Phenolic Constituents , Anti-Inflammatory and Antidiabetic Activities of Cyperus laevigatus L

Background: Cyperus species are well known traditional plants and used for several diseases around the world. Aim of the Study: Our study aimed to identification of the phenolic constituents in addition to evaluation of different extracts of Cyperus laevigatus L as antioxidant, antiinflammatory and antidiabetic agents. Materials and Methods: The phenolic constituents were identified using spectroscopic techniques. The antioxidant activity was evaluated using in vitro DPPH assay. Total extract, methanol and EtOAc fractions were evaluated for their antiinflammatory activity using RAW 264.7 macrophages assay. Antidiabetic activity of the total extract was examined biochemically and histopathologically using streptozotocin-induced diabetic rats. Results: A new flavone, chrysoeriol 7-O-β-(6′′′-O-acetyl-β-D-glucopyranosyl)-(1→4) glucopyranoside (1), along with seven knowns (2-8) were isolated from Cyperus laevigatus L. The structures of isolated compounds were established depending upon 1D, 2D-NMR and HR-ESI-MS. The MeOH and EtOAc fractions exhibited significant antioxidant activity while the isolated flavonoids exhibited from moderate to weak antioxidant activity. The total extract, MeOH and EtOAc fractions exhibited significant anti-inflammatory activity using LPS-stimulated RAW 264.7 macrophages model by decreasing of NO accumulation by 76 – 66% and 84 – 67%, of the original accumulation values with increasing concentrations in comparison with the reference drug, dexamethasone. The total extract exhibited antidiabetic activity in streptozotocin-induced diabetic rats and this effect was manifested by decreasing serum levels of glucose, glucagon and NO. It also increased level of insulin and promoted paraoxonase activity. Conclusion: These results proved that this plant may be multiple sources for medicinal natural drugs especially for anti-inflammatory and antidiabetic.


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 before 12 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% CO 2 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. 13Cells (0.5 × 10 5 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% CO 2 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 × 10 5 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. 13itrite 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.

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. 15Serum glucagon and insulin were performed according to previous reported methods. 15,16NO was determined according to the reported method 17 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 ºC.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

Statistical analysis
All data were expressed as mean ± standard error.Data were analyzed using one-way ANOVA using SPSS (Version 16).Duncan's new multiplerange test was used to assess differences between means.A significant difference was considered at the level of P < 0.05.

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.
m with the acetyl carbonyl group at δ C 172.3 (J 3 ).Moreover, a correlation between the methyl proton signal at δ H 2.15 s and the carbonyl group at δ C 172.3 (J 2 ) was observed.Also the HMBC correlation between the methyl proton at δ H 3.91 s and C-3' at δ C 148.6 (J 3 ) 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). 22,23The structure of this compound was confirmed by mass fragments; at m/z 367.

Antioxidant activity
The total extract and MeOH fractions showed moderate DPPH radical scavenging activity with IC 50 23.39±0.72 and 24.28±0.56,respectively but EtOAc, and n-hexane showed weak activity with IC 50 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. 24The 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

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,   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. 2010 27 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. 2013 28 that found that beta-cells comprise the major of islets' cells of rat's pancreas.
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).STZinduced diabetes may be due to the selective destroying of pancreatic β-cells, which is responsible for the insulin production from endocrine cells. 29The 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.

Figure 2 :
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.

Figure 3 :
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 :
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).