Introduction

Whiteflies, Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae), is one of the most important cotton sucking pests. This sucking pest causes direct and indirect losses in productivity by sucking sap from plants and transferring several viruses. It produces honeydew on their leaves, which promotes the formation of sooty mould and lowers the nutritional value as well as the harvested crops’ viability for the market. Honeydew dropping on open bolls, the lint becomes sticky, that causes difficulties while ginning [1].

Doubtlessly insecticides are frequently used to manage and regulate pests in quality crop preservation and can play a significant role in guaranteeing food security and agricultural productivity [2]. The excessive usage of several synthetic insecticides causes significant threats to others such as humans, domestic birds, essential terrestrial insects, animals, wild and aquatic life and the ecosystem as a whole [3, 4].

The secondary metabolites can serve as a viable substitute for synthetic pesticides because of their easy biodegradability, minimal residuals, and low negative impacts to other non-target organisms, and mammals [5].

Citrus aurantifolia Christm. Swingle (Rutaceae) from the genus Citrus that consists of over 160 genera and 1700 species [6]. C. aurantifolia is well-known as acid lime, Key lime [7], and especially is called Banzhair lime in Egypt [8]. It has spread all over the world, from Southeast Asia to Brazil [9] and can thrive widely in hot tropical and subtropical areas [10]. This plant grow with smooth brown-to-gray barks and with numerous branches and irregular thorns and reach to a height of 3–6 m [9, 11].

A number of studies have discovered that C. aurantifolia has biological activities include insecticide [12], anticancer, antidiabetic [13], antioxidant, antimicrobial [14, 15], anti-inflammation and analgesic effects [16], besides, anti-hypertensive, antibacterial, antifungal [17], in addition to anthelminthic, anti-obesity [18, 19], and hepatoprotective properties [20]. Moreover, it can prevent urinary infections and protect bone, liver and heart diseases [17]. Also, it is beneficial in the treatment of Alzheimer’s disease and colds, flu-like symptoms, with potential virucidal activity against HIV [21,22,23]. The previous secondary metabolites studies of C. aurantifolia exposed the presence of Alkaloids, coumarins [24], carotenoids, flavonoids, triterpenoids, essential oils [17], phenolic acids, and limonoids [25]. Also, steroids, tannins, saponins, cardiac glycosides were screened in this species [26].

Many literatures indicated that the chemical composition of compounds found in any plants can be influenced by various factors, such as the environment in which the plant is grown [27, 28], year of harvest [29], cultivar [30], and geographical area of cultivation [27, 31, 32].

Due to its variously distinct biological properties and chemical profiling, C. aurantifolia is possibly considered a miracle fruit and its peel extract could be a remarkable alternative for synthetic insecticides to reduce the risks associated with its application thereof [33].

Finding out new leads from C. aurantifolia fruit-peels’ that could be utilized as natural insecticides against Bemisia tabaci (Genn.) was the main goal of the presented study, as well as conducting an in-depth phytochemical analysis to figure out the major active principles using spectral and chromatographic techniques.

Experimental

Instruments

[α]D was measured on WXG-4 polarimeter. Ati-Unicam-UV/Visible Vision was employed for measuring UV spectra. NMR spectra were recorded on 500 MHz JEOL in CD3OD or CDCl3. Chemical shifts were represented in δ (ppm) considering the residual solvent peak as internal standard substance at Mansoura University’s Faculty of Science. ESI mass spectra were obtained using an UPLC MS-MS “H2O” 3100 “USA” with TQ detector and Bruker micro OTOF.

Chemicals

F254 (230–400 mesh) silica gel or polyamide 6 were used in performing columns chromatography (CC). Thin layer chromatography and preparative TLC were carried out on 0.25 mm thickness silica gel (Kieselgel 60, GF 254). Hexane, chloroform (CHCl3), methylene chloride (CH2Cl2), butanol, ethyl acetate (EtOAc), methanol (MeOH) and anhydrous sodium sulphate were acquired from Loba Company, India.

Plant material

Citrus aurantifolia was collected from Mansoura University, faculty of agriculture Garden, Egypt in September 2021. Identification of the plant was made by Dr. Mahmoud Makram, Associate Professor, Ornamental Department, Faculty of Agriculture, Mansoura University.

