Calotropis procera has innumerous relevant biological activi¬ties found in its vegetative tissues.
There is an increasing interest concerning the plant Calotropis procera due to innumerous relevant biological activi¬ties found in its vegetative tissues. It is easy to obtain Calotropis procera latex where the plant is found because of its extraor¬dinary endogenous production. It would not be surprising Calotropis procera to produce more latex than Hevea brasiliensis in a comparative basis. Twenty milliliters of latex can be collected within 12 min. This certainly contributed to the exploitation of the latex for curative purposes. The plant seems to be deeply inserted in folk medicine in India. In Brazil poor¬est people use the crude latex mainly to combat skin infections by topic application. There is some information of people that ingest the crude latex diluted in water to combat hyperglycemic effects. Such usages may provoke unexpected effects by continu¬ous latex exposure not yet identiﬁed. Rasik et al. (1999) reported that latex of Calotropis procera has healing potential on der¬mal wounds in Guinea pigs. However, adverse effects of latex of Calotropis procera have also been reported as cited before (Alencar et al., 2006). Furthermore, no information is available concerning possible allergenic effects caused by latex contact. We have accumulated efforts to fractionate the whole latex of Calotropis procera in attempt to separate interesting activities from undesired properties. The NDL, DL and RL fractions were obtained after executing an ordinary protocol based on cen¬trifugation and dialysis that gave rise to a fraction (NDL) that anti-inﬂammatory and antinociceptive properties were joined (Alencar et al., 2004; Soares et al., 2005). Recently we have shown that both NDL and DL are also potent in prevent develop¬ment of larvae of Aedes aegypti, the mosquito vector of Dengue virus transmission (Ramos et al., 2006a). Proteins of NDL were also shown to be devoid of acute toxicology and could not be found in fecal material of rats receiving doses by oral route for 35 days (Ramos et al., 2006b). Despite the relevance of these results, it is not known yet whether the latex of Calotropis pro-cera displays adverse allergenic effects and if does, what could be the contribution of the latex proteins in such an undesired event. This question has motivated the present study as well the fact that the latex has been shown to exhibit anti-inﬂammatory and anti-cancer activity when tested by oral route in rats (Arya and Kumar, 2005; Choedon et al., 2006). As it could be expected more expressive immunological responses were veriﬁed in animals that were sensitized by subcu¬taneous route. The DL fraction was unable to amplify antibodies synthesis. However, it has been already determined that all inﬂammatory potency of the whole latex of Calotropis procera is present in this fraction (Alencar et al., 2006). The DL fraction is composed of salts and many other low molecular substances some of such already described (Seiber et al., 1982; Shivkar and Kumar, 2003). In addition we have determined the presence of peptides in DL although any further biochemical or functional characterization has been done yet. The very low concentra¬tion of such molecules in DL could certainly explain absence of detectable immunological response. Even by subcutaneous route NDL as well as RL elicited IgE synthesis that evidences the presence of proteins in these fractions able to induce allergy. Nevertheless, this proﬁle was not repeated in animals receiving NDL or RL by oral route, even considering that repetitive doses were initially done for 10 con¬secutive days. These ﬁndings provide evidences for an inducible tolerance acquired by mice receiving latex proteins by oral route. In a recent study, we