Introduction

Neem (Azadirachta indica A. Juss.), a member of the Meliaceae family, is a fast growing tropical evergreen tree. The neem tree (A. indica) is native to the Indian subcontinent and grows in many countries of the world like Egypt and Kingdom of Saudi Arabia (KSA). A massive cultivation of this tree was occurred in the plains of Arafat, Makkah Almokaramah, KSA. In 1971, Approximately 50,000 Neem trees were cultivated to provide shade for the millions of Muslim pilgrims (Maridha and Suhaibani, 2014). A. indica contains various active constituents with several medicinal properties. It used for treatment of fever, diabetes, and disorders that relate to arthritis and rheumatism, skin diseases and inflammation. It was practiced by different type of vaidyas and traditional healer in almost all the countries in the world, like KSA, China, India, Egypt, Rome and Greek and its proximity with human culture, hence neem has been called “nature’s drug store” and “the wonder tree”.   (Dholi et al., 2011; Sonia Bajaj, and Srinivasan B.P 1999; El-Mahmood et al., 2010; Maragathavalli et al., 2012; Amer et al., 2010; Van der Nat et al., 1991; Chatterjee and Pakrashi 1994; Shibata, 1955; Clayton et al., 1996; Van der Nat et al., 1991; Chopra et al., 1956; Murthy and Sirsi, 1958; Patel et al., 2011; Bailey, 1953; Engler, 1964; Chopra et al., 1958; Koul et al., 1990; Haque et al., 2006; Habila et al.,  2011; Sharma  and Bhattacharyya, 2005; Bandyopadhyay et al., 2002; Sathiyaraj et al., 2010;  Boadu et al., 2011; Patel et al.,  2011; Kirtikar and Basu, 1975; Bansal et al., 2010; Santra  and  Manna , 2009; Denardi et al., 2010; Baral and Chattopadhyay, 2004; Habila et al., 2011; Schmutterer, 1995).

From the above mentioned information, it was concluded that A. indica have various pharmacological activities and used in the various marketed formulation, so it is necessary to throw a light on the phenolic contents and biological activities of A. indica growing in KSA

Materials and methods

Instruments and materials

NMR (¹H- and ¹³C NMR) spectra were recorded at 300 MHz for 1H and 75 MHz for ¹³C on a Varian Mercury 300. The δ-values are reported as ppm relative to TMS (tetramethyl silane) in DMSO-d₆ and J-values recorded in Hz. ESI-MS spectra were measured using mass spectrometer connected to an ESI-II ion source (Finnigan, Lc-MSLCQdeca. Advantage MAX, Finnigan Surveyor LC pump) (Department of Biological Genetics, National Research Center, Cairo, Egypt), the analysis was performed by the help of Dr. Amel Kamal, faculty of pharmacy, Helwan university, cairo, Egypt and as illustrated in previous study (Haggag et al., 2011). Purity of sample were recorded on a Shimadzu UV 240 spectrophotometer, separately as solutions in methanol and diagnosed by different UV shift reagents (Mabry et al., 1970) and with sprayed Naturstoff reagent (Brasseur & Angenot, 1986).

Chemicals

DPPH (1,1-diphenyl-2-picrylhydrazyl) was purchased from Sigma-Aldrich Co. (St Louis, MO). Sodium phosphate, ammonium molybdate, Folin-Ciocalteu’s reagent, ascorbic acid, gallic acid were purchased from Merck Chemical Co (Darmstadt, Germany). All other chemicals, solvents and reagents used in chromatography were of analytical grade.

Cell line and culture medium

HCT₁₁₆ and MCF₇ and Hep-G₂ cells were preserved in RPMI-1640 medium containing 10% (v/v) heat inactivated fetal bovine serum supplemented mixture of 100 μg/ml streptomycin and 100 U/ml penicillin at 37°C under 5% CO₂ in air.

Preliminary Phytochemical Screening

Phytochemical screening methods were used for detection of various phytochemicals by qualitative chemical tests to give general idea regarding the nature of constituents present in crude drug (Trease and Evanse 1996; Parekh J and Chanda SV, 2007).

Determination of total phenolic content

The total phenolic content of 80% EtOH, EtOAc, n-BuOH extracts of A. indica was determined using Folin-Ciocalteu reagent according to the method described by Kumar et al. (2008). Gallic acid was used as standard. All determinations were carried out in triplicate. The total phenolic content was expressed as mg gallic acid equivalent (GAE)/g extract.