Extraction and isolation

The fresh peels material (5.82 kg) was cut into small pieces and extracted by dist. hot water (1 × 15 L) for 15 min. The water extract was filtered, and the Marc was re-extracted again by dist. hot water (1 × 15 L) for 15 min. Filtration was performed and the filtrate was partitioned successively via separating funnel with chloroform and butanol to furnish chloroform (2.28 g) and butanol (11.36 g) fractions.

Chloroform fraction (2.28 g) was exposed to CC over silica gel and eluted using hexane: EtOAc and CH2Cl2: MeOH of raising polarity. Two fractions I and II were obtained, fraction I was also exposed on silica gel PTLC eluted by CHCl3/ hexane (4: 1) to give compound 1 (30 mg, Rf 0.68), 2 (40 mg, Rf 0.55), and 3 (34 mg, Rf 0.42), while fraction II was also further purified on silica gel PTLC eluted by CHCl3/ MeOH (97: 3) to give compound 4 (30 mg, Rf 0.24), 5 (33 mg, Rf 0.31), 6 (37 mg, Rf 0.44) and 7 (35 mg, Rf 0.60).

Butanol fraction (11.36 g) was subjected to polyamide-S6 column chromatography and eluted using mixture of distilled H2O/ dist.H2O: MeOH / MeOH / MeOH: Acetone/ Acetone/ Acetone: Ammonia/ Ammonia solvent system. Four fractions were obtained. Fraction I was further chromatographed on silica gel PTLC eluted by EMW (EtOAc/ MeOH/ H2O) (41: 6: 3) to give compound 8 (42 mg, Rf 0.45), and 9 (32 mg, Rf 0.61).

Bergapten 1 White crystals, 1H NMR (400 MHz, CDCl3), δ value in ppm, (J value in Hz): 8.16 (1H, d, 9.8, H-4), 7.59 (1H, d, 2.4, H-2’), 7.14 (1H, s, H-8), 7.02 (1H, d, 2.4, H-3’), 6.28 (1H, d, 9.8, H-3), 4.27 (3 H, s, 5-OCH3).

Limettin (citropten) 2 Pale-yellow Crystals, 1H NMR (400 MHz, CDCl3), δ value in ppm, (J value in Hz): 7.96 (1H, d, 9.6, H-4), 6.40 (1H, d, 2.2, H-8), 6.27 (1H, d, 2.2, H-6), 6.14 (1H, d, 9.6, H-3), 3.88 (3 H, s, 5-OCH3), 3.84 (3 H, s,7-OCH3).

Isopimpinellin 3 White Crystals, 1H NMR (400 MHz, CDCl3), δ value in ppm, (J value in Hz): 8.13 (1H, d, 9.8, H-4), 7.63 (1H, d, 2.4, H-2’), 7.00 (1H, d, 2.4, H-3’), 6.29 (1H, d, 9.8, H-3), 4.17 (3 H, s, 5-OCH3), 4.16 (3 H, s, 8-OCH3). 13C NMR (100 MHz, δ, ppm): 160.66 (C-2), 113.04 (C-3), 139.57 (C-4), 144.50 (C-5), 114.82 (C-6), 150.02 (C-7), 128.08 (C-8), 143.82 (C-8a), 107.66 (C-4a), 145.28 (C-2`), 105.24 (C-3′), 61.89 (5-OCH3), 61.00 (8-OCH3).

Oxypeucedanin hydrate 4 Pale-yellow residue, 1H NMR (500 MHz, CD3OD), δ value in ppm, (J value in Hz): 8.43 (1H, d, 9.8, H-4), 7.80 (1H, d, 2.4, H-2’), 7.23 (1H, d, 2.4, H-3’), 7.20 (1H, br s, H-8), 6.29 (1H, d, 9.8, H-3), 4.39 (1H, dd, 9.8, 8.5, H-1’’a), 4.80 (1H, dd, 9.8, 2.4, H-1’’b), 3.82 (1H, dd, 8.5, 2.4, H-2’’), 1.30 (3 H, s, H-4’’), 1.24 (3 H, s, H-5’’).

