HPLC-DAD-MS/MS profiling of phenolics from Securigera securidaca flowers and its anti-hyperglycemic and anti-hyperlipidemic activities Academic research paper on "Chemical sciences"

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Original Article

HPLC-DAD-MS/MS profiling of phenolics from Securigera securidaca flowers and its anti-hyperglycemic and anti-hyperlipidemic activities

qi Rana M. Ibrahim3, Ali M. El-Halawanya'b'*, Dalia O. Salehc, El Moataz Bellah El Naggard, Abd El-Rahman O. El-Shabrawya, Seham S. El-Hawarya

a Pharmacognosy Department, Faculty of Pharmacy, Cairo University, Kasr-El-Ainy Street, Cairo 11562, Egypt b Department of Natural Products, Faculty of Pharmacy, King Abdulaziz University, Jeddah 21589, Saudi Arabia c Pharmacology Department, National Research Centre, Dokki, 12622 Cairo, Egypt d Department of Pharmacognosy, Faculty of Pharmacy, Damanhour University, Damanhour City, Egypt

ARTICLE INFO

ABSTRACT

20 21 22

Article history: Received 22 December 2014 Accepted 21 February 2015 Available online xxx

Keywords:

Securigera securidaca Flowers

HPLC-DAD-MS/MS

Antidiabetic

Anti-hyperlipidemic

Securigera securidaca (L.) Degen & Doefl., Fabaceae, has been widely used in the Iranian, Indian and Egyptian folk medicine as antidiabetic and anti-hyperlipidemic remedy. Phenolic profiling of the ethanolic extract (90%) of the flowers of S. securidaca was performed via HPLC-DAD-MS/MS analysis in the positive and negative ion modes. The total polyphenols and flavonoids in the flowers were determined colorimet-rically, and the quantification of their components was carried out using HPLC-UV. Total phenolics and flavonoids estimated as gallic acid and rutin equivalents were 82.39 ±2.79 mg/g and 48.82 ±1.95 mg/g of the dried powdered flowers, respectively. HPLC-DAD-MS/MS analysis of the extract allowed the identification of 39 flavonoids and eight phenolic acids. Quantitative analysis of some flavonoids and phenolics (mg/100g powdered flowers) revealed the presence of isoquercetrin (3340 ±2.1), hesperidin (32.09±2.28), naringin (197.3±30.16), luteolin (10.247±0.594), chlorogenic acid (84.22±2.08), cat-echin (3.94 ±0.57) and protocatechuic acid (34.4 ±0.15), in the extract. Moreover, the acute toxicity, hypoglycemic and hypolipidemic effects of the extract were investigated using alloxan induced diabetes in rats in a dose of 100,200, and 400 mg/kg bwt. The ethanolic extract was safe up to a dose of 2000 mg/kg. All tested doses of the flower extract showed marked decrease in blood glucose level by 31.78%, 66.41% and 63.8% at 100, 200 and 400 mg/kg bwt, respectively, at p <0.05. Regarding the anti-hyperlipidemic effect, a dose of 400 mg/kg of the flower extract showed the highest reduction in serum triacylglycerides and total cholesterol levels (68.46% and 51.50%, respectively at p<0.05). The current study proved the folk use of the flowers of S. securidaca as anti-diabetic and anti-hyperlipidemic agent which could be attributed to its high phenolic content.

© 2015 Sociedade Brasileira de Farmacognosia. Published by Elsevier Editora Ltda. All rights reserved.

23 Introduction

24 Diabetes mellitus is a complex disorder that characterized

25 by chronic hyperglycemia and disturbances of fat and protein

26 metabolism associated with malfunction in insulin secretion and/or

27 insulin action. The utilization of impaired carbohydrate leads to

28 accelerated lipolysis, resulted in hyperlipidemia (Kim et al., 2006).

29 The Middle East and Northern Africa has the highest prevalence of

30 diabetes as a world region with 34 million diabetic persons accord-

31 ing to international diabetes federation (IDF Diabetes Atlas, 2012).

q2 * Corresponding author at: Pharmacognosy Department, Faculty of Pharmacy, Cairo University, Kasr-El-Ainy Street, Cairo 11562, Egypt.

E-mail: ali.elhalawany@pharma.cu.edu.eg (A.M. El-Halawany).

In recent years, there is growing evidence that plant polyphenols 32

including flavonoids are unique nutraceuticals and supplementary 33

treatments for various aspects of type 2 diabetes mellitus. Plant 34

polyphenols can modulate carbohydrate and lipid metabolism, 35

attenuate hyperglycemia, dyslipidemia, insulin resistance, alleviate 36

oxidative stress and prevent the development of long-term diabetic 37

complications (Bahadoran et al., 2013). Nowadays, there is a grow- 38

ing interest in the analysis and identification of medicinal plants' 39

phenolic constituents aiming at finding new sources for these com- 40

pounds and to establish their structure activity relationship. 41

Securigera securidaca (L.) Degen & Dorfl., Fabaceae, has been 42

widely used in the Iranian, Indian and Egyptian folk medicine 43

as antidiabetic and anti-hyperlipidemic remedy (Ali et al., 1998; 44

Azarmiy et al., 2009; Porchezhian and Ansari, 2001). Chloroformic 45

extract of S. securidaca decreased fasting serum glucose level, 46

http://dx.doi.org/10.1016/j.bjp.2015.02.008

0102-695X/© 2015 Sociedade Brasileira de Farmacognosia. Published by Elsevier Editora Ltda. All rights reserved.

