Traditional uses , Chemistry and Pharmacology of Croton species ( Euphorbiaceae )

Croton é um gênero de Euphorbiaceae com cerca de 1300 espécies, amplamente distribuídas em regiões tropicais do Novo e Velho Mundos. Várias espécies há muito tempo desempenham papel importante nos usos tradicionais de plantas medicinais na África, Ásia e América do Sul. Tais usos incluem tratamento de câncer, constipação intestinal, diarréia e outros problemas digestivos, diabetes, feridas externas, febre, hipercolesterolemia, hipertensão, inflamação, vermes intestinais, malária, dor, úlceras e obesidade. Várias espécies de Croton têm um látex vermelho que contém, em algumas espécies, pró-antocianidinas e/ou alcalóides. Os alcalóides de Croton são a taspina ou substâncias relacionadas a benzilisoquinolinas. Os diterpenos são muito comuns em Croton, correspondendo a clerodanos, cembranóides, halimanos, cauranos, labdanos, ésteres do forbol, traquilobanos e sarcopetalanos. Algumas espécies são aromáticas, devido à presença de óleos voláteis. Representantes de novas classes de substâncias (fenilbutanóides, alcalóides glutarimidínicos e diterpenos sarcopetalanos) têm sido isoladas de espécies de Croton. Enquanto laticíferos têm sido descritos em espécies de Croton, até o momento não há estudos anatômicos sobre estruturas secretoras de óleos voláteis. Parece haver afinidades químicas no gênero relacionadas à geografia, agrupando espécies com (i) cauranos e/ou labdanos, (ii) traquilobanos e (iii) alcalóides. Os ensaios farmacológicos freqüentemente vêm corroborando os usos tradicionais de espécies de Croton. Grande parte dos ensaios farmacológicos trata do clerodano trans-desidrocrotonina, envolvendo uma grande diversidade de efeitos, incluindo hipolipidêmico, hipoglicêmico, antiestrogênico e anticâncer. Efeitos citotóxicos também vêm sendo observados em ensaios com alcalóides (taspina) e com diterpenóides secocaurenos, labdanos e cembranóides. Vários outros efeitos de substâncias de Croton têm sido relatados, incluindo anti-hipertensivos, antiinflamatórios, antimaláricos, antimicrobianos, antiespasmódicos, antiulcerogênicos, antivirais e mio-relaxantes.

Croton is a genus of Euphorbiaceae comprising around 1,300 species, widespread in tropical regions of the Old and New Worlds.Several species have a long role in the traditional use of medicinal plants in Africa, Asia and South America.Popular uses include treatment of cancer, constipation, diabetes, digestive problems, dysentery, external wounds, fever, hypercholesterolemia, hypertension, inflammation, intestinal worms, malaria, pain, ulcers and weight-loss.Several species of Croton have a red sap, in some species containing proanthocyanidins and/or alkaloids.The latter may be taspine or some of several benzylisoquinoline-like compounds.Diterpenes are very common in Croton, corresponding to clerodanes, cembranoid, halimanes, kauranes, labdanes, phorbol esters, trachylobanes and sarcopetalanes.Some species are aromatic due to the possession of volatile oils.Representatives of new classes of compounds (phenylbutanoids, glutarimide alkaloids, sarcopetalane diterpenes) have been isolated from Croton species.While laticifers have been described in Croton species, so far there are no anatomical studies about secretory structures of volatile oil.Few studies about flavonoids have been carried out with Croton species.Chemical affinities are apparent in the genus, grouping species with (i) kauranes and/or labdanes, (ii) trachylobanes and (iii) alkaloids.Pharmacological assays have frequently corroborated the traditional uses of Croton species.A great part of pharmacological assays with Croton substances dealt with the clerodane trans-dehydrocrotonin, a wide diversity of effects having been noticed, including hypolipidemic, hypoglycaemic, antioestrogen and anti-cancer.Cytotoxic effects also have been observed in assays with alkaloids (taspine) and with secokaurene, labdane and cembranoid diterpenes.Several other effects of Croton substances have been registered, including anti-hypertensive, anti-inflammatory, antimalarial, antimicrobial, antispasmodic, antiulcer, antiviral and myorelaxant.

Introduction
The World Health Organization estimates that around 80% of the world population in developing countries relies on traditional plant medicines for primary healthcare needs, of which a major proportion corresponds to plant extracts or their active principles. 1 Many Euphorbiaceae are well known in different parts of the world as toxic and/or medicinal.The high diversity of the described effects is a reflex of the high chemical diversity of this plant group.Croton is a large genus of Euphorbiaceae, comprising around 1,300 species of trees, shrubs and herbs distributed in tropical and subtropical regions of both hemispheres.In the state of Rio de Janeiro alone 39 species have been identified and some have been used in popular medicine for many applications, including cancer. 2 The genus is rich in constituents with biological activities, chiefly diterpenoids such as phorbol esters, clerodane, labdane, kaurane, trachylobane, pimarane etc. 3 Croton is also rich in active alkaloids. 4,5Several species of the genus are aromatic, indicating the presence of volatile oil constituents. 6,7As most Euphorbiaceae, Croton species may contain latex, which is red-colored in some species, a characteristic usually associated with medicinal properties. 8,9his paper gives an account of the species used in traditional medicine, their chemistry and the pharmacological activities of crude extracts and pure compounds.The data collected are based on papers published up to March 2006.The data bases assessed include Biological and Chemical Abstracts, and ISI Web of Science.