(12R, 12’R)-aurantifolin 5 Yellow crystals, [α]21D +80° (c = 0.01, MeOH), The UV (MeOH) λmax (log ε): 399 (4.46), sh 329 (4.53), sh 290 (4.61), and 252 (4.69) nm. The ESI-MS (positive mode) m/z 691[M + CH3OH + K]+ and m/z 662 [M + CH3CN + H]+, (negative mode) m/z 669 [M + CH3OH + H2O-H]−, (Calcd for C33H32O12, 620). 1H NMR (500 MHz, CD3OD); 13C NMR (125 MHz, CD3OD) (see Table 1).

21,23-dihydro-23-methoxy-21-oxolimonin 6 White residue, 1H NMR (500 MHz, CD3OD), δ value in ppm, (J value in Hz): 7.40 (1H, t, 1.3, H-22), 5.92(1H, t, 1.4, H-23), 5.36 (1H, t, 1.3, H-17), 4.95 (1H, d, 13.2, H-19β), 4.58 (1H, d, 13.2, H-19α), 4.18 (1H, d, 4.0, H-1β), 4.11 (1H, s, H-15), 3.08 (1H, dd, 14.7, 15.7, H-6β), 2.88 (1H, dd, 1.5, 16.6, H-2α), 2.78 (1H, dd, 4, 16.6, H-2β), 2.35 (1H, dd, 3.4, 14.7, H-6α), 1.37 (1H, m, H-12α) and 2.06 (1H, m, H-12β). (3.53(s), 1.08 (s), 1.24(s), 1.18(s), 1.12(s)).

21,23-dihydro-23-methoxy-21-oxonomilin 7 White residue, 1H NMR (500 MHz, CD3OD), δ value in ppm, (J value in Hz): 7.42 (1H, t, J 1.3 Hz, H-22), 5.94 (1H, t, J 1.3 Hz, H-23), 5.36 (1H, t, J 1.3 Hz, H-17), 5.02 (1H, d, J 7.3 Hz, H-1β), 3.88 (1H, s, H-15), 3.57 (3 H, s, 23-OCH3), 3.53 (1H, t, J 14.1 Hz H-6β), 2.60 (1H, dd, J 3.6, 14.3 Hz, H-6α), 2.02 (3 H, s, CH3CO), (1.14 (s), 1.42(s), 1.38(s), 1.60(s), 1.24(s)).

Chrysoeriol 8-C-glucoside (scoparin) 8 Yellow amorphous powder, 1H NMR (500 MHz, CD3OD), δ value in ppm, (J value in Hz): 7.64 (1H, dd, 8.1, 2.8, H-6’), 7.54 (1H, br s, H-2’), 7.05 (1H, d, 8.1, H-5’), 6.58 (1H, s, H-3), 6.27 (1H, s, H-6), 5.04 (1H, d, 8.0, H-1’’), 4.11 (1H, t, 9.6, H-2’’), 3.98 (1H, br d, 12.4, H-6’’a), 3.95 (3 H, s, 3’- OCH3), 3.88 (1H, br. m, H-6’’b).

Isorhamnetin 3-O-β-D-rutinoside (narcissin) 9 Yellow solid residue, 1H NMR (500 MHz, CD3OD), δ value in ppm, (J value in Hz): 7.95 (1H, d, 1.8, H-2’), 7.63 (1H, dd, 8.4, 1.8, H-6’), 6.91 (1H, d, 8.4, H-5’), 6.35 (1H, d, 2.0, H-8), 6.15 (1H, d, 2.0, H-6), 5.17 (1H, d, 7.3, H-1’’), 4.50 (1H, d, 1.3, H-1’’’), 3.95 (3 H, s, 3’-OCH3), 1.1 (3 H, d, 6.3, H-6’’’).

Insect collection and rearing

By using aspirator, the whiteflies (Bemisia tabaci) adults were collected from the Mansoura University Faculty of Agriculture’s farm. B. tabaci colony was kept alive on untreated cotton plants put in cages made of muslin material (1.5 × 1.5 × 1.5 m) in the greenhouse and were kept at 25–35 °C, 55–75% RH, and day light.

Bioassays

By using spray method technique, biological tests of the fractions and isolated compounds along with the positive control Okios 3.2% EC (azadirachtin) within lab setting were performed on B. tabaci 3rd instar nymphs following the method of Mostafa et al., 2019 [1].

Statistical analysis

Mortality percentages were adjusted using Abbott’s formula [34]. Finney, 1971, states the values of LC50, LC90, and slope [35]. The toxicity index of C. aurantifolia fractions and its isolated compounds was determined according to Sun’s equation [36].

