Journal of Pharmacognosy and Phytotherapy Volume 9 Number 5, May 2017 ISSN 2141-2502

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Dr. Baojun Bruce Xu Dr. Adibe Maxwell Ogochukwu Beijing Normal University-Hong Kong Baptist Clinical Pharmacy and Pharmacy Management, University United International College Zhuhai, Faculty of Pharmaceutical Sciences, Guangdong Province, University of Nigeria, Nsukka China Enugu state, Nigeria. Dr. Hamad H. Issa Department of Physical Sciences, Dr. Odukoya, Olukemi Abiodun School of natural Sciences, Department of Pharmacognosy, The University of Dodoma, Faculty of Pharmacy Tanzania University of Lagos. Nigeria. Dr. Gagan Deep Department of Pharmaceutical Sciences Dr. Qinxue Richard Ding School of Pharmacy, Medical Center at Stanford University, University of Colorado Denver, Palo Alto, Colorado, USA USA Dr. Sulejman Redžic Dr. Fengguo Xu Faculty of Science of the University of Sarajevo Dept of Epidemiology and Public Health, 33-35 Zmaja od Bosne St., Yong Loo Lin School of Medicine, Sarajevo, National University of Singapore, Bosnia and Herzegovina Singapore Dr. Michal Tomczyk Dr. Haitao Lv Medical University of Bialystok, Medicine and Endocrinology, Faculty of Pharmacy, Albert Einstein College of Meidicine, Department of Pharmacognosy, Yeshiva University, Poland USA Dr. Ugur Çakilcioglu Hassane MEKHFI Firat University, University Mohammed the First, Faculty of Science and Arts, Faculty of Sciences, Department of Biology, Department of biology, Elazig Laboratory of Physiology and Ethnopharmacology, Turkey Morocco

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Journal of Pharmacognosy and Phytotherapy

Table of Contents: Volume 9 Number 5 May 2017

ARTICLES

Phytochemical and biological investigation of Eugenia uniflora L. cultivated in Egypt 57 Riham O. Bakr, Shaza A. Mohamed and Nermien E. Waly

Chemical constituents and biological activities of different solvent extracts of Prosopis farcta growing in Egypt 67 Amal Mohamed Saad, Mosad Ahmed Ghareeb, Mohamed Sayed Abdel-Aziz, Hassan Mohamed Fawzy Madkour, Omar Maajal Khalaf, Ahmed Kamel El-Ziaty and Mamdouh Abdel-Mogib

Vol. 9(5), pp. 57-66, May 2017

DOI: 10.5897/JPP2017.0443 Article Number: 966E82B63903 Journal of Pharmacognosy and ISSN 2141-2502 Copyright © 2017 Phytotherapy Author(s) retain the copyright of this article http://www.academicjournals.org/JPP

Full Length Research Paper

Phytochemical and biological investigation of Eugenia uniflora L. cultivated in Egypt

Riham O. Bakr1, Shaza A. Mohamed2 and Nermien E. Waly3*

1Department of Pharmacognosy, Faculty of Pharmacy, MSA University, Giza 11787, Egypt. 2Department of Pharmacognosy, Faculty of Pharmacy, Al-Azhar University (Girls), Cairo, Egypt. 3Department of Physiology, Faculty of Medicine, Helwan University, Helwan 11795, Egypt.

Received 3 January, 2017; Accepted 24 March, 2017

Eugenia uniflora L. (Myrtaceae) is a plant species used in folk medicine for treatment of various disorders. This study aims to quantify the phenolic and flavonoid contents of E. uniflora aqueous methanolic extract (AME), identification of its major constituents, as well as the evaluation of its biological activity. Quantification was performed using colorimetric assays. Column chromatographic separation was used for isolation of the major phenolic constituents while their structures were elucidated by 1D- and 2D Nuclear magnetic resonance spectroscopy (NMR) spectra. Alkaloids were identified using gas chromatography-mass spectrometry (GC-MS) analysis. The antidepressant activity of E. uniflora AME in mice was evaluated using the tail suspension test (TST). The weight control effect was evaluated by serial weighing. The results showed high phenolic and flavonoid contents of E. uniflora AME. The chromatographic investigation identified a new flavonoid, myricetin 3-O-(4'', 6''- digalloyl) glucopyranoside, for first time in this genus, along with four flavonoids and phenolic acids. Integerrimine alkaloid was identified through GC/MS analysis. Administration of E. uniflora AME significantly reduced the immobility time in mice (P value of < 0.0001) in a dose- dependent manner for doses of 1, 10, 50 and 100 mg/kg. Also, a non-significant weight reduction in mice chronically treated with the E. uniflora AME was observed. Our study reports the isolation of myricetin 3-O- (4'', 6''- digalloyl) glucopyranoside from E. uniflora. It confirms that E. uniflora leaf extract has an antidepressant and anti-obesity effect.

Key words: Eugenia uniflora, flavonoid, alkaloid, depression, obesity.

INTRODUCTION

Obesity and depression are escalating global and secretion and metabolism result in obesity (McArdle et societal problems (McArdle et al., 2013; WHO, 2006). al., 2013). There is an increasing interest by researchers Associated environmental factors such as poor dietary to identify food items or dietary components that may habits, and sedentary lifestyle, that impair hormone control weight. On the other hand, conventional therapy

*Corresponding author. E-mail: [email protected].

Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0 International License

58 J. Pharmacognosy Phytother.

of depression has been reported to be effective only in (Whatman Ltd., Maidstone, Kent, England) were used. The solvent 50% of cases due to this, thus the necessity for a new systems were: S1: n-BuOH-HOAc-H2O (4:1:5, upper layer) and S2: treatment has risen (Kessler et al., 2003). The use of 15 % aqueous HOAc. All solvents used for separation processes were of analytical grade. herbal therapy may be preferred in depression as it could provide a safer and potentially a more effective Plant material therapeutic option (Elhwuegi, 2004; Halliwell, 2006). The genus Eugenia is considered one of the largest Fresh leaves of E. uniflora L. were harvested in April 2014 from El genera belonging to family Myrtaceae with about 500 Zohria garden, Cairo, Egypt and were identified according to Prof. species of trees and shrubs in tropical and subtropical Abdel-Haleem Abdel-Mogaly, Department of Plant Taxonomy, America (Faqueti et al., 2013). Eugenia uniflora L. Herbarium of Horticultural Research Institute, Agricultural Research Centre, Dokki, Cairo, Egypt. A voucher specimen (RS 0018) was (Pitanga cherry or Brazilian cherry) is a putative deposited in the herbarium of the Faculty of Pharmacy, MSA candidate for management of both diseases. E. uniflora University. L. is a semi-deciduous shrubby tree with edible, cherry- like fruits, native to Brazil. The leaves and fruits of E. Extraction and isolation uniflora have been used in folk medicine for treatment of diarrhea, inflammation, rheumatic pains, fever, stomach Fresh leaves of E. uniflora (1 kg) were dried, ground and defatted in problems and hypertension (Almeida et al., 1995; petroleum ether under reflux. The extracts were combined and Consolini et al., 1999). In addition, it was reported to dried under vacuum, while the leaves were refluxed with 70% methanol till exhaustion. The extracts were filtered off, combined, possess a wide range of medicinal properties including and dried under reduced pressure, giving rise to a crude aqueous alleviation of mood disorders, antibacterial, antifungal, methanolic extract (AME) (110 g). Seventy grams of the AME were cytotoxic as well as radical scavenging activities (Alves, applied on a cellulose column (1.5 m × 10 cm, 1000 g), elution 2008; Auricchio et al., 2007; Auricchio and Bacchi, 2003; being carried out with water-methanol mixtures of decreasing Franco and Fontana, 2004; Rattmann et al., 2012; Samy polarity and monitored by paper chromatography and detection by et al., 2014a; Saravanamuttu, 2012; Schapoval et al., UV light. Fractions of 100 mL each were collected. The similar fractions were gathered yielding four main fractions according to 1994). their chromatographic properties (fluorescence-UV light, and Among its wide range of biological activities, E. uniflora responses towards different spray reagents on PC). Fraction I (0- showed hypoglycemic as well as hypolipidemic effects 20% MeOH, 15 g) was phenolic free. Fraction II (30-40% MeOH, 4 (Saravanamuttu, 2012). Also, oral administration of g) was subjected to Sephadex LH-20 column using 50% MeOH to essential oils (EO) from other Eugenia species (E. yield compound 1 (20 mg). Fraction III (40-60% MeOH, 2 g) was purified on a Sephadex column using saturated butanol for elution, brasiliensis, E. catharinae and E. umbelliflora) produced to yield compound 2 (15 mg). Fraction IV (80-100% MeOH, 2.5 g) antidepressant-like effects in mice (Colla et al., 2012; was chromatographed on a Sephadex column with 50% MeOH to Victoria et al., 2013). yield compounds 3, 4 and 5 (7 mg, each). For alkaloid estimation, The leaves of E. uniflora were the subject of many ten grams of the AME extract was mixed with 200 mL distilled phytochemical reports and the plant itself is deemed of water, acidified with 5% sulfuric acid solution, and then fractionated interest as it is rich in tannins, flavonoids, triterpenpoids with dichloromethane. The aqueous extract was further alkalinized with ammonia, then extracted with dichloromethane. The process and alkaloids. The identified alkaloids have been was repeated until the dichloromethane extract was negative to correlated with its anti-diabetic activity (Auricchio and Dragendorff’s reagent (Yubin et al., 2014). The combined Bacchi, 2003; Consolini et al., 1999; Fortes et al., 2015; dichloromethane extract was concentrated under vacuum and kept Lee et al., 1997; Samy et al., 2014a; Saravanamuttu, for GC/MS analysis. 0.25 g of total alkaloid extract was obtained 2012). with a percentage yield 2.5%.

