INTRODUCTION
Cyperaceae is a largest family in the monocotyledons that includes more than 100 genera. Cyperus genus is the largest genus in this family that contains more than 600 species.1 Cyperus species are widely used as weeds in traditional medicines.2 In 1983, Buolus reported that Cyperus species were traditionally used as an emollient to treat analgesic, diuretic, carminative and others.3 Pharmacological studies on Cyperus sp. indicated a myriad of biological effects such as anti-inflammatory, hepatoprotective, gastroprotective, anti-malarial and anti-diabetic activities.4-7 Several secondary metabolites were reported from Cyperus sp. Including quinones, flavonoids, sesquiterpenes, steroids and essential oils.2-8-10 Herein we report the phenolic constituents of the aerial parts of Cyperus laevigatus (Family: Cyperaceae) as well as the pharmacological effects of the different extracts and isolated compounds.
MATERIALS AND METHODS
General Experimental procedures
Optical rotation was measured using JASCO P1020 polarimeter (JASCO International Co. Ltd., Tokyo, Japan). NMR spectra were recorded on JEOL AL-400 NMR spectrometer (JEOL Inc., Tokyo, Japan). HR-ESI-MS were recorded by a Shimadzu LC-MS-IT-TOF-MS spectrometer (Shimadzu Inc., Kyoto, Japan). An OMM 7070E Shimadzu visible recording model UV 200 and 240 spectrophotometers (Shimadzu Inc., Kyoto, Japan) were used for UV spectra.
Plant material
Aerial parts of Cyperus laevigatus L., were collected from Baltim, Kafr Elsheikh, Egypt, in April 2013 and kindly identified by Asoc. Prof. Ahmed M. Abdel Gawad. A voucher specimen (PHG-P-CL179) was deposited in Ain Shams University herbarium, Cairo, Egypt.
Extraction and isolation
Air-dried powder aerial parts of C. laevigatus (2.0 kg) were extracted by 70% MeOH, filtered, and dried under vacuum to give dark black gum (85.0 gm). The dry extract was successively fractionated using n-hexane (12.0), CH2Cl2 (10.0), EtOAc (11.0) and MeOH (52.0), respectively. The MeOH fraction was subjected to polyamide CC and eluted with H2O: EtOH in gradient afforded 8 major fractions (CL-1:CL-8). Fraction CL-3 (160.0 mg; 25% MeOH) was subjected to preparative paper chromatography that afforded two subfractions CL-3 (A, 112 mg; Rf: 0.4 AcOH) and (B; 43 mg; Rf: 0.65 AcOH). Subfractions CL-3-B was eluted on Sephadex LH-20 by 40% MeOH afforded compound 1 (8.3 mg). The other seven flavonoids (2-8) were purified using different chromatographic techniques such as preparative paper and Sephadex LH-20 column. Complete acid hydrolysis followed by PC investigation was preceded.11
Spectroscopic data of new flavonoid (1)
Yellow amorphous solid (8 mg), {[α]
–16° (c 0.01, MeOH)}, UV λmax nm (MeOH): 208, 266, 347; NaOAc: 213, 260, 348, 368; + Boric acid: 215, 258, 348; AlCl3: 211, 279, 306.6, 355.4; + HCl: 216.2, 277.2, 300.8, 347.4; H3BO3: 209.2, 269.6, 336.6; +NaOAc: 221.8, 270.8, 334.8. HR-ESI-MS m/z: 689.6365 [M+Na]+, ESI-MS m/z [M-H]-: 666.1. 1H- (400 MHz); 13C- (100 MHz; DMSO-d6) NMR (see Table 1).
Table 1
1H- (400 MHz) and 13C-NMR (100 MHz) of compound 1 (DMSO-d6).
Antioxidant activity of C. laevigatus extracts
The antioxidant activity of the different extracts of C. laevigatus aerial parts along with the isolated flavonoids 1-8 were evaluated in terms of DPPH radical-scavenging ability, as described before12 in a comparising with a reference drug, ascorbic acid.
Anti-inflammatory activity of different extracts of C. laevigatus
The anti-inflammatory of total extract, MeOH and EtOAc fractions was evaluated at different concentrations (12.5, 25, 50 and 100 μg/ml) using LPS-stimulated RAW264.7 macrophages model with a reference drug, dexamethasone as described previously.13
Cell culture
Raw murine macrophages (RAW 264.7) were purchased from the American Type Culture collections and cultured in RPMI-1640 supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, containing 100 U/mL penicillin G sodium, 100 U/mL streptomycin sulphate, and 250 ng/ml amphotericin B. Cells were maintained in humidified air containing 5% CO2 at 37 °C (Cambrex BioScience, Copenhagen, Denmark).
