HPLC-UV Fingerprint Profile and Bioactivity of Citrus Aurantium Var

Home , Bitter orange, Grapefruit juice

Vol. 11(15), pp. 284-295, 17 April, 2017 DOI: 10.5897/JMPR2017.6361 Article Number: 9726FDA63917 ISSN 1996-0875 Journal of Medicinal Plants Research Copyright © 2017 Author(s) retain the copyright of this article http://www.academicjournals.org/JMPR

Full Length Research Paper

HPLC-UV fingerprint profile and bioactivity of Citrus aurantium var. deliciosa fruits: peel and seeds on certain plant-parasitic nematodes

Sabah Hussein El- Gayed1, Abeer M. El Sayed1*, Ahlam M. Al-Ghonaimy2 and AbdelWahab Samia M.1

1Pharmacognosy Department, College of Pharmacy, Cairo University, Kasr El-Einy Street, 11562, Cairo, Egypt. 2Plant Protection Department, Nematology Unit, Desert Research Center, Cairo, Egypt.

Received 15 February, 2017; Accepted 28 March, 2017

A potential approach to valorize citrus peel (P) in addition to seed (S), which remain wastes of the juice abstraction manufacturing, is to custom them as accepted bionematicides. However, information on the limonoids as well aspolyphenolics contents of the peel and seeds of Citrus aurantium var. deliciosa were limited. In the present study, methanolic extracts of the peel and seeds of C. aurantium were analyzed using high performance liquid chromatography (HPLC). For activity of P and S against egg emerging and transience of juveniles of root-knot nematode, Meloidogyne incognita, reniform nematode, Rotylenchulus reniformis and citrus nematode, Tylenchulus semipenetrans was investigated. HPLC analysis of limonoids for the tested extracts was performed. Total limonoids in (S) is nearly twice its amount in P. Both extracts contained nomilin, intense bitter limonoid. Nomalin amounted 288.77 and 514.67 mg/100 g extracts of P and S, respectively. Moreover, limonin was detected in P and S (57.85 and 195.74 mg/100 g, respectively). Peel was rich in phenolics contents than seed (49.80 and 10.54 mg/100 g, respectively) as gallic acid. Also, peels and seeds exhibited comparable flavonoids contents. Moreover, HPLC analysis of phenolics and flavonoids revealed that P was rich in flavones (apigenin, luteolin) and glycosylated flavanones (naringen, hesperidin) than S. Both extracts at the tested concentrations inhibited egg hatching at each exposure period for each nematode species compared to those of the control. Egg hatching was maximum in control. Generally, the percentages of hatching gradually increased with time and decreased with concentration. In other words, the percentages of hatching were maximum at 96 h and minimum at 24 h. Exposure to 1000 ppm extract caused the least percentages hatching of egg, followed by those of 750 and 500 ppm. As for juvenile mortality, the percentages mortality was concentration- and exposure period-dependent as the percentage nematode mortality increased with increasing the concentration and time of exposure.

Key words: Mandarin, limonoids, phenolics, peel, seed, plant-parasitic nematodes.

INTRODUCTION

Usage of plant extracts for narrow plant parasitic Nowadays, increasing pressure is to use alternative safe nematodes is flattering interesting due to environmental and inexpensive biocontrol methods. Usefulness of pollution as a result of the use of chemical pesticides. different plant extracts in nematode control has been El- Gayed et al. 285