Results and discussion

Identification of compounds (1–9)

The use of botanical insecticides was considered to be one of the most effective and less harmful biological method in controlling insect pests [37]. Our interest in detecting new leads that could be served as eco-friendly acceptable botanical insecticides has inspired us to evaluate the insecticidal potential of C. aurantifolia secondary metabolites on B. tabaci 3rd instar nymphs.

Bioassay-guided fractionation of chloroform and butanol fractions led to isolate and identify of nine secondary metabolites (Fig. 1) using chromatographic (CC and PTLC) and spectrophotometric analyses (1H, 13C NMR, HSQC, HMBC, NOESY and ESI-MS).

Compounds 1–4 were isolated from the CHCl3 fraction. The 1H NMR spectrum of 1 clearly confirmed the existence of α,β-unsaturated lactone of the pyrone ring of coumarins. Comparing with authentic spectra indicated that compound 1 is the 5-substituted linear furanocoumarin, bergapten [38] that was reported previously by Ramírez-Pelayo C from lime C. aurantifolia peels [39]. The 1H NMR spectrum of compound 2 exhibited a simple coumarin pattern, which was verified by matching its spectra with those previously published [40] as limettin (citropten) from lime C. aurantifolia peels [39]. The 1H NMR spectra of 3 revealed signals comparable to the pattern of 1 with the replacement of the aryl proton singlet by an additional methoxyl group in agreement with 5,8-dimethoxy linear furanocoumarin, that was confirmed by 13C NMR and HMBC as isopimpinellin [41, 42] which was previously published from lime C. aurantifolia peels [39]. The 1H NMR spectrum of compound 4 exhibited a furocoumarin pattern with H-5 has substituted with a prenyl side chain, which was identified as oxypeucedanin hydrate [43] that was reported previously from Kabosu (C. sphaerocarpa Hort. ex Tanaka) fruits [44] and from West Indian lime (C. aurantifolia) oil [45].

Fig. 1
figure 1

Isolated compounds from C. aurantifolia fruit peels

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Compound 5 was isolated as yellow crystals also from the CHCl3 fraction. (Rf 0.31). The UV spectrum showed absorption maxima at 399, sh 329, sh 290 and 252 nm. The positive mode ESI MS spectra of 5 exhibited a quasi-molecular ion peaks at m/z 691 and m/z 662 due to the adducts [M + CH3OH + K]+ and [M + CH3CN + H]+, respectively, while the negative mode spectrum displayed a quasi-molecular ion peaks at m/z 669 [M + CH3OH + H2O-H]− all typically with the molecular formula C33H32O12. The 1H, 13C NMR and HSQC spectra of 5 (Table 1) indicated the existence of two sets of protons for two C-8-substituted linear furanocoumarin units at [δH (6.30 ppm (1H, d, J 9.8 Hz, H-3)/δC 114.95, (δH 8.26 (1H, d, J 9.8 Hz, H-4)/δC 141.51, (δH 7.24 (1H, d, J 2.3 Hz, H-9)/ δC 106.44 and δH 7.84 (1H, d, J 2.3 Hz, H-10)/δC 146.78], while the second mono-substituted furanocoumarin unit showed signals at [δH 6.39 ppm (1H, d, J 9.6 Hz, H-3’)/δC 113.05, δH 8.04 (1H, d, J 9.6 Hz, H-4’)/δC 146.78, δH 7.58 (1H, s, H-5’)/δC 114.92, δH 6.97 (1H, d, J 2.2 Hz, H-9’)/ δC 107.97, δH 7.89 (1H, d, J 2.2 Hz, H-10’)/δC 148.52]. Furthermore, two pairs of oxymethylene protons appeared at δH 4.57 (1H, dd, J 10.3, 2.8 Hz, H-11a), δH 4.29 (1H, dd, J 10.3, 8.1 Hz, H-11b)/δC 76.79] and δH 4.75 (1H, dd, J 10.3, 2.7 Hz, H-11’a), (δH 4.45 (1H, dd, J 10.3, 8.1 Hz, H-11’b)/δC 76.46, two oxymethines protons at δH 3.84 (1H, dd, J 8, 2.7 Hz, H-12)/δC 78.26 and δH 3.86 ppm (1H, dd, J 8.1, 2.6 Hz, H-12’)/δC 78.26, beside four methyl groups at δH 1.28 (3H, s, H-14)/δC 26.71, δH 1.22 (3H, s, H-15)/δC 25.12; δH 1.26 (3H, s, H-14’)/δC 26.65 and δH 1.23 (3H, s, H-15’)/δC 25.12. In addition, one methoxyl group appeared at δH 4.22 (3H, s)/δC 61.41 ppm. Thus compound 5 was suggested to contain two unites of prenylfurocoumarins, comprising (R)-heraclenol [46] and (R)-byakangelicin [47]. Careful examination of the long-range couplings in HMBC spectrum (Fig. 2) has indicated cross peaks of δH 4.22 (-OCH3) to C-5 (146.08) and of the oxymethylene proton signals at δH 4.29 (H-11b) and 4.57 (H-11a) to C-8, C-12 and C-12’ which has confirmed the location of 5-OCH3 and the side chain at C-8 in the (R)-byakangelicin unit. Also, the observed cross peaks of δH 4.45 (H-11’b) and 4.75 (H-11’a) with C-8’, C-12 and C-12’ which has established the location of the other side chain at C-8’ in the (R)-heraclenol. This has confirmed the ether linkage between the oxymethine carbons in both units. In the NOESY spectrum of 5 (Fig. 2), the correlation of both H-12’ and H-12 with CH3-14, CH3-14’, CH3-15 and CH3-15’ as well as the correlation to each other indicated the relative stereochemistry of both protons to be α-oriented. The absolute configuration of C-12 and C-12’ was assigned as R for both based on comparing the NMR data with those of (R)-heraclenol [46] and (R)-byakangelicin [47]. Thus, compound 5 was identified as a new compound named (12R, 12’R)-aurantifolin and to the best of our knowledge it wasn’t reported previously from any natural source.

