Grupo de Alelopatía, Departamento de Química Orgánica, Facultad de Ciencias, Universidad de Cádiz, C/ República Saharaui s/n, Apdo. 40, 11510-Puerto Real (Cádiz), Spain. email@example.com
The discovery of new allelochemicals from plants or microbes has attracted much attention in wood decay since humans began building with wood thousands of years ago. When trees with natural durability were available, they were commonly used, but the scarcity of durable timbers in some areas of the world coupled with a need to make the wood products and structures enduring, led to the development of techniques to preserve wood. The most useful of these processes consist of adding adequate concentrations of different substances that upgrade wood resistance to biological attacks. Nowadays, it is important to replace synthetic wood preservatives by more environmentally friendly natural products. Furthermore, there is an enormous problem about the elimination of the vegetal waste from the agricultural and food industries in Europe. Thus, the interest to obtain new natural wood preservatives from these residues is increasing.
Volatile constituents and flavonoids of extracts of Citrus fruits have been suggested to act as defences against fungus and insects. In the present study, the essential oil from the fruit peels of different varieties of Citrus sinensis was collected by water stream. The non-volatile solids were subsequently extracted with hexane, dichloromethane and methanol. All the extracts were tested for activity with the etiolated coleoptiles bioassay. The active extracts were chromatographed to obtain several polymethoxylated flavones. Further bioassays will be performed in order to determine the possible use of these compounds as wood preservatives.
Citrus essential oils, polymethoxylated flavones, bioactivity
Defence systems in living organisms are based in many cases in autoprotection by means of selective natural biocides. Plant kingdom offers the biggest scientific potential for these biocides.
The majority of trees produce natural compounds, usually in form of resins or oils, which impregnate their wood and give them resistance against xylophagous organisms (fungus and/or insects). In 1979, Hart and Shrimpton investigated the application of extracts of woods with biocidal effect in other species woods. Results were not satisfactory due to the loss of bioactivity of extracts due to interactions wood/extract. Nevertheless, Bultman et al. (1986, 1987) achieved promising results using extracts of guayule's resin (Parthenium argentatum).
Nowadays, there are some European projects under the European CRAFT research programmes that are led by CIRAD (France) and CIDEMCO (Spain), where biocidal behaviour of different extracts from wood species (as Cupressus sempervirens, Cedrus atlantica ) is being studied. Further research has shown that different compounds with high bioactivity can be found in Citrus fruits. These compounds are flavones (El-Shafae 2002), phenolic compounds and essential oils. The figure below shows one example of each compound found in Citrus
The BCN Research Laboratories, Inc. located in Tennessee (US) conducted antimicrobiological studies of Citrus extracts and confirmed their activity against bacteria Gram Positive and Gram Negative (Vargas 1999; El-Sayed ST 2001).Accordingly, OMRI (Organic Materials Review Institute) in Oregon (US) approved the commercialisation of the Citrus extract product CITROBIO, for disinfection in agrifood industries (Ezeonu 2001).
The use of Citrus waste as insecticide and/or fungicide has not been developed until now. Its activity is still in an initial stage of knowledge (Strange 1993), but some studies published during the last years allow to foresee that essential oils, flavones and some phenolic compounds from Citrus show some kind of biocide activity (Chkhisvishali 1995; Shalaby 1998; Singh 2001; El Shafae 2002; Del Río 2000;2004).
Agrifood industries that work with fruit and vegetables as raw materials generate a lot of waste, meaning in some cases more than 60% of the initial weight (e.g. juice and/or marmalade industries). Actual exploitation techniques of this waste are not well solved. The main use, as compost for field fertilisation, has showed to negatively affect crop germination, nutrient availability, infiltration and water evaporation, plagues and microbial activity.
The main objective of the current work is to extract a natural biocide, alternative to the existing ones, from Citrus waste in order to be used as insecticide and/or fungicide in new wood preservatives.