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increased food consumption, body weight and glycogen content of the liver in rats (Zahedi-Asl et al., 2005). Total aqueous extract of the seeds showed significant decrease in blood glucose level (-35%) in glucose loaded mice (Ali et al., 1998). In addition, S. securidaca seed suspension has a protective effect against alloxan-induced hyperglycemia and oxidative stress in rats (Mahdi et al., 2011). Hydroalcoholic extract of S. securidaca seeds produced a significant reduction in the level of triglyceride, LDL as well as decrease in lipid peroxidation (Fathi et al., 2010). Total seed extract improved endothelium dependent vasodilation in high fat fed rats by lowering lipid formation around the aorta in hypercholesterolemic rats and decreasing atherosclerotic lesions (Azarmiy et al., 2009). The hypoglycemic effect of the seed was estimated to be related to its flavonoid content (Hosseinzadeh et al., 2002). Concerning the flowers of S. securidaca, two flavonoids, kaempferol and astragalin were isolated from its aqueous extract (Ali et al., 1998) but no reports were found regarding the effect of the flowers on plasma glucose and lipids in diabetic rats.

In the present study, the acute toxicity, anti-diabetic, anti-hyperlipidemic effect of the alcoholic extract of S. securidaca flowers was evaluated in alloxan induced diabetes model in rats. The total polyphenols and flavonoids in the flowers were determined and the phenolic composition of the extract was described using HPLC/DAD (high-performance liquid chromatographic/diode array detector) coupled with ESI-MS (electrospray ionization/mass spectrometric) to identify the major phenolic compounds present in the extract. In addition, HPLC-DAD quantification of major phenolic acids and flavonoids was carried out.

Material and methods

Chemicals

Reagents for spectrophotometric determination of phenolic compounds, Folin-Ciocalteu's reagent: was obtained from Loba-Chemie (Mumbai, India), sodium carbonate and t-butyl hydroquinone were obtained from Sigma, USA.

Regarding HPLC analysis of phenolic compounds; acetonitrile and methanol used were of HPLC grade, and were purchased from Sigma-Aldrich (Steinheim, Germany). o-Phosphoric acid used was of analytical grade from Sdfine Chemlimited (Mumbai, India) and formic acid was purchased from E-Merck (Darmstadt, Germany). Distilled water was further purified using a Milli-Qsystem (Millipore, MA, USA). Acetonitrile and acidulated water were filtered through a 0.45 pm membrane filter (Pall Gelman Laboratory, USA) and degassed in an ultrasonic bath prior to HPLC analysis. Isoquercetrin, rutin, gallic acid, trans-cinnamic acid, salicylic acid, naringin, protocatechuic acid, ellagic acid, luteolin, quercetin, caffeic acid, hesperetin, p-coumaric acid, kaempferol and hesperidin were purchased from sigma Co. (St. Louis, MO, USA).

Plant material

Samples of S. securidaca (L.) Degen & Dörfl, Fabaceae, were collected during the years (2010-2013) from The Experimental Station of Medicinal and Aromatic Plants, Pharmacognosy Department, Faculty of Pharmacy, Cairo University, Giza. Plant was kindly authenticated by Botany specialist, Dr. Mohamed El-Gebaly, Department of Botany, National Research Center (NRC), Giza, Egypt and a voucher specimen was kept at the Herbarium of the Department of Pharmacognosy, Faculty of Pharmacy, Cairo University (no. 11-6-2013-2).

Determination of total phenolics and flavonoids contents

Quantification of phenolic compounds was carried out using Folin-Ciocalteau's method as reported by Siger et al. (2008). Briefly; 1 g of dried powdered flowers was homogenized with 40 ml of 80% methanol using a pestle and mortar, filtered through Watmann No. 1 filter paper and transferred into a volumetric flask (100 ml) with 80% methanol. 0.2 ml of the methanolic extract was placed in a volumetric flask (10 ml) and 0.5 ml Folin-Ciocalteu reagent (2 N) was added. After 3 min, saturated sodium carbonate (1 ml) (20% in distilled water) was added and the volume was completed with distilled water. After 1 h, absorbance of blue color was measured at ^max 725 nm against a blank (distilled water) using Unicam UV-visible Spectrometer. Gallic acid was used to compute the standard calibration curve (20, 40, 60, 80, 100mg/ml). Determinations were carried out in triplicates; results were represented as the mean values ± standard deviations and expressed as mg gallic acid equivalents per gram dry weight (mg GAE/ g D.W.).

Total flavonoids were extracted according to the method of Hertog et al. (1992), 1 g of dried powdered flowers was homogenized with 40 ml of 62.5% methanol with 0.1 g t-butyl hydroquinone (w/v) and 10 ml of 6 N hydrochloric acid was added carefully and the mixture refluxed at 90°C for 2h. After cooling, the supernatant was filtered and transferred to a volumetric flask (100 ml) with 62.5% methanol. Sample (1 ml), Folin-Ciocalteau's reagent (2N) (0.5ml) and Na2CO3 (200mg/ml) (3ml) were added, vor-texed and then allowed to stand for 15 min at room temperature in a dark place and absorbance was measured at 725 nm. Rutin was used as standard and the equivalents (w/w) were determined from a standard concentration curve (20, 40, 60, 80, 100mg/ml) (Meenakshi et al., 2009). Determinations were carried out in triplicates; results were represented as the mean values ± standard deviations and expressed as mg rutin equivalents (RE) per gram dry weight.

