Journal ABSTRACT INTRODUCTION - Misr University

BUTYL- ISO-BUTYL PHTHALATE AS AN ORBANCHE CRENATA SEED GERMINATION ACTIVATOR SECRETED BY ROOTS OF VICIA FABA

Journal J. Biol. Chem. Environ. Sci., 2013, Vol. 8(4):157-167 www.acepsag.org

Rasha, A. Mohammed1; Khaled, M. A. Ramadan2, Sameh, E. Hassanien1, Ragy R. Francis2 and Ahmed Z. Abdel Azeiz3 1

Bioinformatics Department, Agricultural Genetic Engineering Research Institute (AGERI), Agricultural Research Center (ARC), Giza, Egypt. 2 Department of Agricultural Biochemistry, Faculty of Agriculture, Ain Shams University, Cairo, Egypt 3 Colleges of Biotechnology, Misr University for Science and Technology (MUST), 6th October City, Egypt.

ABSTRACT Broomrape (Orbanche sp.) is a holoparasitic plant that causes dramatic decrease in worldwide Vicia faba production. Several plants which produce broomrape seeds germination stimulators belong to the strigolactones group. Use of such compound can be a useful method to control this parasite, due to the fact that the germinated broomrape seeds die if it can’t attach with the host plant during few days. Therefore, the present study aimed to structurally identify a broomrape seed germination stimulator produced by the root of Vcia faba. The root exudates were collected and extracted having different solvents with gradient polarity and screened for broomrape germination stimulation activity. The active extract was purified by silica gel column and the chemical structure of the pure compound was elucidated by spectroscopic 1D and 2D-NMR, IR and MS analyses. One pure germination activator compound was separated and structurally identified as butyl, iso-butyl phthalate. Key words: Orobanche sp., broomrape, root exudates, Vicia faba, butyl phthalate.

INTRODUCTION Broomrape (Orobanche sp.) is a holoparasitic plant which it lakes of chlorophyll. It attaches to dicotyledonous host plants using a special intrusive multicellular organ, the haustorium, and deprive water and

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BUTYL- ISO-BUTYL PHTHALATE AS AN ORBANCHE CRENATA SEED

nutrients from them. Parasitic plants sense their host plant through the recognition of secondary metabolites released by its roots (Yoder, 1999). Due to the fact that the germinated seeds of broomrape die if it can’t attach with identify natural seeds germination stimulators. These activators are produced in the root exudates of several plants. These compounds activate developmental programs such as germination, growth of the radical towards the host root, attachment-organ development and the creation of bridge tissue connecting the vascular tissues of the host and parasite. Most of them are belonging to the strigolactones, isoflavanones and sesquiterpene lactones (Fischer et al., 1989; Pe´rez de-Luque, 2000; Keyes et al., 2001; Bouwmeester et al., 2003; Tsanuo et al., 2003 and Xiaonan et al., 2009). Furthermore, some of the parasitic seeds activators were reported to be produced by microorganisms. Yoneyama et al. (1998) reported that the fungal metabolites Cotylenins and fusicoccins stimulate seed germination of Striga hermonthica and Orobanche minor. Antonio et al. (2006) also reported that of several fusicoccin (phytotoxins) derivatives had the ability to stimulate the seed germination of the parasitic species Orobanche ramose. On the other hand, several studies were conducted to identify broomrape seeds germination inhibitors. Mohamed and Khaled (2010) reported that several Fusarium spp. produce some of these inhibitors in their broth. Some of these compounds were identified by Andolfi et al. (2005) as macrocyclic trichothecenes, namely, verrucarins A, B, M, and L acetate, roridin A, isotrichoverrin B, and trichoverrol B. The main metabolite produced by F. compactum was neosoloaniol monoacetate. The present study aimed to separate and structurally elucidate a broomrape seed germination stimulator produced by the root of Egyptian varieties of Vcia faba.