Our objectives were to identify the major constituents of E. uniflora aqueous methanolic extract (AME) using GC/MS analysis different chromatographic techniques, quantifying its The analysis was carried out using a GC (Agilent Technologies phenolic and flavonoid contents, as well as investigating 7890A) interfaced with a mass-selective detector (MSD, Agilent its antidepressant and weight control activity in mice. 7000) equipped with a nonpolar Agilent HP-5ms (5%-phenyl methyl poly siloxane) capillary column (30 m × 0.25 mm i.d. and 0.25 μm film thickness). The carrier gas was helium with the linear velocity of MATERIALS AND METHODS 1 mL/min. The identification of components was based on the comparison of their mass spectra and retention time with those of General experimental procedure the authentic compounds and by computer matching with NIST and WILEY libraries as well as by comparison of the fragmentation The NMR spectra were recorded at 400 (1H) and 100 (13C) MHz, on pattern of the mass spectral data with those reported in the Varian Mercury 300, Bruker APX-400 and JEOL GX-500 NMR literature (Santana et al., 2013). spectrometers and δ-values are reported as parts per million. (ppm) relative to TMS in the convenient solvent. For column Quantitative colorimetric estimation of phenolic and flavonoid chromatography, Sephadex LH-20 (Pharmacia, Uppsala, Sweden), contents microcrystalline cellulose (E. Merck, Darmstadt, Germany) was used. For paper chromatography, Whatman No. 1 sheets The total phenolic content of E. uniflora AME was quantified using

Bakr et al. 59

Folin-Ciocalteau Reagent (FCR) and gallic acid as standard motionless. Conventional antidepressants decrease the immobility (Sellappan et al., 2002) measured at λmax of 765 nm. Calculations time in this test (Cunha et al., 2008; Steru et al., 1985). were based on gallic acid calibration curve where the total phenolics were expressed as milligram of gallic acid equivalents (GAE) per gram dry extract. The total flavonoid content was Statistical analysis determined using aluminum chloride colorimetric assay (Kosalec et All experimental results are given as the mean ± S.E.M. al., 2004) where the measurement was performed at λmax of 415 nm. Calculations were based on quercetin calibration curve and the Comparisons between experimental and control groups were total content was expressed as milligram of quercetin equivalent performed using one-way ANOVA. A value of P of 0.05 was (QE) per gram dry extract. All measurements were carried out in considered to be significant. triplicate.

RESULTS HPLC analysis Phytochemical investigation Identification of flavonoids in E. uniflora AME was performed using HPLC (Mattila et al., 2000). Dry plant extract (0.1 g) was mixed with Column chromatographic investigation of E. uniflora AME 5 mL methanol and centrifuged at 10,000 rpm for 10 min and the supernatant was filtered through a 0.2 µm Millipore membrane filter, results in the isolation of five compounds identified as: after which then 1 to 3 mL was collected in a vial for injection into Myricetin 3-O-(4'', 6''-digalloyl glucopyranoside (1), HPLC Hewlett-Packard (series 1050) equipped with auto-sampling myricetin 3-O-glucopyranoside (2) (Figure 1), quercetin injector; solvent degasser, ultraviolet (UV) detector set at λmax of (3), gallic acid (4) and ellagic acid (5). Compounds (1) 330 nm and quarter horsepower pump (series 1050). The column and (2) were identified by comparing their spectroscopic temperature was maintained at 35°C. Gradient separation was data with that reported (Samy et al., 2014b) while carried out with methanol and acetonitrile as a mobile phase at flow rate of 1 mL/min. Phenolic compounds were determined according compounds 3 to 5 were identified using Co- to Goupy et al.,(1999) with the same sample preparation with the chromatography, by comparing with standards. exception that the UV detector was set at λmax of 80 nm and quarter HP pump (series 1100). Flavonoids and phenolic acid standards from Sigma Co. were dissolved in a mobile phase and Myricetin 3- O-(4'', 6''-digalloyl glucopyranoside (1) injected into HPLC. Retention time and peak area were used for calculation of flavonoids and phenolic acid concentrations by the Yellow amorphous powder, purple fluorescent turned into data analysis of Hewlett-Packard software. 1 yellow by long UV light. H NMR (400 MHz, CD3OD): ppm 3.2-4.3 (sugar protons), 5.3 (1H, br s, H-1ʹʹ), 6.12 Antidepressant activity (1H, d, J=2Hz, H-6), 6.31 (1 H, d, J=2Hz, H-8), 7.14 (2H, d, J=2Hz, H-2ʹ-6ʹ), 7.22 (2 H, s, H-2ʹʹʹ/6ʹʹʹ), 7.29 (2H, s, H- Animals 13 2ʹʹʹ/6ʹʹʹ),. C-NMR (100 MHz, CD3OD): 63 (C-6ʹʹ), 71.5 (C- Forty male albino mice (4 to 5 weeks) were used for this study. 4ʹʹ), 72.1 (C-2ʹʹ), 74 (C-5ʹʹ), 75 (C-3ʹʹ), 94.6 (C-8), 99.8(C- Animals were housed randomly in-groups of 4 rats per cage, kept at 6), 101 (C-1ʹʹ), 107.06 (C-10), 109.6 (C-2ʹ-6ʹ), 109.6 (C- room temperature and provided with rodent chow and water ad 2ʹʹʹ-6ʹʹʹ), 121 (C-1ʹʹʹ), (122 (C-1ʹ), 136 (C-3), 137 (C-4ʹ), libitum. Mice were kept under a 12:12 h light: dark cycle (lights on at 141.1 (C-4ʹʹʹ), 143.4 (C-3ʹ-5ʹ), 143.4 (C-3ʹʹʹ-5ʹʹʹ), 156.92 (C- 07:00 h). Mice were allowed to acclimatize to the holding room for 9), 159 (C-2), 163 (C-5), 165 (C-7), 168 (C-7ʹʹʹ), 168.2 (C- at least 24 h before the behavioral procedure. All manipulations were conducted in the light phase, with each animal used only once 7ʹʹʹ), 179 (C-4). (n = 7 animals per group). Animal care and handling was performed in conformance with approved protocols of the MSA research ethics committee. Myricetin 3-O-glucopyranoside (2)