South America
Croton cajucara Benth., popularly known as "sacaca", grows in the Brazilian Amazonian region.It is a traditional medicinal plant in the region, its leaves and stem bark being used in the form of tea or pills for the treatment of diabetes, high blood cholesterol levels and gastrointestinal disturbances, 10 as well as hepatic disturbances and weight loss. 11. celtidifolius Baill., commonly known as "sanguede-adave", is a tree found in the Atlantic forest of southern Brazil, mainly in Santa Catarina.Its bark and leaf infusions have been popularly used for the treatment of inflammatory diseases, leukemia, ulcer and rheumatism.12 C. eluteria Bennett., commonly known as "cascarilla", is a tropical shrub or small tree in the West Indies and northern South America.Its bitter bark has been widely C. zehntneri Pax.et Hoffm. is an aromatic plant native in northeastern Brazil, where it is popularly called "canelade-cunhã" and used in traditional medicine mainly as sedative, appetite stimulating, antianorexigen and for the relief of gastrointestinal disturbances; its bark and leaves are used as sweeteners in foods and drinks.28,29 2.2.North America and Central America C. arboreous Millsp. is native in Mexico, where it is known as "cascarillo".In Tabasco and Chiapas a beverage is prepared from the aerial parts and used as an auxiliary anti-inflammatory in respiratory ailments.30 C. californicus Müll.Arg. is a pale olive-green perennial herb growing usually in sandy areas of the Mojave Desert.31 Indians used to make a hot poultice from its powdered leaves as pain reliever for rheumatism. 32. draco Cham.& Schltdl. is a shrub native in Mexico and Central America.It is one of the "sangre-de-drago" plants, bearing a red sap widely used in traditional medicine, including internal use for cough, flu, diarrhea and stomach ulcers, and topically as wound healing for cuts, open sores, herpes, anti-septic after tooth extraction and for oral sores.33

Africa
C. macrostachys Hochst.ex Rich. is a medium-sized deciduous tree of East Africa.Its roots are used in Tanzania as antidiabetic 34 and the seeds are widely used in Somalia as purgative. 35. zambesicus Müll.Arg.(syn.C. amabilis Müll.Arg.; C. gratissimus Burch.) is native in tropical west and central Africa and used to treat fever, dysentery and convulsions. 36he leaf decoction is used in Benin as anti-hypertensive, anti-microbial (urinary infections) and to treat malarialinked fever. 3,374.Asia C. kongensis Gagnep. is popularly known in Thailand as "Plao Ngeon" or "Plao Noi".It is frequently used in folk medicine for dysmenorrhoea.38 C. oblongifolius Roxb., popularly known as "chucka", is a middle-sized tree and grows in India and Thailand.The leaves are used as tonic, the flowers against flat worms, the fruits to treat dysmenorrhoea, the seeds as purgative, 39 the bark to treat dyspepsia and the roots to treat dysentery.40 The bark is also used to treat chronic enlargement of the liver and remittent fever.It is applied externally to the hepatic region in chronic hepatitis.41 In Thai traditional medicine C. oblongifolius has been used in association with C. sublyratus to treat gastric ulcers and gastric cancers.42 Tribal people in India used various parts of C. roxburghii NP Balakr. agnst snake poisoning and to treat infertility, fever and wounds.43 C. sublyratus Kurz is a tropical plant growing extensively in southeastern Asian countries.In Thailand, the plant is a popular medicine used as anti-helminthic 44 and to treat dermatological problems.45 C. tiglium L. is an Asian shrub or tree (up to 12 m tall), used in Chinese medicine as laxative.46 Other traditional uses include tumors and cancerous sores (www.hort.purdue.edu/newcrop/duke).The seeds are source of a commercially available oil (croton oil), used as purgative.
C. tonkinensis Gagnep., popularly called "Kho sam Bac Bo", is a shrub native to northern Vietnam.The dried leaves are prescribed for stomach-ache and have been used in Vietnamese traditional medicine to treat burns, abscesses, impetigo, dyspepsia and gastric/duodenal ulcers.They are a component of recipes used to treat urticaria, leprosy and psoriasis. 47

Chemistry
Croton chemistry is considerably diverse.Terpenoids are the predominant secondary metabolite constituents in the genus, chiefly diterpenoids, which may belong to the cembranoid, clerodane, neoclerodane, halimane, isopimarane, kaurane, secokaurane, labdane, phorbol and trachylobane skeletal types.Triterpenoids, either pentacyclic or steroidal, have frequently been reported for Croton species.Volatile oils containing mono and sesquiterpenoids, and sometimes also shikimate-derived compounds are not rare in the genus.Several species have been reported as sources of different classes of alkaloids, a fact that enhances considerably the importance of the genus from the medicinal point of view.Phenolic substances have frequently been reported, among which flavonoids, lignoids and proanthocyanidins predominate.

Volatile oils and their constituents
Although most Euphorbiaceae are plants not known as aromatic, some Croton species contain volatile oils.Other species have not been reported as bearing volatile oils, although they were shown to possess sesquiterpenes commonly found in volatile oils.The volatile oils of several species contain phenylpropanoids and terpenoids (mono and sesquiterpenes), while from other species only terpenoids have been obtained.

