Euphorbia-Derived Natural Products with Potential for Use in Health Maintenance

Euphorbia genus (Euphorbiaceae family), which is the third largest genus of angiosperm plants comprising ca. 2000 recognized species, is used all over the world in traditional medicine, especially in the traditional Chinese medicine. Members of this taxa are promptly recognizable by their specialized inflorescences and latex. In this review, an overview of Euphorbia-derived natural products such as essential oils, extracts, and pure compounds, active in a broad range of biological activities, and with potential usages in health maintenance, is described. The chemical composition of essential oils from Euphorbia species revealed the presence of more than 80 phytochemicals, mainly oxygenated sesquiterpenes and sesquiterpenes hydrocarbons, while Euphorbia extracts contain secondary metabolites such as sesquiterpenes, diterpenes, sterols, flavonoids, and other polyphenols. The extracts and secondary metabolites from Euphorbia plants may act as active principles of medicines for the treatment of many human ailments, mainly inflammation, cancer, and microbial infections. Besides, Euphorbia-derived products have great potential as a source of bioactive extracts and pure compounds, which can be used to promote longevity with more health.

However, some Euphorbia plants, especially their latex or milky sap (e.g., E. hirta, E. helioscopia, E. royleana Boiss. among others), are considered as irritating materials for skin, mouth, and throat, causing burning sensation, acute inflammation (even blisters), and nausea [58]. In veterinary medicine, E. milii Des Moul. and E. nivulia is used to treat diarrhoea and wounds in livestock, respectively, but other Euphorbia species can produce irritations [6].

Euphorbia Plants: Essential Oil Composition and Activities
Researchers from various countries worldwide have studied the chemical composition of essential oils (EOs) from different Euphorbia species. An overview of their most abundant components (the content higher than 5%) along with the most relevant biological activities to health maintenance (when available, and when the biological activity of a positive standard compound was also presented) is given in Table 1. The chemical structure of the major constituents of EOs from Euphorbia species whose content is higher than 25% is depicted in Figure 1. The Table 1 data show that EOs were obtained mainly from aerial parts (39%) and inflorescences (29%), in addition to leaves (18%), roots (11%), and stems (3%), by using basically two extraction methods-hydro-distillation (HD) (52%) and steam distillation (SD) (45%). The oil yield ranged from 0.07% to 1.52% (w/v) in E. cotinifolia (syn. E. caracasana) and E. fischeriana, and from 0.08% to 0.84% (w/w) in E. pilosa and E. densa. Microwave-assisted extraction (MAE) was reported only once (3%) with faster extraction time (3:1) and higher oil yield (1.2% vs. 0.7% w/v) than conventional techniques (MAE vs. HD) [74]. Qualitative and quantitative analyses were performed by gas chromatography (GC) or GC coupled to mass spectrometry (GC-MS). Samples were found to contain from 8 to 83 phytochemicals representing 81.7-99.9% of the oils content. Oxygenated sesquiterpenes (up to 86.1% of the oil in E. teheranica) characterize EOs of Euphorbia species, followed by sesquiterpene hydrocarbons (up to 34   Toxic to brine shrimp larvae (LC 50 = 21.7 µg/mL). Radical scavenging activity similar to ascorbic acid, lower than BHA but higher than α-tocopherol at 250 µg/mL. [77] a Compounds with content higher than 5%.
As reported by Lokar et al. [63], different habitats can influence the quantitative composition of EO from the same species. For example, EO of E. fragifera growing in a xeric habitat was richer in aromatic terpenes than that obtained from plants collected in shady and moist soils (e.g., 61.55% vs. 3.36% of carvacrol) being the last ones characterized by great quantity of acyclic compounds (e.g., 1.24% vs. 59.65% of geraniol). Moreover, variation in the components of EOs may occur due to the season, geographical area, and date of collection [63].
From Table 1, it appears that most of the EOs of Euphorbia species studied exhibit antioxidant properties, especially by the radical scavenging mechanism. Note that some of them are more active than ascorbic acid, BHT, or BHA compounds well known for their antioxidant properties and are widely used in the food industry as a preservative.