MATERIALS AND METHODS Plant materials: Two resistant Faba bean species (Masr-1 and Giza 843) and two sensitive species (Nubaria and V) for broomrape infection and Orbanche crenata seeds were obtained from Legume Crops Research Institute, Agricultural Research Center, Giza, Egypt.

J. Biol. Chem. Environ. Sci., 2013, 8(4), 157-167

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Seeds sterilization: Faba bean and broomrape seeds were surface-sterilized with ethanol 70% for 10min, rinsed thoroughly with sterile distilled water and dried for 15-min in a laminar air flow cabinet. Viability test: Viability of broomrape seeds was assessed by detecting respiratory activity through staining with 2, 3, 5-triphenyltetrazolium chloride (TTC) (AOSA, 2002). Conditioning of broomrape seeds: The broomrape seeds were placed separately on 12 discs of 2-cm diameter glass fiber filter paper (GFFP), obtained from Sigma, in Petri dishes. The seeds were moistened with 1.5 ml, of sterilized distilled water and incubated in the dark at 25 ˚C for one week in order to promote the necessary conditioning for germination (Fernandez et al., 2008). Collection of root exudates: Seeds of Faba bean were maintained in plastic pots containing sterilized sand under growth chamber conditions (25 °C, 16 h light). Plants were irrigated with Hoagland nutrient solution (twice/week). After 3 weeks the plants were removed from the sand and their roots were washed with sterilized distilled water, immersed in a 500ml conical flask containing 100ml of sterilized distilled water (10 plants/ flask) and left at 24 °C for one day in a growth chamber. During this period the roots exudates were released in the water. Extraction of the active compounds from the root exudates solution: The secreted compounds were extracted from the root exudates solution by the following solvents with gradient polarity: hexane, methylene chloride and ethyl acetate. Each solvent extract was evaporated to dryness by rotary evaporator at 40◦C. Stimulation of broomrape seeds germination: The residue remaining from each solvent extract of the root exudates solution was dissolved in a little amount of Dimethyl sulfoxide (DMSO). Each solution or the crude root exudates solution was added on a disk of GFFP followed by addition of broomrape seeds (conditioned and nonconditioned seeds). The disks were then placed on a moistened, sterilized filter paper in a Petri dish and left at 24°C for 7 days in incubation chamber at 25C˚, in dark. A negative control was made by water and DMSO. Germination was scored by determining the number of germinated seeds out of 100 seeds for each solution by using a binuclear.

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Seeds were considered as germinated when the radical was visible through the seed coat. HPLC/MS analysis: It was conducted with a LC MS/MS instrument (WATERS), TQ DECECTOR fitted with an ODS (C18) column (INERTSIL RP-18, 4.6*50 mm). The crude extracts were dissolved in 60% methanol and filtered through 0.45 μm pore size filter. Five μl were injected. The mobile phase was 60% methanol in water and was changed to 100% methanol 30 min after injection. The column was then washed with 100% methanol for 20 min. The flow rate was 0.2 ml min–1 and the column temperature was set to 40°C. (Kaori, et al., 2008). HPLC analysis: The active extract was scanned by Shimadzu UV-1800 spectrophotometer for identification of the detection wavelength. The HPLC analysis was conducted by using Agilent HPLC 1050 equipped with binary pump and variable wave length detector. Two methods were tested: 1) ODS (25 X 4.6 X 5μm) column eluted with acetonitrile and 2) silica gel column (25 X 4.6 X 5μm) eluted with ethyl acetate: hexane (75:25). The detection wavelength was 280nm. Purification of the active stimulator: The ethyl acetate extract was purified by using glass column packed with silica gel (Merck, G-50), eluted with two different mobile phase systems in two separate experiments: 1) stepwise elution with different mixtures of hexane: ethyl acetate, (100: 0), (75: 25), 50:50), (25: 75) and finally 100% ethyl acetate, 2) 20-ml of acetone followed by 20-ml of methanol. All of the collected fractions were analyzed by HPLC. Spectroscopic analysis: The NMR spectra (1H, 13C, APT, COSY and HMQC) were recorded on a Varian Mercury VX-300 NMR spectrometer. 1H spectra were run at 300 MHz and 13C spectra were run at 75.46 MHz in deuterated dimethyl sulphoxide (DMSO-d6). The analysis was performed in Department of Chemistry, Faculty of Science, Cairo University. The mass spectrometer analysis was performed using Direct Inlet unit (DI-50) of Shimadzu GC/MS- QP5050A at the Regional Center for Mycology and Biotechnology, AL-Azhar University. The IR analysis was performed by FTIR, IR Affinity, Shimadzu.