Extraction procedures

The air-dried powdered S. securidaca flowers (1.6 kg) were exhaustively extracted with 90% ethanol by cold maceration. The total alcoholic extract was combined and evaporated under reduced pressure at a temperature not exceeding 50°C, yielding 250 g of dry residue. For biological study the extract dissolved in bi-distilled water by the aid of an ultrasonic bath just prior to the investigation.

Preparation of the extract for HPLC-DAD-MS/MS analysis

The previously prepared ethanolic extract of the flowers (20 mg) was dissolved in HPLC grade methanol (2 ml). The methanolic extract was placed in ultrasonic bath for 5 min and filtered through 0.4 |im membrane filter. Aliquot of 10 |il was injected into the LC/DAD/MS analysis system.

HPLC-DAD-ESI-MS apparatus

The analysis was performed using a Hewlett-Packard 1100 (Waldbronn, Germany) composed of a quaternary pump with an on line degasser, a thermostated column compartment, a photodiode array detector (DAD), an auto sampler, and 1100 ChemStation software, coupled with electrospray ionization (ESI) interfaced Bruker Daltonik Esquire-LC ion trap mass spectrometer (Bremen, Germany) and an Agilent HP1100 HPLC system equipped with an autosampler and a UV-vis absorbance detector.

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Conditions for HPLC-DAD-MS/MS analysis offlavonoids

The HPLC separation was performed on Eclipse XDB C18 column (50 mm x 2.1 mm, 1.8 |im, Agilent Company, USA). Mobile phase consisted of two solvents, (A) methanol and (B) 0.2% formic acid. Separation of compounds was carried out with gradient elution profile: 0min, A: B 10:90; 36min, A: B 70:30; 50min, A: B 100:0; 60 min. Chromatography was performed at 30 °C with a flow-rate of 0.8 ml/min. UV traces were measured at 254,360 and UV spectra (DAD) were recorded between 190 and 900 nm.

Mass spectrophotometric conditions

The ionization parameters were as follows: capillary voltage 4000 V, end plate voltage -500 V; nebulizing gas of nitrogen at 35.0 p.s.i.; drying gas of 10l/min nitrogen at 350°C. Mass analyzer scanned from 50 to 1300 | . The MS-MS spectra were recorded in auto-MS-MS mode. The fragmentation amplitude was set to 1.0 V. Mass spectra were simultaneously acquired using electrospray ion-ization in the positive and negative ionization modes.

Quantitative determination of phenolic compounds by HPLC

Quantitative determination of phenolic compounds was performed using HPLC apparatus, Agilent Series 1200 apparatus (Agilent, USA) equipped with autosampling injector, solvent degasser, quaternary HP pump (series 1200) and ultraviolet (UV) detector (set at 280 nm for phenolic acids and 330 nm for flavonoids). The analysis was achieved on a zobrax ODS C18 column (particle size 5 |im, 250 mm x 4.6 mm 0). Column temperature was maintained at 35 °C. Flavonoid separation was done adopting the method of Mattila et al. (2000), using a mobile phase consisting of 50 mM H3PO4, pH 2.5 (solution A) and acetonitrile (solution B) acetic acid (40:60, v/v) in the following gradient: isocratic elution 95%A:5%B, 0-5 min; linear gradient from 95%A:5%B to 50%A:50%B, 5-55 min; isocratic elution 50%A:50%B, 55-65 min; linear gradient from 50%A:50%B to 95% A:5%B, 65-67 min. The flow rate of the mobile phase was 0.7 ml/min. Phenolic acids separation was done adopting the method of Goupy et al. (1999) with a solvent system consisting of A (aqueous acetic acid 2.5%), B (aqueous acetic acid 8%) and C (acetonitrile) in the following gradient: at 0 min, 5% B; at 20 min, 10% B; at 50 min, 30%B; at 55 min, 50%B at 60 min, 100%B; at 100 min, 50% B and 50% C; at 110 min, 100% C until 120 min. The solvent flow rate was 1 ml/min. The injection volumes were 5 |l. Standard flavonoids and phenolic acids were prepared as 10 mg/50 ml solutions in methanol and they were diluted to make concentrations (20-40 |g/ml) and injected into HPLC. Quantification of compounds was performed based on peak area computation (external standard method). The analysis was run in triplicates and the concentrations of the identified compounds were expressed as (mg ± SD/100g dry weight) and listed in Table 2.

Experimental animals

Adult Wister male albino rats, weighing 180-250 g, were obtained from the Animal House Colony of the National Research Center (Dokki, Giza, Egypt), and were housed under conventional laboratory conditions throughout the period of experimentation. The animals were fed a standard rat pellet diet and allowed free access to water.

Drugs and kits

Alloxan monohydrate powder (Sigma-Aldrich, St. Louis, MO, USA), Gliclazide (Servier, Egypt) were used in the present investigation. The biochemical kits used in the study were glucose kits

(Biodiagnostic, Egypt), cholesterol kits (Biodiagnostic, Egypt) and triacylglycerides kits (Biodiagnostic, Egypt).

Acute toxicity

Acute oral toxicity of the ethanolic extract of the flowers of S. securidaca L. was performed following the method of Lorke (1983).