On the other hand, the data presented also show that there are many Euphorbia species whose EOs are still not yet studied, thus evidencing a knowledge gap about the potential of these species.

Antibacterial and Antifungal Activity of Euphorbia Plants
Plants belonging to the genus Euphorbia are also of the great interest in the matter of their antimicrobial activity [51,52,[78][79][80]. In fact, these plants are also widely used in the traditional medicine in the microbial infections [81,82], and some Euphorbia plants are believed to be a promising source of phytochemicals used in the pharmacy and food industries [83]. Since consumers prefer healthy products without synthetic raw materials, the constantly growing interest in the natural and ecologically friendly antimicrobial agents is still being observed, and therefore research on the antimicrobial activity involving Euphorbia species is relevant. The most recent studies in this subject and with greater impact is summarized in Table 2. This is not an exhaustive list of all studies performed, and only those that meet as a minimum requirement the indication of at least one statistical parameter associated with the average value of activity level (e.g., standard deviation) are included.
According to Ashraf et al. [84], the hexane extract of E. royleana, when compared with methanol and water extracts, is the one with highest phenolic and flavonoid contents and the best antimicrobial agent. In fact, it exhibits antifungal activity against Aspergillus niger and antibacterial activity against the gram-positive bacteria Bacillus subtilis, about half that of the reference compounds (rifampicin and terbinafine). * Express as diameter of inhibition zone (mm), as minimum inhibitory concentration, MIC or as minimum bactericidal concentration (MBC) (µg/mL). When available, the antimicrobial activity of the reference compound is presented.
The methanol extracts of E. hirta L. and E. tirucalli L. exhibit similar activity against a broad spectrum of bacteria and fungi associated with skin infections (zones of growth inhibition ranged from 9.0 mm (B. subtilis) to 14.0 mm (P. aeruginosa)) [85]. These results support the use of these species in traditional Indian medicine, and they can be used as an easily accessible source of natural antimicrobial agents [85].
The results obtained by Kumara Swamy et al. [86] showed that, independent of the extract prepared and the microorganism tested, E. neriifolia exhibits low antibacterial and antifungal activity, being the less interesting Euphorbia species as a source of antimicrobial agents.
In another study, the chloroform extract of E. paralias L. stems seems to be the most interesting extract, since it exhibits similar activity against fungi, gram-positive, and gram-negative bacteria (MIC = 15 µg/mL against C. albicans, B. subtilis, and E. coli strains, Table 2), while the chloroform extract of leaves only exhibits identical activity against C. albicans. The acetone extracts exhibit low activity against all the microorganisms tested [87].
According to the study conducted by Awaad et al. [88], the ethanolic extract of E. hirta aerial parts was the most active against all the bacteria and fungi tested when compared with E. granulata and E. helioscopia ethanol extracts, exhibiting an antifungal activity against M. canis similar to the amphotericin B in the same experimental conditions. Moreover, the authors demonstrated that heptacosan-1-ol, isolated from the active extract, could be the main metabolite responsible to the antimicrobial activity of E. hirta extract.
The study conducted by Ogbulie et al. [89] revealed that ethanolic extracts of E. hirta leaves showed higher activity against the gram-positive bacteria S. aureus (MIC = 22.55 mg/mL) than against the other microorganisms tested, but it was much less active than the ethanol extract of aerial parts against the same S. aureus strain (MIC= 1.95 µg/mL).
Unlike the ethanolic extract from the leaves of E. hirta, that does not inhibit the growth of Salmonella typhi, the ethanolic extract of aerial part from this species used in the study of Perumal et al. [90] that exhibited the strongest antimicrobial activity against Salmonella typhi with MIC value of 31 µg/mL, an activity higher than the chloramphenicol activity against the same strain (Table 2). Additionally, the same extract (the ethanolic extract of aerial part) also exhibits, against the gram-negative bacteria Proteus vulgaris, higher activity (MIC = 250 µg/mL) than the reference compounds gentamicin and chloramphenicol (MIC = 500 µg/mL) [90]. On the other hand, hexane extract was not active against all the tested microorganisms, except a weak activity against P. vulgaris [90].