Determination of total flavonoid content

The total flavonoid content of 80% EtOH, EtOAc, n-BuOH extracts of A. indica was determined using the procedure described by Kumaran and Karunakaran (2006) using Quercetin as a standard. All determinations were carried out in triplicate. The total flavonoid in each plant extract was determined as mg quercetin equivalents (QEs)/g extract.

DPPH radical scavenging activity

The ability of 80% EtOH, EtOAc, n-BuOH extracts of A. indica to scavenge DPPH radicals was evaluated according to the procedure described by Mensor et al. (2001).

Reducing power assay

Reducing power of 80% EtOH extract of A. indica as well as two of the isolated pure compounds (4&6) was determined according to the method of Oyaizu (1986) using ascorbic acid as standard. Three replicates were made for each tested sample. Increased absorbance of the reaction mixture indicated increased reduction power.

Cytotoxic assay

The cytotoxic activity of 80% EtOH extract of A. indica as well as the isolated pure compounds 4 and 6 was carried out according to the method described by Skehan et al. (1990) using colourimetric assay (SRB) and as described in our previous study (Haggag et al., 2011). The IC50 values were calculated from the prism program obtained by plotting the percentage of surviving cells vs. the concentration interpolated by cubic spine (Boyed, 1997).

Plant material

Leaves samples of neem (Azadirachta indica) were freshly collected from Bahra in KSA. The samples were collected in November 2011.

Extraction and Isolation

Air-dried ground leaves (1000 g) were extracted with hot 80% aqueous ethanol (2.5×5L) under reflux (70°C). After evaporation of solvent under reduced vacuum, the residue (115 g) was defatted with pet.ether (60-80°C) under reflux (1.5 L×5L. 60°C). The residue (110 g) after evaporation of pet.ether was fractionated on a polyamide column (Ø 5.5×120 cm) and was eluted water/methanol mixtures of decreasing polarities to afford 25 fractions. Those fractions were concentrated under vacuum and purified on Sephadex LH-20 column and or Cellulose columnsusing different eluting systems. The homogenity of the fractions was tested on 2D-PC and Comp-PC using Whatmann No. 1 paper (systems S₁ & S₂); S₁: n-BuOH-HOAc-H₂O (4:1:5, top layer) and S₂: 15% aqueous HOAc. The compounds were visualized by spraying with naturstoff (flavonoids) and nitrous acid or KIO₃-reagents (tannins).

Data analysis

All values in this study are expressed as mean ± standard deviation of the mean (M+SD). Data were analyzed by one-way analysis of variance (ANOVA). When variation among groups was found significant, Tukey-Kramer multiple comparisons test was carried out to compare between groups. Differences were considered significant when p value was < 0.05.

Results

Phytochemical Screening

Phytochemical screening tests for A. indica leves extract resulted in detection of alkaloids, flavonoids, reducing sugars, steroids, carbohydrates, glycosides and tannins, while Anthraquinones and cardiac glycosides were not detected from the extract.

Total phenolic, flavonoid content and antioxidant activities

Phenolic Content of the Extracts

Ethanolic (80%), Ethyl acetate and butanol extracts from the leaves of A. indica were standardized for their contents of phenolic compounds. As shown in Figure 1 The calibration curve showed linearity for gallic acid in the range of 12.5-400 μg·ml^–1, with R₂ (correlation coefficient) of 0.999. Ethanolic contained the highest content of phenolics (69.17 ±1.57 mg·g^–1), followed by ethyl acetate extract (38.13 ± 1.25 mg·g^–1) and Butanol extract (24.38 ± 3.13 mg·g^–1).

Flavonoid content of extracts

Ethanolic (80%), Ethyl acetate and butanol extracts from the leaves of A. indica were standardized for their contents of phenolic compounds. The calibration curve showed linearity for Quercetin in the range of 12.5-100 μg·ml^–1, with a correlation coefficient (R2) of 0.999 (Figure 2). Ethanolic contained the highest content of flavonoids (42.435±0.8 mg·g^–1), followed by ethyl acetate extract (25.256±0.89 mg·g^–1) and Butanol extract (14.87±1.35 mg·g^–1).