Anti-hyperglycemic activity and anti-hyperlipidemic activity

Rats were weighed and injected intraperitoneally with alloxan (150mg/kg) dissolved in distilled water. After 48 h blood samples were withdrawn from the retro-orbital venous plexus under light ether anesthesia and the serum was separated by centrifugation for the determination of glucose level. Only rats with serum glucose levels more than 250 mg/dl were selected and considered as hyper-glycemic animals according to method of Neshwari et al. (2012). The hyperglycemic rats were then divided into five groups (10 rats each). The first group of diabetic rats served as control; the second to fourth groups received alcoholic extract of the flowers at doses 100,200 and 400 mg/kg orally for 10 consecutive days; and the fifth group of diabetic rats received Gliclazide (antidiabetic standard) at dose of 5 mg/kg bwt orally for 10 consecutive days. The extract and Gliclazide were started 48 h after alloxan injection at which time hyperglycemia was confirmed. Twenty-four hours after the last dose of either drug treatment, a blood sample was withdrawn from the retro-orbital venous plexus from 18 h food-deprived rats and was centrifuged at 3000 rpm for 10 min. The serum was obtained for determination of the serum glucose level, triacylglycerides and total cholesterol level.

Determination ofserum glucose level

Glucose level was determined as quinineamine using a test reagent kit (Biodiagnostic, Egypt) according to the method of Trinder (1969). The absorbance was measured at 510 nm and the results were expressed as mg/dl.

Determination ofserum triglyceride level

Triacylglycerides were estimated by enzymatic methods using diagnostic kit (Biodiagnostic, Egypt) according to the method of Fossati and Prencipe (1982). The absorbance was measured at 510 nm and the results were expressed as mg/dl.

Determination ofserum total cholesterol level

Total cholesterol was estimated by enzymatic methods using diagnostic kit (Biodiagnostic, Egypt) according to the method of Allain et al. (1974). The absorbance was measured at 500 nm and the results were expressed as mg/dl.

Statistical analysis

Statistical analysis was carried out by one way analysis of variance (ANOVA) followed by Tukey test. Results are expressed as means ±SEM (n = 10).

Results and discussion

Total phenols and flavonoids contents of S. securidaca flowers were investigated. The value of total phenolics was 82.39 ± 2.79 mg gallic acid equivalent (GAE)/g (D.W.) and that of the total flavonoids was 48.82 ± 1.95 mg rutin equivalent (RE)/g (D.W.).

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Table 1

Peak assignmenet of metabolites in ethanolic extract of S. securidaca using LC-DAD/MS in positive and negative ionization modes.

No. RT [M-H]- m/z [M+H]+ m/z Fragment ions Identified compounds

1 0.9 147 - 147, 62 trans-Cinnamic acid

2 1.2 - 139 137, 93 Salicylic acid

3 1.5 - 216 170 [(M+H)-HCOOH], 125, 97 Gallic acid

4 1.8 - 152 153, 109 Protocatechuic acid

5 2.8 - 292 289, 271, 245, 227, 205,179,125 Catechin

6 11.1 447 - 327[(M-H)-120], 285[(M-H)-162], 284, 255, 227 Kaempferol-7-O-glucoside

7 11.7 448 - 447, 357 [(M-H)-90], 327[(M-H)-120], Orientin

285[(M-H)-162]

8 12.4 625 - 505[(M-H)-120], 463[(M-H)-162],300, Quercetin-3,7-di-O-glucoside

301[(M-H)-162]

9 12.7 447 - 327[(M-H)-120], 284[(M-H)-H-162],255 Luteolin-3'-O-glucoside

[aglycone-2H-CO], 243, 241, 217, 213, 199,175,149

10 12.8 447 - 327[(M-H)-120], 285[(M-H)-162], 284, Luteolin-7-O-glucoside

257[aglycone-CO], 243, 241, 217, 213, 199,175,149

11 13.2 593 - 503[(M-H)-90], 473[(M-H)-120], 431[(M-H)-162], Isovitexin-4'-O-glucoside

311[(M-H)-120-162], 283

12 13.6 609 - 489[(M-H)-120], 447[(M-H)-162], Isoorientin-4'-O-glucoside

357[(M-H)-90-162], 327[(M-H)-120-162]

13 14.2 593 - 575 [(M-H)-18], 503[(M-H)-90], 473[(M-H)-120], Apigenin 6,8-di-C-glucoside

383[(M-H)-90-120],353[(M-H)-120-120] (vicenin-2)

14 14.9 593 - 503[(M-H)-90], 473[(M-H)-120], 431, 341,311 Isovitexin-7-O-glucoside

[(M-H)-120-162] (saponarin)

15 15 - 595 449, 433, 284 Kaempferol-O-

neohesperidosideee

16 15.4 623 - 503[(M-H)-120], 461[(M-H)-162], Isorhamnetin-3-O-glucoside-

341[(M-H)-120-162], 315[(M-H)-146-162], 297, 7-O-rhamnoside

195, 179,161,153, 135

17 15.9 448 - 447, 429,357 [(M-H)-90], 327 [(M-H)-120], Iso-orientin

285[(M-H)-162]

18 15.9 564 - 545, 503[(M-H)-60], 473[(M-H)-90], Apigenin-6-C-pentoside-8-C-

443[(M-H)-120], 425, 413,383,353[(M-H)-120-90], hexoside

19 16.1 639 - 639(M-H)-, 519[(M-H)-120], 477[(M-H)-162], 459 Isorhamnetin-O-sophoroside

[(M-H)-180], 357[(M-H)-120-162], 314,

315[(M-H)-162-162]

20 16.3 593 - 503 [(M-H)-90], 473 [(M-H)-120], 447 [(M-H)-146], Isoorientin-2"-O-rhamnoside