measured (Akhtar, 1999; Kali and Gupta, 1980; Rossner Citrus wastes are still rich in a variety of biologically and Zebitz, 1986; Netscher and Sikora, 1990; Agbenin et secondary metabolites linked with human health. Citrus al., 2005; Joymati, 2009). peel contains a high content of polymethoxylated Several studies have fixated on Citrus secondary flavones and flavanones, including primarily hesperidin, metabolites as well as bioactivities and have been neohesperidin and naringin (Khan et al., 2010; Marzouk, intended to expand new chemotherapeutic or 2013). complementary medication in current decades (Lv et al., The objective of this study was to evaluate limonoid 2015). Citrus-derived secondary metabolites, counting contents (nomilin, limonin) and quantification of flavonoids, alkaloids, limonoids, coumarins, carotenoids, polyphenolic compounds in mandarin peel and seeds phenolic acids and essential oils, are of crucial using HPLC method. Moreover, evaluate the effect of the significance to human healthiness due to their active methanolic extracts of mandarin peel and seed on eggs properties. Seeds are the major bases of limonoids. and juveniles of certain plant-parasitic nematodes. Large amount of limonoids were detected in Citrus junos seeds (Minamisawa et al., 2014). Limonin and limoninglucoside are the most plentiful limonoids for MATERIALS AND METHODS majority of Citrus species. Plant Citrus tissue and juice contain actual superior concentrations of limonoid glycosides (Hasegawa et al., Fruits of Citrus aurantium var. deliciosa, Family Rutaceae were 1989). Citrus limonoids have been screened for a number collected in April 2015 from Agriculture Research Center, Ministry of of biological activities, including antitumor (Kim et al., Agriculture and Land Reclamation, Giza. These fruits were 2012, 2013), anti HIV, and cholesterol lowering properties selected, weighed, peeled and the seeds were separated. Peel and (Manners, 2007). Limonoids have some important seed separately were dried in the air (at 25°C in the dark). The plants were authenticated by Dr. Reem Samir Hamdy, Professor of biological actions such as antifeedant action against Plant Taxonomy, Botany Department, Faculty of Science, Cairo insects (Klocke and Kubo, 1982; Alford and Bentley, University, Giza, Egypt. Voucher samples of the plants were 1986). deposited at the Museum of the Pharmacognosy Department, Citrus-derived flavonoids in rinds prevailed by two Faculty of Pharmacy, Cairo University in codes number 2242015. classes of compounds referred to as flavanone HPLC analysis of limonoids, flavonoids and phenolics were carried out at Food Technology Research Institute, Agriculture Research glycosides and polymethoxylated flavones (Benavente‐ Center, Ministry of Agriculture and Land Reclamation, Giza, Egypt García et al., 1997). Amongst the aglycone forms, in 2015. apigenin, hesperetin, naringenin, nobiletin, tangeretin and quercetin are broadly detected. For glycoside forms, O-, C-glycosides, rutinosides, glucosides and Extraction and hydrolysis neohesperidosides are widespread (Bocco et al., 1998). Hesperidin was reported as the major component in rind The fresh peel (P) and seed (S) of C. aurantium (100 g each) were tissues of mandarins (Kalt et al., 1999). Owing to this cut into pieces size ranging from (0.5-1 cm long × 1-2 cm wide) and extracted with methanol using cold method of extraction metabolite, Citrus fruits display abundant bioactivities (percolation) (2 × 1 L) till exhaustion. The methanol extract in each counting anti-oxidant and anti-inflammatory. case was filtered, distilled and evaporated under reduced pressure The utilization of Citrus fruits or juice is inversely separately at 40°C using rotary evaporator (Buchi, USA). related with numerous diseases because of its plentiful Concentrated extracts were ground using a pulverizer. The final secondary metabolites. About 33% of the Citrus fruits are yields were 6.94 and 3.96 g of P and S, respectively. For stock solutions, 10 mgml-1 of each sample was dissolved with methanol. technically processed for juice production, conversely, Then, 10 ml of 6 M HCL was added. The combination was stirred about half of processed citrus counting peels, segment cautiously. In each sample, nitrogen was bubbled for ~40 to 60 s. membrane and seeds end up as wastes (Wilkins et al., The extraction mixture was, then, refluxed in a water bath at 90°C 2007). These solid residues are referred to as Citrus for 2 h. Subsequent to cooling, it was filtered and completed up to wastes with estimated worldwide production of 15 million 100 ml with methanol. Prior to the determination by HPLC, the tons per year (Marin et al., 2007). It is valuable to enlarge samples were filtered speedily through a 0.45 µm membrane filter (Millex-HV). more useful recycling approaches of Citrus wastes. The applications particular by Citrus wastes may assist the industrial processors to find new ways of increasing the Qualitative determination of the limonoid contents of C. revenue by recycling bioactive compounds and also aurantium using TLC plummeting the substantial problem of wastes (Marin et al., 2007). Limonoid aglycones were recognized by TLC on silica gel plates

*Corresponding author. E-mail: [email protected] or [email protected].

Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0 International License 286 J. Med. Plants Res.

urbanized in three solvent systems EtOAc:Hexane (6:4), term of quercetin corresponding (mg of QAE/g of sample). EtOAc:CH2Cl2(4:6) and CH2Cl2:MeOH(9.7:0.3). After drying the plates, they were vaporized with Ehrlich’s reagent and exposed to HCL gas (Dreyer, 1965). Typical red colored spots were obtained HPLC analysis and identification of phenolic components for limonoids. Plates were sprayed with 10% sulfuric in MeOH followed by heating at 100°C for 10 min. Limonoids aglycones were The phenolic component analysis was conceded out using an recognized with standards by illustration assessment. Agilent Technologies 1100 series liquid chromatograph (RP-HPLC) attached with a UV-Visible multiwavelength detector. The division was carried out on a 250 × 4.6 mm, 4 μm Hypersil ODS C18 column Quantitative determination of the limonoid contents of C. at ambient temperature. The movable phase consisted of aurantium using HPLC acetonitrile (solvent A) and water with 0.2% sulphuric acid (solvent B). The stream rate was kept at 0.5 ml min-1. The ascent HPLC analysis was carried out using a Perkin Elmer Series pump programme was as follows: 15% A:85% B for 0 to 12 min, then 40% 2000 joined with Perkin Elmer Series 2000Auto sampler and Perkin A: 60% B for 12 to 14 min, followed by 60% A: 40% B for 14 to Elemer 235C diode array detector to quantify limonoid aglycones 18 min. Furthermore, 80% A:20% B for 18 to 20 min, then 90% A: (Ozaki et al., 1991; Sun et al., 2013). Using methanol of HPLC 10% B about 20 to 24 min, finally 100% A for 24 to 28 min grade, the peel and seed extracts were dissolved in a concentration (Bourgou et al., 2008). The inoculation volume was 20 μl, and of 10 mg/ml; then, filtrated through 0.45 µm filter and then aliquant peaks were monitored at 280 nm. The recognition of each of 5 µl subjected to HPLC analysis. Using a C-18 reversed-phase compound was based on a grouping of retention times as well as column (SpherisorbODS-2.5 µm particle size; 250×4.6 mm, Alltech spectral matching to those of authentic samples and relative area Associate, Deerfield, IL, USA) with H2O-MeOH-CH3CN (4.9:4.1:1.0) percentages computed by integration. in isocratic elution for 30 min limonoid aglycones were quantitatively analyzed by HPLC. The flow rate was 1.0 ml/min and absorbance at a UV wavelength 210 nm was monitored, and the column Evaluation of the biological activity temperature was 25°C. Each compound was quantified by its peak area where, each peak was characterized by the procedure of Eggs of each nematode species, root knot nematode, Meloidogyne Herman et al. (1990). Average values from the two injections were incognita; reniform nematode, Rotylenchulus reniformis from used. The compounds were identified by matching the retention infected tomato roots and citrus nematode, Tylenchulus time and absorption spectra with standards. Apaired-sample t test semipenetrans from infected citrus roots used as inocula were gave no significant difference at the p=0.05 level. extracted by clorox (NaOCl 1.0%), then the deferment was poured onto a 500 mesh-sieve and washed by excess tap water (Hussey and Barker, 1973). To determine the effect of the tested materials Determination of total phenolic contents on the percentages of hatching of eggs at the tested concentrations, each material was prepared by soaking (500,750 Spectrophotometric methods were used to determine total and 1000 ppm) weight of each material in distilled water for three phenolics in each sample (Singletion et al., 1999). Each sample at days. The mixture of each material was filtered through Whatman concentration of 1 mgml-1 in methanol was used in the analysis. filter paper No.1. Three concentrations (500, 750 and 1000 ppm) The reaction of mixture was ready by mixing 0.5 ml of each were obtained by adding distilled water. 1 ml of distilled water methanolic sample, 2.5 m of 10% Folin-Ciocalteu’s reagent containing 200 nematode eggs of each nematode species was put dissolved in water and 2.5 ml 7.5% NaHCO3. Concurrently, blank in petri dish with 5 ml water extract of each plant. Control treatment was prepared by mixing 0.5 ml of methanolic sample, 2.5 m of 10% was made by adding 1 ml of distilled water to 5 ml distilled water. Folin-Ciocalteu’s reagent dissolved in water and 2.5 ml 7.5% There were three replicates for each treatment. Observations on NaHCO3. The samples were subsequently incubated in a the number of hatched juveniles from eggs by light microscope thermostat at 45°C for 45 min. The absorbance was determined were made 24, 48, 72 and 96 days after treatment. Data on egg using spectrophotometer at λ max=765 nm (ASYA-HITTECH, UVM- hatching were converted to the percentages of hatching at each 340). Samples for each analysis were prepared in triplicate and the period and concentration as follows: mean value of absorbance resulted. Same practice was repeated for the standard solution of gallic acid and the calibration line was % of egg hatching = Number of the hatched eggs/Total number of construed. Based on the absorbance, the concentration of eggs in control × 100 phenolics was interpreted (mg/ml) from the calibration line; then the contents of phenolics in extracts were articulated in the total as illustrated by Joytami (2009). For determining the effect of two phenolic contents as gallic acid correspondent (mg of GAE/g of different methanolic extracts of mandarin fruit peel (P) and seed (S), sample). they were prepared as mentioned earlier. 5 ml of each extract was added to 1 ml of nematode suspension containing 100 nematode juveniles for each nematode species in petri dish. Control treatment Determination of total flavonoid contents was made by adding 5 ml distilled water to 1 ml of suspension containing the same number of nematodes. Each treatment was The flavonoids contents in each sample were spectrophotometrically replicated three times. Numbers of alive and dead juveniles in each determined (Quettier et al., 2000). The sample enclosed in 1 ml of treatment were counted under light microscope 24, 48, 72 and 96 h methanolic solution of the extract at the concentration of 1 mgml-1 after treatment. Data on nematode mortality were converted to the and 1 ml of 2% AlCl3 solution in methanol. After incubation of the percentages cumulative nematode mortality at each period and samples for an hour at room temperature the absorbance was corrected according to Abbott (1925) formula as follows: determined at λ max 415 nm. Similar procedure was returned for the standard solution of quercetin and the calibration graph was Mortality%= m-n×100 /100-n constructed. Based on the measured absorbance, the concentration of flavonoids was interpreted (mgml-1) on the calibration line and where m and n stand for the percentages of juvenile mortality in then, the content of flavonoids in each sample was articulate as a treatment and control, respectively. El- Gayed et al. 287