Antioxidant Activity of the Extracts

The standardized ethanolic, ethyl acetate, butanol extracts and gallic acid as +ve control were assessed for their capacity to scavenge DDPH free radical. The antioxidant activity data are written as percentage of free radical inhibition in Table 1. The ethanolic (80%) extract of of A. indica levaes exhibited pronounced antioxidant activity at a concentration of 12.5-100 μg·ml^–1, followed by ethyl acetate and butanol extracts when compared by ascorbic acid as standard. Natural polyphenolic compounds are known to have chemopreventive and suppressive activities against cancer cells by inhibition of metabolic enzymes involved in the activation of potential carcinogens or arresting the cell cycle [Newman et al., 2002]. Nevertheless, a compound with strong free radical scavenger activity can also contribute to DNA protection and prevent apoptosis [Rajkumar et al., 2011]. Further studies are therefore required to detect potential anticancer activities of the extracts reported here. So it was interesting to isolate and identify polyphenolic compounds from the highest antioxidant extract i.e. ethanolic extract which contains also the highest contents of phenolic and flavonoidal contents and estimate their reducing powers and their cytotoxic activity.

Figure 1:  Standard calibration curve of gallic acid at concentrations of 12.5, 25, 50, 100, 200 and 400 μg·ml–1. Spec- trophotometric detection was at 765 nm Click here to view full figure

Figure 2:  Standard calibration curve of quercetin standard at concentrations of 12.5, 25, 50 and 100 μg·ml–1. Spec- trophotometric detection was at 415 nm Click here to view full figure

Table 1: percentage of DPPH radical scavenging activity of A. indica extracts.

Identification of Phenolics isolated from A. indica

Compound 1

Yellowish white amorphous powder (20 mg). Chromatographic properties, R_f values: 0.65 (S₁), 0.50 (S₂); shiny violet fluorescent under UV-light turned to deep blue color with FeCl₃ spray reagent. This evidence was supported by the comparison with respective authentic samples and confirmed by Mass analysis. Negative HRESI-MS, 169.08[M-H]^–.

Compound 2

Off white amorphous powder (18 mg). Chromatographic properties: R_f-values 0.41 (S₁), 0.20 (S₂); UV/response with spray reagents: buff fluorescent spot by long and short UV light, turned to dull yellow fluorescence on exposure to ammonia vapours, greenish yellow (naturstoff), faint indigo-red colour (nitrous acid reagent) and  faint blue colour (FeCl₃). UV λ_max (MeOH), nm: 216, 257, 363.  Negative HRESI-MS, 300.9 [M-H]^–.

Compound 3

Isolated as yellow amorphous powder (9 mg), gave chromatographic properties, UV spectral data and responses towards spray reagents similar to Quercetin. This evidence was supported by the comparison with respective authentic samples and confirmed by Mass analysis. Negative HRESI-MS: m/z, 301.04 [M-H]^–.

Compound 4

Brown amorphous powder (36 mg). Chromatographic properties: R_f-values on PC; 0.1(S₁), 0.67 (S₂); UV/response with spray reagents: dark purple fluorescent spot by short UV-light and dull brown under long UV-light, turned to indigo red (NaNO₂/glacial AcOH) and deep blue colour (FeCl₃). UV λ_max (MeOH), nm: 248, 289 sh, 300. -ve HRESI-MS: m/z 933.067[M-H]^–, (MS²) 914.89[M-H-H₂O]^–, 631.063[M-H-ellagic acid]^–, (MS³) 896.95[M-H-2H₂O]^–, (MS⁴) 878.96[M-H-3H₂O]^–, 301.08[Ellagic acid-H]^–. ¹H NMR:δ ppm 6.59(1H, s, H3“` FLG), 6.52 (1H, s, H3““ HHDP), 6.37 (1H, s, H3““` HHDP), 5.48(1H, d, J=5.6 Hz,  H1), 5.42(1H, br d, J=8.1 Hz, H5), 4.94(1H, t-like, J =  8.1 Hz, H4), 4.9(1H, dd, J=11.8, 3  Hz, H6), 4.79(2H, m, H3/2), 3.98(1H, br d, J=11.8 Hz, H6`). <sup>1</sup>H, <sup>1</sup>H-COSY spectrum, Short range coupling HMQC spectrum, Long range coupling HMBC spectrum. <sup>13</sup>C NMR: δ ppm 169.14, 165.21, 165.86, 165.80, 162.87(C7““/C7““`HHDP, C7“`/C7“/C7`FLG), 146.67, 144.91, 144.85, 144.73, 144.46(C4“`FLG, C6““/6““`/4““/4““` HHDP, 4`/4“FLG), 144.12, 143.72, 142.47(C6`/6“/6“`FLG), 136.98, 135.80, 135.58(C5“`FLG, C5““/5““`HHDP), 135.10(C5“ FLG), 133.26(C5“` FLG), 125.90(C2“ FLG), 124.79, 123.52(C2““/2““`HHDP), 123.43(C2“` FLG), 121.14(C2` FLG), 115.26, 114.59, 114.47, 114.36, 114.3(C1`/1“/1“`FLG, C1““/1““`HHDP and C3`FLG), 111.69(C3“FLG), 106.73, 105.66, 105.59(C3“`FLG, C3““/3““`HHDP), 72.69(C2), 69.79(C5), 68.07(C4), 66.12(C1), 65.74(C3), 65.58(C6).