Pisano et al. [91] carried out relatively extensive studies on the antimicrobial activity of the aqueous and ethanolic extracts from leaves, stems, and flowers of E. characias L. ( Table 2). The results showed that most of these extracts are non-active against the organisms tested (MIC > 1.25 mg/mL), with the exception of the ethanolic extract from leaves against gram-positive bacteria Bacillus subtilis (MIC = 312.5 µg/mL).

Other Biological Activity of Euphorbia Plants
The Euphorbia plants have been used as medicinal plants for the treatment of many human ailments besides to those caused by bacteria and fungi. In fact, as will be demonstrated in the items below, Euphorbia extract and compounds have been evaluated as potential natural drugs with many other activities such as anti-inflammatory, antiviral, and antitumour.

In Vitro Studies
Studies on in vitro biological activity of several Euphorbia plants have been reported, involving both extracts and pure compounds.
The cytotoxicity of the crude extract of the stem bark of E. umbellata (Pax) Bruyns and its fractions (hexane, chloroform, ethanol, and methanol) were studied using in vitro assay and by applying the leukemic cells Jurkat clone E6-1 [92]. The study revealed that the CHCl 3 fraction had the highest cytotoxicity (IC 50 = 7.72 µg/mL), an activity value much lower than the reference compound vincristine (IC 50 = 0.0031 µg/mL), but below the limit 30 µg/mL. According to NCI criteria [93], the activity of extracts is considered interesting to be studied in more detail. The established mechanism responsible for this action was associated with the promotion of cell cycle arrest at G0/G1 phase and apoptosis, which was related to two main classes of compounds in this fraction-steroids and triterpenes [92].
More recently, ethanolic extract of latex from E. umbellata has been evaluated for its anti-HIV properties. The study demonstrated in several models (in resting CD 4 +T cells, in cells from HIV-1 + individuals, and in primary non-human primate CD4+T cells) that this extract is able to reactivate the HIV virus in latency, acting on three factors with synergistic effect [94].
The ethanolic extract of E. lunulata Bunge (this name is a synonym of E. esula L.), at 10-20 µg/mL, had a significant anti-proliferative, anti-migration, and anti-invasion effect on multidrug resistant human gastric SGC7901/ADR cells, being able to arrest cell cycle progression and to induce cell apoptosis [95]. The same study further revealed that the cell cycle was arrested in G 2 /M phase, while significant apoptotic morphological changes was observed.
Methanolic extract of E. hirta aerial parts exhibited 90% growth inhibition against Plasmodium falciparum at 5 µg/mL and low toxicity against KB 3-1 cells, demonstrating its potential as antimalarial agent [96]. From this extract and by a bio-guided methodology, the authors isolated three flavonol glycosides-afzelin, quercitrin, and myricitrin ( Figure 2)-that exhibit the ability to significantly inhibit the proliferation of the protozoan responsible for malaria disease Plasmodium falciparum strains FCR-3 (cycloguanil-resistant from Gambia) and CDC1 (chloroquine sensitive), with similar IC 50 values 2.5 to 11.6 µM [96]. Quercitrin is able to arrest irreversibly the life cycle of the parasite at the trophozoite stage [96].