Protocols for preparation of raw material
Laranja do Algarve Lda. (LARA) is a Portuguese SME, that focuses its activity on the production and commercialisation of orange juice and its by-products. It produces natural juice, pasteurised and without artificial colours or preservatives. By-products from orange juice processing (pulp, peel, seeds) are crushed, pressed, dried and commercialised for animal feeding. Two different varieties of orange wastes (Citrus sinensis) “Naveline” and “Valencia Late” were selected. For each variety, “FRIP” was the name given to the first waste obtained from the juice production (high pulp and peel content waste, no seeds) and “SEMFIN TOTAL” was the name for the waste obtained at the end of the production chain (rich in seeds and peel waste, but also with pulp).
Collection of orange wastes.
Orange processing was as follows. The fruit was washed in a tank with 50 thousand liters of water to remove all the dirtiness. After laundering, oranges were selected and calibrated in different sizes.
Depending on its size, they passed through appropriate tools for juice extraction. After that, the juice was filtered to obtain an uniform texture and was pasteurized and stored. The waste of the process was packed in 10 kg black plastic bags and placed into a container. When material was to be processed (dried) within 24 hours, it was stored at room temperature. Otherwise, it was refrigerated (4±1ºC) or frozen (-18ºC).
For our study, 60kg of Naveline orange waste and 60kg of Valencia Late orange waste (30kg of frip and 30kg of semfin wastes) were stored in a fridge. Material was defrosted at room temperature for 24 hours. Around 20 kg of fresh raw material was placed on a plastic mesh and distributed on several holed trays in a computer assisted air-forced drying oven. Material was dried for a week at a temperature of 25-35ºC, and a relative humidity of 10%. Drying process finished when raw material reached a final weight of 6-10% the initial sample weight. Dried material was packed into opaque sealed containers and stored at room temperature until be analysed.
Extraction and isolation
Steam distillation has been, together with solvent extraction, the most widely used conventional technique for the extraction of essential oils plants. Waste samples of Valencia Late frip were ground. Steam distillation was performed in an apparatus containing 200g of waste with 200mL of water until the obtention of 50mL of essential oil-water mixture. Extraction of this mixture with 2mL of diethyl ether was performed and ethereal phases with essential oils (256mg) were stored at 4ºC in the dark.
The extract was separated on silica gel using n-Hexane/EtOAc mixture of increasing polarity, and was purified by HPLC with a LiChrosphere Si60 (50µ)/250-4 column in normal phase.
An immersion extraction of Valencia Late frip was performed in vessels containing 200g of waste in 1:3 w/v of solvent n-Hexane and DCM. The different extracts were evaporated under vacuum in a cold water bath, to avoid of volatiles compounds. This technique yielded 1.8663g for DCM extract and 5.6g for n-Hexane. These extracts were separated on silica gel using n-Hexane/EtOAc mixtures of increasing polarity again and after purified by HPLC. Figure 1 shows the different compounds obtained from DCM extract.
Evaluation of extract bioactivity
The objective of this task consists in testing the bioactivity of the crude extracts obtained through the different extraction techniques. Evaluation of the activity of each extract was made using the etiolated wheat coleoptiles bioassay (Cutler, 1984). The bioassay was carried out in 10 mL test tubes. Five etiolated coleoptiles obtained from the apical part of three days-old wheat seedlings were placed in 2 mL of phosphate/citrate buffered solution adjusted at pH 5.6 containing the extract. The solution was kept in the darkness under constant agitation during 24 h at 22ºC.
Test solutions were prepared from the initial extract through successive dilutions until final concentrations of 1000, 500, 250, 125 and 75 ppm. Each assay was repeated three times, including the control. Wheat coleoptile length was measured using Photomed®, a digital device developed by our group to obtain accurate measurements in the range of 4-10 mm (typical length of a coleoptile). Data were automatically processed and statistically analysed by the Studentt test. Data are presented as percentage differences from the control, where zero represents the control, positive values stimulation of the coleoptile growth and negative values inhibition of the coleoptile growth.
Figure 1. Main compounds isolated from steam distillation extract (1-5) and immersion DCM extract (6-11) of Valencia Late frip.
Selective extraction of bioactive metabolites.