have submitted groups of rats to receive doses of NDL by oral route during 35 consecutive days to eval¬uate sub-chronic effects. Surprisingly, white blood cell proﬁle was altered in which lymphocytes were found to be augmented (unpublished results). Another previous study had already cited similar observation, although the whole latex was the sample assayed instead of the richest protein fraction of the latex (Dada et al., 2002). These results and those shown in Figs. 3A and 5A suggest that latex proteins may act as an immune stimulant. The fact that results found with RL fraction were fully similar to those determined to NDL reinforces the idea that immuno¬logical and allergenic responses displayed by RL should be attributed to the presence in RL of proteins naturally found in NDL. The present study describes immunological and allergenic effects induced by latex fractions of Calotropis procera in mice. Allergenic effects could be detected in anti-sera of mice that were sensitized subcutaneously while such effects were not detected when the animals were treated by oral route. The fact that treat¬ment of rats with latex fraction (NDL) by oral route did not induce detectable allergenic response ought to be considered as a relevant point. This opens the question if allergenic patients could be treated by receiving oral doses of latex in attempt to further develop tolerance when exposed to latex proteins. This exciting question will remain to be investigated. Considering that latex is also used as topic agent, it remains to be investigated if this contact could induce undesired effects by long-term usage. Of similar relevance, skin reactions provoked by latex exposure should be investigated taken as sample the latex fractions described here. The anti-inﬂammatory and anti¬cancer activities found to be present into the latex were already reported to be effective by oral administration of the latex (Arya and Kumar, 2005; Choedon et al., 2006). 5. Conclusion The results presented here, regarding absence of allergenic effects when the latex proteins were administrated by oral route represent new relevant data to the use of latex constituents as putative drugs. Presently, anti-inﬂammatory and antinociceptive activities of the NDL fraction of the latex are been evaluated by oral route and will clarify the remaining question; if such activ¬ities will be retained when tested by oral route. Contributions of different research groups have progressively supported the Ethnopharmacology of this species. Acknowledgements Studies on biochemical, functional and applied properties of the latex from Calotropis procera has been supported by grants of the following agencies: MCT/PADCT, CNPq, FUN¬CAP, RENOBIO and CAPES from Brazil and International Foundation for Science (IFS) from Sweden. References • Alencar, N.M.A., Figueiredo, I.S.T., Vale, M.R., Bitencurt, F.S., Oliveira, J.S., Ribeiro, R.A., Ramos, M.V., 2004. Anti-inﬂammatory effect of the latex from Calotropis procera in three different experimental models: peritonitis, paw edema and hemorrhagic cystitis. Planta Med. 70, 1144–1149. • Alencar, N.M.N., Oliveira, J.S., Mesquita, R.O., Lima, M.W., Vale, M.R., Etchells, J.P., Freitas, C.D.T., Ramos, M.V., 2006. Pro-and anti¬inﬂammatory activities of the latex from Calotropis procera (Ait.) R. Br. are trigged by compounds fractionated by dialysis. Inﬂamm. Res. 55, 559– 564 • . Al-Mezaine, H.S., Al-Rajhi, A.A., Al-Assiri, A., Wagoner, M.D., 2005. Calotropis procera (ushaar) keratitis. Am. J. Ophthalmol. 391, 199–202. • Arya, S., Kumar, V.L., 2005. Antiinﬂammatory efﬁcacy of extracts of latex of Calotropis procera against different mediators of inﬂammation. Mediat. Inﬂamm. 