Alkaloids
Alkaloids are not common in Euphorbiaceae, but some Croton species are notable for their alkaloids.The most frequent Croton alkaloids are compounds identical or similar to substances found in Ranunculales, i.e. alkaloids biogenetically related to benzylisoquinolines, such as morphinandienones and tetrahydroprotoberberine alkaloids.Glutarimide alkaloids and a new class of sesquiterpene guaiane-type alkaloids have recently been obtained from Croton species.
Taspine (14), an unusual alkaloid with a dilactone structure resembling elagic acid and one nitrogen atom not included in a heterocyclic ring, was found in the red latex of three species, C. draco, 33,108 C. lechleri 9,109 and C. palanostigma. 110Taspine has been obtained also from plant sources of benzylisoquinolines and biogenetically related alkaloids, such as Berberidaceae and Magnoliaceae.From leaves of C. lechleri other alkaloids, probably related biogenetically to taspine, have also been isolated, such as glaucine (15), isoboldine, magnoflorine, norisoboldine, thaliporphine 5 and sinoacutine. 111Tetrahydroprotoberberine alkaloids have been reported for C. hemiargyreus Müll.Arg. and C. flavens L. From leaves and stems of the former Amaral and Barnes 4 isolated 2,10-dihydroxy-3,10dimethoxy-8β-methyldibenzo[a,g]-quinolizidine (hemiargyrine) (16), in addition to glaucine, oxoglaucine, salutaridine (17) and norsalutaridine.The tetrahydroprotoberberine alkaloids scoulerine and coreximine and the morphinanedienone alkaloids salutaridine and salutarine, in addition to sebiferine, norsinoacutine and flavinantine, were isolated from specimens from Barbados of C. flavens by Eisenreich et al. 112 From leaves of C. flavens, Charris et al. 113 isolated (-)-amuronine.From shoots of C. salutaris Barnes and Soeiro 114 isolated salutarine and salutaridine, the latter a biosynthetically precursor of morphine. 115Isoboldine and laudanine were found in the ethanolic extracts of leaves and twigs from C. celtidifolius. 116Stuart and Graham 117 verified that C. linearis synthesizes crotonosine through linearisine.Murillo et al. 33 obtained two known aporphines from the bark and wood of C. draco.The β-carboline alkaloids 2ethoxycarbonyltetrahydroharman and 6-hydroxy-2methyltetradydroharman (18) were obtained from plants of C. moritibensis Bail., a species from northeastern Brazil. 118The aerial parts of C. cuneatus yielded the new glutarimide alkaloids julocrotol, isojulocrotol and julocrotone (19), in addition to julocrotonine. 119Anabasine and the novel guaiane-type alkaloids muscicapines A, B and C (20) were isolated from roots of the Northeastern Brazilian C. muscicapa Müll.Arg. 120

Proanthocyanidins
Tannins are polyphenols virtually ubiquitous in plants.They are medicinally important if occurring in high proportions in the plants.They may be formed by the combination of catechin monomers (the so-called proanthocyanidins), or by ester bounded units of glucose, gallic and/or ellagic acid (hydrolysable tannins).So far, only proanthocyanidins have been characterized in Croton species.

Flavonoids and other phenolic substances
Flavonols and/or flavones are ubiquitous in vascular plants, at least in green tissues.From the red latex of C. draco 108 and C. panamensis 126 myricitrin (myricetin-3-O-rhamnoside) was isolated.Leaves of C. cajucara yielded kaempferol-3,7-dimethyl ether and 3,4',7trimethyl ether, 50 while shoots of C. schiedeanus contain quercetin-3,7-dimethyl ether. 24From leaves of C. betulaster Müll.Arg., Barbosa et al. 106 obtained 5hydroxy,7,4'-dimethoxyflavone, and from leaves of C. hovarum Leandri, Krebs and Ramiarantsoa 62 isolated the flavone C-glycoside vitexin (21).From leaves and stems of C. brasiliensis, Palmeira et al. 59 isolated the 3-methoxyflavones artemetin (22), casticin, chrysosplenol-D and penduletin.The n-hexane extract of the Caribbean and Central American C. ciliatoglanduliferus Ort.yielded the highly methoxylated flavonols retusin and pachypodol. 128Flavonoid data is still too scarce to establish a pattern for Croton.The available data suggest a common presence of flavonols and flavones as free highly methoxylated aglycones.It is important to note, however, that no investigation directed specifically to flavonoids, which is currently made in hydro-alcoholic extracts, 129 has been carried out with Croton species.Hence, it is quite possible that flavonol and/or flavone glycosides (present in green tissues of most angiosperm species), have not been detected in the so far reported Croton chemical analyses.

Other classes of compounds
Scopoletin (a coumarin) was obtained from the wood extract of C. draco; the seeds of the same species contain p-hydroxybenzaldehyde and p-methoxybenzoic acid, while the dried sap yielded 3,4,5-trimethoxycinnamic alcohol. 33henylbutanoids, an interesting class of compounds known to occur in some genera of angiosperms, were obtained from shoots of C. schiedeanus by Puebla et al. 132 These authors isolated (2S)-7,9-dimethoxyrhododendrol ( 25), (2S)-acetoxy-7,9-dimethoxyrhododendrol and (2S)-2,8diacetoxy-7,9-dimethoxyrhododendrol.The formation of this class of phenolics has been proposed to occur via decarboxylative condensation of 4-coumaroil-CoA with malonyl-CoA to produce C 6 C 4 skeletons. 133The novel compounds 4-(2-hydroxyethyl)-benzoic acid and 2,5dihydroxy-phenylethanol were isolated from the red sap of C. panamensis. 126Lichexanthone was obtained from the aerial parts of C. cuneatus. 119From the same source, Hernandez and Delgado 77 obtained a mixture of polyprenols, castaprenol-11 being the major compound.Using hightemperature high resolution gas chromatography coupled to mass spectrometry (HT-HRGC-MS), Pereira et al. 2,134 characterized in shoots of C. hemiargyreus more than 100 compounds, including alkaloids, amino acids, terpenoids and wax esters.Among the compounds detected is a high molecular mass triterpenoid (simiarenol) and esters of amyrine with fatty acids containing carbon chains above 20 atoms.The polyalcohols 1L-1-O-myo-inositol and neoinositol were isolated from shoots of C. celtidifolius. 135enzoyl-methylpolyols were isolated from C. betulaster and Croton luetzelburgii Pax & K. Hoffm. 136Furanoarabinogalactan, a polysaccharide, is the main component found in the gum exsudate from C. urucurana. 137The peptide derivatives aurentiamide acetate and N-benzoylphenylalanine were isolated from shoots of C. hieronymi. 102yclopeptides were reported for the red latex of C. draco. 108