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RESULTS AND DISCUSSION Among the tested faba bean varieties, the maximum broomrape seeds germination percentage (12%) was obtained from the root exudates solution of the Faba bean Masr-1 variety, with the conditioned broomrape seeds; while the non-conditioned seeds showed only 6% germination percentage. The germination percentages for the other faba bean varieties were ranged from 2 to 4% only. The root exudates solution of Faba bean, variety Masr-1, was extracted with several solvents with increasing polarity. The ethyl acetate extract showed the highest broomrape seeds germination activity (fig.1).

Fig. 1: A germinated broomrape seed as affected by the ethyl acetate extract of the Faba bean root exudates solution. HPLC analysis: Striglactones were not detected in the HPLC/MS analysis of the ethyl acetate extracts. Therefore, the HPLC method had to be optimized firstly to enable following up the compound purity. This was conducted by using two HPLC columns (silica and ODS) and two different mobile phases at detection wavelength 280 nm. as identified from the spectrophotometeric scanning. The ODS column showed absence of any separated compounds, while the silica gel column showed only one pure compounds at retention time 1.4 min. (fig. 2). This result explores absence of any compounds in the HPLC/MS separation, where the separation was performed using ODS column. Therefore, this result reflects the non-polar nature of the active compound, since it bind with the ODS column and didn't elute by the used mobile phase.

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Fig. 2: The HPLC chromatogram of the ethyl acetate extract separated by silica gel column and eluted with hexane: ethyl acetate (25: 75), detected at 280nm. To ensure the compound purity, the extract was exposed to silica gel column purification and elution with two different mobile phase systems in two different experiments. The HPLC analysis of the fractions obtained from the step-wise elution system showed presence of the same compound in all fractions, except fraction 1 (eluted by 100% hexane), in which there was no any compound. Therefore, the active compound was soluble in all of the used mixtures of hexane: ethyl acetate, but with different degree of solubility. Therefore, we suggested using another elution system (acetone followed by methanol). The acetone fraction showed presence of one pure compound with the same retention time. This fraction was selected for the spectroscopic analysis and chemical structure elucidation. Spectroscopic analysis: The IR showed presence of aliphatic hydrogen at 2860 to 2960 cm-1 and carbonyl group at 1714cm-1. The mass spectrum showed the molecular ion peak at m/z 279 in addition to fragments at m/z 167and 149 which are characteristic for phthalate derivatives (Rowshanul and Rezaul, 2009). The H1 NMR spectrum showed some common main signals regions indicating presence of methyl groups at 0.88 and 0.91 ppm, methylene groups at 1.36 and 1.65 ppm, O-CH2 at 4.02 and 4.2 ppm and aromatic ring at 7.6-7.7 ppm.


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The molecular formula of the active compound was deduced as C16H22O4, which is corresponding with the molecular weight 278.3. By excluding the phthalate moiety (MW 164) the remaining weight will be 114 which are corresponding to two butyl moieties (2 X 57). This result was confirmed from the NMR analysis where it showed two O-CH2 groups at 4.22 and 4.02 ppm. The COSY data interpretation showed that the first group at 4.22 ppm (triplet) was connected with another CH2 group at 1.65 ppm (pentet), connected with CH2 group at 1.36ppm (sextet), connected with methyl group at 0.88 (triplicate). All of these groups are corresponding to a butyl chain. The second O-CH2 group at 4.02 ppm was connected with CH group at 1.86 (nonatet) which was connected with two methyl groups at 0.91ppm (isobutyl chain). The C13 and APT spectra confirmed presence of carbonyl group at 186.9 ppm, aromatic ring at 126.6 to 131.3 ppm, and the other mentioned groups (table 1). The HMQC spectrum exactly confirmed the COSY data interpretation. From these results the compound was assigned as butylisobutyl phthalate (fig. 3). Table 1: NMR spectral data of the O. crenata seeds germination stimulator:

