Pharmaceutical potential of phorbol esters from Jatropha curcas oil

Article abstract of DOI:10.1080/14786419.2012.716057

Phorbol esters (PEs) are diterpenes present in Jatropha curcas L. seeds and have a myriad of biological activities. Since PEs are toxic, they are considered to be futile in Jatropha-based biodiesel production chain. In the present study, the extracted PEs from Jatropha oil were used as a starting material to synthesise pharmacologically important compound, prostratin. The prostratin synthesised from Jatropha showed identical mass with that of the reference standard prostratin, as determined by Nano-LC-ESI-MS/MS. Considering the rapid growth in Jatropha biodiesel industry, potential exists to harness large amount of PEs which can be further utilised to synthesise prostratin as a value added product.

Full text of DOI:10.1080/14786419.2012.716057

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Pharmaceutical potential of phorbol
esters from Jatropha curcas oil
Rakshit K. Devappa a , Chandi C. Malakar b , Harinder P.S. Makkar a
& Klaus Becker b
a Institute for Animal Production in the Tropics and Subtropics,
(480b), University of Hohenheim , Stuttgart – 70599 , Germany
b Institut für Chemie, Universität Hohenheim , Stuttgart ,
Germany
Published online: 22 Aug 2012.
To cite this article: Rakshit K. Devappa , Chandi C. Malakar , Harinder P.S. Makkar & Klaus Becker
(2013) Pharmaceutical potential of phorbol esters from Jatropha curcas oil, Natural Product
Research: Formerly Natural Product Letters, 27:16, 1459-1462, DOI: 10.1080/14786419.2012.716057
To link to this article: http://dx.doi.org/10.1080/14786419.2012.716057
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Natural Product Research, 2013
Vol. 27, No. 16, 1459–1462, http://dx.doi.org/10.1080/14786419.2012.716057
SHORT COMMUNICATION
Pharmaceutical potential of phorbol esters from Jatropha curcas oil
a
b
a
*
Rakshit K. Devappa , Chandi C. Malakar , Harinder P.S. Makkar and
Klaus Beckerb
aInstitute for Animal Production in the Tropics and Subtropics, (480b), University of Hohenheim,
b
Stuttgart – 70599, Germany; Institut fur Chemie, Universitat Hohenheim, Stuttgart, Germany
¨
¨
(Received 17 November 2011; final version received 24 July 2012)
Phorbol esters (PEs) are diterpenes present in Jatropha curcas L. seeds and have a
myriad of biological activities. Since PEs are toxic, they are considered to be futile
in Jatropha-based biodiesel production chain. In the present study, the extracted
PEs from Jatropha oil were used as a starting material to synthesise pharmaco-
logically important compound, prostratin. The prostratin synthesised from
Jatropha showed identical mass with that of the reference standard prostratin, as
determined by Nano-LC-ESI-MS/MS. Considering the rapid growth in Jatropha
biodiesel industry, potential exists to harness large amount of PEs which can be
further utilised to synthesise prostratin as a value added product.
Keywords: by-product; Jatropha; phorbol esters; prostratin; toxicity
1. Introduction
Phorbol esters (PEs) are tigliane diterpenes present in the genus Euphorbiaceae. These
groups of compounds have an array of biological activities, exhibiting toxicity at higher
doses and beneficial or pharmaceutical effects at lower doses (Goel, Makkar, Francis, &
Becker, 2007). In Jatropha curcas L. (Euphorbiaceae), six different types of PEs (Jatropha
factors C1–C6) are present (Haas, Sterk, & Mittlebach, 2002). The Jatropha kernel, screw
pressed oil and screw pressed cake generally contains PEs in the range of 2–6 g kg�1
,
3–6 g kg�1 and 0.5–4 g kg�1, respectively (Makkar & Becker, 2009; and unpublished data
from our laboratory). Although present in lower concentrations, they exhibit severe
toxicological symptoms upon oral and topical exposure to different parts of Jatropha plant
including seeds. Several studies report the need to remove these compounds to increase the
potential use of byproducts such as protein rich seed cake (Goel et al., 2007; Makkar,
Becker, Sporer, & Wink, 1997). Due to toxicity, these compounds are generally regarded
as futile compounds in the Jatropha biodiesel production chain. In addition, the removal
of PEs from Jatropha-based products and their further utilisation in agropharmaceutical
applications could enhance the economic sustainability of the Jatropha biodiesel
production chain. In the present study, the pharmaceutical potential of PEs obtained
from Jatropha oil was evaluated by converting them into a high-valued compound, which
is nontoxic and has high biological activity. The studies were focused on the synthesis of
prostratin from Jatropha PEs.
*Corresponding author. Email: Harinder.Makkar@fao.org
© 2013 Taylor & Francis

