Terpenoid Compounds in the Latex of Euphorbia Azorica from Azores

Plants of Euphorbia genus are well known for their irritant milky latex rich in
bioactive terpenoids and their use in traditional medicine worldwide for treatment
of several diseases, including cancer that is the second leading cause of death in the
world. This research evaluates, for the first time, the major terpenoid compounds from
the latex of Azorean E. azorica. Five triterpene alcohols of the euphane, lanostane and
cycloartane-type skeleton (namely euphol, obtusifoliol, lanosterol, cycloartenol and
24-methylenecycloartenol) were isolated from the n-hexane fraction. In addition, the
diterpene alcohol ingenol was obtained from the ether fraction following to alkaline
hydrolysis, which indicates the presence of potential bioactive ingenane diterpenoid
esters. Their structures were elucidated by physical, chemical and spectroscopic
methods (1H NMR, 13C NMR, IR and mass spectra) and comparison with literature data.
The referred triterpenols have been recently reported to possess anti-inflamatory and/
or antitumor activities. Furthermore, ingenol constitutes the core of a recently approved
anticancer drug. Thus, the terpenoid composition of E. azorica latex may indicate their
chemopreventive and chemotherapeutic potentials on anticancer strategies, which
should be investigated.


Short Communication
Euphorbia (Euphorbiaceae), commonly named spurge, is among the largest genera of Angiosperms, comprising about 2000 species that are characterized by specialized inflorescences (cyathia) and by a caustic and toxic milky latex, which plays defensive roles against herbivores and pathogens. Its worldwide distribution, remarkably diverse growth forms and traditional medicine use have attracted human interest since ancient times. In fact, the genus Euphorbia is of great importance to researchers and pharmaceutical industries due to their numerous medicinal uses attributed to its richness in valuable bioactive metabolites that could provide a reservoir for drug discovery [1]. Euphorbia species, and particularly its latex, are well known to produce a large variety of diterpenoid and triterpenoid compounds that show anticancer and chemopreventive properties as well as ant-inflammatory, antioxidative, antiviral, antibacterial and antifungal ones [1][2][3].
A good example could be the diterpene ingenol mebutate, isolated from the latex of E. peplus (subgenera Esula), that is the active principle of the new drug Picato for topical treatment of actinic keratosis, a common skin lesion in adults which usually occurs on chronically photoexposed areas and considered as a precancerous lesion or a superficial squamous-cell carcinoma [4].
Also, triterpenic compounds found in latex, including euphane, lanostane and cycloartane-types, can become an alternative method for treating cancer because of their cytotoxic properties and chemopreventive activities [3,5]. As a part of our continuing phytochemical investigation of Azorean plants and also to extend the knowledge towards the Euphorbia species [6], the present study aimed at the isolation and characterization of major terpenoid compounds from the latex produced by E. azorica (subgenera Esula), which causes irritation of the skin and eyes, as other Euphorbia species. No studies on the chemistry or pharmacology bioactivity of this species have been published. Acetilation with Ac2O/pyridine was carried out in order to convert the natural alcohols into the corresponding acetates. Hydrolysis of diterpenoid esters was carried out according to the procedure described by Lima, et al. [2].

Sample Collection and Fractionation
One hundred mL of latex was collected from E. azorica Hochst. The combined extracts (11g) were suspended in 85% aq. MeOH and partitioned with n-hexane to give a non-polar residue of 5g. The hydroalcoholic residue was partitioned between water and ether (1:1), with the latter residue yielding 910mg. An aliquot (819mg) was hydrolyzed and then extracted with CH2Cl2 yielding a residue of 245mg.

Isolation of Tetracyclic Triterpenes from The N-Hexane Extract
The n-hexane extract (5g) was subjected to CC on silica gel (150g) using a gradient of n-hexane-EtOAc. The 80 eluated fractions of 50mL were combined in five fractions (A-E). Fraction B (nos. 19-29; 0.5g; n-hexane-EtOAc, 98:2; white powder) was acetylated and then dissolved in hot Me2CO. On cooling, the ppt. was filtered off (18mg) and was identified as euphol acetate (Rt 14.05 min). The mother liquor was conc. yielding a white powder (100mg) that was purified by prep. TLC on 20% AgNO3/silica gel 60 plate eluted

Isolation of the Tetracyclic Diterpene Alcohol Ingenol from Ether Extract
The hydrolysate (245mg) from the ether extract, obtained of the hydroalcoholic residue, was acetylated and then purified by

Results and Discussion
This research evaluates the major terpenoid compounds from the latex of E. azorica, endemic to the archipelago of Azores.
Five triterpenols (euphol, obtusifoliol, lanosterol, cycloartenol and 24-methylenecycloartenol) were isolated from the n-hexane fraction, obtained of the acetone extract of the latex by partition in n-hexane/85% aq. MeOH. In addition, ingenol was isolated from the ether fraction, obtained of the hydroalcoholic residue by partition between water and ether (1:1), following to hydrolysis. Thus, this screening procedure, involving hydrolysis, as referred, followed by acetylation and GC analysis of the products, revealed the potential occurrence of ingenane-type esters in the latex of E. azorica, which may contributes to its irritant action on skin and eyes. The structures of the isolated compounds were elucidated by physical, chemical and spectroscopic methods, and comparison with literature data [2]. The above referred tetracyclic triterpenes have been reported to possess anti-inflamatory and/or antitumor activities in vitro and in vivo studies [7][8][9][10], presenting euphol the most potent effects among the tested compounds with several reports suggesting this compound as a novel potent antineoplastic agent, namely for glioblastoma that is the most frequent and aggressive type of brain tumor [11]. Furthermore, ingenol constitutes the core of a recently approved anticancer drug for topical treatment of actinic keratosis [3]. Thus, the terpenoid composition of E. azorica latex may indicate their chemopreventive and chemotherapeutic potentials that should be investigated.

Conclusion
Plants from Euphorbia genus are highly reputed for their use in medicine to treat several illnesses, including cancer that was responsible for 9.6 million deaths in 2018, and is expected to rise in the coming decades according to estimates from WHO. The medicinal value of these plants has been mainly attributed to the presence of unique terpenoid metabolites. In this study, the latex of Azorean E. azorica has been reported, for the first time, to contain several known nonpolar bioactive triterpenoids, such as euphol (a potential novel anticancer drug), together with ingenane derivatives (not identified). A more detailed investigation on the phytochemical profile of the latex of E. azorica is needed, together with biological activity studies in order to evaluate its value as a source of natural products of pharmaceutical interest or industrial application, such as crude extracts and its fractions or pure compounds.