Compound 5

Brownish amorphous powder (16 mg). Chromatographic properties: R_f-values on PC; 0.21 (S₁), 0.9 (S₂); UV/response with spray reagents: dark purple fluorescent spot by short UV-light, turned to indigo-red (NaNO₂-glacial AcOH) and deep blue colour (FeCl₃). UV λ_max (MeOH), nm: 246 with 286 sh and 300. Negative ESI-MS/MS: m/z 481.31[M-H]^–, (MS²) 301.19[Ellagic acid-H]^–, (MS³) 257.08[Ellagic acid-H-CO₂]^–, 229.26[Ellagic acid-H-CO₂-CO]^–. ¹H NMR: δ ppm 6.42(1H, s, H3“), 6.19(1H, s, H3`), 5.17 and 4.84(1/2H, s, H1α and β), 5.13 and 4.88(1H, t-like, J=10 Hz,  H3α and β), 4.70 and 4.50(1H, d, J=10 Hz, H2α and β ), 3.72-2.72(broad band, hidden by H<sub>2</sub>O-signal represents remaining sugar protons). <sup>13</sup>C NMR: δ ppm 169.24(C7“α,β), 168.63, 168.40(C7`α,β), 144.49(C6`/6“α,β), 144.42(C4`/4“α,β), 134.97, 134.85(C5`/5“α,β), 125.48, 125.29 , 124.89(C2“α,β/ C2`α,β), 113.79, 113.71, 113.67, 113.51(C1“α,β/ C1`α,β), 105.30, 104.95(C3`/3“α,β), 92.88, 88.97(C1β/ C1α), 79.61(C3α), 77.27(C3β), 76.95, 76.77(C2α/2β), 74.24(C4β), 72.17(C5α), 67.24(C4α), 66.89(C5β), 61.03(C6α,β).

Compound 6

Yellow amorphous powder (37 mg). Chromatographic properties: R_f-values 0.41 (S₁), 0.49 (S₂); UV/response with spray reagents: deep purple fluorescent spot by long UV light turned to orange (naturstoff), green (FeCl₃). UV λ_max: (MeOH), nm: 260, 297(sh), 352; (+NaOMe) 271, 329(sh), 404; (+NaOAc) 272, 318 (sh), 362; (+NaOAc/H₃BO₃) 271, 402; (+AlCl₃) 272, 303 (sh), 337(sh), 422; (+AlCl₃/HCl) 267, 300(sh), 362 (sh), 400. Negative HRESI-MS: m/z 433.08[M-H]^–, 867.32[2M-H]^–, 300.15[M-pentoside]^–₌[quercetin-H]^–, -Ve ESI-MS: m/z 301.05[quercetin-H]^–, 179.00(C₉H₇O₄, cinnamoyl fragment), 151.00(C₆H₃O₄, benzoyl fragment) (MS³). ¹H NMR: δ ppm 12.63(1H, s, OH5), 7.57(1H, m, Hz, H6`), 7.48(1H, d, J = 2.3 Hz, H2`), 6.86(1H, d, J= 8.4 Hz, H5`), 6.41(1H, d, J = 2.3 Hz, H8), 6.21(1H, d, J=2.3 Hz, H6), 5.58(1H, d, J = 1.4 Hz, H1“), 4.17-3.24(remaining sugar protons). <sup>13</sup>C NMR: δ ppm 177.56(C4), 164.15(C7), 161.18(C5), 156.95(C2), 156.43(C9), 148.53(C4`), 145.04(C3`), 133.21(C3), 121.77(C1`), 121.01(C6`), 115.54(C2`/5`), 108.04(C1“), 104.01(C10), 99.03(C6), 93.572(C8), 85.57(C4“), 82.08(C2“), 77.03(C3“), 60.65(C5“).