MTT cell viability assay
The mitochondrial-dependent reduction of MTT to formazan was used to measure cell respiration as an indicator of cell viability.13 Cells (0.5 × 105 cells/ well) in serum-free media were plated in a flat bottom 96-well microplate, and treated with 20 μL of different concentrations of the tested samples for 24 h at 37 °C, in a humidified 5% CO2 atmosphere. After incubation, the media were removed and 40 μL MTT solution / well was added and incubated for an additional 4 h. MTT crystals were solubilized by adding 180 μL of acidified isopropanol/well and the plate was shacked at room temperature, followed by photometric determination of the absorbance at 570 nm using 96 wells microplate ELISA reader.
Inhibition of nitric oxide (NO) production
Raw murine macrophages (RAW 264.7) were seeded in 96-well plates at 0.5 × 105 cells / well for 2 h in RPMI without phenol red. The cells were stimulated with LPS with final concentrations of 10 μg/mL. The stimulated cells after 2 extra h were treated with serial concentrations of the tested samples, dexamethasone (50 μg/mL) or left with the LPS alone. Untreated cells were used as a negative control.13
Nitrite accumulation was used as an indicator of NO production using a microplate assay based on the Griess reaction. In each well of flat bottom 96 well-microplates, 40 μL of freshly prepared Griess reagent was mixed with 40 μL cell supernatant or different concentrations of sodium nitrite ranging from 0-100 μmol/L. The plate was incubated for 10 min in the dark and the absorbance of the mixture at 540 nm was determined using the microplate ELISA reader. The amount of nitrite in the media was calculated from NO standard curve.
Antidiabetic activity of total alcoholic extract of C. laevigatus Chemicals and animals
Streptozotocin (STZ) was purchased from Sigma Chemical Co. St. Louis, MO, USA. The study was conducted on 60 adult albino rats (200-210 g) (National Research Centre (NRC), Dokki, Giza, Egypt). Rats were performed in accordance with the Ethics Committee of the NRC. Rats were divided into 4 groups (15 rats in each) as follow: control group, rats received intragastric C. laevigatus MeOH fraction (50 mg /kg b.w. day) dissolved in distilled water, Diabetic group, diabetes were induced by single subcutaneous injection of streptozotocin (50 mg/kg b.w.) The animals were considered diabetic if fasting glucose level was 200 mg/dL after 48 hours of the injection, treated group, diabetic rats received intragastric C. laevigatus total extract (50 mg/kg b. w. day).14
Biochemical Analysis
Serum glucose was performed according to the method of Passing, 1983.15 Serum glucagon and insulin were performed according to previous reported methods.15,16 NO was determined according to the reported method17 where nitrite, stable end product of nitric oxide radical, is mostly used as indicator for the production of NO. The activity of paraoxonase was measured spectrophotometrically in supernatants using phenyl acetate as the substrate. In this assay, aryl esterase/paraoxonase catalyzes the cleavage of phenyl acetate, resulting in phenol formation. The rate of phenol formation is measured by monitoring the increase in absorbance at 270 nm at 25 °C. Absorbance at 270 nm was taken every 15 s for 120 s using UV Spectrophotometer.18
Histopathological evaluation
The pancreatic tissues were dissected out immediately, fixed in 10% normal formalin dehydrated in series of alcohol and then to xylene each for 1 h followed by embedding in wax at 60°C. Paraffin blocks of the tissues were sectioned to 5 μm thickness. The sections were then stained with hematoxylin and eosin for histopathological evaluation.19
RESULTS AND DISCUSSION
A new acylated flavone diglucoside namely, chrysoeriol 7-O-β-(6‴-O-acetyl-β-D-glucopyranosyl)-(1→4) glucopyranoside (1) and seven knowns, apigenin (2), apigenin 7-O-β-glucopyranoside (3), luteolin (4), luteolin 7-O-β-glucopyranoside (5), chrysoeriol (6), chrysoeriol 7-O-β-glucopyranoside (7), and tricin (8)20,21 were isolated for the first time from the aerial parts of C. laevigatus (Figure 1A). The structures of isolated compounds were established using spectroscopic methods, including UV, 1D, 2D-NMR and HR-ESI-MS.