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Fig. 3: Butyl, isobutyl phthalate To confirm our obtained result, the pure compound was injected into GC/MS and the data analysis of the obtained mass spectrum confirmed this compound as bis-isobutyl collected and extracted with ethyl acetate. This extract was directly analyzed by GC/MS. The separated compounds were primary identified by the MS library software as di-octylphthalate and and di-nonylphthalate. One of these compounds was purified by the silica gel column, as previously discussed in the method section, and analyzed by H1 and COSY NMR analysis. It was identified as diethoxybutyl phthalate. Several phthalate derivatives have been discovered in several living organisms including plants, algae and microorganisms. Rowshanul and Rezaul (2009) isolated Di-(2-ethylhexyl) Phthalate from Calotropis gigantea Flower that has antimicrobial activity against Gram positive and negatice bacteria and cytotoxicity against Artemia salina. The dibutyl phthalate was also isolated from the stem of Ipomoea carnea by Khatiwora et al. (2012). It was found to have antibacterial activity. Abdul Alim et al. (2006) identified the antimicrobial compound produced by Streptomyces bangladeshiensis as dioctyl phthalate. Farida et al. (2011) identified the antimicrobial compound produced by an actinomycete strain of Actinoalloteichus sp. as dioctyl phthalate. Mohammed (2012) isolated Di-(2-ethylhexyl) Phthalate from the culture of Streptomyces mirabilis Strain NSQu-25 that has antimicrobial and cytotoxic activity. It was clearly appeared from the literature that several plants produce several phthalate derivatives. The major role of these derivatives is it works as antimicrobial agents. It is logically that although the root of Faba bean has been penetrated by Orobanche spp, the root was not


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infected by fungi! The reason now becomes clear after this investigation; since the Faba bean root secretes antimicrobial compounds, which are the phthalate derivatives. These derivatives, at the same time, enhance the Orobanche spp. seeds germination. Conclusion: Germination of parasitic weed Orobanche spp. seeds are usually stimulated by several compound related to strigolactones, which are produced in the root exudates of the host plants. In the present study an Orobanche crenata. seeds germination stimulator was separated, purified and identified by spectroscopic techniques as butyl, isobutyl phthalate.

REFERENCES Abdul Alim, M. Al-Bari, M. Abu Sayeed, M. Sazedur Rahman and M. Ashik Mossadik. (2006). Characterization and antimicrobial activities of a Phthalic acid derivative produced by Streptomyces bangladeshiensis, a novel species collected in Bangladesh. Research Journal of Medicine and Medical Sciences, 1(2): 77-8. Andolfi, A., A., Boari, A., Evidente, and M., Vurro,(2005). Metabolites inhibiting germination of Orobanche ramosa seeds produced by Myrothecium verrucaria and Fusarium compactum. J Agric Food Chem., 53(5):1598-603. Antonio Evidente, Anna Andolfi, Michele Fiore, Angela Boari and Maurizio Vurro, (2006). Stimulation of Orobanche ramosa seed germination by fusicoccin derivatives: A structure–activity relationship study. Phytochemistry, 67 (1): 19–26. AOSA (Association of Official Seed Analysts, (2002). Tetrazolium testing handbook. In Contribution No. 29 (ed. J. Peters), Association of Official Seed Analysts Lincoln, NE, USA Bouwmeester, H.J., R.,Matusova, S., Zhongkui, M.H., Beale, (2003). Secondary metabolite signaling in host–parasitic plant interactions. Current Opinion in Plant Biology 6: 358–364. Farida B, Ab ZitouniI, Florence MathieuII, A. LebrihiII, and N. Sabaou, (2011). Taxonomic study and partial characterization of antimicrobial compounds from a moderately halophilic strain of the genus Actinoalloteichus Braz. J. Microbiol., 42 (4): 835-845. Fernandez, A.M., A., Andolfi, A., Evidente, A.Pe´rez-de-Luque, and D., Rubiales, (2008). Fenugreek root exudates with Orobanche species specific seed germination stimulatory activity. Weed Research 48: 163–168.