Table 1. Results of HPLC of Limonoids in peel and seeds of Citrus aurantium var. deliciosa.

Limonoids aglycones (mg/100 g)a Extract Total Nomallin Limonin Peel 288.77 57.85 346.62 Seed 514.67 195.74 710.41

aAverage of two analyses.

Nomilin Limonin

Hesperidin Naringin

P-coumaric acid Cinnamic acid

Figure 1. Structure of some predominate constituents in both the seed and peel of C. aurantium .

Statistical analysis and 2). Both extracts enclosed nomilin, limonin intensely bitter limonoids. The threshold of nomilin bitterness in SPSS 10.0 for Windows (SPSS Inc. Chicago, IL) were used for carrying out the statistical analyses. Data were subjected to orange juice is similar to that of limonin (Rouseff, 1982). analysis of variance (ANOVA). Duncan's multiple‐range tests were Nomilin was the major identified limonoid aglycone in second-hand to evaluate the significant differences in the mean both tested samples. Nomilin was presented in peel and values (P ≤ 0.05). seed of C. aurantium by 288.77 and 514.67 mg/100 g extract, respectively. Nomilin has been reported to be

RESULTS present in grapefruit juice, ranging from 0.1 to 1.6 ppm (Rouseff, 1982). Nomilin yet at little concentration, could Identification and quantification of limonoids be predictable to enhance bitterness. While, limonin was presence of limonoids delaying bitterness, mainly in the All samples investigated were bitter in taste signifying the measured in the methanolic extract of peel and seed to seed extract. Total limonoids in seed extract is nearly be 57.85 and 195.74 mg/100 g, respectively. Limonoids twice its amount in the peel extract (Table1 and Figures 1 concentration in mandarin juices averaging 220 ppm 288 J. Med. Plants Res.

