Pharmaceutica Analytica Acta

Experiments conducted with photodynamic substances yielded very promising results. In a recent work performed by Abdel-Kader et al. [10] it was demonstrated that larvae of Culex are sensitive against hematoporphyrine (0.07 μmol/ml). Also larvae of Musa domestica could be killed with this substance (10 μmol/ml). In the course of his PhD thesis Allah el Tayeb, [11] could demonstrate the effectiveness of hematoporphyrine against Culex and eggs of the snail Lymnea natalensis (vector of the trematode Fasciola hepatica). In this work the penetration of the photosensitizer inside the organism was shown by means of fluorescence microscopy as well as the pronounced effects on the ultrastructure of inner organs and muscles [10,11].

Wohllebe et al. [12], under laboratory conditions, the use of chlorophyllin/pheophorbid as photodynamic substances for pest control in water bodies promises to be not only effective and ecologically beneficial but also cheap. The LD₅₀ (50% of mortality in the tested organisms) value in Culex sp. larvae was about 6.88 mg/l, in Chaoborus sp. larvae about 24.18 mg/l, and in Daphnia 0.55 mg/l. The LD₅₀ values determined for pheophorbid were 8.44 mg/l in Culex, 1.05 mg/l in Chaoborus, and 0.45 mg/l in Daphnia, respectively. In some cases, chlorophyllin and pheophorbid were also found to be (less) active in darkness. The results presented in this paper show that chlorophyllin is about a factor of 100 more effective than methylene blue or hematoporphyrine, which were tested earlier for the same purpose. It is also much cheaper and, as a substance found in every green plant, it is 100% biodegradable.

Erzinger et al. [13] was demonstrated that mosquito larvae can be killed by means of photodynamic processes after the larvae have incorporated the photosensitizer chlorophyllin or pheophorbid, and were treated with light. The water-soluble substances were applied to and incorporated by the larvae in darkness. With Chaoborus sp. a dark incubation of about 3 h is sufficient to yield mortality of about 90% and ≥6 h resulted in almost 100% mortality during subsequent illumination. Temperature did not influence mortality of the larvae significantly in a treatment of 6 h dark incubation and subsequent 3 h illumination. At 10°C, 20°C, or 30°C, between 80% and 100% of the treated larvae died when the light intensity from a solar simulator was above 30 W/m². Lower irradiances were less effective.

Wohllebe et al. [14] have demonstrated for the first time that the photodynamic treatment of C. crystallinus larvae with chlorophyllin induces necrosis and apoptosis in these organisms. The intrinsic pathway of apoptosis plays a key role in this model.

Preservation of photodynamic activity of chlorophyllin

Erzinger et al. [13] described a major problem for the use of chlorophyll and its derivatives are related allomerization reactions. Allomerized chlorophyll a differs from the original material in that the product of hot alkaline hydrolysis is a mixture of unstable chlorins instead of the initial product chlorin. The same unstable chlorins are obtained from unallomerized chlorophyll or the magnesium-free compounds (pheophytin a or methyl pheophorbide a by saponification with alcoholic alkali at or below room temperature). Allomerization of chlorins is a dehydrogenation caused by oxygen of the air. It is interesting that the dehydrogenation of a very easily oxidizable group in chlorophyll is without pronounced effect on its color, although marked color changes, as a rule, accompany the oxidation of colored substances. The unusual behavior of chlorophyll in this respect is due to the fact that the chromophoric group is not the most easily oxidized group [15-17] (Figure 1).

Figure 1: Chlorophyll and metabolic derivates.

One of the alternatives considered were to make structural changes in chlorophyll derivatives by substitution of magnesium or in the process of conservation. The LD₅₀ value of magnesium chlorophyllin was about 22.25 mg/l and for Zn chlorophyll 17.53 mg/l, while Cu chlorophyll (LD₅₀ 0.1 mg/l) was shown to be toxic also without light. Chlorophyllin, which was lyophilized immediately after extraction, was far more lethal to the larvae (LD₅₀0 14.88 mg/l) than air-dried Mg chlorophyllin.Lyophilization immediately after isolation increases photodynamic activity of chlorophyllin and ensures its long-term stability [13].

Development of a new product

In 2010 Erzinger and Häder [18] developed and patented at INPI (National Institute of Intellectual Property) a new Bioinsecticide Nontoxic Biodegradable from a new semi-synthetic derivative of chlorophyll and in conjunction with a formulation system was able to get a product with high stability front light and maintained the same lethal power of chlorophyll and chlorophyllin for mosquito larvae described in previous work. Currently is being done to study the possible environmental impacts of the continued use of this new product.

The derivates of chlorophyll is a very interesting substance for photodynamic pest control. For this reason we conducted some primarily experiments in which we tested the effects on larvae of Chaoborus and Culex flies. We extracted chlorophyll from different plant sources (spinach, grass, dandelion etc.). Some experiments we conducted with chlorophyll converted into pheophytin or into a watersoluble chlorophyllide. We have developed a cheap new strategy for reducing the number of mosquito larvae, which may be very important for reducing the amount of mosquito bites and thereby the incidence of malaria and other parasitic diseases in the future.

Prof. Dr. Donat Peter Häder is a postdoctoral research fellow of the FAU. The several studies presented in this paper were financially supported by Research Support Fund of UNIVILLE and DAAD, Germany. Dr. P. Richter, Department of Biology, Cell Biology, Friedrich-Alexander-University Erlangen-Nuremberg, Germany and S. Wohllebe, Bavarian State Research Centre for Agriculture, Institute for Fisheries, Starnberg, Baden-Württemberg, Germany for the fruitful collaboration.