Yellow amorphous powder, purple fluorescent turned into Treatment 1 yellow by long UV light. H NMR (400 MHz, CD3OD):3.2- The E. Uniflora AME was dissolved in distilled water and 4.3 (sugar protons), 5.05 (1H, d, J=7.6 Hz, H-1ʹʹ), 6.1 (1H, administered orally acutely (just once at the beginning of the d, J=2Hz, H-6), 6.31 (1 H, d, J=2Hz, H-8), 7.23 (2H, d, 13 experiment) or chronically (for 10 days) at doses 1, 10, 50 and 100 J=2Hz, H-2ʹ-6ʹ), C-NMR (100 MHz, CD3OD):61 (C-6ʹʹ), mg/kg by oral route (p.o.) 60 min before the tail suspension test. A 70 (C-4ʹʹ), 72 (C-2ʹʹ), 73 (C-5ʹʹ), 76 (C3ʹʹ), 94 (C-8), 99 (C- control group received distilled water only. 6), 104 (C-1ʹʹ), 105 (C-10), 109 (C2ʹ,6ʹ), 109 (C-2ʹʹʹ-6ʹʹʹ), 122 (C-1ʹ), 136 (C-3), 138 (C-4ʹ), 146 (C-3ʹ-5ʹ), 158 (C2, Tail suspension test (TST) C-9), 162 (C-5), 166 (C-7), 179 (C-4).

The total duration of immobility induced by tail suspension was measured according to the method described previously (Steru et Compound 3, 4 and 5 al., 1985). Mice were suspended about 50 cm above the floor by adhesive tape placed around 1 cm from the tip of the tail. Immobility time was recorded during a 6 min period. Mice were considered These were identified as quercetin, gallic acid and ellagic immobile only when they hung passively or stay completely acid by Co-PC.

60 J. Pharmacognosy Phytother.

OH

OH

HO O OH

3

O

OH O OH 6''' 4''' 5''' O HO 1''' HO 2''' OH 3''' Myricetin 3- O-(4'', 6''-digalloyl) glucopyranoside Myricetin 3-O-glucopyranoside.

Figure 1. Chemical structures of E. uniflora main constituents.

GC/MS analysis phenolic and flavonoid contents based on comparison of spectra with those of available standards. Results were Investigation of the dichloromethane fraction resulted in presented in Tables 2 and 3. The results showed that the identification of ten compounds (Figure 2 and Table gallic acid (10.85 mg/g) was the most abundant phenolic 1) including one main alkaloid, nitrogenated derivatives acid followed by benzoic acid (5.23 mg/g) then isoferulic and some fatty acids. The identification was based on acid (2.96 mg/g), while flavonoid analysis revealed comparison of the MS characteristics and molecular quercetrin (2.17 mg/g) as the main identified flavonoid weight (MW) with the mass spectra of the available followed by naringin (1.92 mg/g). databases and previous reports. Dodecanoic, oleic acid, in addition to hexadecanoic, heptadecanoic and octadecanoic acid derivatives have been identified. Antidepressant activity Compound 10 was identified as Integerrimine according to its fragmentation pattern (Figure 3). Administration of E. uniflora AME reduced the immobility time in the TST in a dose-dependent manner for doses 1 and 10 mg/kg. The average immobility time for control Quantitative colorimetric estimation of phenolic and mice was 3.7 ± 0.4 seconds, while it was 2.3 ± 0.8 and flavonoid contents 0.9 ± 0.3 s for doses 1 and 10 mg/kg consequently. This reduction was statistically significant with the P value of < E. uniflora appeared rich in phenolic content expressed 0.0001. At doses of 50 and 100 mg/kg the extract still as 98.17±0.35 mg/g GAE (standard curve equation: y = 2 significantly reduced (P value of < 0.0001) the immobility 0.0011x + 0.0009, r = 0.9867). In addition, flavonoid time to 1.6 ± 0.4, and 2.4 ± 0.3 seconds respectively content represented 8.1±0.27 mg/g QE (standard curve 2 (Figure 4). equation: y = 0.005x - 0.0198, r = 0.9774). On the other hand, administering E. uniflora AME

chronically for 10 days significantly (P <0.0001) reduced HPLC analysis the immobility time in TST at all doses tested. The immobility times for doses 1, 10, 50 and 100 mg/kg were HPLC analysis was employed to identify the main 1.4 ± 0.2, 2.3±0.3, 2.9 ± 0.5 and 2.1 ± 0.4 s, respectively.

Bakr et al. 61

Figure 2. Fragmentation pattern of integerrimine identified in E. uniflora dichloromethane fraction.

Table 1. Identification of the main constituents in the dichloromethane fraction (GC/MS analysis).