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Fischer, N.H., J.D. Weidenhamer, and J.M.,Bradow, (1989). Dihydroparthenolide and other sesquiterpene lactones stimulate witchweed germination. Phytochemistry 28: 2315–2317. Kaori Y, X. Xie, H. Sekimoto, Y. Takeuchi, S. Ogasawara, K. Akiyama, H. Hayashi and K. Yoneyama, (2008). Strigolactones, host recognition signals for root parasitic plants and arbuscular mycorrhizal fungi, from Fabaceae plants. New Phytologist, 179: 484– 494. Keyes, W.J., J.V., Taylor, R.P. Apkarian, and D.G., Lynn, (2001). Social controls in parasitic plant development. Plant Physiology 127: 1508–1512. Khatiwora, E., V. B., Adsul, M., Kulkarni, N. R. Deshpande, and R.V.Kashalkar, (2012). Antibacterial activity of Dibutyl Phthalate: A secondary metabolite isolated from Ipomoea carnea stem. Journal of Pharmacy Research,. 5 (1): 150-156. Mohamed, A. A. and A. E. Khaled, (2010). Fusarium spp. suppress germination and parasitic establishment of bean and hemp broomrapes. Phytopathol. Mediterr.49, 51–64. Mohammed, H. El-Sayed. (2012). Di-(2-ethylhexyl) Phthalate, a major bioactive metabolite with antimicrobial and cytotoxic activity isolated from the culture filtrate of newly isolated soil streptomyces (Streptomyces mirabilis Strain NSQu-25). World Applied Sciences Journal 20 (9): 1202-1212. Pe´rez-de-Luque, A., J.C., Galindo, F.A. Macı´as, and J.Jorrı´n, (2000). Sunflower sesquiterpene lactone models induce Orobanche cumana seed germination. Phytochemistry 53: 45–50. Rowshanul, M. H. and M. K. Rezaul, (2009). Antimicrobial and cytotoxic activity of Di-(2-ethylhexyl) phthalate and anhydrosophoradiol-3-acetate Isolated from Calotropis gigantea (Linn.) Flower. Mycobiology 37(1): 31-36. Tsanuo MK, A,Hassanali. AM.Hooper (2003). Isoflavanones from the allelopathic aqueous root exudate of Desmodium uncinatum. Phytochemistry 64: 265–273. Xiaonan, X., Y., Kaori, H., Yuta, F.,Norio, Y.,Yoichi, I., Satoshi, Y.,Takao, T.Yasutomo, and Y.Koichi, (2009). Fabacyl acetate, a germination stimulant for root parasitic plants from Pisum sativum. Phytochemistry 70: 211–215 Yoder, J.I. (1999). Parasitic plant response to host plant signals: a model for subterranean plant–plant interactions. Current Opinion in Plant Biology 2: 65–70

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‫‪Yoneyama, K., Y., Takeuchi, M., Ogasawara, M., Konnai, Y.,‬‬ ‫‪Sugimoto, T. Sassa, (1998). Cotylenins and fusicoccins stimulate seed‬‬ ‫‪germination of Striga hermonthica (Del.) Benth and Orobanche minor‬‬ ‫‪Smith. J. Agric. Food Chem., 46: 1583-1586.‬‬