Compound 7

Yellowish amorphous powder (17 mg). Chromatographic properties: R_f-values 0.47 (S₁), 0.60 (S₂); UV/response with spray reagents: deep purple fluorescent spot by long UV light turned to yellowish orange (naturstoff), and green by FeCl₃. UV λ_max: (MeOH), nm: 259, 355; (+NaOMe) 273, 412; (+NaOAc) 266, 373; (+NaOAc/H₃BO₃) 261, 375; (+AlCl₃) 250 (sh), 250, 271, 405; (+AlCl₃/HCl) 250 (sh), 262, 271, 362. -Ve HRESI-MS: m/z 463.1[M-H]^–, 301.1[M-hexoside]^–₌[quercetin-H]^–, (MS³) 178.9(C₉H₇O₄ represent cinnamoyl fragment), 150.9(C₆H₃O₄ represent benzoyl fragment). ¹H NMR: δ ppm 12.61(1H, s, OH5), 7.67(1H, m, H6`), 7.59(1H, d, J=2.4 Hz, H2`), 6.84(1H, d, J= 8.7 Hz, H5`), 6.41(1H, d, J=3 Hz, H8), 6.21(1H, d, J=3 Hz, H6), 5.37 (1H, d, J=8Hz, H1“), 3.67-3.26 (remaining sugar protons). <sup>13</sup>C NMR: δ ppm 177.33(C-4), 164.28(C-7), 161.19(C-5), 156.30(C-2), 156.26(C9), 148.43(C4`), 144.78(C3`), 133.51(C3), 121.23(C1`), 121.08(C6`), 115.80(C5`), 115.19(C2`), 104.01(C10), 101.19(C1“), 98.51(C6), 93.68(C8), 73.8(C3“), 73.32(C5“), 71.25(C2“), 67.53(C4“), 60.30(C6“).

Reducing power

Compounds 4 and 6 represents the major comounds have been solated from the extract so it was be interesting to evaluate their reducing power in comparison with the ethanol extract. Compound 4 exhibited highest activity followed by compound 6, then the ethanolic extract (Table 2), when compared to ascorbic acid as standard. Based on the obtained data, it is interesting to carry out the cytotoxic assay.

Table 2: Reducing power assay for ethanolic extract and isolated compound 4 and 6 of A. indica

Cytotoxic activity

Significant antioxidant and reducing power activity of the ethanolic extract and compound 4 and 6 directed us to test their cytotoxic activity against certain cell lines such as MCF₇, Hep-G₂ and HCT₁₁₆ using the SRB method (Skehan et al., 1990). The ethanolic extract exhibited cytotoxic activity against HCT₁₁₆, MCF₇ and Hep-G₂ (IC₅₀ value 72.29, 61.86 and 128.7μg/ ml respectively). Compound 4 was the most active compound (IC₅₀ =20.38, 42.52 and 86.75 μg/ml respectively). Compound 6 exhibited cytotoxic activity (IC₅₀=39.25, 47.28 and 99.24 μg/ml, respectively). Accordingly, compound 4 is the most active followed by the compound 6 and finally the ethanolic extract.

Discussion and conclusion

Phytochemical screening for active substances of A. indica resulted in detection various compounds such as glycosides, tannins, alkaloids, flavonoids, reducing sugars, steroids and carbohydrates, while anthraquinones and cardiac glycosides were absent from the extract, which are in accordance with previous studies (Imran et al., 2010;  Ejoba, 2012). It is well known that there is a strong relationship between phenolic content of plants and the antioxidant activity, as phenols possess strong scavenging activity for free radicals due to their phenolic hydroxyl groups (Wojdylo et al., 2007; Bendini et al., 2006; Dlugosz et al., 2006 Yildirim et al., 2000; Mohamed et al., 2008; Singh et al., 2009). This study also proved that the extracts of neem (i.e. ethanolic extract, ethyl acetate and butanol extracts) have significant free radical scavenging activity. This may be attributed to their polyphenolic and flavonoidal contents where ethanol extract of the neem showed the highest DPPH inhibition activity followed by ethyl acetate extract then butanol extract and that was parallel to their phenolic and flavonoidal contents (Figures 3 and 4). Owing to the high antioxidant activity of 80% EtOH extract (Fig. 5), it was subjected to chromatographic fractionation.

Figure 3: Flavonoid contents of 80% ethanol, ethyl acetate and n-butanol extracts of the leaves of  A. indica ( n =3) Click here to view full figure

Figure 4: Total phenolic contents of 80% ethanol, ethyl acetate and n-butanol extracts of the leaves of A. indica (n =3) Click here to view full figure

Figure 5: Percentage of DPPH radical scavenging activity of A. indica extracts Click here to view full figure