The seeds of E. lathyris contain many natural macrocyclic diterpenes with a lathyrane skeleton [46,99], five of which were evaluated against A549, MDA-MB-231, MCF-7, KB, and KB-VIN cancer cell lines [99]. The compound EFL-9 ( Figure 2) exhibits the strongest cytotoxicity against all cell lines (IC 50 values ranging from 5.7 to 8.4 µM against four of the five cell lines tested). The most important is its similar toxicity against the multidrug-resistant cancer cell line KB-VIN and the parental cell KB line (IC 50 values of 5.7 and 6.1 µM, respectively), while the anticancer taxol exhibits a highly differentiated toxicity to these cell lines (IC 50 values of 1.9 µM and 6.7 nM, respectively) [99]. The nicotinate ester at C-7 on the EFL-9 compound seems to play a crucial role in the cytotoxicity against the KB-VIN cell line. Additionally, the cytotoxicity of EFL-9 is due to its action on actin filament aggregation as well as on partial disruption of microtubules networks [99]. Several diterpenes isolated from stem bark of E. neriifolia exhibit anti-HIV-I activity [97]. Among these diterpenes, euphorneroid D and ent-3-oxoatisan-16α,17-acetonide ( Figure 2) were the most active compounds, with EC50 values of 34 μM and 24 μM respectively, although much less active than the reference compound (zidovudine, EC50 = 0.0019 μM). Besides these diterpenes, the ingenol derivative EK-16A (Figure 2), isolated from the E. kansui S.L.Liou ex S.B.Ho, can also be useful as antiviral agent against HIV-1 [98]. In fact, this compound is a PKCγ activator, 200-fold more potent (IC50 = 3.53 to 4.06 nM) than prostratin (IC50 = 768 to 865 nM) in reactivating latent HIV-1 without exerting detectable cytotoxicity on C11 and the J-Lat 10.6 cells viability and T cell activation. In addition, this compound exhibits a synergistic effect with other activators of HIV-1 from latency like prostratin, 5-azacytidine, and romidepsin [98].
The seeds of E. lathyris contain many natural macrocyclic diterpenes with a lathyrane skeleton [46,99], five of which were evaluated against A549, MDA-MB-231, MCF-7, KB, and KB-VIN cancer cell lines [99]. The compound EFL-9 ( Figure 2) exhibits the strongest cytotoxicity against all cell lines (IC50 values ranging from 5.7 to 8.4 μM against four of the five cell lines tested). The most important According Safwat et al. [101], quercetin-3-O-β-d-glucoside (Figure 2), a safe compound isolated from 70% methanol extract of E. paralias whole plant, in addition to exhibiting moderate toxicity against HepG2 and A549 cancer cell lines (IC 50 values of 41 and 36 µM, respectively), exhibits the ability to inhibit the glutamine synthetase enzyme (IC 50 = 0.048 µM). Taking into account that this enzyme is identified as a potential target in the development of new antimycobacterial agents, once it plays a significant role as virulence factor of Mycobacterium tuberculosis, Safwat et al. [101] showed that quercetin-3-O-β-d-glucoside could be used as an antituberculotic agent.
The main component of E. umbellata and E. tirucalli latex is the tetracyclic triterpene alcohol euphol ( Figure 2) [104,105]. This compound exhibits cytotoxic effect against several leukemia, colorectal, melanoma and glioma cancer cell lines [104,105]. When tested against Jurkat, HL-60, K-562 B16F10 and HRT-18 cell lines, euphol is toxic mainly against K-562 cell line (34.44 µM) by apoptosis induction, with non-toxicity to blood cells (SI > 2), but low selectivity to 3T3 cell line (SI = 0.55) [104]. On the other hand, euphol is particularly active against glioma cells (including primary, paediatric and adult glioma cell lines), with IC 50 values range from 5.98 to 31.05 µM, much more active than the temozolomide, a clinical drug used to treat some brain cancers (IC 50 values range from 97.00 µM to 1 mM) and with higher selective cytotoxicity index (0.64-3.36) than temozolomide (0.11-1.13) [105].

In Vivo Studies
The first significant in vivo studies reported on Euphorbia extracts or pure compounds were published in the 1990s. For example, the lyophilized E. hirta decoction exhibits antidiarrheal activity on diarrhoea induced by castor oil, arachidonic acid, and prostaglandin E2, but is inactive when the diarrhoea is induced by magnesium sulphate [106]. The flavonoid quercitrin (Figure 2) isolated from the previously reported lyophilized E. hirta decoction, exhibits antidiarrheal activity (at doses of 50 mg/kg), in castor oil-and PGE2-induced diarrhoea in mice [107], although the same authors showed that quercitrin activity is due to the glycone quercetin released in the intestine [107]. On the other hand, the water extract of E. hirta leaves, in adult Wister rats at 50-100 mg/kg dose, exhibits diuretic effect and increase the excretion of Na + , K + and HCO 3 [108].
