 11 (12) double bond retains their activities and that the higher number of hydroxyl groups does not enhance antiosteoclastogenesis [100].

Anti-HIV Activities
Twelve ent-isopimarane diterpenes isolated from stem barks of E. neriifolia were evaluated in vitro for the anti-HIV properties in HIV-1 NL4-3 infected MT4 cells, with zidovudine (AZT) as the positive control. All the tested compounds showed significant anti-HIV activities. Eupneria J (42) and eurifoloid H (24) [69] reported potent activities, with IC 50 values of 0.31 µg/mL and 6.70 µg/mL, respectively, while others showed insignificant activities with an IC 50 value of fewer than 25.00 µg/mL. Further investigation of the structure-activity relationship (SAR) of the eupneria J (42), eupneria K (43), eupneria M (45), eupneria P (48), and oryzalexin F (50) [73] presumed from the observations that βoriented hydroxyl group at C-4 could be linked to their activity. The comparative analysis of the SAR of eupneria O (47), eurifoloid I (49), and eurifoloid H (24) revealed that the acetoxy group at C-18 contributes to the anti-HIV activities, rather than at C-3 [69,70].
In another study, the phytochemical analysis of E. lathyris ethanol crude extracts resulted in the isolation of ingenane and lathyrane type diterpenoids. All the isolated compounds (282-295) [31] were evaluated for their anti-HIV activities against HIV-1 and MT4 cells. None of the tested compounds showed anti-HIV activities compared to zidovudine positive control, nonetheless, the ethanol crude extracts showed significant activities with an EC 50 value of 0.33 µg/mL against the HIV-1 [31] This showed that the compounds were potent due to synergy. Analysis of isolated diterpenoids from E. neriifolia for ant-HIV activities revealed that ent-16α,17-dihydroxyatisan-3-one, and eurifoloid R showed potent anti-HIV-1 activities with EC 50 values of 6.32 µg/mL and 6.45 µg/mL respectively [69,70]. In related studies, two ent-atisanes, including ebractenone A and bractenone B possessing a rare 2-oxopropyl moiety, displayed good antiviral activities against human rhinovirus 3, with an IC 50 value of 25.27 µM [117].

Melanin Synthesis
Biological studies on six new lathyrane, ent-abietane and known ingenols diterpenoids, from E. antiquorum, revealed that ingenol diterpenoids had better activities on melanin synthesis. Among them, euphonoid A (117), euphorantin I (123), and euphorantin J (127) [34] displayed better inhibition abilities of 124.38%, 203.11%, and 177.43% as compared to the positive control (8-MOP; 124.38%) at 50 µM. The ingenol diterpenoids were found to be almost twice better than the positive control, with euphorantin I (123), showing the highest value at 203.10% against B16 cells. It was therefore deduced that this compound could be a promising agent for the treatment of vitiligo diseases [34].

Conclusions and Prospects
In recent years, there has been growing interest in Euphorbia species to discover new diterpenes with promising biological activities and which possess an intriguing structural framework. Due to the emergence of new structurally diverse Euphorbia diterpenes with a wide range of pharmacological activities, it was remarkable to review the latest information on their isolation, structures, biological activities, and the structure-activity relationship. In the course of our survey, it was established that over 350 new diterpenes were isolated for the first time in roots, stems, seeds, stem barks, and whole plant of Euphorbia species, each bearing different skeletal structures. Particularly, jatrophanes, lathyranes, and ingenanes possessing structurally unique polyoxygenated derivatives were predominant in most species. These diterpenes are promising compounds for multidrug resistance reversal abilities and showed the ability to act as anti-inflammatory agents both in vivo and in vitro.
These properties might open new insights and perspectives in designing and developing new anti-inflammatory drugs. It is also noteworthy that, some diterpenoids with unusual skeletal frameworks like meroterpenoids, were reported for the first time in Euphorbia species with promising cytotoxic, antibacterial, anti-HIV, anti-influenza, multidrug resistance reversal abilities and anti-inflammatory activities. Specifically, jatrophanes and lathyranes diterpenoids were found to inhibit the P-glycoprotein thus inducing multidrug resistance-reversal abilities. The anticancer activities of these diterpenes were largely investigated. Conversely, SAR studies on the isolated diterpenes and their analogs revealed the significance of hydroxyl functionality within the structures. Esterification of this functionality was shown to enhance the activities in some analogs and lowered or showed no effect in others. For instance, jatrophanes diterpenoids having 11,12-diol groups showed significant activity, while mysrinanes possessing trans-fused 5/7/6 ring system occurring in an angular shape was relevant to their activity; as well, the free hydroxyl group at C-8 was found to be beneficial to the activity of these compounds. It was established that acetylation of the hydroxyl group at C-3 and C-8 in ingol and lathyranes type diterpenoids improved activity. The SAR studies of these diterpenes are essential as they can help to synthesize and discover lead compounds with low toxicity, good solubility, and high potency. It is significant to note that diterpenoids possessing unusual skeletal structures showed significant cytotoxic activities. It is observed that, despite the wide isolation of these diterpenoids, there is little publication on their total or semi-synthesis, that isolation from medicinal plants remains the only source of obtaining them notwithstanding, the unique skeletal structures and frameworks exhibited by Euphorbia diterpenes that can be precursors in synthetic endeavors to construct new derivatives with improved activities. Furthermore, few studies on these diterpenoids have reached clinical trials and for the few in vitro studies conducted, emphasis was focused on only limited pharmacological studies. It is also surprising to note that, despite tigliane (phorbol esters) reporting better activity, they have been isolated only in few species of the genus recently. This could be due to their complex nature that hinders their isolation and identification. In addition, little has been investigated to evaluate the toxicities of these diterpenes and their mechanisms of action. Therefore, to obtain more comprehensive information about the isolated diterpenes, there is a need for further studies to determine their mode and mechanisms of action. Also, more attention should be directed to their latex and water-soluble components, as limited study on these extracts is reported. It is also fascinating to note that over 380 new diterpenes were isolated in slightly over 30 Euphorbia species of more than 2000 species in the genus. This shows the structural diversity of Euphorbia diterpenes yet to be isolated. These diterpenes will give insights and understanding of the taxonomic relationship of Euphorbia species, and their chemotaxonomic significance. Hence, the current review shows the potential of the genus Euphorbia as a promising source of new bioactive compounds that will provide possible lead compounds for pharmaceutical applications, such as anticancer and anti-inflammatory agents.

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