429 [(M-H)-146-H2O], 413, 395,383,353, 329, 299

21 17.3 431 - 431, 353,341[(M-H)-90], 311[(M-H)-120], 269 Isovitexin

22 17.6 577 - 577, 413[(M-H)-146-18], 457[(M-H)-120], 341,311, Isovitexin-2"-O-rhamnoside

293[aglycone+ 41-18]-, 173

23 17.7 - 580 579, 459, 271, 235 Naringin

24 17.9 463 - 301[(M-H)-162], 300, 271, 255,179, 151 Isoquercetrin

25 18.1 - 303 301, 258,143 Hesperetin

26 18.1 - 464 343, 301,179,151 Hyperoside

27 18.2 - 303 257, 229,185 Ellagic acid

28 18.9 578 - 431[(M-H)-146], 308, 285, 269[(M-H)-162-146] Apigenin-7-O-rutinoside

29 19.2 608 - 301,281,237,326 Hesperidin

30 19.3 577 - 431[(M-H)-146], 285[(M-H)-146-146] Kaempferol-3,7-dirhamnoside

(kaempferitrin)

31 19.5 - 609 301,464,179,151 Rutin

32 19.5 448 - 285[(M-H)-162], 284, 255, 267, 257, 256, 241, 229, Kaempferol-3-O-glucoside

213, 163 (astragalin)

33 19.5 607 - 608[(M-H)-H], 463[(M-H)-146], 447[(M-H)-162], Quercetin-3-O-glucoside-7-O-

299[(M-H)-162-146] rhamnoside

34 19.7 - 287 287, 285,217, 241,175 Luteolin

35 19.7 593 - 593 (M-H), 447[(M-H)-146], 285[(M-H)-146-162] Kaempferol-3-O-glucoside-7-

rhamnoside

36 19.9 477 - 357[(M-H)-120], 315[(M-H)-162], 314, 286, 285, 271, Isorhamnetin-3-O-glucoside

243, 299

37 20.1 479 - 357[(M-H)-120], 315[(M-H)-162], 314, 286, 285, 271, 299 Isorhamnetin-7-O-glucoside

38 23.3 479 - 477, 301[(M-H)-176], 273, 257,179, 193,151 Quercetin-3-glucuronide

39 24.9 625 - 449, 461[(M-H)-162], 447[(M-H)-176], 337, 287, Luteolin-7-O-glucuronide-3-O-

285[(M-H)-162-176] glucoside

40 25.4 609 - 449[(M-H)-162], 431[(M-H)-180], 301, Luteolin di-O-glucoside

287[(M-H)-162-162]

41 26.4 563 - 440 [(M-H)-120], 323, 269[(M-H)-132-162] Apigenin-O-pentosyl-hexoside

42 33 - 301 301, 151,179 Quercetin

43 34.3 - 286 285, 257,151,169, 241 Kaempferol

44 38.5 - 179 179, 135,107 Caffeic acid

45 38.5 593 - 473[(M-H)-120], 447[(M-H)-146], Quercetin-3,7-dirhamnoside

301[(M-H)-146-146], 299

46 38.8 - 353 353, 191,190 Chlorogenic acid

47 43.5 - 165 163, 119 p-Coumaric acid

q4 a Compounds reported in other Coronilla species. b Compounds reported in S. securidaca L.

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Intens x105 В

A = (-) BPC at 254

678g10

3^_3L3g3gX_.-1^ д.

Г I" -r—r

45 Time [min]

■ BPC 50-1300 -AII MS

x105 2.5 B = (-) BPC at 360 nm

1.5 1В

0.5 0.0 LIU 1g 15..................................

45 Time [min]

■ BPC 50-1300 -AII MS

Intens x105

.4 I - 1|1.....Ц][„..Г Ajr

20 25 30

45 Time [min]

■ BPC 300-400 +AII

Fig. 1. HPLC-ESI-MS/MS base peak chromatograms (BPC) of the crude ethanolic extract of the flowers of S. securidaca L. recorded at 254 nm (A) and 360 nm (B), negative ion q3 mode, and at 230 nm (C), positive ion mode. Peak numbers follow those listed in Table 1.

269 HPLC-DAD-MS/MS analysis of phenolic compounds

270 The HPLC-DAD-MS/MS analysis was carried out in both neg-

271 ative and positive ionization modes. The HPLC-DAD base peak

272 chromatograms (BPC) recorded at 254 nm (A), 360 nm (B) in the

273 negative mode and BPC in the positive mode obtained at 230 nm

274 (C) are shown in Fig. 1. The identities, retention times and observed

275 molecular and fragment ions for individual compounds are pre-

276 sented in Table 1.

277 A total of 47 phenolic compounds have been tentatively iden-

278 tified by comparing retention times and MS data of the detected

279 peaks with that reported in the literature and by searching

280 phytochemical dictionary of natural products database (CRC).

281 Identified compounds belonged to various classes (Table 1) includ-

282 ing eight phenolic acids and 39 flavonoids. The phenolic acids

283 included three hydroxybenzoic acids (salicylic acid, gallic acid and

284 protocatechuic acid) and four hydroxycinnamic acid derivatives

285 (trans-cinnamic acid, caffeic acid, chlorogenic acid and p-coumaric

286 acid), in addition to ellagic acid. Flavonoids were present mostly

287 as flavones (including both O- and C-glycosides with apigenin or

288 luteolin as aglycone) and flavonols (derived from the aglycones;

289 quercetin, kaempferol and isorhamnetin), flavanones (naringenin

and hesperitin and their glycosides). While, only one flavan-3-ol (catechin) was identified. Sugar moieties consists of hexosides, deoxyhexosides and pentosides as deduced from the loss of 162 146and 132respectively.