Table 2. Total flavonoids and phenolics peel and seeds of Citrus aurantium var. deliciosa.

Sample Total Phenolics mg/g as GAE Total flavonoids as mg/g as quercetin Peel 49.80 11.10 Seeds 10.54 13.32

Average of two analyses.

Table 3. Phenolics identified by HPLC, at =280 nm, in the methanol extract of the peel and seeds of Citrus aurantium var. deliciosa.

Conc. (ppm) No. Identified phenolics Rt (min) Peel Seed 1 Gallic acid 7.227 41.94 102.16 2 Pyrogallol 7.327 3203.65 4246.93 4 Protocatechuic acid 8.535 221.23 604.09 5 Catechin 8.628 320.79 1061.56 6 Chlorogenic acid 9.410 86.58 564.74 7 Catechol 9.490 592.25 801.05 8 Epicatechin 9.606 140.1 529.08 9 P-Hydoxy benzoic acid 9.939 203.08 347.28 10 Caffeic acid 10.172 205.19 77.79 11 Ferulic acid 11.816 75.44 75.39 12 Isoferulic acid 12.624 333.54 300.81 13 Resveratrol (stilbenoidcpd) 13.256 220.25 83.83 14 Ellagic acid 13.643 1062.40 2528.62 15 Vanillic acid 13.719 1577.71 6903.05 16 3,4,5-Methoxy-cinnamic 14.631 42.20 109.63 17 Salicylic acid 14.987 261,94 275.37 18 p-Coumaric acid 15.400 158.20 450.37 19 Trans-cinnamic acid 16.660 20.42 58.78 - Total phenolic acids - 8766.91 19120.53 - Percentage - 0.88 1.91

Rt: Retention time; Conc.: concentration, average of three determinations.

(Ozaki et al., 1995) was much lower than those of peel and seed was performed and presented in Tables 2 marketable orange juices which averaged 320 ppm (Fong and 3. The chromatogram of mandarin peel and seed et al., 1989). Natural debittering of limonoids through methanolic extracts as compared to authentic principles glycosidation of the limonoid aglycones occurred in the of phenolic acid and flavonoid, profiles allowed fruit tissues and seeds at not on time stages of fruit identification of forty one phenolic compounds development and maturation (Hasegawa et al., 1989). constituting 60.90 and 23.86 mg/g in the peel and seed of The low conversation of aglycones to their respective C. aurantium, respectively (Table 3). Nineteen phenolic glycosides through maturation could be due to low action acids were represented with gallic, pyrogallol, of limonoid glycucosyltransferase in the fruit tissue during protocatechuic acid, catechin, vanillic, p-coumaric, ferulic, maturation. chlorogenic, rosmarinic, p-coumaric, and trans- cinnamicacids as shown in Table 4, whereas, phenolic acids in the peel and seed amounted about 0.88 and Identification and quantification of phenolic 1.91%, respectively. Major phenolic acid detected was components vanillic acid. Twenty-two flavonoid compounds (naringin, rutin, hesperedin, and flavones-C-glycosylated) were Quantitative determination of polyphenol in C. aurantium detected in the peel extract. El- Gayed et al. 289

Figure 2. HPLC -Uvchromatogram : (a )– the limonoid contents of the peel ; (b ) the limonoids of the seed of C. aurantium . monitored at 210 nm.

Pyrogallol, vanillic and ellagic acids were the majority any of the tested concentrations of S after 24 h. On the abundant phenolic compounds of C. aurantium prevailed other hand, the maximum percentage egg hatching was 0.4, 0.2 and 0.6%, respectively, in the seed extract and evidenced at 96 h, whereas minimum ones occurred at 0.3, 0.1 and 0.2%, respectively, in the peel. Amongst 24 h. flavonoids, leuceninII was the most plentiful compound constituting 1.4% of the extract of the peel and 0.7% of the peel. Naringen was detected nearly in comparable Mortality bioassay amount (in the peel and seed extracts). Data in Table 6 illustrated the percentage of mortality of three nematode species as affected by different Hatching bioassay concentrations of bitter orange peel (P) and seed (S) after different durations. In general, the percentages When using P and S extracts, the percentage of egg mortality was concentration- and exposure period- hatching was concentration-exposure period- dependent dependent as the percentage nematode mortality as the percentage egg hatching decreased with increased with rising the concentration and point in time increasing the concentration (Table 5). Egg hatching was of exposure. The concentration of 1000 ppm induced the maximum in the control. On this basis, exposure to 1000 highest percentages mortality (92.3 and 69.1%) after ppm of both extracts caused the minimum percentages of exposure period of 96 h for citrus and reniform egg hatching, followed by those of 750 and 500 ppm nematodes, respectively by using (P). 1000 ppm elicited except after 24 h, no egg hatching occurred at 1000 and the highest effect (73.7%) for juveniles of reniform 750 ppm either by using P or S. In case of citrus and nematode and achieved (60.5 and 46.8%) mortalities for reniform nematodes, no egg hatching occurred by using juveniles of citrus and root knot nematodes, respectively 290 J. Med. Plants Res.