- extraction yield depended on the solvent used, being higher for more polar solvents. Immersion in DCM extraction showed a better performance than the immersion in hexane extraction, and valencia late variety provided a bigger extractive quantity than Naveline. Therefore, different compounds were isolated (essential oils, flavones and phenolic compounds) depending on the orange cultivar, the solvents and the extraction methods used.
Evaluation of extract bioactivity
The results of generalist bioactivity from Valencia Late extract are shown in Figure 2 and allow us to propose for studies of bioactivity on termites and fungus of this variety. This cultivar was the most active in the wheat coleoptiles bioassay and with all the methods employed (Immersion: I/H,I/DCM, I/Acetone, I/MeOH,I/Water; Steam –Distillation: AV; Microwaves: MAE/Acetone,MAE/MeOH, MAE/Water and Ultrasonic: US/Diethylether).The best activities shown in the different extractions methods were with Immersion in Hexane, Immersion in DCM and Steam-Distillation (Figure 2).
The compounds isolated from these extracts present potential fungicide and /or insecticide activities. It needs to do bio effective test to see if there are bioactive molecules in each extracts.(Huamin, 2002).
Fig. 2. Results of wheat coleoptiles bioassay extracts of Valencia Late Frip
This research was supported by European Project entitled: “New Natural Wood Preservatives from Citrus Waste” (CIPRES) (G1ST-CT-2002-50313)
Chkhisvishcili ID and Gogiya NN (1995). Flavonoids of Citrus fruit refuse and their fungistatic action on the fungus Phoma tracheiphila. Prikladnaya Biokhimiya I Mikrobiologiya, 31, 341-5.
Cutler HG (1984). Afresh look at the wheat coleoptile bioassay. Proceeding of the 11TH Annual Meeting of the Plant Growth Regulator Society of America.1-9.
Del Rio J A, Arcas M C, Botia J M, Ortuño AM (2000). Involvement of phenolic compounds in the antifungal defense mechanisms of Olea europea L. and Citrus sp. Recent Research Developments in Agricultural and Food Chemistry 4, 331-41.
Del Río J A, Gómez P, Arcas MC, Botia JM, Ortuno A (2004). Changes in the Leaves of Polymethoxyflavones and Flavanones as Part of the Defense Mechanism of Citrus sinensis (Cv. Valencia Late) Fruits against Phytophthora citrophthora. Journal of Agricultural and Food Chemistry 52, 1913-17.
El-Sayed ST, Jwanny EW, Salem AM, Shehata AN (2001). Bioactivity of Egyptian orange (Citrus sinensis) peel against some microorganisms. Al-Azhar Journal of Microbiology 51, 83-98.
El-Shafae Azza M (2002). Bioactive polymethoxyflavones and flavanone glycosides from the peels of Citrus sinensis. Chinese Pharmaceutical Journal 54, 199-206.
Ezeonu CF, Chidume GI, Udedi SC (2001). Insecticidal properties of volatile extracts of orange peels; Bioresource Technology, 76, 273-274.
Huamin H,Juntao F,Anliang Ch, Xing Z (2002). Estudies on the bioactivity of essential oils against insects.Tianran Chanwu Yanjiu Yu Kaifa, 14, 27-30.
Shalaby AA, Allam KA, Mostafa A (1998). Insecticidal properties of citrus oil against Culex pipiens and Musca domestica. A Journal of the Egyptian Society of Parasotology, 28, 595-606.
SinghGurdip, Singh Om Prakash, Rao P, Singh PK, Pandey KP (2001). Studies on essential oils, part 29: Insecticidal activity of some volatile oils and monoterpenoids against white termite (Odontotermes obesus Rhamb). Sugar Cane International, 18-20.
Strange Richard R, Midland Sharon L, Sims James J, (1993). An antifungal compound produced by grapefruit and Valencia orange after wounding of the peel. Journal of Natural Products 56, 1627-9.
Vargas I, Sanz I, Moya P, Prima-Yúfera E (1999). Antimicrobial and antioxidant compounds in the non-volatile fraction of expressed orange essential oil. Journal of Food Protection 62, 929-32.