The dried residue of 80% EtOH extract, which was extracted with chloroform for defatting and extracting of aglycones (Marzouk et al., 2009) was chromatographed on a polyamide column followed by successive purification on Sephadex LH-20 and/or cellulose columns getting seven pure compounds, among which was compound 1, which show chromatographic properties of gallic acid, This evidence was supported by the comparison with respective authentic samples and confirmed by Mass analysis. Negative HRESI-MS, 169.08[M-H]^–. Resulted in Gallic acid (compound 1) which isolated before from A. indica. (Singh et al., 2005). Compound 2, this compound gave more or less the same chromatographic properties (R_f-values, colour and fluorescence changes on exposure to ammonia vapours and with the different spray reagents) of the ellagic acid authentic sample. Based on its intrinsic δ-electronic conjugation over the total C-skeleton, it showed the characteristic UV-absorption bands of ellagic acid (Tanaka et al., 1986 a and b).  Negative HRESI-MS of compound 2 exhibited a molecular ion peak at m/z 300.9 [M-H]^–, that corresponding to the MF C₁₄H₅O₈ of ellagic acid. Accordingly, compound 2 was established as ellagic acid (Tanaka et al., 1985), which is isolated before from A. indica (Garima et al., 2014). Compond 3, UV spectral data and responses towards spray reagents similar to Quercetin. This evidence was supported by the comparison with respective authentic samples and confirmed by Mass analysis. Negative HRESI-MS: m/z, 301.04 [M-H]^–. Accordingly, this compound was established as quercetin. (Mabry et al., 1970). Which isolated before from A. indica (Garima et al., 2014). Compound 4 showed more or less the same chromatographic behavior of C-glycosidic ellagitannin. It exhibited also, very broad strong UV absorption maxima with the characteristic shoulder at 289 nm.  Upon acid hydrolysis it gave ellagic acid in the organic phase and an unknown ellagitannin. The absence of the glucose in the aqueous hydrolysate was a confirmative evidence for the C-glycosidic nature of Compound 4 (Okuda et al, 1983). -Ve HRESI-MS spectrum showed a molecular ion peak at m/z 933.067 [M-H]^– representing  C₄₁H₂₅O₂₆. At high fragmentation energy MS/MS spectra, several diagnostic fragment ions were recorded; the most important ones are at 631.063 and 301.08 corresponding to the loss of a HHDP moiety and the negative ellagic acid ion, which was indirect indication for the other C-glycosidic phenoyl moiety as flavogallonoyl. The ¹H NMR spectrum exhibited three singlets, each one proton assigned at δ 6.59, 6.52 and 6.37 ppm for a C-glycosidic flavogallonoyl attached to C-1, C-2, C-3 and C-5 and HHDP at C-4 and C-6 of open chain glucose.  The δ– and J-values of the glucose moiety, especially that of H-1 at δ 5.48 with J₁₂ of 5.6 Hz, were confirmative for full substituted open glucose with anomeric axial OH-group (Vivas et al, 1995; Okuda et al, 1983; Moharram et al, 2003).  Like in the previous C-glycosidic ellagitannin, ¹³C NMR spectrum showed 6 upfield typical carbon resonances of a C-glycosidic glucose and 14 resonances of a HHDP moiety together with 21 of flavogallonoyl forming C-glycosidic linkage with C-1 glucose. Characteristic downfield shift of C-3` at δ 114.3 (≈+10 ppm) and upfield shift of the carbonyl carbon C-7` at 162.87 (≈-7 ppm) of the flavogallonoyl moiety. The δ– and J₁₂– values of H-1 glucose proved that the absolute configuration at the anomeric position should be of axial OH and equatorial H, which was completely, agree with those of castalagin (Vivas et al., 1995; Okuda et al., 1983 and Moharram et al, 2003). All assigned ¹H and ¹³C signals were confirmed by 2D NMR spectral analysis. Accordingly, Compound 4 was finally identified as 2,3,5-(S)-flavogalonoyl-4,6-(S)-hexahydroxydiphenoyl-D-glucose (Castalagin) (figure 6), (Vivas et al., 1995). Compound 4, was isolated for the first time from A. indica leaves. Compound 5 appeared as deep purple fluorescent spot by short UV-light changed into deep blue colour with FeCl₃ and indigo-red colour with HNO₂ spray reagent characteristic for hexahydroxydiphenoyl esters (ellagitannins), (Gupta et al., 1982). The UV-spectrum showed intrinsic broad absorption band with a shoulder at λ_max 286 nm characteristic for the ellagitannins. On complete acid hydrolysis, compound 5 gave ellagic acid in the organic phase and glucose in the aqueous one (CoPC with authentic phenolic acid and sugars samples). Accordingly, it was expected that the structure of compound 5 is a hexahydroxydiphenoyl ester of glucose. The -ve ESI-MS spectra gave a molecular ion peak at m/z 481.31[M-H]^– corresponding to a mono-hexahydroxydiphenoyl-glucose. In addition at high fragmentation energy, three ellagic acid fragment ions have been detected at 301.19 [Ellagic acid-H]^– (MS²), 257.08 [Ellagic acid-H-CO₂]^–, 229.26 [Ellagic acid-H-CO₂-CO]^– (MS³) in MS/MS spectra. These fragments were confirmative for the presence of a hexahydroxydiphenoyl group (HHDP). The positions of the attachment of HHDP onto glucose and its stereochemistry were concluded from NMR analysis. ¹H NMR exhibited two singlet signals, each one proton, at δ ppm 6.42 and 6.19 of one HHDP group (H-3”, 3′). In the aliphatic region, duplication of all assigned ¹H-resonances was indicative to a free anomeric-OH and the presence of compound 5 in the form of α/β-anomers (Okuda et al., 1989).  The downfield assignment of H-3 and H-2 in both anomers at 5.13, 4.88 (3α/β) and 4.70, 4.50 (2α/β) indicated the attachment of the HHDP-group at C-2 and C-3. As well as, the large J-values (9-10.1Hz) for all sugar signals together with H-1α and H-1β at 5.17 and 4.84 (J = 8.3 Hz) were confirmative evidences for an α/β–⁴C₁-pyranose structure of the glucose moiety (Gupta et al., 1982, Okuda et al., 1983). Therefore, compound 5 was identified as 2,3-hexahydroxydiphenoyl-(α/β)-D-⁴C₁-glucopyranose. The ¹³C NMR spectrum showed also the duplication of all aliphatic and aromatic signals to confirm the α/β-configuration of the glucose, especially those of the anomeric carbon at 92.88 and 88.97 for C-1α and C-1β. The remaining ¹³C-signals were assigned by the comparison with published data of structural similar compounds (Okuda et al., 1982). Thus, compound 5 was confirmed as 2, 3-(S)-hexahydroxydiphenoyl-(α/β)-D-glucopyranose (figure 6). This isolated for the first time in A. indica leaves.