Chemotaxonomy
Given the huge dimension of the genus and the so far minute sampling of species chemically studied, no definite conclusions can be drawn about chemical relationships among Croton species.However, the present picture of the distribution of compound classes is suggestive of chemical patterns in the genus (Figure 1) and encourages proposals of chemical affinities, which in turn may stimulate further work on prospects of Croton biologically active compounds.
The most common class of compounds of Croton is represented by diterpenoids.Apparently, clerodane is the widest spread class of diterpenoids in Croton, which has been found in species from America (e.g. C. cajucara), Africa (e.g. C. macrostachys) and Asia (e.g. C. oblongifolius).Phorbol esters (such as 11) seem also to be widespread in Croton, having been found in species from Asia (e.g. C. tiglium), Africa (e.g. C. macrostachys) and America (e.g. C. californicus).Thus, the common possession of clerodanes and phorbol esters by groups of species should be viewed as retention of an ancient characteristic, rather than a mark of natural affinity.
Instead, kauranes, labdanes and trachylobanes seem to be more restricted in their distribution.insularis in Australia (Figure 1).A recent molecular phylogeny of Croton has been published, 138 but unfortunately few chemically studied species were included.Nonetheless, it is interesting that C. kongensis and C. zambesicus are grouped in a same clade.Other classes of diterpenoids, such as cembranoid, pimarane and isopimarane diterpenes seem to be highly restricted.In Croton, cembranoids have been found only in C. oblongifolius, which so far is the sole known halimane diterpenoid bearer.In Croton, isopimaranes are so far exclusive of C. zambesicus and pimaranes of C. joufra (Figure 1).Up until now, C. sarcopetalus is the sole known source of sarcopetalane diterpenoids.
Assuming the possession of diterpenes as an ancient chemical feature in Croton, the apparent absence of diterpenoids in extant Croton species indicates that the ability to produce or accumulate such compounds was lost in the evolutionary process.It has been claimed that terpenoid production by plants is more costly than other secondary metabolites. 139It seems that loss of diterpenoids in Croton occurred during the spread of Croton species into America.Benzylisoquinoline-like alkaloids (15-17) are an important group of secondary metabolites found so far exclusively in American Croton species, namely C. celtidifolius, C. draco, C. flavens, C. hemiargyreus, C. lechleri, C. linearis, C. palanostigma and C. salutaris (Figure 1).Again, it is worth noting that C. lechleri and C. flavens emerge inside a same clade in Croton molecular phylogeny. 138Alkaloids from other classes also occur in American Croton species, such as glutarimide alkaloids in C. cuneatus (Venezuela), β-carbolines in C. moritibensis (Brazil) and anabasine and guaiane-type alkaloids in C. muscicapa (Brazil).There seems to be in Croton a nearly mutual exclusiveness between diterpenes and alkaloids.Among the mentioned alkaloid-bearer species only C. salutaris has been reported to bear alkaloids and diterpenoids.C. lechleri and C. palanostigma are akin also due to the common possession of taspine (14), high amounts of proanthocyanidins and the lignoid 3',4-dimethylcedrusin (24) (Figure 1).As commented above, lignans are common in plant groups that produce benzylisoquinolines.In addition, these two species, plus C. celtidifolius, have red latex.Red sap occurs also in non-alkaloidal species, such as C. cajucara and C. urucurana, which share the possession of the rare triterpenoid acetyl aleuritolic acid (12).
Another trend that possibly occurred in the chemical evolution of Croton is the loss of diterpenoids and the substitution by mono and sesquiterpenes, mainly as volatile oil constituents, accompanied sometimes by shikimate derived volatiles (e.g.methyl eugenol, and anethole  1).Among these species only C. arboreous, C. cajucara, C. draco, C. sarcopetalus and C. zambesicus have been reported to possess diterpenoids and volatile oils.Volatile oils seem to be absent from alkaloidal species; so far only C. cuneatus was shown to bear both volatile oil and alkaloids, the latter represented by the rare glutarimide-type alkaloids.Most aromatic species occur in America, the exceptions being C. oligandrum (Africa), C. stellulifer (S.Tomé and Príncipe, Gulf of Guinea, west of Africa) and C. zambesicus (Africa).
A molecular phylogeny of Croton, including plants with known chemistry, would be extremely helpful to clarify trends in the chemical evolution of the genus.

Antilipidemic
Extracts of C. cajucara leaves showed significant reductions in the serum total cholesterol, low-density lipoprotein cholesterol and triglyceride levels, as well as a significant elevation in the HDL/total cholesterol ratio in treated rats compared with the control group. 140xperiments treating rats with water extracts 11 gave support to the popular use of C. cajucara bark in loss-weight programs.Treated rats had lower weight gain than control rats, the sensitivity of the lipolytic responses to isoprenaline and adrenaline being significant higher in adipocytes from treated rats.

