A New Kaempferol Glycoside with Antioxidant Activity from Chenopodium ambrosioides Growing in Egypt

The current study aimed to identify the chemical constituents of Chenopodium ambrosioides (Linn.), and the assessment of the in vitro antioxidant activity of the different extracts and pure isolates. Methods: The antioxidant activity was estimated via free radical scavenging and phosphomolybdenum assays. Structure elucidation of pure compounds was achieved via UV, IR, 1H & 13C-NMR, 1H-1H COSY, HMQC, and HMBC, spectroscopy. Bioassay-guided fractionation and isolation of the n-butanol fraction led to the isolation of a new kaempferol glycoside namely; kaempferol 3-O-α-L-C4-rhamnosyl-(1’’’→2’’)-β-DC1-xylopyranoside (1), together with five known compounds identified as; kaempferol 3-O-α-L-C4–rhamnopyranoside (afzelin) (2), kaempferol 7-O-α-LC4–rhamnopyranoside (3), caffeic acid (4), 1,2-benzopyrone (coumarin) (5), and kaempferol (6). Compound (1) showed in vitro antioxidant activity of SC50 12.45 μg/ml, compared to ascorbic acid (AA) with SC50 of 7.50 μg/ml. It can conclude that the leaves of C. ambrosioides can be used as promising natural antioxidant agents.


INTRODUCTION
The genus Chenopodium constituted of approximately 120 species, 45 of which are known to be distributed all over the world, and nine of which are distributed in Egypt 1,2 .Different species belonging to this genus showed numerous pharmacological properties 3,4 .Also, the previous chemical investigations carried out on this genus revealed the presence of flavonoids [5][6][7] , terpenes 8 , sesquiterpene pygmol 9 , xyloside 10 , coumarins 11 , and essential oils 12 .Chenopodium ambrosioides Linn (Family Chenopodiaceae) is native to temperate America 13 .For a long time C. ambrosioides was used traditionally for solving many health problems i.e., inflammation, diabetes, parasites, cough, typhoid, and influenza 14,15 .Reviewing the literature revealed that the different parts of C. ambrosioides showed numerous biological activities i.e., antimicrobial 16,17 , cytotoxicity 18 , antioxidant 19 , larvicidal 20 , antidiabetic 4 , antiparasitic 21 , antiviral 22 , and molluscidal 23 .Moreover, from the phytochemistry point of view C. ambrosioides is known to contain secondary metabolites i.e., saponins 24 , terpenoids 2,25,26 , flavonol glycosides 27,28 , flavone glycosides 29 , and chenopodiumamines & chenopodiumoside 30 .The accumulation of the reactive free radicals in our bodies led to phenomena known as oxidative stress which represent the imbalance between the endogenous antioxidant molecules in our bodies and the extent of such species.Consequently, any excess amounts of such species led to harmful effects, and in such case should be fixed via supplying the human body by external antioxidants agents in the form of nutrients or food additives 19 .Therefore, the aims of our current research work were to evaluate the in vitro antioxidant activity of C. ambrosioides leaves growing in Egypt, accompanied by the chromatographic separation and identification of its bioactive pure isolates.

Plant materials
Leaves of Chenopodium ambrosioides Linn (Family Chenopodiaceae) were collected from Giza Governorate, Egypt in February 2015.The plant was identified by Dr. Threase Labib, Consultant of Taxonomy at the Ministry of Agriculture.A voucher specimen (Ca-2015) was kept at the Herbarium of our department.

Extraction and fractionation
The dried leaves (2.5 Kg) of C. ambrosioides were extracted via percolation in 70% methanol (5 L) at room temperature 25±2°C, and then repeated till complete extraction process.The combined extracts were concentrated via rotatory evaporator at 45°C, to afford a dark brown residue (230 g) from 70% methanol extract, then was dissolved in petroleum ether (60-80°C) (2 L) to remove the lipoidal materials i.e., fats and sterols, to afford a semi oily residue (6.06 g).The defatted 70% methanol (210 g) was suspended in distilled water and then successively fractionated with methylene chloride (2 L), ethyl acetate (2.5 L), n-butanol (3 L), to afford methylene chloride (31.89 g), ethyl acetate (3 g), n-butanol (27 g), and water (126 g) dry fractions.The resulting fractions were chromatographically compared via PC using solvent systems (S 1 & S 2 ), which guide us to select the n-BuOH fraction for further chromatographic separation.

Antioxidant assays Free radical scavenging antioxidant activity assay
The free radical scavenging antioxidant activity of different fractions and pure compounds was evaluated according to the reported method 31 .

Phosphomolybdenum assay
The total antioxidant capacity was evaluated by the reported method with some minor modifications 32 .

Statistical analysis
The antioxidant data were presented as mean±standard deviation (S.D.) of triplicates (n=3) using SPSS 13.0 program (SPSS Inc.USA).

Chromatographic isolation of n-BuOH fraction
Twenty five grams of the n-butanol fraction were subjected to chromatographic isolation using polyamide column chromatography (CC.) (100 X 6 cm, 300 gm).A gradient elution was started with 5% MeOH and the polarity was gradually increased by methanol to pure MeOH at the end.Fractions (250 ml each) were collected, concentrated and examined (PC, S 1 &S 2 , 5% AlCl 3 and 1% FeCl 3 , UV light for detection).Similar fractions were pooled according to their pattern upon paper chromatography, to afford five main fractions (1-5)

Complete acid hydrolysis
The compound (3-5 mg) was refluxed using dilute hydrochloric acid HCl in MeOH at 100°C for 3 hrs, then the hydrolysate was exhaustively extracted with ethyl acetate in separating funnel.Aglycones identified via Co-PC with authentic aglycone sample.The aqueous phase was neutralized with 5% sodium bicarbonate and used for investigation of the sugar moieties via Co-TLC with authentic sugar markers 33 .

