Azadirachta indica as a Source for Antioxidant and Cytotoxic Polyphenolic Compounds

1Departments of Pharmacognosy and 2Pharmaceutics, Faculty of Pharmacy, Umm Al-Qura University, Makkah 21955, Saudi Arabia. 3Department of Pharmacognosy, Faculty of Pharmacy, Helwan University, Cairo 11795, Egypt. 4Department of Pharmacognosy, Faculty of Pharmacy, Al-Azhar University, Cairo, Egypt. 5Department of Pharmaceutics, College of Pharmacy, Mansoura University, Mansoura, Egypt. 6Pharmaceutics Department, Faculty of Pharmacy, Minia Universuty, Egypt. 7Biochemistry Department, Genetic Engineering and Biotechnology Division, NRC, Cairo, Egypt.

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 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

Instruments and materials
NMR ( 1 H-and 13 C NMR) spectra were recorded at 300 MHz for 1H and 75 MHz for 13 C on a Varian Mercury 300.The δ-values are reported as ppm relative to TMS (tetramethyl silane) in DMSO-d 6 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).

Cell line and culture medium
HCT 116 and MCF 7 and Hep-G 2 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% CO 2 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.

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.

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 R 2 (correlation coefficient) of 0.999.Ethanolic contained the highest content of phenolics (69.17 ±1.57mg•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±0.8mg•g -1 ), followed by ethyl acetate extract (25.256±0.89 .

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.

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.

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 MCF 7 , Hep-G 2 and HCT 116 using the SRB method (Skehan et al., 1990).The ethanolic extract exhibited cytotoxic activity against HCT 116 , MCF 7 and Hep-G 2 (IC 50 value 72.29, 61.86 and 128.7µg/ ml respectively).Compound 4 was the most active compound (IC 50 =20.38,42.52 and 86.75 µg/ml respectively).Compound 6 exhibited cytotoxic activity (IC 50 =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
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., 2006Yildirim 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.
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 fvalues, 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 δelectronic 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 C 14 H 5 O 8 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 C 41 H 25 O 26 .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 1 H NMR spectrum exhibited three singlets, each one proton assigned at ´ 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 ´-and J-values of the glucose moiety, especially that of H-1 at δ 5.48 with J 12 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, 13 C 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 Cglycosidic linkage with C-1 glucose.Characteristic downfield shift of C-3' at δ 114.3 (H"+10 ppm) and upfield shift of the carbonyl carbon C-7' at 162.87 (H"-7 ppm) of the flavogallonoyl moiety.The δand J 12 -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 andMoharram et al, 2003).All assigned 1 H and 13 C signals were confirmed by 2D NMR spectral analysis.Accordingly, Compound 4 was finally identified as 2,3,5-(S)-flavogalonoyl-4,6-(S)-hexahydroxy diphenoyl-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 FeCl 3 and indigo-red colour with HNO 2 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 monohexahydroxydiphenoyl-glucose.In addition at high fragmentation energy, three ellagic acid fragment ions have been detected at 301.19 [Ellagic acid-H] -(MS 2 ), 257.08 [Ellagic acid-H-CO 2 ] -, 229.26 [Ellagic acid-H-CO 2 -CO] -(MS 3 ) 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 1 Hresonances 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-4 C 1 -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-4 C 1glucopyranose.The 13 C 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 13 C-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.UVspectrum 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 3 spectra after the addition of H 3 BO 3 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 20 H 18 O 11 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 1 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 J 12 =1.4Hz was characteristic for α-L-arabinofuranoside moiety (Servettaz, 1984) with the other sugar protons. 13 (Agrawal, 1989).In addition, five highly strained and downfield shifted 13 C 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-±-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 AlCl 3 spectra after the addition of H 3 BO 3 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) C 21 H 20 O 12 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 metacoupled 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-4 C 1 -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-4 C 1 -glucopyranoside (figure 6), which is isolated before from A. indica (Monirul et al., 2012;Chakroborty et al., 1989;Babu et al., 2008).
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 (HCT 116 SC50= 20.38

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