2005, 228–232. • Bradford, M.M., 1976. A rapid and sensitive method for the quantiﬁcation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 12, 248–254. • Choedon, T., Mathan, G., Arya, S., Kumar, V.L., Kumar, V., 2006. Anticancer and cytotoxic properties of the latex of Calotropis procera in a transgenic mouse model of hepatocellular carcinoma. World J. Gastroenterol. 12, 2517– 2522. • Dada, Y.O., Lamidi, M.T., Eghianruwa, K.I., Adepaju, F., 2002. Effects of oral administration of the latex of Calotropis procera on weights, hematology and plasma biochemistry of rats. Trop. Vet. 20, 218–225. • Handa, F., Sadana, J.K., Sharma, P.K., 1984. Allergic contact dermatitis due to the plant Calotropis procera (Vern: AK): a case report. Indian J. Dermatol. 29, 27–29. • Iqbal, Z., Lateef, M., Jabbar, A., Muhammad, G., Khan, M.N., 2005. Anthelmintic activity of Calotropis procera (Ait.) ﬂowers in sheep. J. Ethnopharmacol. 14, 256–261. • Kumar, S., Dewan, S., Sangraula, H., Kumar, V.L., 2001. Anti-diarrhoeal activity of the latex of Calotropis procera. J. Ethnopharmacol. 76, 115–118. • Laemmli, U.K., 1970. Cleavage of structural proteins during the assembly of the bacteriophage t4. Nature 227, 680–685. • Larhsini, M., Markouk, M., Jaouhari, J.T., Bekkouche, K., Lazrek, H.B., Jana, M., 2002. The anti-pyretic activity of some Moroccan medicinal plants. Phytother. Res. 16, 97–98. • Melo, V.M.M., Xavier-Filho, J., Lima, M.G.S., Danon-Prouvost, A., 1994. Aller¬genicity and tolerance from Brazil Nut (Bertholletia excelsa H. B. K.). Food Agric. Immunol. 6, 185–195. • Mota, I., Wong, D., 1969. Homologous and heterologous passive cutaneous anaphylactic activity of mouse anti-sera during the course of immunization. Life Sci. 8, 813–820. Ramos, M.V., Moreira, R.A., Oliveira, J.T.A., Cavada, B.S., Roug´e, P., 1996. Structural similarities among Diocleinae lectins. In: Van Driessche, E., Roug´ • e, P., Beeckmans, S., Bog-Hansen, T.C. (Eds.), Lectins: Biology, Bio¬chemistry, Clinical Biochemistry, vol. 11. Textop, Hellerup, Denmark, pp. 44–49. • Ramos, M.V., Bandeira, G.P., Freitas, C.D.T., Nogueira, N.A.P., Alencar, N.M.N., Sousa, P.A.S., Carvalho, A.F.U., 2006a. Latex constituents from Calotropis procera (R.Br.) display toxicity upon egg hatching and larvae of Aedes aegypti (Linn). Mem. Inst. Oswaldo Cruz 101, 503–510. o M.V. Ramos et al. / Journal of Ethnopharmacology 111 (2007) 115–122 • Ramos, M.V., Aguiar, V.C., Xavier, A.A.S., Lima, M.W., Bandeira, G.P., Etchells, J.P., Nogueira, N.A.P., Alencar, N.M.N., 2006b. Latex proteins from the plant Calotropis procera are partially digested upon in vitro enzymatic action and are not immunologically detected in fecal material. Fitoterapia 77, 251–256. • Rasik, A.M., Raghubir, R., Gupta, A., Shukla, A., Dubey, M.P., Srivastava, S., Jain, H.K., Kulshrestha, D.K., 1999. Healing potential of Calotropis procera on dermal wounds in Guinea pigs. J. Ethnopharmacol. 68, 261–266. • Soares, P.M., Lima, S.R., Matos, S.G., Andrade, M.M., Patroc´ınio, M.C.A., Freitas, C.D.T., Ramos, M.V., Criddle, D.N., Cardi, B.A., Carvalho, K.M., Assreuy, A.M.S., Vasconcelos, S.M.M., 2005. Antinociceptive activity of Calotropis procera latex in mice. J. Ethnopharmacol. 99, 125–129. • Seiber, J.N., Nelson, C.J., Lee, S.M., 1982. Cardenolides in the latex and leaves of seven Asclepias species and Calotropis procera. Phytochemistry 21, 2343–2348. • Shivkar, Y.M., Kumar, V.L., 2003. Histamine mediates the pro-inﬂammatory effect of latex of Calotropis procera in rats. Mediat. Inﬂamm. 12, 299– • 302. Shivkar, Y.M., Kumar, V.L., 2004. Effect of anti-inﬂammatory drugs on pleurisy induced by latex of Calotropis procera in rats. Pharmacol. Res. 50, 335–340. Sussman, G.L., Beezhold, D.H., Liss, G., 2002. Latex allergy: historical per¬spective. Methods 27, 3–9. • Yeang, H.Y., Arif, S.A.M., Yusof, F., Sunderasan, E., 2002. Allergenic proteins of natural rubber latex. Methods 27, 32–45.