In vitro antioxidant activities (DPPH, TAC)
In the current study, the Chenopodium ambrosioides leaves were examined for their antioxidant activities using 2,2' diphenyl-1 -p i c r y l h y d r a z y l r a d i c a l ( D P P H • ) a n d Phosphomolybdenum assays.The results in Table 1 revealed that n-butanol extract exhibited a potent activity with SC 50 value 2.98 mg/ml, which nearly twice the value of standard ascorbic acid 7.50 mg/ml.While, the ethyl acetate extract exhibited activity with SC 50 value 16.48 mg/ml, followed by a moderate activity of the 70% methanol extract with SC 50 value 55.33 mg/ml, while the remaining teste extracts (petroleum ether and methylene chloride) showed very weak activity >100 mg/ ml.Furthermore, in the phosphomolybdenum assay the n-butanol extract was also the most potent of TAC (554.54 ± 2. 27

Structural elucidation
Compound (1): Was obtained as a yellow needles, mp 208-210°C, Rf-values 0.56 in (S 1 ),   2).The 3-O-subistituted kaempferol moiety was confirmed from its characteristic 13 resonances in the 13 CNMR spectrum as it was interpreted above,   chemical and NMR spectral data to kaempferol except for the NMR spectral data assignable to the sugar moiety.Acid hydrolysis of compound 2 afforded rhamnose in the aqueous phase and kaempferol in the organic phase.The 1 HNMR at δ 5.54 (assignable to α-anomeric protons as brs) and at 1.05 (for CH 3 -6'') were characteristic for α-L-rhamnopyranosyl moiety at OH-3.Compound 6 showed six 13 C-resonances of O-rhamnopyranoside moiety among which C-4'', C-3'', C-2'' and C-5'' of δ-values around 70 ppm and CH3-6'' as the most upfield signal at 17.4 ppm.Glycosidation at OH-3 was proved by the relative upfield shift of C-3 to 134.9 (Δ≈-3-4 ppm) and downfield shift of C-2 at 157.7 ppm.Assignment of all other 13 C resonances was proved by their comparison with the reported data in the literature 37 .

In vitro antioxidant activity of the isolated compounds (1-6)
The antioxidant activities of the compounds (1-6) were evaluated using free radical scavenging assay.Compound 4 exhibited potent antioxidant activity of SC 50 4.20 μg/ml, followed by compound 6 of SC 50 6.35 μg/ml, while the remaining compounds 1, 2, 3, and 5 showed moderate antioxidant activities of SC 50 9.41, 12.45, 9.20, and 10.84 μg/ml separately, with respect to ascorbic acid of SC 50 7.50 μg/ml.The high antioxidant activity of 4 and 6 may be return to the presence of the characteristic structural criteria for effective free radical scavenging activity including; the presence of cinnamoeyl moiety and ortho dihydroxy groups in case of compound 4, and also the presence of an 2, 3 unsaturated double bond, 4-oxo group (ring-C), and 5-OH (A-ring), in case of compound 643.Moreover, mono and di kaempferol glycosides 1 and 2 were less active than their aglycone 6, which may be due to steric hindrance offered by a bulky glycosidic moiety at position 3 (ring-C).On the other hand the slightly high activity of 3 than 1 and 2 due to the location of the glycosidic moiety in position 7 at ring-A which not affected on the conjugation process between ring-B and 2,3 D.B. & 4-carbonyl group of the aromatic C-ring 44 .

Table 1 : Antioxidant activities of the different fractions of C. ambrosioides. Sample DPPH free radical Total antioxidant capacity scavenging activity (mg ascorbic acid equivalent SC 50 (mg/ml) 1 AAE /g dry extract) 2
361) mg ascorbic acid equivalent/g dry extract) (Table1).The results obtained can serve as a significant indicator for the antioxidant activity of the n-butanol extract which prompted us to subject it for further chromatographic isolation to identify its chemical constituents which may be responsible for such activity.Reviewing the literature revealed, several Chenopodium species were investigated for their in vitro antioxidant activities, i.e.,Amri et al.  (2015)reported that the antioxidant activity of the 80% methanol extract of Moroccan Chenopodium ambrosioides equal to 73.80%34.In addition, the 80% MeOH of C. ambrosioides leaves growing in Egypt showed superoxide anion scavenging activity of 55.78% and iron chelating activity 73.37%35.Moreover,Barros et al. (2013)reported that the activity of the methanolic extract of Portuguese C. ambrosioides in EC 50 was equal to 0.62 mg/ml19.Further more, the activity of the methanolic extract of C. ambrosioides growing in Yemen was 31.9% at 100 mg/ml36.Actually, no reports are accessible on the total antioxidant capacity of the C. ambrosioides.
1 SC 50 : concentration from sample required for scavenging of 50% of radical; 2 Total antioxidant capacity (TAC) was evaluated by the phosphomolybdenum assay in mg ascorbic acid equivalent AAE /g dry extract.1

Table 2 : 1 H & 13 C NMR spectral data (300/75 MHz-DMSO-d 6 ), 1 H-1 H COSY and HMBC assignments of compound 1
13e sugar moieties were deduced to have β-4 C 1 -, or α-1 C 4 -pyranose stereo structure in the case of the xylosyl and rhamnosyl moieties, respectively, on the basis of the J-values of the anomeric protons and δ-values of their13CNMR resonances (Table2).All other 1 H and13C resonances were also confirmed by the 1 H-1 H-COSY, HMQC and HMBC spectra and by comparison with previously reported data for structurally related compounds 1 H: Chemical shifts δ in (ppm). 2 Coupling constants J in (Hz).