On the basis of its chromatographic properties and UV spectral data, compound 6 was suggested to be quercetin-3-O-glycoside. UV-spectrum in MeOH showed the two characteristic bands for a quercetin aglycone.  The presence of a free 4`-OH group was deduced from the bathochromic shift in band I (+52 nm) with the increase in the intensity (Mabry et al, 1970). On addition of NaOAc, a bathochromic shift in band II (~+10 nm) was an evidence for a free 7-OH. Moreover, the remaining of the bathochromic shifts occurred in band I and II on both NaOAc and AlCl<sub>3</sub> spectra after the addition of H<sub>3</sub>BO<sub>3</sub> and HCl revealed the ortho 3`,4`-dihydroxy-function and the free OH-5. Complete acid hydrolysis produced quercetin in organic phase and arabinose in aqueous phase. -Ve HRESI-MS spectrum exhibited a molecular ion peak at m/z 433.08, corresponding to MF C<sub>20</sub>H<sub>18</sub>O<sub>11</sub> for a quercetin pentoside.  As well as, this evidence was further supported by the fragment ions at m/z 301.05, 179.00 and 151.00. The <sup>1</sup>H NMR, showed in the aromatic region the two characteristic spin coupling systems i.e. ABX (H-2`d, H-6` dd, H-5`d) and AM (H-8 d; H-6 d) for 3`,4`-dihydroxy B- and 5,7-dihydroxy A- ring protons.  In the aliphatic region, a doublet at 5.58 with J₁₂ =1.4 Hz was characteristic for α-L-arabinofuranoside moiety (Servettaz, 1984) with the other sugar protons. ¹³C NMR spectrum showed 15 carbon resonances among which the key signals at δ 177.56 (C-4), 148.53 (C-4`), 145.04 (C-3`) and 133.21 (C-3) for a 3-O-glycosyl-quercetin (Agrawal, 1989). In addition, five highly strained and downfield shifted ¹³C resonances particulary those at 108.04, 85.57 and 82.08 assignable to C-1“, C-4“ and C-2“ of an arabinofuranoside moiety (Agrawal, 1989).  So Compound 6 was identified as quercetin3-O-α-L-arabinofuranoside (Avicularin) (figure 6), which is isolated for the first time from A. indica leaves.