Wound healing, gastric effects and protection
Extracts of the bitter bark of cascarilla (C.eluteria) has been shown to strengthen the histamine-stimulated gastric acid secretion, giving experimental support to the use of cascarilla in bitter preparations aimed to improve digestion. 141The "sangre de grado" of C. lechleri has shown wound-healing activity 142 in cutaneous disorders and, orally, in a dilute form to facilitate the healing of gastric ulcers, reducing ulcer size and bacterial content of the ulcer. 143he volatile oil from the bark of C. cajucara has been shown to exert gastric ulcer healing activity, as well as protection of the gastric mucosa. 144Low molecular weight sesquiterpenes appear to be important active constituents of the volatile oil. 95Efficacy of the oil seems to be based on its ability to stimulate local mucus synthesis and prostaglandin production by the gastric mucosa. 145raditional uses, Chemistry and Pharmacology of Croton species J. Braz.Chem.Soc.

Anti-diarrheic
The red sap from C. urucurana showed promising potential for the control of pathologies associated with secretory diarrhea. 122Proanthocyanidin SP-303 (from sangre de drago of C. lechleri) is a potent inhibitor of cholera toxin-induced fluid accumulation and chloride secretion. 146

Immunomodulatory, antibacterial, antifungal and antiviral
The red latex of C. lechleri showed antioxidant or prooxidant properties and stimulated or inhibited the phagocytosis.In addition, sangre de drago has immunomodulatory activity, inhibiting the proliferation of activated T-cells. 9The latex is also known to have antioxidant, free radical scavenging 147 and antiviral activities. 124In addition, it has revealed anti-bacterial properties. 142The red latex from C. urucurana inhibited the growth of the fungi Tricophyton tonsurans, Tricophyton mentagrophytes, Tricophyton rubrum, Microsporum canis and Epidermophyton floccossum, showing a potential utility as an alternative treatment for dermatophytosis. 148Phytochemical analyses demonstrated in the product the presence of catechins, such as gallocatechin and epigallocatechin, substances previously known to exert antifungal activity.The red latex of C. draco and its ethyl acetate and ethyl ether extracts exhibited high inhibition on the classical activation pathway of the complement system using hemolytic assay. 108

Antimalarial
Several Croton species are traditionally used as antimalarials throughout endemic malarial areas.Antiplasmodial activity was demonstrated in vitro for C. pseudopulchellus Pax., a species from southern Africa. 149Shoot methanolic extracts from aerial parts of C. lobatus L. (a widespread species in tropical America, from Florida to Argentina) were active toward Plasmodium falciparum 3D7 chloroquine sensitive strains, while root methanolic extracts inhibited growth of K1 resistant strains. 150

Shoots of C. hieronymi have shown strong activity
against lung A-549 carcinoma cells and mouse lymphoma and some activity against human colon carcinoma. 102The dichloromethane extract of leaves of C. zambesicus showed in vitro cytotoxicity against human cervix carcinoma cells. 37The red latex of C. lechleri has been shown to have anti-tumor activity. 142

Antimutagenic
C. lechleri sap possess antimutagenic properties and may inhibit the proliferation of human leukemic cells. 151

Mutagenic
The red sap of C. lechleri showed mutagenic activity for strain TA1535 of Salmonella typhimurium, in the presence of metabolic activation, and weak mutagenic activity for strain TA98; mutagenic activity was also noted in the haploid Saccharomyces cerevisiae strain XV185-14c. 152Methanolic extracts of C. cajucara bark were tested in mice for mutagenicity and antimutagenicity. 153No statistical significant results were obtained, indicating that, at the concentrations tested, the extracts have no mutagenic effect.

Antioxidant
C. celtidifolius bark has anti-inflammatory and antioxidant activity, which results from the direct action of constituents on specific targets, such as cyclooxygenase. 12. urucurana red latex has antioxidant effect against lipid peroxidation and free radical scavenging activity. 26C. lechleri sap possesses significant antioxidant activity against the oxidative damages induced by apomorphine and hydrogen peroxide in Saccharomyces cerevisiae and maize plantlets. 152Leaf extracts of C. cajucara were observed to exert antioxidant effects against the free radical DPPH and in paraquat treated yeast cells. 154

Myorelaxant, antispasmodic and anti-hypertensive
The volatile oil of the bark and leaves of C. nepetaefolius, which contains mainly 1,8-cineole, methyleugenol and terpineol, exerted antispasmodic effect on gastrointestinal tissues and antihypertensive activity in the cardiovascular system. 155Intravenous treatment with the volatile oil decreases mean aortic pressure and heart rate in either anaesthetized or nonanaesthetized rats. 156The myorelaxant activity is coherent with the use of C. nepetaefolius in traditional medicine as antispasmodic.Its large efficacy as a myorelaxant agent associated with its low acute toxicity makes it an agent of therapeutic potential. 91Aqueous and ethanolic extracts of C. schiedeanus have a decreasing effect on blood pressure, probably by means of an antihypertensive rather than hypotensive effect. 23,157The antihypertensive activity and vasodilatory effects of C. schiedeanus are attributed to a synergistic activity among flavonoids and terpenoids. 24he volatile oil of C. zehntneri has relaxing effect on smooth muscle, which supports the use of C. zehntneri in traditional medicine as a gastrointestinal antispasmodic, an activity that may in part be attributed to estragole. 28,29