Identification of flavonoids

Seventeen flavones were identified on the basis of their MS/MS fragmentation. Three compounds were mono-C-glycosyl flavones (peaks 7,17 and 21) producing MS fragmentation patterns characteristic to C-glycosides flavonoids including dehydration and cross ring cleavage of the glucose moiety that producing 0, 2 cross ring cleavage [(M-H)-120] and 0, 3 cross ring cleavage [(M-H)-90] (Figueirinha et al., 2008). Compounds 7, 17 and 21 showed pseu-domolecular ions at m/z 448, m/z 448 and m/z 431, respectively, and exhibited typical fragmentation patterns ofC-glycosides, hence they were assigned as orientin, isoorientin and isovitexin, respectively.

Peaks 13 and 18 were di-C-glycosyl flavones and showed fragmentation pattern of [(M-H)-18], [(M-H)-90], [(M-H)-120], [aglycone +113], and [aglycone + 83] found in negative mode MS/MS spectra (Zhang et al., 2011). Vicenin-2 (13) showed a

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310 pseudomolecular ion at m/z 593 and characteristic fragment ions

311 at 575 [(M-H)-18], 503[(M-H)-90], 473[(M-H)-120], and 383

312 [(M-H)-90-120].

313 Compound 18 was identified as isochaftoside (6-C-pentosyl-8-

314 C-hexosyl apigenin) (Figueirinha et al., 2008; Zhang et al., 2011).

315 It showed a pseudomolecular ion at m/z 564 and a fragmen-

316 tation pattern typical of the asymmetric di-C-glycosides. MS2

317 data showed fragments at m/z 473[(M-H)-90] and 443 [(M-H)-

318 120], indicating the presence of a C-hexosyl unit. In the same

319 spectrum a fragment was observed at m/z 503 [(M-H)-60], cor-

320 responding to the fragmentation of a pentose. The base peak at

321 m/z 473 [(M-H)-90] and the high abundance of the fragment

322 at m/z 503 [(M-H)-60] revealed the presence of a 6-C pentosyl

323 unit. The ions at m/z 353 (aglycone+83) and 383 (aglycone + 113)

324 supported the conclusion that apigenin (MW 270) was the

325 aglycone.

326 Six compounds were identified as O-glycosyl flavones (peaks 9,

327 10,28,39,40 and 41) and showed fragmentation pattern beginning

328 with the cleavage of the O-sugarbond (Zhang et al., 2011). Five com-

329 pounds were identified as O-, C-glycosyl flavones (compounds 11,

330 12, 14, 20 and 22) producing a characteristic fragment ions of O-,

331 C-glycosyl flavones at [(M-H)-120], [(M-H)-90] and [(M-H)-162]

332 or [(M-H)-146] (Figueirinha et al., 2008; Zhang et al., 2011). For

333 example, isoorientin-2"-O-rhamnoside (20) had a pseudomolecu-

334 lar ion at m/z 593 that reveals a luteolin glycoside with a hexose and

335 deoxyhexose. Fragments at m/z 447 [(M-H)-146], corresponding

336 to loss of one deoxyhexose and another at m/z 429 [(M-H)-146-

337 H2O] corresponding to the loss of rhamnose + H2O were observed.

338 In addition, the absence of the aglycone ion is consistent with

339 an O-, C-diglycoside structure (Figueirinha et al., 2008). MS2 data

340 also exhibited fragments at m/z 473 [(M-H)-120] (base peak) and

341 a minor ion at m/z 503 [(M-H)-90], which indicated the pres-

342 ence of a C-glucosyl unit. Compound 11 (isovitexin-4'-O-glucoside)

343 exhibited a pseudomolecular ion at m/z 593 with a fragmenta-

344 tion pattern of apigenin dihexoside and characteristic fragment

345 ions of O-, C-glycosyl flavones with the loss of a O-hexosyl moiety

346 (-162

347 Eighteen flavonols were identified in the ethanolic extract

348 of the flowers of S. securidaca. The identification of these com-

349 pounds was facilitated by the analysis of fragmentation pathways

350 of (M-H)-/(M+H)+ ions in the negative and positive ion modes

351 and the observation of glycosidic residues (rhamnosyl (146

352 and glucosyl (162 were cleaved sequentially and generated

353 characteristic aglycone fragments compared to the available lit-

354 erature. Among these compounds five compounds were identified

355 as kaempferol glycosides (6, 15, 30, 32 and 35) and seven were

356 identified as glycosides of quercetin (8, 24, 26, 31, 33, 38 and 45).

357 In addition, four isorhamnetin glycosides (16, 19, 36 and 37) were

358 identified.

359 Flavanones usually occur as O-glycosyl derivatives, with the

360 sugar moiety bound to the aglycone hydroxyl group at either C-7 or

361 C-3. Among these compounds, the O-diglycosides are a dominant

362 category and their structures are usually characterized by the link-

363 age of either neohesperidose or rutinose to the flavonoid skeleton.

364 Compound 23 was found to be the neohesperidoside naringin. The

365 precursor and product ions of this compound were m/z 579 and

366 271, respectively, indicating the loss of O-diglycoside (m/z 308)

367 (Zhang et al., 2011) and compound 29 was found to be the ruti-

368 noside hesperidin with precursor and product ions of m/z 609 and

369 299, respectively.