However, the highest incidence of in vivo studies involving Euphorbia species has occurred in the last few years.
The 80% hydroethanolic extract of E. tirucalli latex at 0.250 mg (daily for 21 and 35 days) seems to be able to induce significant increases in T H 1 cytokines (GM-CSF, IL-2), T H 2 cytokines (IL-6), and chemokines (IL-1β, RANTES) and thus incite immunological stimulation and improve the immune system on adult male Sprague Dawley rats [109].
A steroid and terpenoid-rich fractions were isolated from the hydroethanolic extract of E. tirucalli root that, at 60 mg/kg, exhibits better protection against peripheral nociceptive pain on acetic acid induced abdominal constrictions mice model than the analgesic aceclofenac sodium and better anti-inflammatory activity in carrageenan-induced mice model than indomethacin [110].
On the other hand, the 70% hydroethanolic extract of E. supina Raf. (this name is a synonym of E. maculata L.), at 10 mg/mL, is able to significantly reduce the ear thickness and number of inflammatory cells on the Propionibacterium acnes-induced skin inflammation by inhibition of pro-inflammatory cytokines expression and the MAPK signalling pathway [111].
The  [112]. The authors showed that the methanol extract of E. characias subsp. wulfenii was the most active, significantly reducing the linear incision wound (43.04%) while the circular excision wound decreased 65.24%, with a significant decrease in wound inflammation (34.74%) in relation to the control groups. These activities were further linked with the quercetin glycosides identified in the extract [113].
A comparative study with several species, including E. helioscopia, E. lactea Haw., and E. nivulia Buch.-Ham. showed that the ethanol extract from the latter (at 100 mg/kg) was the one that caused the greatest anticonvulsant effects and highest reduction in the progression of epileptogenesis in pentylenetetrazole-induced kindling model of epilepsy in mice [114].
It was well demonstrated that the lyophilized decoction of fresh whole E. hirta had great potential against dengue virus since, after 14 days of administration at 100 mg/kg dose, it significantly increased platelet counts in rats, also causing a decrease in coagulation time and bleeding [115].
As shown above and in a recent review on the subject [116], several reports have suggested that Euphorbia plant possesses, in in vitro assays, considerable cytotoxic potential. Some in vivo studies also corroborate the anticancer potential and safety of Euphorbia extracts and compounds. For example, the methanolic extract of E. triaculeata Forssk., when tested in albino mice at 250 to 1000mg/kg body weight, is not genotoxic or clastogenic, while the pre-treatment with this extract inhibits the clastogenicity induced by cyclophosphamide without impairing its cytotoxic potential [117].
Euphornin L (Figure 3), a relatively abundant jatrophane diterpenoid from E. helioscopia, exhibits outstanding lipid-lowering effect on golden Syrian hamsters fed with a high-fat diet at a well-tolerated dose of 30mg/kg (p.o.) [118]. In fact, the authors observed a significant decrease on CHOL and LDL-C levels in the treatment group when compared with control group, while other parameters (HDLC, TG, and body weight) did not suffer significant changes [119].  Euphorbia factor L2 (EFL-2, Figure 3), a secondary metabolite isolated from seeds of E. lathyris, was tested to treat lipopolysaccharide (LPS) induced acute lung injury (ALI) in mice [119]. EFL-2 at concentration of 40 mg/kg causes an attenuation of the pathological changes in the lung by significant suppression of the recruitment and transmigration of inflammatory cells, specifically neutrophils [119]. The same compound was tested, using SMMC7721 xenograft BALB/c nude mice, to evaluate its effect on human hepatocellular carcinoma [120]. EFL-2, administered at 25 and 50 mg/kg for 14 days, is able to inhibit the growth of hepatocellular carcinoma in BALB/c athymic nude mice, causing a decrease in tumour volume and weight, mainly by targeting STAT3 and AKT inactivation during TGF-β-induced EMT and metastasis [120].