Anti-inflammatory and antinociceptive
Crude leaf extracts of C. cajucara exhibited significant antinociceptive effect in rats. 10The red latex of C. lechleri revealed strong anti-inflammatory activity.Although taspine is probably an important and active constituent of the red latex, it cannot be considered the main responsible for these activities, and other constituents, probably proanthocyanidins, should be also involved. 9Orally administered, the volatile oil of C. nepetaefolius promoted a dose-dependent antinociceptive effect in hot-plate test. 158he aqueous extract of the aerial parts of C. cuneatus had significant activity against plantar inflammation induced by bovine serum albumin. 134The volatile oil of C. sonderianus had antinociceptive effect in tests with oral administration, but was inactive in hot-plate tests. 160An antinociceptive effect of the volatile oil of C. zehntneri was evidenced, most likely associated with antiinflammatory activity. 6The aqueous extract of C. malambo bark administered intraperitoneally showed antinociceptive and anti-inflammatory effects, comparable to acetylsalicylic acid and sodium diclofenac. 161C. celtidifolius bark has anti-inflammatory activity. 12

Central depressive
The volatile oil from the bark and leaves of C. zehntneri produced central depressive effects in rats without anxiety alterations. 162

Reliever of insect bites swelling
The red latex of C. lechleri relieves swelling of insect bites. 143

Anti-diabetic, anti-hyperlipidemic, anti-hypercholesterolemic
Pharmacological studies carried out with the terpenoids t-DCTN (1), trans-crotonin, acetyl aleuritolic acid (12)  and with plant extracts showed a striking correlation with the traditional therapeutic use of C. cajucara species in the Amazon region for the control of hyperlipidemy and associated pathologies. 50,163Hypolipidemic effects were observed by in assays with t-DCTN from C. cajucara. 164,165n addition to hypolipidemic action, t-DCTN exhibited hypoglycemic effect in alloxan-induced diabetic, but not in normal rats. 140

Tumor promotion
Phorbol esters (such as 11) are known co-carcinogenic agents, being involved in the transformation of normal human epithelial cells through infection by Epstein-Barr virus. 166he commercially available oil of Croton derives from the Asiatic C. tiglium.This oil is the source of phorbol esters, tetracyclic diterpenes isolated from the seeds, whose cocarcinogenic properties have been the object of numerous investigations.9][170] C. tiglium is still utilized in homoeopathy and in combination with some sort of acupuncture (as a constituent of Baunscheidt oil).Its main irritant component, 12-O-tetradecanoylphorbol-13acetate (TPA) was used as a standard tumor promoter in mice in experimental cancer research, 88 but Castagna et al. 171 and Nishizuka 172 noted that phorbol-12-myristate-13-acetate is also effective.In addition, the latter compound is hardly metabolized by the cell. 173Several phorbol esters were prepared with polar functional groups terminating their C12 and/or C13 acyl chains.Such phorbol ester lipophilic domain is thought to be responsible for redistributing a phorbolprotein complex to the plasma membrane. 174Biotransformation of phorbol esters by intestinal bacteria has been successfully achieved. 1753.Anticancer t-DCTN (1) exhibits anti-tumor efficacy and immunomodulatory actions in vivo, which may be related to its chemical structure.176 DCTN and its synthetic derivative dimethylamide-crotonin (DCR) inhibit HL60 cells growth in vitro partly by apoptosis induction and cell differentiation, but do not cause serious damage to immune cells.177,178 However, t-DCTN is not cytotoxic (and also not genotoxic) to bone marrow cells of Swiss mice submitted to acute intraperitoneal treatment in vivo 179 and antimutagenic with regard to cyclophosphamide, in particular if administered by gavage.180 Correa et al. 181 developed a β-cyclodextrin complex to improve delivery of DCTN.A lower cytotoxicity of the complex β-cyclodextrin-DCTN to V79 fibroblasts and rat cultured hepatocytes, compared to free t-DCTN, suggests that such complex may be useful for in vivo dehydrocrotonin administration.
Tannins are known to complex with proteins, acting as multidentate ligands and thus being able to bind simultaneously to more than one point to the protein surface. 189Proanthocyanidin SP-303 has been extensively studied and has shown activity against a variety of virus: respiratory syncytial virus, parainfluenza type-1 and type-3, and influenza A and B viruses; 190 herpes virus; 191 genital and anogenital herpes virus, in treatment of patients with AIDS; 192 several DNA and RNA virus, including hepatitis viruses. 143C. tiglium seeds contain anti-HIV-1 phorbol esters, 12-O-acetylphorbol-13-decanoate (11) and 12-Odecadienoylphorbol-13-(2-methylbutyrate) that inhibit the cytopathic effect of HIV-1; 12-O-tetradecanoylphorbol-13-acetate (TPA) is even more active than the mentioned phorbol esters against HIV-1. 89,90atechin and acetyl aleuritolic acids, compounds obtained from C. urucurana, are effective against S. aureus and Salmonella typhimurium; acetyl aleuritolic acid showed minimum inhibitory concentration ten fold higher than catechin. 27Alviano et al. 98 observed that the volatile oil from leaves of C. cajucara, composed mostly by linalool, inhibits the growth of Candida albicans, Lactobacilus casei, Porphyromonas gengivalis, S. aureus and Streptococcus mutans, all involved in diseases of the oral cavity.Among these microorganisms, the authors noted that linalool is active almost exclusively against Candida albicans, and that the volatile oil is not toxic to mammalian cells.
The phenylpropyl benzoates 3'-(4"-hydroxy-3",5"dimethoxyphenyl)-propyl benzoate, 3'-(4"-hydroxyphenyl)propyl benzoate and 3'-(4"-hydroxy-3"-methoxyphenyl)-propyl benzoate, obtained from stems of C. hutchinsonianus, were shown to exert effect against Candida albicans. 130erivatives of phorbol esters have been evaluated as inhibitors of proliferation of HIV-1.Among them 12-Oacetylphorbol-13-decanoate has been shown to be the most potent. 193,194.5.Wound-healing, gastric effects and protection t-DCTN (1) showed strong antiulcerogenic activity. 195,196he A-ring of both crotonin and DCTN is not directly involved in the antiulcerogenic activity. 197t-DCTN has good antiulcerogenic activity; a long term use of which, however, may induce liver damage. 198A semi-synthetic crotonin, namely 4SRC, was synthesized and it was shown to have a significant preventive effect against gastric ulcer induced by different agents. 199Presence of a lactone moiety or Michael acceptor is probably essential for the antiulcerogenic effect, a mechanism of gastric cytoprotection being mediated by an action on prostaglandin biosynthesis, and by a Michael reaction between the SH-containing compounds of the mucosa on the Michael acceptors present in antiulcerogenic compounds. 200hen previously administered orally the volatile oil from the bark of Croton cajucara significantly reduced the gastric injury induced in rats. 95,144launotol is an anti-peptic ulcer agent, 201 commercially available under the name Kelnac. 45The anti-ulcer effect of plaunotol is probably linked to its activity against Helicobacter pilory. 202,203