‫ﺑﻴﻮﺗﻞ – اﻳﺰوﺑﻴﻮﺗﻞ ﻓﻴﺜﺎﻻت آﻤﻨﺸﻂ ﻻﻧﺒﺎت ﺑﺬور اﻟﻬﺎﻟﻮك واﻟﻤﻔﺮز ﺑﻮاﺳﻄﺔ ﺟﺬور‬ ‫ﻧﺒﺎت اﻟﻔﻮل اﻟﺒﻠﺪي‬ ‫رﺷﺎ ﻋﺒﺪ اﻟﻘﺎدر)‪ - (1‬ﺧﺎﻟﺪ ﻣﺤﻤﺪ اﻣﻴﻦ رﻣﻀﺎن)‪ - (2‬ﺳﺎﻣﺢ اﻟﺴﻴﺪ ﺣﺴﻨﻴﻦ)‪ - (1‬راﺟﻲ رﻳﺎض‬ ‫)‪(3‬‬ ‫ﻓﺮﻧﺴﻴﺲ)‪ - (2‬اﺣﻤﺪ زﻳﻦ اﻟﻌﺎﺑﺪﻳﻦ ﻋﺒﺪ اﻟﻌﺰﻳﺰ‬ ‫)‪(1‬ﻗﺴﻢ اﻟﻤﻌﻠﻮﻣﺎﺗﻴﻪ اﻟﺤﻴﻮﻳﻪ ﻣﻌﻬﺪ ﺑﺤﻮث اﻟﻬﻨﺪﺳﺔ اﻟﻮراﺛﻴﺔ ‪ -‬ﻣﺮآﺰ اﻟﺒﺤﻮث اﻟﺰراﻋﻴﺔ ‪ -‬ﺟﻴﺰة ‪ -‬ﻣﺼﺮ‪.‬‬