Fig. 2
figure 2

HMBC and NOESY correlations of compound 5

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Table 1 1H, 13C and HMBC data of 5
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1H NMR data of compound 6, which was isolated from CHCl3 fraction, displayed limonin-like signals, with the exception of the furan ring being absent and instead the existence of α-substituted γ-methoxybutenolide group [48]. Thus 6 was defined as 21,23-dihydro-23-methoxy-21-oxolimonin, which was isolated previously from Satsuma Orange (Citrus reticulata) peels [48]. Compound 7 showed signals in the 1H NMR spectrum almost similar to nomilin [48] with the exception of the furan ring being absent and instead the existence of same group as 6. Thus, 7 was identified as 21,23-dihydro-23-methoxy-21-oxonomilin, previously identified from Satsuma Orange (Citrus reticulata) peels [48].

Careful examination of 1H NMR data of 8 and 9, which were isolated from butanol fraction, showed proton signals pattern characterized for flavone and flavonol glycoside moieties respectively. After comparing these spectral data to those found in the literature, 8 was identified as chrysoeriol 8-C-glucoside (scoparin), which was identified previously from C. aurantifolia peels by using LC-UV, LC-MS and MS/MS techniques [49, 50] and identified 9 as narcissin that was detected previously from C. aurantifolia peels [50, 51].

Insecticidal efficacy of C. aurantifolia fractions to whiteflies B. tabaci 3rd instar nymphs

According to several previously studies, C. aurantifolia has been studied as ecofriendly natural insecticides, larvicide, and repellent and the potential of replacing the high risky synthetic chemical insecticides to manage crop pests was established [12].

The chloroform and butanol fractions of C. aurantifolia were assessed for their toxic effect against B. tabaci 3rd -instar nymphs after 24 h of exposure using laboratory conditions in comparison with the positive control azadirachtin (Okios 3.2% EC) (Table 2). The chloroform fraction was the most potent at LC50 value followed by butanol fraction and Okios 3.2% EC. The recorded LC50 (Toxicity index) were 37.1 (100%), 41.7 (88.94) and 45.5 ppm (81.54), respectively. Also, after 72 h of treatment (Table 3), chloroform was the most potent followed by butanol fractions and Okios 3.2% EC. The recorded LC50 (Toxicity index) were 9.6 (100%) 18.6 (51.8) and 27.4 ppm (35.04), respectively.