Like compound 6, Compound 7 had chromatographic properties and UV spectral data of a quercetin-3-O-glycoside. As in case of compound 6_, the bathochromic shift with increase in the intensity of band I upon addition of NaOMe was confirmative to a free 4`-OH.  This evidence was also deduced from the remaining of the bathochromic shifts recorded in both NaOAc and AlCl₃ spectra after the addition of H₃BO₃ and HCl. Acid hydrolysis of compound 7 afforded quercetin as an

aglycone and galactose as sugar moiety, were identified by CoPC in different convenient solvent systems (Mabry, 1970). Negative HRESI-MS spectrum showed a molecular ion peak at m/z 463.09 corresponding to molecular formula (MF) C₂₁H₂₀O₁₂ of a quercetin hexoside, together with other fragment ion peaks at 301.02, 178.9 and 150.9 confirming the presence of the quercetin as an aglycone. ¹H NMR, showed in its aromatic region two characteristic spin coupling systems. ABX system was recorded as a doublet of doublet at 7.67, a meta doublet at 7.59 and an ortho doublet of one proton at 6.84, which were assignable to H-6`, 2` and 5` of 3`, 4`- dihydroxy B-ring. The AM system was recorded as meta-coupled doublets at 6.41 and 6.21 for H-8 and H-6 of 5, 7-dihydroxy A-ring, respectively (Harborne, 1984). ¹³C NMR spectra showed more or less the same 15 carbon resonances of a quercetin-3-O-glycoside, which were assigned in case of the previous compound 6. The sugar moiety was finally proved to be glucoside in a β–⁴C₁-pyranose stereostructure due to its typical six signals, particularly those of C-5“ and C-3“, which have a difference less than 1 ppm (Agrawal, 1989). Thus, compound 7 was identified as quercetin 3-O–β-D-⁴C₁-glucopyranoside (figure 6), which is isolated before from A. indica (Monirul et al., 2012; Chakroborty et al., 1989; Babu et al., 2008).

Figure 6: Structures of compounds isolated from the leaves of Azadirachta indica Click here to view full figure

EtOH extract and the major isolated compounds (i.e. compound 4 and comound 6) evaluated as reducing power activity. In this assay, the ability of 80% EtOH extract, compound 4 and compound 6 of the leaves of A. indica to reduce ferric to ferrous was evaluated at 700 nm. From Figure 7, it is evident that the tested samples have reducing power. Higher absorbance at 700 nm corresponds to higher reducing power. When compared the reducing power of the extracts to standard reference (ascorbic acid), it was appeared that the 80% EtOH extract, compound 4 and compound 6 exhibited significant reducing power.

Recent studies prove that the efficacy of chemotherapeutic agents can be enhanced and lower their toxicity to normal tissues when combined with plant derived chemopreventive agents to prevent the action of generation of free radicals (Silici et al., 2011; Takaki-Doi et al., 2009). In our study neem exhibited significant cytotoxic activity against the tested cell lines and its extract has high antioxidant activity and that was supported by the previous reported data where Neem is one of those candidate plants which have chemoprotective effect and strong antioxidant potential (Sithisarn, et al., 2005; Chattopadhyay, 2003; Biswas et al., 2002). Castalagin (compound 4), avicularin (compound 6) and ethanolic extract exhibited significant cytotoxic activity against the tested cell lines (Figures 8, 9 and 10). Castalagin represents an important compound where it supressed human promyelocytic leukemia cell line HL-60 (Yang et al., 2000). Castalagin exhibited marvelous cytotoxic activity against colorectal carcinoma (HCT₁₁₆ SC50= 20.38 μg/ml).  As Colorectal cancer (CRC) ranks first and third in males and females, respectively among all cancers in Saudi Arabia (KSA) and high incidence of rectal cancer in Egypt [Abdul et al., 2011; Darlene et al., 2012], so Neem and castalagin could introduce a help for suppression of colorectal carcinoma.

Figure 7: Reducing power of 80% ethanol, ethyl acetate and n-butanol extracts of the leaves of  A. indica relative to  ascorbic acid ( n =3) Click here to view full figure

Figure 8:  Cytotoxic activity of 80% ethanol extract, compound  4 and compound  6 of  A. indica  against HCT116 cell line ( n =3) Click here to view full figure

Figure 9: Cytotoxic activity of 80% ethanol extract, compound  4 and compound  6 of  A. indica  against MCF7 cell line ( n =3) Click here to view full figure

Figure 10: Cytotoxic activity of 80% ethanol extract, compound  4 and compound  6 of  A. indica  against Hep G2 cell line ( n =3) Click here to view full figure

Acknowledgment

The authors would like to thank the Deanship of Scientific Research and the Institute of Scientific Research and Revival of Islamic Heritage at Umm Al-Qura University, KSA (Project ID: 4331014) for the financial support.

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