370 One flavan-3-ol was identified, compound 5, which produced a

371 protonated molecular ion peak at m/z (292) and yielded fragment

372 ions at m/z 245, 205, and 179 characteristic for (+)-catechin. The

373 fragment ion at m/z 245, corresponding to [M+H-44]+, was pro-

374 duced by the loss of a (CH)2OH group from the catechin molecule

375 (Sun et al., 2007).

Identification of phenolic acids 376

Eight phenolic acids belonged to various classes have been 377

identified by comparing their retention times and fragmentation 378

patterns with that reported (Sanchez-Rabaneda et al., 2003; Sun 379

et al., 2007). In the positive ion mode hydroxybenzoic acids pro- 380

duced a protonated [M+H]+ molecule and a [M+H-44]+ fragment ion 381

via loss of a CO2 group from the carboxylic acid moiety (Sun et al., 382

2007). Three hydroxybenzoic acids have been identified; salicylic 383

acid, gallic acid, and protocatechuic acid (2, 3, and 4). 384

Four hydroxycinnamic acids were identified; trans-cinnamic 385

acid, caffeic acid, chlorogenic acid and p-coumaric acid (1, 44, 46 386

and 47). Caffeic and p-coumaric acids produced protonated molec- 387

ular ions at m/z 179 and 165, respectively, and MS2 spectra due to 388

loss of CO2 group from the carboxylic acid function (fragment ions 389

at m/z 135 and 119, respectively, [(M-H)-44]) (Sun et al., 2007). 390

Chlorogenic acid showed a molecular ion peak at (m/z 353) and a 391

fragmentation ion that corresponding to the deprotonated quinic 392

acid (m/z 191) (Sun et al., 2007). Compound 27 had an [M+H]+ ion 393

at m/z 303 which yielded a major ion at m/z 301 and minor ions at 394

m/z 284, 257, and 229 characteristic of ellagic acid fragmentation 395

(Sandhu and Gu, 2010). 396

Quantitative determination of some phenolic compounds in S. 397

securidaca flowers 398

Absolute quantification of phenolics using the available 399

standards was carried out (Table 2). Eight phenolic acids i.e. trans- 400

cinnamic acid (2.36 ± 0.98 mg/100 g), salicylic acid (15.54 ± 1.91), 401

protocatechuic acid (34.4±0.155), ellagic acid (13.47±3.95), caf- 402

feic acid (5.4 ±1.43), chlorogenic acid (84.22 ±2.08), p-coumaric 403

acid (7.58 ±1.51) and gallic acid (9.5 ±0.14) were determined. In 404

addition to seven flavonoids i.e. isoquercetrin (3340 ± 2.1), naringin 405

(19.73 ±3.016), hesperidin (32.098 ±2.28), luteolin (10.247± 406

0.594), quercetin (1.16 ±0.022), kaempferol (0.62 ±0.129), cate- 407

chin (39.44 ± 5.73) and hesperetin (0.109 ± 0.013). 408

Acute toxicity 409

The ethanolic extract of the flowers of S. securidaca was found 410 to be safe up to a dose of 2g/kgbwt with no mortality or signs of 411

behavioral changes or toxicity observed which suggests its safety 412

(Osweiler et al., 1985). 413

Table 2

Quantifications of some phenolic compounds identified in S. securidaca using HPLC q5 analysis.

Compound 'Concentration (mg/100g)

trans-cinnamic acid 2.36 ± 0.98

Salicylic acid 15.54 ± 1.91

Protocatechuic acid 34.4 ± 0.15

Naringin 19.73 ± 3.01

Ellagic acid 13.47 ± 3.95

Luteolin 10.24 ± 0. 59

Isoquercetrin 3340 ± 2.1

Quercetin 1.16 ± 0.02

Kaempferol 0.62 ± 0.12

Caffeic acid 5.4 ± 1.43

Catechin 39.44 ± 5.73

Hesperidin 32.09 ± 2.28

p-Coumaric acid 7.58 ± 1.51

Hesperetin 0.10 ± 0.01

Gallic acid 0.95 ±0.014

Chlorogenic acid 8.42 ± 2.08

Average concentration of three HPLC determinations ± SD. C = (+)BPC at 230 nm. B = (-)BPC at 360 nm. A = (-)BPC at 254 nm.

ARTICLE IN PRESS

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A. Flavonoids

Cpd. R1 R2 R3

Apigenin H H OH

Luteolin H OH OH

Kaempferol OH H OH

Quercetin OH OH OH

Isorhamnetin OH OCH3 OH

Cpd. R1 R2

NaringeninH OH

HesperetinH OCH3

Catechin

B. Phenolic acids

Salicylic acid Protocatechuic acid Ga||ic acid

Cinnamic acid Caffeic acid

P-coumaric acid

Ellagic acid

Chlorogenic acid

Fig. 2. Biological activities of the ethanolic extract of the flowers of S. securidaca L. (A) Effect on serum glucose level in alloxan-induced hyperglycemic rats. (B) Serum triacylglycerides and cholesterol levels in alloxan-induced hyperglycemic rats. @Significant difference from normal rats p < 0.05. 'Significant difference from hyperglycemic rats p < 0.05.

Anti-hyperglycemic activity

The ethanolic extract of the flowers showed marked anti-diabetic activity on blood glucose levels in alloxan-induced diabetic rats at all tested doses 100, 200 and 400mg/kgbwt with potencies of 31.78, 66.41 and 63.8% respectively (Fig. 2A). The most potent reduction in serum glucose level was recorded with a dose of 200 mg/kg bwt compared to Gliclazide at a dose of 5 mg/kg bwt (64.85%).

