Isolation of biologically active constituents from Moringa peregrina (Forssk.) Fiori. (family: Moringaceae) growing in Egypt

Background: Moringa peregrina is a wild plant that grown in the eastern desert mountains in Egypt. Although, this plant is native to Egypt, no details studies were traced on its chemical composition and biological activity. Materials and Methods: The different fractions of the ethanolic extract of the dried aerial parts of the plants were subjected to fractionation and purification on various silica and sephadex columns for the isolation of the major compounds which were tested for there anticancer activity. The aqueous and ethanolic extract as well as its different fractions were tested for antihyperglycemic effect on Streptozitocin-induced diabetes in rats. Results: Investigation of the different fractions of the ethanolic extract of the aerial parts of M. peregrina yielded lupeol acetate (1), β -amyrin (2), α -amyrin (3), β -sitosterol (4), β -sitosterol-3-O-glucoside (5), apigenin (6), rhamnetin (7), neochlorogenic acid (10), rhamnetin-3-O-rutinoside (12), and 6-methoxy-acacetin-8-C- β -glucoside (13) which were isolated for the first time from the plant. Compound (13) was isolated for the first time from genus Moringa . In addition, quercetin (8), chryseriol-7-O-rhamnoside (9) and quercetin-3-O-rutinoside (11) were also isolated. Identification has been established by spectral data (UV, MS, IR, 1H, 1H -1H COSY, and 13C-NMR). The major isolated compounds were found to have valuable cytotoxic activities against breast (MCF 7) and colon (HCT 116) cancer cell lines and their activities were comparable to the reference drug doxorubicin. On the other hand, the aqueous and ethanolic extracts as well as the n-hexane fraction were found to have potent antihyperglycemic effect on Streptozitocin-induced diabetes in rats. Conclusion: The Egyptian plant M. peregrina is rich in biologically active ingredients which showed potent cytotoxic activity and also its ethanolic extraxt exert a significant antihyperglycemic effect.


INTRODUCTION
On reviewing the literature of Moringa peregrina (Forssk.) Fiori, a single report was found about the antihyperglycemic effect of the ethanolic extract of the defatted aerial parts from which quercetin, quercetin-3-O-rutinoside, chryseriol-7-O-rhamnoside, and 6,8,3′,5′-tetramethoxy apigenin were isolated. [1] In this article, the separation and characterization of 10 compounds isolated for the first time from M. peregrina is reported, in addition to 3 compounds that were isolated before from the same plant. Also, the acute toxicity and antihyperglycemic effect of the aqueous extract, ethanolic extracts, and its fractions, as well as the cytotoxic effect of the different fractions and the major isolated compounds are presented.

MATERIALS AND METHODS
UV spectra were measured using a Shimadzu UV 240 (P/N 204-58000) spectrophotometer (USA). Mass spectra were measured using Shimadzu QP-2010 Plus (USA). NMR spectra were recorded at 300 ( 1 H) and 75 MHz ( 13 C) on a Varian Mercury-300 instrument (Switzerland). The NMR spectra were recorded in CDCl 3 or DMSO-d 6 , and chemical shifts were given in δ (ppm) relative to TMS (Trimethylsulphoxide) as internal standard. Electrothermal 9100 (United Kingdom) was used for the determination of melting points (mp) (uncorrected).
Authentic sterols and triterpenes were obtained from E. Merck (Darmstadt, Germany). Silica gel H (E-Merck, Darmstadt, Germany) for vacuum liquid chromatography (VLC) and silica gel 60 (Fluka, 70-230 mesh ASTM, Germany) and Sephadex LH-20 (Pharmacia, Uppsala, Sweden) for column chromatography were used. Thin-layer chromatography (TLC) was performed on silica gel GF 254 precoated plates (Fluka, Germany). The chromatograms were visualized under UV light (at 254 and 366 nm) before and after exposure to ammonia vapor, as well as spraying with anisaldehyde-sulfuric acid spray reagent.

Plant material
The aerial parts of M. peregrina (Forssk.) Fiori (Family: Moringaceae) were collected in the spring from the eastern desert mountains, Egypt. The plant was identified by Prof. Dr. Ahmed Hegazy, Head of the Department of Botany, Faculty of Science, University of Cairo, Egypt. The collected material was air-dried, reduced to powder and kept for extraction.

Extraction and isolation
The air-dried aerial parts (650 g) were powdered and then extracted by percolation with 95% ethanol (4 × 7 L) to yield (250 g) ethanolic extract residue. The residue (200 g) was suspended in distilled water and partitioned between n-hexane, chloroform, ethyl acetate, and n-butanol (saturated with water). The solvents were separately evaporated under reduced pressure to yield 6, 3, 4.7, and 5 g, respectively.

Measurements of cytotoxicity by sulfrhodamine B assay
Cytotoxicity was tested using the method of Skehan et al. [2] on 2 human cell lines, colon cancer cell line (HCT116), and breast cancer cell line (MCF-7). The IC 50 (dose of the extract, which reduces survival to 50%) and IC 10 (dose of the extract, which reduces survival to 10%) for each tested sample were calculated and recorded in Table 1 and compared with the standard drug doxorubicin.