UV spectroscopic data of compound (1) indicated a flavone with free 5 and 4’-hydroxyl groups.11 HR-ESI-MS spectrum showed a molecular ion peak [M+Na]+ at m/z 689.6365, corresponding to the molecular formula of C30H34O17. The 1H-NMR spectral data (Table 1) showed a downfield shift of H-6 at δH 6.41 (d, J = 2.0), H-8 at 6.77 (d, J = 2.0 Hz) suggested the presence of C-7-O-substitution. A characteristic signal of methoxy group at δH 3.91 s was detected. Additionally, the spectrum showed two anomeric protons at δH 5.11 (d, J = 7.2) and at δH 5.08 (d, J = 7.2). The signals appeared at δH 3.16 – 4.11 were attributed to the protons of the remaining sugar. A characteristic proton signal of one methyl for acetyl group was observed at δH 2.15 s. 13C-NMR spectrum showed 30 carbon resonances (Table 1), two of which were carbonyls which appeared at δC 182.3 for C-4 and at δC 172.3 for characteristic for an acetoxy group. Dept-145 experiment suggested the presence of two methylene, sixteen methine signals, one metyl of methoxy group (at δC 56.1); ome methyl for acetoxy group (at δC 21.1) and ten quaternary carbon signals. The C-7-O-sabstitution was suggested by the downfield shift carbon signal of C-7 at δC 163.4. Ten carbon signals for two glucosyl moieties were showed at δC 73.2 (C-2’’), 73.5 (C-3’’), 79.5 (C-4’’), 74.2 (C-5’’), 62.3 (C-6’’), 75.3 (C-2’’’), 76.6 (C-3’’’), 72.1 (C-4’’’), 73.5 (C-5’’’), 63.3 (C-6’’’), two anomeric carbons at δC 99.8 (C-1’’), δC 100.0 (C-1’’’). The assignment of the protonated carbons was established by HSQC experiment. The 7-O-glucosidic linkage was confirmed by HMBC correlation of the anomeric proton at δH 5.11 (d, J = 7.20, H-1’’) and C-7 at 163.4 (J3). The very clear downfield shieft of C-4’’ at δC 79.5 in addition to the HMBC correlation of the anomeric proton at δH 5.08 (d, J = 7.20, 1’’’) with the C-4’’ (J3) deduced the 4’’ → 1’’’ diglucoside linkage 22. Also the downfield shieft of the C-6’’’ by approximately 1 ppm at δC 63.3 suggested the acetylated C-6’’’ that supported by HMBC correlation of H-6’’’ at δH 3.16 m with the acetyl carbonyl group at δC 172.3 (J3). Moreover, a correlation between the methyl proton signal at δH 2.15 s and the carbonyl group at δC 172.3 (J2) was observed. Also the HMBC correlation between the methyl proton at δH 3.91 s and C-3’ at δC 148.6 (J3) indicated methoxylation of C-3’ (Figure 1B). The β orientation of the glucosidic linkage was confirmed was by the large coupling constant of the anomeric proton (7.2 ppm).22,23 The structure of this compound was confirmed by mass fragments; at m/z 367.1056 characteristic to 6’’’-O-acetyl-β-D-diglucopyranoside; at m/z 301.1413 characteristic to chrysoeriol and at m/z 461.2 in the negative mode ESI-MS characteristic to chrysoeriol-7-O-glucoside. The acid hydrolysis of 1 afforded an aglycone and a sugar moiety, which was composed of chrysoeriol and glucose. From the above mentioned data, the structure of 1 was characterized as chrysoeriol 7-O-β-(6‴-O-acetyl-β-D-glucopyranosyl)-(1→4) glucopyranoside (1)
Antioxidant activity
The total extract and MeOH fractions showed moderate DPPH radical scavenging activity with IC50 23.39±0.72 and 24.28±0.56, respectively but EtOAc, and n-hexane showed weak activity with IC50 57.89±0.71, and 76.98±0.73, respectively. Also the isolated flavonoids exhibited from moderate to weak antioxidant activity by the order of compound 4>2>5>6>3>7>8>1 (Table 2). The moderate antioxidant activity of both the total extract and MeOH fraction was attributed to the high flavonoid content. It was reported that flavonoids possess antioxidant activity especially luteolin derivatives and methoxylated flavonoids.24 The DPPH reaction mechanism with the compounds that exhibited activity depending upon the free OH groups in B-ring, so the flavons exhibited more activity that the flavons 7-O-glycosides.25
Table 2
Antioxidant activity of C. laevigatus extracts and isolated flavonoids (1-8).