Anti-hyperlipidemic activity

The ethanolic extract of the flowers exhibited potent hypolipi-demic effect on the elevated serum triacylglycerides and cholesterol levels in alloxan induced hyperglycemic rats (Fig. 2B) with 39.68% and 41.46% decreases in serum triacylglycerides and cholesterol levels at dose 200 mg/kg, 68.46% and 51.50% for dose 400 mg/kg, compared to gliclazide at dose of 5 mg/kg bwt showing reduction in serum triacylglycerides and cholesterol levels with 73.01% and 62.20%, respectively.

Diabetes mellitus is a complex disorder that characterized by chronic hyperglycemia and dyslipidemia. The disease becomes a real problem of public health in developing countries, where its prevalence is increasing steadily and adequate treatment is often expensive or unavailable. Results of the present study revealed that alloxan-induced hyperglycaemia is associated with metabolic changes. Alloxan induces chemical diabetes in rats by damaging insulin secreting pancreatic P-cells leading to decrease in insulin release (Kim et al., 2006).

Hyperlipidaemia is a major characteristic of diabetes (Pushparaj et al., 2000). DM induced hyperlipidaemia is attributable to excess mobilization of fat from the adipose tissue due to the utilization of the glucose. Moreover, studies suggested that hyperlipidemia is one of the most common features in alloxan-induced hyperglycaemia in experimental rats (Krishnakumar et al., 2000). In this study, an increase in the levels of total cholesterol and triglycerides has been observed in alloxan-induced hyperglycaemic rats. Plants used in traditional medicine to treat diabetes mellitus represent a valuable alternative for the control of this disease (Kumar and Verma, 2011). In the present study, we evaluate the acute toxicity, anti-hyperglycemic and hypolipidemic effects of the ethanolic extract of the flowers ofS. securidaca L. Moreover, the phenolic composition ofthe extract was characterized using HPLC-DAD-ESI/MS technique to help in chemical profiling and standardization of the extract. Oral

treatment of hyperglycemic rats with the ethanolic extract of the flowers (100, 200 and 400 mg/kg bwt) significantly decreased the elevated serum glucose level as well as serum total cholesterol and triglycerides levels in diabetic rats with potencies comparable to gliclazide. Hence, we could say that extract had beneficial effects on carbohydrate metabolism in hyperglycaemic rats.

HPLC-DAD-ESI/MS analysis revealed that the ethanolic extract contains complex mixture of phenolic compounds including different classes of phenolic acids and flavonoids. Most of the detected phenolic compounds were reported to have anti-diabetic effect though different mechanisms. Isovitexin, luteolin 7-O-glucoside, hyperoside and isorientin were reported to possess antihyper-glycemic action (Brahmachari, 2011; del Pilar Nicasio-Torres et al., 2012; Folador et al., 2010). Vicenin-2 was reported to be an antioxidant that strongly inhibited a-glucosidase and exhibited potent anti-glycation properties (Islam et al., 2014). Isoquercetrin and astragalin were found to be glycation inhibitors having comparable activity to that of aminoguanidine (Brahmachari, 2011). Rutin was reported to possess potent hypoglycemic and hypolipidemic activities by enhancing peripheral glucose utilization by skeletal muscle and stimulation of P-cells (Jadhav and Puchchakayala, 2012).

The respective aglycones, quercetin and kaempferol were found to improve insulin-stimulated glucose uptake in mature adipocytes (Figueirinha et al., 2008). Isorhamnetin-3-O-glucoside was reported to lower serum glucose concentration, sorbitol accumulation in the lenses, red blood cells by exerting potent inhibitory activity against rat lens aldose reductase, leading to improved diabetic complications (Brahmachari, 2011). Rutin and hesperidin were reported to prevent the progression of hyperglycemia by increasing hepatic glycolysis, glycogen concentration and lowering hepatic gluconeogenesis (Jung et al., 2004). Through a docking study, catechin showed potential agonist characteristic to insulin receptor (insulin mimetic) (Pitchai and Manikkam, 2012). Gallic acid was reported to have hypoglycemic and hypolipidaemic effects against streptozotocin induced diabetic rats (Latha and Daisy, 2011), while chlorogenic acid was reported to exhibit hypoglycemic, hypolipidemic, and antioxidant properties (del Pilar Nicasio-Torres et al., 2012).

Conclusion

In this work we have evaluated the anti-hyperglycemic and anti-hyperlipidemic activity of the ethanolic extract of S. securidaca flower. Phenolic acids and flavonoids extracted with 90% ethanol

lü^^H ARTICLE IN PRESS

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497 have been identified and quantified. The ethanolic extract of the

498 flowers was safe up to dose of 2 g/kg. The extract showed potent

499 anti-diabetic and hypolipidemic effect in alloxan induced hyper-

500 glycemic in rats. The current results indicate that the flavonoid-

501 and phenolic acid-rich extract ofS. securidaca flowers is a promising

502 natural pharmaceutical for combating diabetes.

503 Conflicts of interest

504 The authors declare that they have no competing interests.

505 Author's contributions

506 RI wrote the manuscript, carried out extraction procedures and

507 analyzed data. AM wrote the manuscript, planed the work and ana-

508 lyzed data. DS carried out biological activity. EN carried out LCMS

509 analysis and interpreted data. AE revised the manuscript and super-

510 vised work. SE suggested the point and revised the manuscript.

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