S/N Rt1 Constituent M+ Mass spectral data (m/z2, intensity %) 1 5.28 Pyridine 79 79 (100%) 52 (80%) 51 (60%) 50 (48%) 2 5.75 3 methyl Pyrrol 81 81 (75%) 80.1 (100%) 53 (30%) 3 6.33 2 butoxy ethanol 118 57.1 (100%) 45 (46%) 87.1 (30%) 75 (15%) 100 (10%) 4 15.70 Dodecanoic acid 200 55.1 (100%) 43 (92%) 73 (90%) 60 (85%) 129 (30%) 85 (30%) 5 16.66 Dihydromethyl jasmonate 226 83 (100%) 55 (30%) 67 (20%) 105 (20%) 155 (15%) 74 (100%) 87.1 (80%) 43 (60%) 55 (50%) 298.3 (30%) 143.1 6 21.38 Heptadecanoic acid methyl ester 298 (10%) 109 (10%)

55.1 (100%) 69 (70%) 83 (69%) 97.1 (50%) 111.1 (30%) 129 7 21.76 Oleic acid 282 (15%) 264.2 (15%) 226.1 (10%)

43 (100%) 83.1 (90%) 97.1 (80%) 111.1 (50%) 125.1 (20%) 8 22.14 Octadecyl acetate 312 252.2 (10%) 139.1 (10%) 165.1 (10%)

Hexadecanoic acid bis 129 (100%) 57 (50%) 112 (50%) 70.1 (30%) 83 (27%) 147 9 23.73 370 (2ethylhexyl)ester (27%) 241 (8%) 259.1 (8%)

120.1 (100%) 136 (98%) 94.1 (80%) 80.1 (50%) 43 (22%) 67 10 23.86 Integerrimine 335 (20%) 220.1 (20%) 335.1 (20%) 246 (18%)

Rt = Retention time; m/z = Mass to charge ratio; M+ = Molecular ion.

The immobility time for the control was 2.1±0.8 s (Figure observed. The average weight for the control mice was 5). 17.75 ± 0.7 while for the treated groups it was 16.8 ± 0.9, In addition, a non-significant weight reduction in mice 18.3 ± 0.6, 17.1 ± 0.8 and 16.4 ± 0.8 g for doses of 1, 10, chronically treated with the E. uniflora AME was 50 and 100 mg/kg respectively (Figure 6). For all

62 J. Pharmacognosy Phytother.

Figure 3. Fragmentation pattern of integerrimine identified in E. uniflora dichloromethane fraction using HPLC.

Table 2. Phenolic acid identified in E. uniflora AME using HPLC Table 3. Flavonoid content in E. uniflora AME using HPLC analysis. analysis.

Phenolic compound Concentration (mg/g) Flavonoid Concentration (mg/g) Gallic acid 10.85 Naringin 1.92 Protocatechuic acid 0.238 Rutin 0.38 Chlorogenic acid 1.364 Hesperidin 0.196 Catechol 0.474 Quercetrin 2.17 Catechin 0.289 Quercetin 0.024 P- hydroxy-benzoic acid 0.26 Naringenin 0.069 Caffeic acid 0.019 Hesperetin 0.38 Vanillic acid 0.969 Apigenin 0.023 Ferulic acid 0.433 7-OH- flavone 0.007 Isoferulic acid 2.96

Ellagic acid 1.383

Alpha coumaric acid 0.169 Compound 1, Myricetin 3- O-(4'', 6''-digalloyl) Benzoic acid 5.23 glucopyranoside was identified for first time in this Salicylic acid 0.441 species, compared with myricetin 3-O-(4''-O-galloyl) α-L- 3, 4, 5 methoxy-cinnamic acid 0.066 rhamnopyranoside, previously identified (Samy et al., P-Coumaric acid 0.162 2014a). 1H-NMR and 13C-NMR showed closely related Cinnamic acid 0.037 data to myricetin 3 O-glucopyranoside (compound 2) except for the presence of additional signals at δ 7.22

and 7.29 ppm referring to the galloyl moieties. The experiments N=7 for each experimental group. location was determined to be at C-6'' and C-4'' on the basis of the downfield shift of C-6'' and C-4'' glucose compared with the resonance of the corresponding DISCUSSION carbon in the spectrum of the free glucopyranose present in compound 2. The two equivalent Galloyl protons H-2 This study confirmed high phenolic and flavonoid and H-6 appeared as sharp singlet integrated for two contents evaluated through HPLC and colorimetric protons at δ 7.22 and 7.29 ppm. Our data are similar to assays, in agreement with reported literature (Suhendi et previous literature (Suhendi et al., 2011). al., 2011). Beside the previously tentatively identified Integerrimine, the main alkaloid identified through its constituents, five compounds were isolated and identified. fragmentation pattern (Figure 3), was compared with the

Bakr et al. 63

Figure 4. The effect of acute treatment with E. uniflora AME on the tail suspension test (TST) in mice. Administration of E. uniflora AME significantly reduced the immobility time in TST in mice treated for 60 min before the test at doses of 1, 10, 50 and 100 mg/kg. Results expressed as mean ± SEM and analyzed using one-way ANOVA; (*** = P value was < 0.0001).