Antispasmodic and anti-diarrheic
The volatile oils of some South-American Croton species are antispasmodic.Cineole, methyleugenol and terpineol, constituents of C. nepetaefolius volatile oil, have been reported to have myorelaxant and antispasmodic effects in laboratory animals. 153Experimental results suggest that C. nepetaefolius volatile oil induces relaxation of guinea-pig ileum. 204Anethole and estragole, major components of the volatile oil of C. zehntneri, are effective relaxants of skeletal muscles. 205ischer et al. 147 described a method to optimize the preparation of an extract from the bark latex of C. lechleri.The extract was named SB-300 and contains on average 70.6% by weight of the proanthocyanidin SP-303.The authors suggest its use for the treatment of fluid loss in acute watery diarrhea.SP-303 is the active agent of Provir, a C. lechleri derived product with antisecretory properties 206,207 and hence useful to control fluid loss and diarrhea.SP-303 has been indicated particularly for patients of AIDS, common victims of diarrhea. 208
The volatile oil of C. zehntneri was shown to have antinociceptive activity in mice, 6 and the volatile oil of C. cajucara has anti-inflammatory and antinociceptive effects in rodents. 209Cajucarinolide, a diterpene from C. cajucara, was shown to possess anti-inflammatory activity and to inhibit bee venom phospholipase A2 in vitro. 210.211A chromatographic analysis from the ethyl acetate soluble fraction of the crude extract of the stem bark of C. celtidifolius showed predominantly proanthocyanidins, which exhibited antinociceptive effects in tests using intraperitonial injections of chemical stimulants. 1278.Vasorelaxant, hypotensive and bradycardiac t-DCTN (1) was shown to reduce the mean arterial pressure and heart rate in a dose-dependent manner in normotensive rats and to relax the tonic contraction in isolated rat aortic rings induced by phenylephrine.212 The neo-clerodanes (12R)-12-hydroxycascarillone, 5βhydroxy-cis-dehydrocrotonin, cis-and trans-dehydrocrotonin from C. schiedeanus relaxed aort rings.57 A cooperactivity between diterpenoids and quercetin-3,7dimethyl ether (ayanin, the predominant flavonoid in C. schiedeanus leaves) was observed in the relaxing effect.Flavonoids have been assigned a role as protectors against vascular accidents.A vasorelaxant activity of quercetin-3,7-dimethyl ether from C. schiedeanus was observed, the activity probably being influenced by hydroxylation at positions 3' and 4' of the B ring. 24

Central anti-depressive
While the volatile oil of C. zehntneri exhibited depressive effects in models using rats (see above), methyleugenol, its major constituent, was shown to have antidepressive effect in experiments based on the same models. 21310.Anti-oestrogenic t-DCTN (1) obtained from C. cajucara was tested for anti-oestrogenic activity using immature rats for bioassay of oestrogen and regularly cycling rats of proven fertility for antiimplantation effect.The compound prevented increases of uterine wet weights, but did not affect blastocyst implantation.214

Insect growth inhibition and insecticidal
The diterpene fraction from C. linearis showed lethal effect on insects. 84A prenylbisabolane diterpene from C. linearis has insecticidal effect. 215The same comment applies to hardwickiic acid, a diterpene present in C. aromaticus and C. californicus. 76

Anti-leishmanial and anti-malarial activity
The linalool-rich volatile oil from leaves of C. cajucara was shown to be a potent agent against Leishmania amazonensis.The DL 50 for promastigotes is 8.3 ng mL -1 and for amastigotes, 8.7 ng mL -1 .The inhibitory concentration for L. amazonensis promastigotes growth is extremely low (MIC 85.0 pg mL -1 ), and the oil has no cytotoxic effects against mammalian cells. 978,9-Secokauranes obtained from C. kongensis were shown to have anti-malarial activity with IC 50 1.0-2.8μg mL -1 . 38