From E. helioscopia aerial parts were isolated several secondary metabolites that exhibit vasodepressive activity, being the euphornin ( Figure 3) the most active, causing a significant reduction in direct blood pressure (42 mmHg) in an adult male Wistar albino rat model during 70 min [121].

Clinical Studies
There are very few clinical studies involving Euphorbia species. In a clinical trial whose results were published in 2011 [122], skin cancer lesions (basal cell carcinomas, squamous cell carcinomas, and intraepidermal carcinomas) were treated topically with 100-300 μL of E. peplus latex (daily for three days). The results showed this latex exhibits a clinical response that is comparable to existing non-surgical treatments used in clinical therapeutics against human non-melanoma skin cancers. Euphorbia factor L2 (EFL-2, Figure 3), a secondary metabolite isolated from seeds of E. lathyris, was tested to treat lipopolysaccharide (LPS) induced acute lung injury (ALI) in mice [119]. EFL-2 at concentration of 40 mg/kg causes an attenuation of the pathological changes in the lung by significant suppression of the recruitment and transmigration of inflammatory cells, specifically neutrophils [119]. The same compound was tested, using SMMC7721 xenograft BALB/c nude mice, to evaluate its effect on human hepatocellular carcinoma [120]. EFL-2, administered at 25 and 50 mg/kg for 14 days, is able to inhibit the growth of hepatocellular carcinoma in BALB/c athymic nude mice, causing a decrease in tumour volume and weight, mainly by targeting STAT3 and AKT inactivation during TGF-β-induced EMT and metastasis [120].
From E. helioscopia aerial parts were isolated several secondary metabolites that exhibit vasodepressive activity, being the euphornin ( Figure 3) the most active, causing a significant reduction in direct blood pressure (42 mmHg) in an adult male Wistar albino rat model during 70 min [121].

Clinical Studies
There are very few clinical studies involving Euphorbia species. In a clinical trial whose results were published in 2011 [122], skin cancer lesions (basal cell carcinomas, squamous cell carcinomas, and intraepidermal carcinomas) were treated topically with 100-300 µL of E. peplus latex (daily for three days). The results showed this latex exhibits a clinical response that is comparable to existing non-surgical treatments used in clinical therapeutics against human non-melanoma skin cancers. The active ingredient of E. peplus latex has been identified as ingenol mebutate, the active principle of the clinical medicine Picato®used in the treatment of actinic keratosis [24].
The E. hirta extract is able to cause an increase in the number of platelets in dengue patients from 30-55 age group and a decrease in flu-like symptoms in 70% of the patients. However, no identical effect was observed in the 14-25-age group [123].
A clinical study on phase 2 to determine the safe and efficacious dose of E. prostrata for control of per rectal bleeding in patients with first-and second-degree haemorrhoids is now complete (Clinical Trials Identifier: NCT01041911) [124]. In that trial, 82% of the patients had complete cessation of bleeding at the end of two weeks and thus demonstrate that E. prostrata can be used as an effective therapeutic agent and without adverse effects in the treatment of early stage haemorrhoids [125].
A clinical trial involving E. kansui (an E. kansui extract powder prepared as tea) is currently in the recruitment phase and intends to evaluate the immune response of HIV+ individuals to this herbal supplement and its dependence with the dose applied. Also, the safety of the herbal preparation is evaluated (Clinical Trial Identifier: NCT02531295) [124].

Conclusions
The Euphorbia species are plants well known for their applications, especially its latex, in traditional medicine around the world. Their chemical composition may vary according to the species, to the part of the plant, and the applied extraction methodology. This variability could also be influenced by the different habitats, seasons, and dates of collection. Quantitative and qualitative analysis have been developed for the compounds' identification, being the major constituents of essential oils oxygenated sesquiterpenes followed by sesquiterpene hydrocarbons.
Additionally, several studies reported and discussed above, confirmed, in vitro, in vivo, and in clinical trials, the biological activities of Euphorbia extracts and pure compounds. These compounds and extracts could be applied to the treatment of different diseases mainly related to microbial infections, as well as inflammation and cancer.
Euphorbia plants have great potential as a source of bioactive extracts and pure compounds, which may lead to the development of new drugs for clinical use.