Figure 5. The effect of chronic treatment with E. uniflora AME on tail suspension test (TST) in mice. Administration of E. uniflora AME significantly reduced the immobility time in TST in mice treated for 10 days before the test at doses of 1, 10, 50 and 100 mg/kg. Results expressed as mean ± SEM and analyzed using one-way ANOVA; (*** =P value was < 0.0001).

reported literature (El-Shazly et al., 1996; Zhu et al., pyrrolizidine alkaloid, where the ion fragment at m/z 220 2015). The series of ions at m/z 136, 120, 119, 93, 94 is due to the cleavage of the weak allylic ester bond. and 80, are characteristic of 1, 2- unsaturated Gallic acid was identified with HPLC analysis in high pyrrolizidine diesters. While the presence of a base peak concentration in agreement with Schumacher et al. at m/z 120 and 138 is denoting a retronecine-type of (2015), while benzoic acid derivatives have been

64 J. Pharmacognosy Phytother.

Figure 6. The effect of chronic treatment with E. uniflora AME on weight in mice. Administration of E. uniflora AME reduced the weight in TST in mice treated for 10 days at doses of 1, 10, 50 and 100 mg/kg. Results expressed as mean ± SEM and analyzed using one-way ANOVA.

identified in E. polyantha (Lelono et al., 2013). Quercetrin factor (BDNF) levels and attributed to the myricetin- which was the highest identified flavonoid was previously mediated anti-oxidative stress in the hippocampus (Ma et identified by Rattmann et al. (2012). al., 2015). Further studies however are needed to confirm With respect to the antidepressant activity of E. uniflora this suggestion. AME extract the result of this study show that the extract On the other hand, the different results obtained by significantly reduced the immobility time in a dose- Colla et al. (2012) who studied the hydro-alcoholic extract dependent manner at the doses of 1 and 10 mg/kg. At of different Eugenia species could be attributed to the higher doses the effect was less pronounced although difference in the plant constitution. Also, we used the reduction was still significant when compared with the different species of mice and only male mice, which can control. Our results come in agreement with Victoria et account for different responses to the extract. Further al., (Victoria et al., 2013) where they studied the studies are required to confirm the effective dose antidepressant-like effect of E. uniflora essential oil in the required for the antidepressant activity of the E. uniflora TST at doses of 10 and 50 mg/kg. Furthermore, the in different animal species as well as in human to detect administration of E. uniflora extract for one month still had the difference in response between males and females. antidepressant like activity (Figure 4) with the dose 1 Although statistically non-significant, the E. uniflora mg/kg being highly significant compared to the other AME produced a degree of weight loss in animals that doses. These results could explain the traditional use of received the extract for one month. This result could E. uniflora in folk medicine for the treatment of symptoms suggest a potential role for this extract in treatment of related to depression, as well as its use by Guarani obesity especially that it has been reported that it has Indians as a tonic stimulant (Alonso, 1998; Greinger, anti-hyperlipidemic and hypoglycemic effect 1996). The stepwise fashion of the decrease in the (Ramalingum and Mahomoodally, 2014). reduction of immobility time at higher doses than 10 mg/kg could in fact support a receptor mediated Conclusion mechanism for the antidepressant effect of E. uniflora AME (Colla et al., 2012). This study reports for the first time the identification of The antidepressant activity demonstrated by E. uniflora myricetin 3- O-(4'', 6''-digalloyl) glucopyranoside and AME can be attributed to its myricetin content. Previous confirmed the high phenolic and flavonoid contents of report suggested the ability of myricetin to attenuate the Egyptian grown E. uniflora AME, beside the presence of depressant-like behaviors in mice exposed to repeated alkaloid and fatty acids. In addition, this work supports restraint stress by restoring the brain derived neurotropic the role for E. uniflora AME in the management of