Concluding Remarks: Points Deserving Further Work
Table 1 summarizes traditional uses and effects observed in pharmacological assays with species of Croton and respective substances.Taking into account the magnitude of the genus, the list of species is still too scant.However, a quick examination of the table contents suffices to give an idea of the wide diversity of (i) health problems, which people from many parts of the world treat with the aid of Croton plant parts, (ii) classes of Croton secondary metabolites, and (iii) pharmacological effects experimentally observed, using crude extracts and pure compounds.For these reasons, much chemical and pharmacological work is needed about Croton.It is important to note that figures much higher would emerge if articles were also counted without Croton in their titles, but dealing with chemical or pharmacological aspects of the genus.Hence, Croton species and their substances are among the highly studied themes in chemistry of natural products, pharmacology and ethnopharmacology, and new data are rapidly accumulating.It is expected that many novelties will rapidly enlarge the current knowledge about Croton species, their substances and corresponding pharmacological effects.
Some trends in Croton research should be rechecked.It is evident from the Reference list of the present work that some species have been the object of relatively many investigations, such as C. cajucara, C. lechleri, C. urucurana and C. zehntneri, whilst most Croton species have so far been neglected by chemists and pharmacologists.
A lot of further work is also necessary about traditional uses of Croton species, not only in Brazil, but also in other tropical regions of both New and Old Worlds.Indeed, the number of Croton species reported in the literature as sources of traditional medicines is too small, if it is considered the high diversity of Croton species, chemistry and pharmacology.
For several species in Table 1, there is good agreement between popular uses and experimentally observed effects, in harmony with Fairnsworth's observation 216

C. zehntneri
Nervous disturbances, anorexia, Volatile oil → intestinal muscle relaxant, 28,29 depressor central effect, 162 gastrointestinal disturbances, sweetener antinociceptive; 6 anethole and estragole → intestinal muscle relaxant; 28 anethole → anti-carcinogenic 186 antimalarial effects of secokaurane diterpenes of C. kongensis, the cytotoxicity of taspine, the hypolipidemic and hypoglycaemic effects of C. urucurana and the cytotoxicity of trachylobane diterpenes of C. zambesicus (Table 1).Another aspect that needs investigation deals with the secretory structures involved in the production and accumulation of some Croton extractives, such as latex and volatile oils.With the exception of a study by Rudall 217 about laticifer distribution in Crotonoideae and other Euphorbiaceae, nothing has been reported about structures involved in the production and accumulation of volatile oils in Croton.In angiosperms, volatile oils may be found inside oil cells (e.g.Lauraceae), secretory cavities (e.g.Myrtaceae), secretory ducts (e.g.Asteraceae) and secretory trichomes (e.g.Lamiaceae), the latter being regarded as evolutionary more recent. 218his is certainly a point deserving research by plant anatomists.
It has been said above (see section Chemical Affinities) that, among Croton species containing alkaloids, only C. salutaris is known to possess also diterpenes.It was said also that most species with volatile oils have not been reported as diterpene-and alkaloid-bearers.Is this apparent mutual exclusiveness between classes of secondary metabolites a reality in Croton or just a delusion resultant of insufficient sampling?Relationships between the occurrences of classes of secondary metabolites in Croton are a point deserving consideration, because it may end up useful in future prospects of pharmacologically active substances.

Figure 1 .
Figure 1.Chemical and geographical affinities among Croton species.

Antonio
Salatino got his Ph.D. from the University of São Paulo in 1979 and carried out postdoctoral programs with Drs.Tom Mabry at the University of Texas and David E. Giannasi at the University of Georgia.He is currently Full Professor at the Department of Botany (which he headed in 2001-2004) of the Institute of Biosciences of the University of São Paulo.His research interests are chemotaxonomy and applied botany, including studies about the effects of pollution, analysis of seed oils, medicinal plants and propolis, and more recently molecular phylogeny and gene expression.Author of nearly 100 papers in national and international scientific journals.Maria Luiza Faria Salatino received her Ph.D. from the University of São Paulo and completed postdoctoral programs with Drs.Tom Mabry and David E. Giannasi.Her research deals with the analysis of plant secondary metabolites to explore their taxonomic and economic potential, and analysis of DNA with interests in molecular systematics.Presently, she is a faculty member at the Institute of Biosciences, Department of Botany and head of its Laboratory of Phytochemistry and Molecular Systematics.Author of 40 papers, including book chapters and articles of national and international scientific journals.Giuseppina Negri obtained a Ph.D. in Organic Chemistry from the State University of Campinas (UNICAMP).She gained expertise in organic synthesis and mass spectroscopy working under the supervision of Prof. Dra.Concetta Kascheres.She held a post-doctorate position at the University of São Paulo, Department of Botany, when she gained familiarity with analysis of natural products.GN has carried out investigations on plant secondary metabolites, with emphasis in analysis of constituents of medicinal plants and propolis.Presently, she integrates the group led by Dr. Elisaldo Luiz de Araujo Carlini at CEBRID/UNIFESP, being involved in several projects dealing with medicinal plants with activity on the central nervous system.Author of 30 papers, including book chapters and articles of national and international scientific journals.
C. draco, C. kongensis, C. sublyratus and C. tonkinensis are chemically akin due to the possession of kauranes.Labdanes occur in C. joufra, C. oblongifolius and C. zambesicus.Trachylobanes have been found in C. insularis, C. macrostachys, C. robustus and C. zambesicus.It is worth noting that, with the exception of C. draco (a species with a highly diverse chemistry), all other species with these diterpene classes do not occur in the New World, most of them occurring in Asia, C. zambesicus in Africa and C.

Table 1 )
that popular uses of medicinal plants frequently are coherent with the pharmacological effects of the main active principles of the respective plant sources.Part of the future pharmacological investigations should center their focus on several popular uses of Croton species, which so far have received no scientific support, as are the cases of C. .On the other hand, experimental work has uncovered potential uses of Croton chemicals with no records in the list of popular uses.A few examples are the cytotoxic, antimycobacterial and