Table 2 Toxic effect of C. aurantifolia fractions’ after 24 h of exposure against B. tabaci 3rd instar nymphs
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Table 3 Toxic effect of C. aurantifolia fractions’ after 72 h of exposure against B. tabaci 3rd instar nymphs
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Insecticidal efficacy of C. Aurantifolia isolated compounds to whiteflies B. tabaci 3rd instar nymphs

Nine pure isolated metabolites were isolated and identified from C. aurantifolia chloroform and butanol fractions using spectral techniques, these metabolites were evaluated for their toxicity against B. tabaci 3rd instar nymphs to find out the active principles of each fraction individually.

The relative susceptibility of the B. tabaci 3rd instar nymphs after 24 h of exposure using laboratory settings to C. aurantifolia isolated compounds were assessed (Table 4). Compound 9 was the most effective at LC50 level followed Okios 3.2% EC, 5, 7, 8, 6, 4, 2, 3 and the least one 1. The recorded LC50 (Toxicity index) were 33.3 (100%), 45.5 (73.19), 55.1 (60.45), 73.9 (45.05), 109.6 (30.38), 121.5 (27.40), 165.3 (20.15), 214.0 (20.15), 305.7 (10.90) and 514.7 ppm (6.47), respectively. After 72 h of exposure (Table 5), the potency of arrangement started by the most effective compound 5 followed by 9, 7, 8, Okios 3.2% EC, 4, 6, 2, 1 and 3, respectively. The recorded LC50 (Toxicity index) were 15.9 (100%), 19.8 (80.22), 21.9 (72.68), 27.3 (58.22), 27.4 (58.03), 27.9 (56.96), 29.3 (56.96), 41.3 (38.53), 67.7 (23.51), 88.3 (18.01), respectively. The slopes of the toxicity lines were calculated to be fluctuated and increased from (0.817) in 7 to (1.907) in Okios 3.2% EC and the other slopes came between these two fractions.

Table 4 Toxic effect of C. Aurantifolia isolated compounds after 24 h of exposure against B. tabaci 3rd instar nymphs
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Table 5 Toxic effect of C. Aurantifolia isolated compounds after 72 h of exposure against B. tabaci 3rd instar nymphs
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As shown by the toxicity index, the study clarified those compounds (12R, 12’R)-aurantifolin and narcissin were the most effective principles amongst the tested metabolites. The nine isolated compounds’ structure-activity connection showed that both class type and substitutions of the natural product were important. Whitefly B. tabaci third instar nymphs were significantly impacted by glycosylated flavonoids (8, 9) and prenylated coumarin (4, 5) classes, then limonoids (6, 7) and coumarin (1–3) classes.

Another conclusion should keep in mind that isoprene substituted coumarin (4, 5) classes is more potent than the unsubstituted ones (1–3). Also, the activity of methyl ester limonoids (7) is more toxic than other derivatives (6).

The synergistic effect could be concluded and observed from the assessment experiment results of chloroform and butanol fractions as the value of LC50 after 72 h of treatment were 9.6 and 18.6 ppm, respectively while, the isolated metabolites recorded toxic effect around 15.9 to 88.3 ppm after 72 h of treatment. So, the presence of multi-bioactive components with a diversity in the structural composition in a fraction could the potential for synergistic interactions and play an important role to reach this toxic effect [52].

The results obtained were in agreement with Mansour (2011) who reported the insecticidal effectiveness of the ethanolic extract of C. aurantifolia against 4th larval instars of Musca domestica, after 24 h of exposure [53]. In addition, the crude aqueous extracts of lemon peels was the most toxic among the tested extracts to Culex pipiens larvae [54]. Also, the lemon fruit peels aqueous extract showed a remarkable toxic effect against rose aphids, Macrosiphum roseiformis under both laboratory and field conditions with no toxicity towards the insect predator, Coccinella septempunctata [55].

A new identified constituent, (12R, 12’R)-aurantifolin as well as eight metabolites were isolated and identified by chromatographic and spectral analyses from C. aurantifolia fruit peels. The insecticidal potency of both isolated fractions and separated compounds was carried out against whiteflies (B. tabaci). The results showed that (12R, 12’R)-aurantifolin and narcissin were the most effective principles amongst the tested metabolites. So, plant natural sources could be used as a valuable source for the production of the natural green insecticides.