Bakr et al. 65

depression and obesity. Further studies are required to PA, Cechinel-Filho V, San Feliciano A, Monache FD (2013). correlate myricetin derivatives with the biological activities Euglobal-like compounds from the genus Eugenia. Nat. Prod. Res. 27:28-31. of E. uniflora, determine the effective dose, and the exact Fortes GAC, Carvalho AG, Ramalho RRF, da Silva AJR, Ferri1 PH, mechanism through which these effects are achieved. Santos SC (2015). Antioxidant Activities of Hydrolysable Tannins and Flavonoid Glycosides Isolated from Eugenia uniflora. L. Rec. Nat. Prod. 9:251-256. Franco IJ, Fontana VL, (2004). Herbs and Plants-Medicine of the ETHICS APPROVAL Simple, eleventh ed. Editora Livraria Vida LTDA, Brazil. Goupy P, Hugues M, Biovin P, Amiot M (1999). Amiot J. Antioxidant Animal care and handling was performed in conformance composition and activity of barley (Hordeum vulgare) and malt with approved protocols of MSA University and Egyptian extracts and of isolated phenolic compounds. J. Sci. Food Argic. 79:1625-1634. Community guidelines for animal care. Greinger CR (1996). Medicinal plants of Seychelles. J. Royal Soc. Health 116(2):107-109. Halliwell B (2006).Oxidative stress and neurodegeneration: where are CONFLICT OF INTERESTS we now? J. Neurochem. 97(6):1634-1658. Kessler RC, Berglund P, Demler O, Jin R, Koretz D, Merikangas KR, Rush J, Walters EE, Wan PS (2003). The epidemiology of major The authors have not declared any conflict of interests. depressive disorder: results from the National Comorbidity Survey Replication (NCS-R). JAMA 289(23): 3095-3105. Kosalec H, Bakmaz M, Pepeljnjak S, Vladimir-kne S (2004). FUNDING Quantitative analysis of the flavonoids in raw propolis from Northern Croatia. Acta Pharm. 54:65-72. Lee MH, Nishimoto S, Yang LL, Yen KY, Hatano T, Yoshida T, Okuda T This research is not supported by any funding agency. (1997). Two macrocyclic hydrolysable tannin dimers from Eugenia uniflora. Phytochemistry 44:1343-1349. Lelono R, Arthur A, Sanro T (2013). Preliminary studies of Indonesian ACKNOWLEDGEMENT Eugenia polyantha leaf extracts as inhibitors of key enzymes for type 2 diabetes . J. Med. Sci. 13(2):103. The authors would like to acknowledge Prof. Richard Ma Z, Wang G, Cui L, Wang Q (2015). Myricetin attenuates depressant- like behavior in mice subjected to repeated restraint stress. Int. J. Hallworth, PhD, Biomedical Sciences Department at Mol. Sci. 16:28377-28385. Creighton University, Omaha, NE, USA, for his invaluable Mattila P, Astola J, Kumpulainen J (2000). Determination of flavonoids editorial assistance. in plant material by HPLC with diode-array and electro-array detections. J. Agric. Food Chem. 48:5834-5841. McArdle MA, Finucane OM, Connaughton, RM, McMorrow AM, Roche HM, (2013). Mechanisms of obesity-induced inflammation and insulin REFERENCES resistance: insights into the emerging role of nutritional strategies. Front Endocrinol (Lausanne) 4: Article 52, 1-23. 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Bioactive compounds from the leaves of Eugenia uniflora. J. Nat. Auricchio MT, Bugno A, Barros SBM, Bacchi EM (2007). Antimicrobial Prod. 7:37-47. and antioxidant activities and toxicity of Eusenia uniflora. Latin Am. Samy MN, Sugimoto S, Matsunami, K Otsuka H, Kamel, MS (2014b). J.Pharm. 26:78-81. Taxiphyllin 6'-O-gallate, actinidioionoside 6'-O-gallate and myricetrin Auricchio MT, Bacchi EM (2003). Eugenia uniflora L. ‘‘Brazilian cherry’’ 2''-O-sulfate from the leaves of Syzygium samarangense and their leaves: pharmacobotanical, chemical and pharmacological biological activities. Chem. Pharm. Bull. 62(10):1013-1018. properties. Rev. Inst. Adolfo Lutz 62:55-62. Santana PM, Miranda M, Payrol JA, Silva M, Hernández V, Peralta E Colla AR, Machado DG, Bettio LE, Colla G, Magina MD, Brighente IM, (2013b). Gas chromatography-mass spectrometry study from the Rodrigues AL (2012). Involvement of monoaminergic systems in the leaves fractions obtained of Vernonanthura patens (Kunth) H. Rob. antidepressant-like effect of Eugenia brasiliensis Lam. (Myrtaceae) in Int. J. Org. Chem. 3:105-109. the tail suspension test in mice. J. Ethnopharmacol. 143:720-731. Saravanamuttu S, Sudarsanam D (2012). Antidiabetic plants and their Consolini AE, Baldini OA, Amat AG (1999). Pharmacological basis for active ingredients. IJPSR 3(10): 3639-3650. the empirical use of Eugenia uniflora L. (Myrtaceae) as Schapoval EE, Silveira SM, Miranda ML, Alice CB, Henriques AT antihypertensive. J. Ethnopharmacol. 66:33-39. (1994). Evaluation of some pharmacological activities of Eugenia Cunha MP, Machado DG, Bettio LE, Capra JC, Rodrigues AL (2008). uniflora L. J. Ethnopharmacol. 44:137-142. Interaction of zinc with antidepressants in the tail suspension test. Schumacher NS, Colomeu TC, De Figueiredo D, Carvalho Vde C, Prog Neuropsychopharmacol. Biol. Psychiatr. 32:1913-1920. Cazarin CB, Prado, MA, Meletti LM, Zollner Rde L, (2015). El-Shazly A, Sarg T, Ateya A, Abdel Aziz A, El-Dahmy S (1996). Identification and antioxidant activity of the extracts of Eugenia Pyrrolizidine Alkaloids from Echium setosum and Echium vulgare. J uniflora Leaves. characterization of the anti-Inflammatory properties . Nat. Prod. 59(3):310-313. of aqueous extract on diabetes expression in an experimental Model Elhwuegi AS (2004).Central monoamines and their role in major of spontaneous type 1 diabetes (NOD Mice). Antioxidants (Basel) depression. Prog Neuropsychopharmacol. Biol. Psychiatr. 28:435- 4(4): 662-680. 451. Sellappan S, Akoh CC, Krewer G (2002). Phenolic compounds and Faqueti LG, Petry CM, Meyre-Silva C, Machado KE, Cruz AB, Garcia antioxidant capacity of Georgia-grown blueberries and blackberries.