Table 4. Phenolics identified by HPLC, at =330 nm, in the methanol extract of the peel and seeds of Citrus aurantium var. deliciosa.

Conc. (ppm) No. Identified phenolics Rt Peel Seed 1 Luteolin- 6,8- di-C-glucoside 10.547 14351.18 7453.09 2 Apigenin- 6,8-di-C-glucoside 11.482 900.34 1893.61 3 Apigenin-6-C-glucoside 12.016 4400.12 4070.81 4 Luteolin 7-O-glucoside 12.100 432.21 456.58 5 Luteolin 12.147 2408.11 1366.45 6 Naringin (Naringenin 7-O-neohesperidoside) 12.201 2128.50 2181.78 7 Rutin (Quercetin rutinoside) 12.317 352.04 347.32 8 Quercetrin-3-O-glucoside 12.338 ND ND 9 Hesperidin (Hesperetin 7-rutinoside) 12.928 4691.19 965.75 10 Rosmarinic acid 12.685 88.85 392.83 11 Apigenin-7-O- neohesperidoside 13.045 233.92 275.73 12 Kaempferol-3,7-O-di-rhamnoside 13.341 1075.36 776.70 13 Apigenin-7-O-glucoside 13.145 565.00 494.17 14 Quercitrin (Quercetin 3-O-a-L-rhamnoside) 13.274 131.78 119.29 15 Quercetin 14.774 197.40 308.09 16 kaempferol 3-O-(3"-O-p-coumaroyl)-glucoside 14.842 772.21 ND 17 Naringenin 15.051 274.45 162.18 18 Hespertin 15.330 305.97 433.56 19 Kaempferol 16.062 50.61 70.93 20 Rhamnetin 16.191 70.86 44.25 21 Apigenin 16.308 21.43 27.88 22 Acacetin 18.200 912.04 651.19 - Total flavonoids - 34363.57 22492.19 - Percentage - 3.44 2.25

Retention time; Conc.: concentration, average of three determinations; ND: not detected.

within 96 h when using the extract of the seed (S). al., 2011; Ntalli and Caboni, 2012). Huge production of Citrus fruits is pertinent to the high utilization. The numerous secondary metabolites DISCUSSION in Citrus, including limonoids, flavonoids and phenolic acids, provide a balanced basis for various biological Plant-parasitic nematodes are at their most susceptible activities. Though, all these energetic metabolites act during their active phase in soil when searching for the synergistically to exhibit anti-oxidative, anti-inflammatory roots of host plants. Just the once endoparasitic species and anti-microbial well as presenting cardiovascular have penetrated a root, control is more complicated as protection, neuroprotective and hepatoprotective effect. nematicidal substances have to be non-phytotoxic and In addition, different species, fruit parts, stages of rather systemic. Anematicide that can be safely practical maturity, ecological conditions during development, to mounting plants and is translocated to the roots in storage circumstances and postharvest treatments can sufficiently large amounts to kill endoparasitic or influence the level of active metabolites and related ectoparasitic nematodes has not yet been detected activities (Committee, 2010). (Hague and Gowen, 1987). Total limonoids in seed extract of C. aurantium is nearly Nematicides are extremely poisonous substances that twice its amount in the peel extract. Citrus limonoids were have very squat LD50 values. The frequency of pesticide established to possess possible biological activities in poisoning and mortality serves as a severe warning of the plummeting the risk of certain diseases. Limonoids are risks that arise when pesticides are widely used under nearby in citrus fruits in the form of aglycones and poor administration (Kottegoda, 1985). Biopesticides and glucosides (Dandekar et al., 2008). particularly bionematicides constitute attractive constituent Peel of the C. aurantium exhibited more phenolic of pest organization knowledge and practices (Ntalli et constituents than the seed. Contrary, the seed prevailed El- Gayed et al. 291