‫)‪(2‬ﻗﺴﻢ اﻟﻜﻴﻤﻴﺎء اﻟﺤﻴﻮﻳﺔ اﻟﺰراﻋﻴﺔ – آﻠﻴﺔ اﻟﺰراﻋﺔ – ﺟﺎﻣﻌﺔ ﻋﻴﻦ ﺷﻤﺲ – اﻟﻘﺎهﺮة – ﻣﺼﺮ‪.‬‬ ‫)‪(3‬آﻠﻴﺔ اﻟﺘﻜﻨﻮﻟﻮﺟﻴﺔ اﻟﺤﻴﻮﻳﺔ – ﺟﺎﻣﻌﺔ ﻣﺼﺮ ﻟﻠﻌﻠﻮم و اﻟﺘﻜﻨﻮﻟﻮﺟﻴﺎ – ﺟﺎﻣﻌﺔ ‪ 6‬اآﺘﻮﺑﺮ – ﻣﺼﺮ‪.‬‬ ‫ﻳﺘﻄﻔﻞ ﻧﺒﺎت اﻟﻬﺎﻟﻮك ﺗﻄﻔﻼ آﺎﻣﻼ ﻋﻠﻰ ﻧﺒﺎت اﻟﻔﻮل اﻟﺒﻠﺪى ﻣﻤﺎ ﻳﺆدى اﻟﻰ اﻧﺨﻔﺎض آﺒﻴﺮ ﻓﻰ‬ ‫اﻧﺘﺎﺟﻪ ﻓﻰ ﺟﻤﻴﻊ اﻧﺤﺎء اﻟﻌﺎﻟﻢ و ﻣﻦ هﻨﺎ ﺗﻢ اﻟﺘﻌﺮف ﻋﻠﻰ ﻣﺮآﺒﺎت ﺗﺘﻨﺘﻤﻰ ﻟﻤﺠﻤﻮﻋﻪ‬ ‫)‪ (striglactones‬اﻟﺘﻰ ﺗﻌﻤﻞ آﻤﺤﻔﺰات ﻻﻧﺒﺎت ﺑﺬور اﻟﻬﺎﻟﻮك و ﺗﻌﺘﺒﺮ هﺬﻩ اﻟﻤﺮآﺒﺎت ﻣﻦ اﺣﺪى‬ ‫اﻟﻄﺮق اﻟﻤﻔﻴﺪة اﻟﻤﺴﺘﺨﺪﻣﺔ ﻟﻠﺘﺤﻜﻢ ﻓﻰ اﻟﺘﻄﻔﻞ وﻳﺮﺟﻊ ذﻟﻚ اﻟﻰ ان ﺑﺬور اﻟﻨﺒﺎﺗﺎت اﻟﻄﻔﻴﻠﻴﻪ اﻟﻨﺎﺑﺘﻪ‬ ‫ﺳﻮف ﺗﻤﻮت اذا ﻟﻢ ﺗﻌﻠﻖ ﺑﺎﻟﻌﺎﺋﻞ ﺧﻼل اﻳﺎم ﻗﻠﻴﻠﻪ ﻟﺬﻟﻚ ﻓﺎن اﻟﻬﺪف ﻣﻦ هﺬﻩ اﻟﺪراﺳﻪ اﻟﺘﻌﺮف ﻋﻠﻰ‬ ‫ﻣﺤﻔﺰات اﻧﺒﺎت ﺑﺬور اﻟﻬﺎﻟﻮك اﻟﻤﻨﺘﺠﻪ ﻣﻦ ﺟﺬور ﻧﺒﺎﺗﺎت اﻟﻔﻮل اﻟﺒﻠﺪى و ذﻟﻚ ﻟﻠﻘﻀﺎء ﻋﻠﻰ ﺑﺬور ﻧﺒﺎت‬ ‫اﻟﻬﺎﻟﻮك اﻟﻤﺘﻌﻤﻘﻪ ﻓﻰ اﻻراﺿﻰ اﻟﺰراﻋﻴﻪ وﺗﻢ هﺬا ﺑﺘﺠﻤﻴﻊ اﻓﺮازات ﺟﺬور ﻟﻨﺒﺎت اﻟﻔﻮل اﻟﺒﻠﺪى ﻣﻦ‬ ‫ارﺑﻌﺔ اﺻﻨﺎف و اﺧﺘﻴﺎر ااﻟﺼﻨﻒ اﻟﺬي ﻳﻌﻄﻲ اﻋﻠﻲ ﻧﺴﺒﻪ اﻧﺒﺎت ﺛﻢ اﺳﺘﺨﻼص ﻧﻮاﺗﺞ اﻓﺮازات‬ ‫اﻟﺠﺬور ﺑﻤﺬﻳﺒﺎت ﻣﺨﺘﻠﻔﻪ اﻟﻘﻄﺒﻴﻪ و اﺧﺘﻴﺎر اﻟﻤﺴﺘﺨﻠﺺ اﻟﺬي اﻋﻄﻰ اﻋﻠﻲ ﻧﺴﺒﻪ اﻧﺒﺎت وﺗﻢ ﺗﻨﻘﻴﻪ هﺬا‬ ‫اﻟﻤﺴﺘﺨﻠﺺ اﻟﻨﺸﻂ ﺑﻌﻤﻮد ﻣﻦ اﻟﺴﻠﻴﻜﺎﺟﻞ و اﻗﺘﺮاح اﻟﺘﺮآﻴﺐ اﻟﻜﻴﻤﻴﺎﺋﻰ ﻟﻬﺬا اﻟﻤﺮآﺐ اﻟﻨﻘﻰ ﺑﻄﺮق‬ ‫اﻟﺘﺤﻠﻴﻞ اﻟﻄﻴﻔﻰ )‪ ( NMR 1,2D-IR-MS‬و ﻣﻦ اﻟﻨﺘﺎﺋﺞ اﻟﻤﺘﺤﺼﻞ ﻋﻠﻴﻬﺎ ﺗﺒﻴﻦ ان هﺬا اﻟﻤﺮآﺐ‬ ‫اﻟﻨﻘﻰ هﻮ )‪(butyl, iso-butyl phthalate‬‬