Anti-inflammatory activity
Cytotoxicity results confirmed that the tested extracts are safe on RAW264.7 macrophages with different concentrations (12.5, 25, 50 and 100 μg/ml). The total extract (12.5, 25, 50, and 100 μg/ml) and MeOH fraction (12.5, 25, and 50 μg/ml) decreased NO% accumulation as the concentration increased reaching 76 - 66% and 84 - 67%, respectively. Also, the EtOAc fraction (12.5, 25, and 50 mg) decreased the NO% accumulation with concentration increased with values ranging from 77 - 66%. The results of the anti-inflammatory assay suggested that the total, MeOH and EtOAc extracts exhibited potent activity at different concentrations and the results were comparable with the reference drug, dexamethasone (Figure 2). A strong correlation has been always noticed between chemical constituents and herbal biological activities. By the same analogy, the anti-inflammatory activity of C. laevigatus can be correlated to its chemical constituents. The phytochemical study of this plant elucidated that it is rich with bioactive compounds. Luteolin, its methoxylated and glycosides derivatives inhibited the NO production and iNOS expression in LPS-stimulated BV-2 microglial cells.26
Figure 2
NO% accumulation in response to; B) total extract; C) MeOH fraction and D) EtOAC fraction of C. laevigatus. The results were compared with the results of LPS stimulated cells, non-treated cells and dexamethasone (50 μg/ml) treated cells. a P ≤ 0.005, b P ≤ 0.0005 compared to LPS stimulated cells.
Antidiabetic activity of total extract
Cyperus laevigatus aerial parts total extract did not exhibit any obvious toxic symptoms or mortality in rats up to 5000 mg/kg b.w. after 14 days. Biochemical markers in the present work exhibited that the diabetic group treated with C. laevigatus extract showed a decrease in the glucose, glucagon, and NO serum levels and promote serum insulin and paraoxonase levels. Zhang et al. 201027 reported that flavonoids exhibited antidiabetic activity by decreasing fasting blood glucose (FBG) and glucagon serum levels while increasing insulin serum levels (Figure 3). Histological examination of the pancreas of the control and extract treated rats indicated normal architecture (Figure 4-A; B). The islets of Langerhans found in the pancreatic tissue were round in shape with normal cell lining. On the other hand, the acini were arranged in a well-organized manner. The interlobular ducts were surrounded with the supporting tissue. These results were consistent with Jarral et al. 201328 that found that beta-cells comprise the major of islets’ cells of rat’s pancreas.
Figure 3
Mean serum glucose, glucagon and NO levels were significantly high and serum insulin and serum praoxnase activity level were significantly low in diabetic group compared to normal group. Mean serum glucose, glucagon and NO levels were significantly low and serum insulin and serum praoxnase activity level were significantly high in treated group compared to diabetic group.
Figure 4
Section of pancreas of A) control group shows the normal structure of exocrine (dense-staining acinar cells) and endocrine pancreas (light-staining islet of Langerhans), B) rat treated with C. laevigatus extract (50 mg/kg b.w./day) shows the normal structure of exocrine and endocrine pancreas, C): diabetic rat shows decrease in pancreatic islet size, atrophy and vacuolation, and connective tissue invasion in the parenchyma of pancreas islet (black arrow) is shown. A reduction in the pancreatic b-cell (blue arrow) numbers compared to the control group, D): diabetic rat received intragastric C. laevigatus extract (50 mg/kg b.w./day) shows normal structure of the pancreas. Few degenerative cells (black arrow) are seen in the islet (H & E stain, Scale Bar: 20 μm).
In the diabetic rats, the pancreas showed a decrease in the pancreatic islet size, atrophy, vacuolation, and connective tissue invasion in the parenchyma of the pancreas islets. The sections revealed a reduced pancreatic β-cell numbers compared to the control group (Figure 4-C). STZ-induced diabetes may be due to the selective destroying of pancreatic β-cells, which is responsible for the insulin production from endocrine cells.29 The pancreas of the diabetic rats treated with C. laevigatus extract showed dramatic suppression of all abnormal histological changes as compared to the diabetic group (Figure 4-D).
The pancreas of the diabetic rats treated with C. laevigatus extract showed dramatic suppression of all abnormal histological changes. Regarding the mechanism by which C. laevigatus can improve β-cells, researchers found that flavonoids, flavonoid glycosides and phenolic acids exhibited a strong contribution as antioxidant agents that can regenerate the changes in the morphology of β-cells.24-30-31
CONCLUSION
A new flavonoid, chrysoeriol 7-O-β-(6‴-O-acetyl-β-D-glucopyranosyl)-(1→4) glucopyranoside (1), and seven knowns (2-8) were isolated and identified from aerial parts of C. laevigatus alcoholic extract. The different extracts and isolated compounds exhibited from moderate to low antioxidant activity. The total acoholic extract, MeOH and EtOAC fractions exhibited significant anti-inflammatory activity using by decreasing of NO accumulation in comparison with dexamethasone as a reference drug using LPS-stimulated RAW 264.7 macrophages model. The MeOH fraction exhibited antidiabetic activity by decreasing levels of glucose, glucagon and NO along with increasing level of insulin and promoted paraoxonase activity in streptozotocin-induced diabetic rats.