Table 5. Effect of mandarin peel and seed extracts on eggs of Meloidogyne incognita, Rotylenchulus reniformis and Tylenchulus semipenetrans.

% hatching % hatching per extract concentration in Duration Treatment Nematode species in control ppm in hours 0 500 750 1000 24 29 4.0 - - 48 52 8.5 7.0 6.5 Meloidogyne incognita 72 73 13.5 12.0 6.5 96 93 18.5 17.0 7.5

24 23 11.5 - - 48 25 12.5 11.5 7.5 M-peel Rotylenchulusreniformis 72 30 15.0 14.0 9.0 96 40 20.0 14.0 9.0

24 25 12.5 - - 48 28 14.0 12.0 6.5 Tylenchulussemipenetrans 72 40 20.0 14.0 7.5 96 45 22.5 19.0 8.0

24 29 7.0 - - 48 52 7.5 7.0 4.5 Meloidogyne incognita 72 73 8.0 7.0 5.5 96 93 9.0 7.0 6.5

24 23 - - - 48 25 7.0 6.5 5.0 M.seed Rotylenchulusreniformis 72 30 13.0 7.5 5.0 96 40 13.0 7.5 6.0

24 25 - - - 48 28 7.0 6.5 5.5 Tylenchulussemipenetrans 72 40 9.0 7.5 6.0 96 45 14.5 7.5 6.0

Values are averages of 3 replicates.

comparable amounts of the flavonoids. Bocco et al. metabolites, indications exist that its involved the fight of (1998) prevailed similar results showing the prevalence of plants against plant-parasitic nematodes (Lindgren et al., phenolic acids in Citrus fruits. Caffeic, p-coumaric and 1992). Family as Meliaceae and Rutaceae received much ferulic acids are characteristic of Citrus (Robbins, 2003). consideration especially because of the occurrence of the Similarly, Gorinstein et al. (2001) found that ferulic, limonoids triterpenes (Schmutterer, 1990). Azadirachtin, sinapic, p-coumaric, and caffeic acids amounts were a tetraanortriterpinoid limonoids found in the Neemtree, is suggestively higher in the peel than in the fruit peeled. registered for nematode control (Javed et al., 2007). The Normally, the methanol extracts of Citrus peels are nematicidal activity of phenolics has been beforehand known for their abundance in phenolic compounds, such stated (Chitwood, 2002; Popa et al., 2008). as phenolic acids and flavonoids (Bocco et al., 1998; Plant resistance to nematodes is based on plant tissue Gorinstein et al., 2001; Abeysinghe et al., 2007). hypersensitivity to nematodes infection. The best known Secondary metabolites have been measured as waste factors involved in vulnerable resistant response of the products for many years ago, with no obvious use for plant to nematodes are the phenolic compounds. There is plants, dissimilarity to primary metabolites which is crucial a direct relation between the degree of plant resistance for the plant’s growth and development (Verpoorte, and the phenolics present in plant tissues by means of 2000). Little attention has been paid to secondary glycosides form of low physiological and chemical action 292 J. Med. Plants Res.

Table 6. Effect of mandarin peel and seed extracts on juveniles ofMeloidogyne incognita, Rotylenchulus reniformis and Tylenchulussemipenetrans.