Determination of LD 50
The LD 50 of the aqueous and ethanolic extracts was calculated according to Karber (1931). [3]

Antihyperglycemic activity Animals grouping and administration of extracts
Healthy albino Wistar rats (males and females) obtained from the animal house were housed throughout the experiment in polycarbonated cages and the housing facility was maintained at standard conditions: temperature (28°C ± 2°C), relative humidity (50% ± 5%), and a 12:12 h light:dark cycle. Water and commercial palletized diet were available to the animals ad libitum throughout the treatment period. The rats were allowed 1 week to acclimatize to pharmacology departmental animal house.

Induction of diabetes Streptozitocin-induction of diabetes
Rats were rendered diabetic by injecting a freshly prepared streptozotocin (60 mg/kg, i.p.; dissolved in 0.1 M acetate buffer; pH 4.5). [4] Experimental design for antihyperglycemic activity Antihyperglycemic activity of M. peregrina ethanolic (E) and aqueous (A) extracts were studied after oral administration of 25 mg/kg body weight (b.wt.) in streptozitocin diabetic rats, and compared with the standard group taking 1.44 mg/kg b.wt. of Diamicron ® tablets orally. And, the antihyperglycemic activity of the n-hexane (HF), chloroform (CF), ethyl acetate (CF), and n-butanol (BF) fractions of the ethanolic extract were tested through intraperitoneal injection of a dose of 50 mg/kg b.wt. and compared with a standard group taking 1 U/kg b.wt. of insulin subcutaneously in rats. Blood samples were collected before treatment and 0.5, 1, 2, and 3 h after material administration. The collected blood samples were centrifuged at 2000 g for 5 min for serum separation. The samples were analyzed for serum glucose content by using glucose-oxidase/peroxidase method [5,6] with optical density measured at 505 nm using a visible spectrophotometer, and the results are recorded in Tables 2 and 3.

Statistical analysis
All data are expressed as mean ± SE and the statistical significance was evaluated by one-way analysis of variance (ANOVA). [7] The values are considered significantly different when P values were less than 0.01.
The IR, UV spectral data, and 1 H and 13 C-NMR spectrum of compound 13 indicated the presence of an acacetin nucleus [15][16][17] with the absence of H-6 and H-8, in addition to a methoxy group and an anomeric proton. Two doublets at δ 6.89 and 7.98 ppm each was integrated as two protons and assigned to H-3′, H-5′ and H-2′, H-6′, respectively. A singlet at δ 6.75 ppm was integrated as 1 proton and assigned to H-3. Finally, a singlet at δ 3.74 ppm was integrated as 3 protons and assigned to the methoxy group at 4′. The appearance of the anomeric proton at δ 4.66 ppm with a large coupling constant 9.6 Hz and the absence of H-8 in the 1 H-NMR spectrum indicated a β-linked sugar and glycosylation was at C-8. [17] This was confirmed from the 13 C-NMR spectrum through the upfield shift of the anomeric carbon C-1″, which appeared at δ 78.83 ppm and the downfield shift of C-8 (by about 9 ppm), which appeared at δ 102.41 ppm. [15] The absence of H-6 and the presence of a singlet at 3.70 integrated as 3 protons assigned to a methoxy group, which appeared in 13 C-NMR spectrum at δ 61.7 ppm suggested the presence of a methoxy group at C-6. This was confirmed through the downfield shift of C-6 (by about 35 ppm), which appeared at 133.07 ppm. [15] From the above data, compound 13 may be 6-methoxy-acacetin-8-C-β-glucoside.
On measuring the acute toxicity, the plant showed no toxic symptoms and no deaths after oral and intraperitoneal administration of the tested doses. The different fractions of the ethanolic extracts of the aerial parts of M. peregrina had a potent cytotoxic activity against the 2 tested human cell lines Colon cancer cell line (HCT116) and breast cancer cell line (MCF-7) [ Table 1]. This is obvious from the small IC 50 of the different fractions, which were comparable to that of doxorubicin. The major isolated compounds were also tested for their cytotoxic activity, and they showed a potent activity against the 2 cancer cell lines.
On the other hand, the aqueous (A) and ethanolic (E) extracts of the aerial parts of M. peregrina significantly decreased blood glucose level of streptozitocin diabetic rats [ Table 2] and their effects were comparable to the oral antidiabetic reference drug Diamicron ® . Accordingly, the n-hexane (HF), chloroform (CF), ethyl acetate (CF) and n-butanol (BF) fractions of the ethanolic extract were also tested for their antihyperglycemic effect through intraperitoneal injection of a dose of 50 mg/kg b.wt. using insulin as a standard antidiabetic [ Table 3]. The n-hexane fraction (HF) was the only fraction that showed a highly significant antihyperglycemic activity. Its effect started 30 min after injection and decreased the blood glucose level by 64%-77.44%. This effect remains significant after 3 h from injection. This effect could be attributed to the lupeol acetate and β-sitosterol, which were isolated from the n-hexane fraction and were reported to possess antihyperglycemic effect. [18]