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R.K. Devappa et al.
Prostratin (12-deoxyphorbol-13-acetate) is a tigliane diterpene, and its presence is
reported in plants such as Pimelea prostrate, Euphorbia cornigera and Homalanthus nutans
(Miana, Bashir, & Evans, 1985). Similar to phorbol-12-myristate-13-acetate (PMA;
synonym, 12-O-tetradecanoylphorbol-13-acetate), the prostratin is a protein kinase C
(PKC) activator. However, prostratin is a potent anti tumour compound while PMA a
tumour promoter. In addition, prostratin was found to inhibit PMA-stimulated tumour
formation (Szallasi & Blumberg, 1991; Szallasi, Krausz, & Blumberg, 1992; Szallasi,
Krsmanovic, & Blumberg, 1993). Thus, it belongs to a discrete subclass of PKC activators
differing in its biological activities when compared with the tumour-promoting PEs, such
as PMA (Ventura & Maioli, 2001). Prostratin has also been found to be a promising
adjuvant in antiviral therapy. Many antiviral treatments have been successful in decreasing
the active viral pool in AIDS patients (HAART). However, the persistence of latent viral
reservoirs limits the complete viral eradication. Prostratin activates this latent virus pool
(Wender, Kee, & Warrington, 2008) in turn acting as an adjuvant in HIV therapy.
Prostratin have been found to induce HIV expression in latently infected cell lines and
primary cells in vitro and also to inhibit HIV entry into target cells by down regulating
CD4 and CXCR4 receptors (Gustafson et al., 1992; Kulkosky et al., 2001).
The prostratin was reported to be the active component present in Samoan mamala
tree (Homalanthus nutans) bark extract which is used for treating hepatitis (Cox, 2001;
Johnson, Banack, & Cox, 2008). In H. nutans, its concentration has been found to vary
from 0.2–52.6 mg g�1 fresh stem wood (Johnson et al., 2008). However, the presence of low
amounts of prostratin in plants, difficult purification steps and the lesser yield limits its
potential to economically harvest in large quantities. In addition, establishment of large-
scale plantation by replacing the native ecosystem to obtain substantial amounts of
prostratin could have significant ecological effects (Johnson et al., 2008; Wender,
Warrington, & Kee, 2009).
Alternatively, prostratin could be obtained by synthetic methods by utilising PEs as a
starting material. Industrial oil crop such as Jatropha curcas provide a promising feedstock
of PEs for prostratin synthesis. Wender et al., (2008) have reported the synthesis of
prostratin from phorbol (obtained from croton oil), which is a basic skeleton in the PEs
such as PMA. They also presumed that prostratin could be synthesised from Jatropha PEs
by hydrolysis, but further investigation has not been carried out. However, the Jatropha
PEs do not contain phorbol as a basic skeleton, instead they contain 12-deoxy 16-hydroxy
phorbol as a basic skeleton. Wender et al. (2008) suggested that 12-deoxy 16-hydroxy
phorbol obtained from the Jatropha PEs could be converted into crotophorbolone, which
can be further subjected to synthetic reactions to obtain prostratin (Figure 1). In the
present study, the possibility of converting Jatropha PEs into crotophorbolone and then
into prostratin was investigated.
2. Results and discussion
Wender et al. (2008) postulated that Jatropha PEs could be a good candidate for
producing highly bioactive compound prostratin. The present study has illustrated that
prostratin could be synthesised from Jatropha PEs. The experimental section is described
in the supplementary material (online only). The synthesis reaction of prostratin from
crotophorbolone is shown in Figure 1. As this was a preliminary study to assess whether
Jatropha PEs from PE-enriched fraction (PEEF) could be used as an intermediate to
synthesise prostratin, the material balance was not considered. The oil and the PEEF had
4.7 and 53 mg g�1 PEs (PMA equivalent) (or 0.11 and 1.3 mg g�1 as factor C1 equivalent).
After treating the PEEF with barium hydroxide in methanol, crotophorbolone was

Natural Product Research
1461
Figure 1. Synthesis of prostratin from Jatropha PEs (Cairnes, Mirvish, Wallcave, Nagel, & Smith,
1981; Wender et al., 2008).
obtained (� 6–10 mg). Similarly, the crotophorbolone was also obtained from Jatropha
PEs by reacting with methanolic sodium methoxide treatment for 2 h at room temperature
(Hirota et al., 1988). The crotophorbolone was the starting material for the reaction to
synthesise prostratin. In our study, prostratin synthesised from crotophorbolone was
recovered after separation by preparative TLC, stored at 4�C for 3 weeks and then mass
was detected by Nano-LC-ESI-MS/MS. The result showed complete degradation of
prostratin obtained from Jatropha PEs, which may be due to prolonged storage (3 weeks)
in dried form. Due to limited sample availability to test the mass, we further subjected the
‘reaction mixture-B’ (which was stored in �80�C) directly to Nano-LC-ESI-MS/MS.
The results showed similar chromatographic peak pattern and had
a similar
molecular weight to that of the reference prostratin obtained from Sigma
(Supplementary Figure S2 – online only).
3. Conclusion
The results suggest that prostratin can be synthesised from Jatropha PEs. However, further
optimisation of conditions to have higher yield is needed. Our assumption is that the
rapidly growing Jatropha biodiesel industry could yield high amounts of oil, about 26
million tons/annum by 2015 (GEXSI, 2008). Considering the average PEs content (our
laboratory observation) of 3–6 g kg�1 oil (PMA equivalent) or 73–145 mg kg�1 oil
(Jatropha factor C1 equivalent), there exists a potential of obtaining 79,500–15,900
tonnes or � 1900–3850 tonnes of PEs, respectively. Overall, the PEs could be obtained as a
by-product in the process of biodiesel production. This means, there will be no
extra burden on the ecosystem for disposing of toxic PEs and even the prostratin
synthesised from Jatropha PEs could be a value added product to the Jatropha biodiesel
industry.

1462
R.K. Devappa et al.
Supplementary material
Figures S1 and S2 and experimental details relating to this paper can be found online.
Acknowledgements
The authors are grateful to the Bundesministerium fur Bildung und Forschung (BMBF), Berlin,
¨
Germany, for financial assistance. The technical assistance of Mr Hermann Baumgartner is
acknowledged.
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