% mortality of Corrected % mortality of juveniles per Duration Treatment Nematode species juveniles in control extract concentration in ppm (h) 0 500 750 1000 24 - 15.7 16.7 20.7 48 - 17.0 18.3 25.7 Meloidogyne incognita 72 9.7 14.9 16.7 28.7 96 9.7 15.6 23.1 28.7

24 - - - - 48 - - 5.3 7.0 M-peels Rotylenchulus reniformis 72 5.0 4.6 6.0 60.4 96 5.0 12.3 35..8 69.1

24 - 7.3 9.3 23.0 Tylenchulus 48 - 27.3 35.0 31.7 semipenetrans 72 9.7 31.7 46.5 39.1 96 9.7 40.0 63.8 92.3

24 - - 6.7 18.7 48 - 8.3 11.7 20.0 Meloidogyne incognita 72 9.7 6.7 6.7 36.2 96 9.7 11.4 11.4 46.8

24 - - - 5.0 Rotylenchulus 48 - 1.7 7.3 10.7 M-seeds reniformis 72 5.0 43.5 42.1 57.6 96 5.0 73.7 68.4 73.7

24 - 5.7 5.7 7.0 Tylenchulus 48 - 16.3 16.3 42.7 semipenetrans 72 9.7 27.3 33.2 42.4 96 9.7 28.4 39.9 60.5

Values are averages of 3 replicates; M: Mandarin.

Activation requires their breakdown to free phenols Limonin, also possessed nematocidal action against M. -1 (Giebel, 1970). Nematodes are able to do this by incognita through LC50 values of 197.37 μgml . Neem discharging β-glycosidases into the plant tissue (Giebel, products, such as seed powder, dry leaf and aqueous 1970). extracts, were reported to exhibit good efficacy against Both Cinnamic and P-coumaricacids are lignin root-Knot nematodes (Ashraf and Khan, 2010). It appears developers and cofactors of IAA-oxidase (Wilski and that azadirachtin action, when used independently, is less Giebel, 1966), prevent a liable response to nematode speedy and inactive (Lynn et al., 2010). Azadirachtin acts attack (Parish, 1969). As a consequence of glycosidase against Meloidogyne species at very high concentration activity in susceptible plant cells damage by nematodes, relating to paralysis properties and biological sequence a system which favors IAA accumulation is stimulated. arrest as it inhibit the movement of the pre adult stage of Conversely, in resistant plants, a system which destroyed R. reniformis. Other limonoids pretentious the mobility of IAA is formed (Giebel, 1970). The close of auxins in the the secondary juvenile stage of M. incognita. plant depends on the action of IAA-oxidase. This action Our results are as good as to those of Tounsi et al. can be tailored by phenolic compounds. Polyphenols (2011) who inveterate the abundance of Citrus juice in usually synergize IAA activity, while monophenols often phenolic acids. Besides, Caro et al. (2004) and antagonize IAA (Tomaszewski and Thimann, 1966). Gorinstein et al. (2004) stated that in addition to vitamin El- Gayed et al. 293

C, a diversity of phenolic combinations, namely, Total limonoids in (S) is nearly twice its amount in P. Peel flavanone glycosides, and hydroxycinnamic acids, are was rich in phenolics contents than seed. Also, peels and present in Citrus fruit as bioactive compounds. seeds exhibited comparable flavonoids contents. Hesperidin and naringin purported Citrus flavonoids, are Moreover, HPLC analysis of phenolics and flavonoids two major flavanone glycosides present in Citrus fruits revealed that P was richin flavones (apigenin, luteolin) (Caro et al., 2004; Kanaze et al., 2003). Wholly, and glycosylated flavanones (naringen, hesperidin) than flavonoids described in Citrus spp. can be categorized S. Both extracts at the tested concentrations inhibited into these groups: flavanones, flavones, and flavonols, egg hatching at each exposure period for each nematode and species are considered by a specific flavanone species compared to those of the control. The tested glycoside arrangement (Nogata et al., 2006). It is samples, considered as a waste product, can be used as significant to annotation that the Citrus flavonoids have a natural bionematicides will, by designation, require been establish to have health-related possessions, which considerable research effort. Innovative awareness is comprise anticancer, antiviral, and anti-inflammatory required for the production of noval nematocidal activities, effects on capillary fragility, and an ability to compounds, and secondary metabolites in waste inhibit human platelet aggregation (Huet, 1982; products can play a major role in discovery for most Benavente-Garcia et al., 1997). compounds for biochemical production. In the current study, the peel and seed extracts caused reticence of hatching of larvae on fresh eggs of M. incognita. The incapacity of the eggs to hatch is as a CONFLICT OF INTERESTS consequence of ingress of plant extracts into the eggs. 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