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Vol. 9(5), pp. 67-76, May 2017

DOI: 10.5897/JPP2017.0452 Article Number: F69CD0163906 Journal of Pharmacognosy and ISSN 2141-2502 Copyright © 2017 Phytotherapy Author(s) retain the copyright of this article http://www.academicjournals.org/JPP

Full Length Research Paper

Chemical constituents and biological activities of different solvent extracts of Prosopis farcta growing in Egypt

Amal Mohamed Saad1, Mosad Ahmed Ghareeb1*, Mohamed Sayed Abdel-Aziz2, Hassan Mohamed Fawzy Madkour3, Omar Maajal Khalaf3, Ahmed Kamel El-Ziaty3 and Mamdouh Abdel-Mogib4

1Medicinal Chemistry Department, Theodor Bilharz Research Institute, Kornaish El-Nile, 12411 Warrak El-Hadar, Imbaba, P. O. Box 30, Giza, Egypt. 2Microbial Chemistry Department, Genetic Engineering and Biotechnology Division, National Research Center, El Behoos Street 33, Dokki-Giza 12622, Egypt. 3Chemistry Department, Faculty of Science, Ain Shams University, El-Khalifa El-Mamoun, 11566 Abbassia, Cairo, Egypt. 4Chemistry Department, Faculty of Science, Mansoura University, Mansoura-35516, Mansoura, Egypt.

Received 7 March, 2017; Accepted 28 March, 2017

Different solvent extracts from the aerial part of Prosopis farcta growing in Egypt have been biologically evaluated by studying their antimicrobial, anticancer and antioxidant activities. Furthermore, the chemical analysis using GC/MS has been performed for the promising extracts n-hexane and methylene chloride, and this analysis led to the identification of twenty six and thirty two compounds respectively from n-hexane and methylene chloride. The major compound identified in the n-hexane is (Z) 9,17- octadecadienal (10.60%) while for methylene chloride is tricosanoic acid (9.24%). In addition, chromatographic isolation of the ethyl acetate and n-butanol extracts resulted in the isolation of four compounds which were identified as; dihydrokaempferol-3-O-α-L-rhamnoside (1), apigenin (2), 4′- methoxyquercetin (tamarixetin) (3) and acacetin-7-O-α-L-rhamnoside (4). n-hexane and methylene chloride showed moderate antimicrobial activities against three microbes for each, that is, Shigella spp., Escherichia coli and Proteus vulgaris for n-hexane and Erwinia spp., Escherichia coli and Staphylococcus epidermis for methylene chloride. On the other hand, the ethyl acetate showed higher antimicrobial activities against Shigella spp., Escherichia coli, and Candida albicans. Likewise n- butanol extract showed higher activity against Shigella spp., Erwinia spp., E. coli, P. vulgaris, S. epidermis and Candida albicans. Moreover, the anticancer activities were evaluated against four human tumor cell lines namely; HepG-2, HeLa, PC3 and MCF-7. The n-butanol extract showed the highest activity against MCF-7 cell line with IC50 of 5.6 μg/ml compared to 5-fluorouracil with IC50 of 5.4 μg/ml, while the ethyl acetate showed the highest activity against Hela cell line with IC50 of 6.9 μg/ml compared to 5-fluorouracil with IC50 of 4.8 μg/ml. Also, the inhibition percentages (I%) of ABTS radical were 83.1, 82.0, 87.2 and 87.0% respectively for the n-hexane, methylene chloride, ethyl acetate and n-butanol extracts, respectively, compared to ascorbic acid with 89.2%. In, conclusion the different extracts of P. farcta aerial part showed promising antimicrobial, anticancer and antioxidant activities, in which may be return to their identified bioactive secondary metabolites.

Key words: Prosopis farcta, antimicrobial, anticancer, antioxidant, chemical constituents.

68 J. Pharmacognosy Phytother.

INTRODUCTION flavonoids (Direkvand-Moghadam et al., 2014). A literature survey revealed that P. farcta has been used in Due to, the rapid development of drug-resistant micro- the treatment of neurological disorders (Mollashahi et al., organisms, appearance of new patients of life-threatening 2013) and cholesterol level (Omidi et al., 2013). infections and the constant return of diseases, the search Moreover, different parts of the plant (seeds, pods and for the discovery of new drugs got strong attention from leaves) were reported to show a wide range of biological researchers (Demain, 2000; Strobel et al., 2004). So, it is activities including; antioxidant (Poudineh et al., 2015), necessary to concentrate on the antimicrobial activity of antibacterial (Sharifi-Rad et al., 2014), hepatoprotective plant derived substances that are being still used as (Alharbi et al., 2017), anti-diabetic (Jafari et al., 2013), to global traditional medicine (Savoia, 2012). Plant reduce cardiac pain (Asadollahi et al., 2010), and secondary metabolites are responsible for the antiparasitic (Gulalp and Karcioglu, 2008). To the best of antimicrobial activity. Vancomycin-resistant enterococci our knowledge, there is little information in the literature (VRE), methicillin-resistant Staphylococcus aureus about the chemical and biological investigations were (MRSA), multi-drug resistant (MDR)- Mycobacterium carried out on the aerial part of P. farcta growing in tuberculosis and MDR Gram-negative bacteria are Egypt. Therefore, the present study is aimed at the documented as the most complicated infections to isolation and identification of the chemical constituents overcome and heal (Dahiya and Purkayastha, 2012). from different solvent extracts of P. farcta aerial part, and Increased illness and death, especially in the third world evaluation of their antimicrobial, anticancer and countries, have been due to the development of broad- antioxidant activities. spectrum β-lactamases (ESBLs) and carbapenemases that target Gram-negative bacteria (WHO, 2002). To overcome the resistance problem, there is a command MATERIALS AND METHODS need to do a renewed effort to screen various medicinal plants for their potential antimicrobial traits, which are due Plant material to compounds synthesized in the secondary metabolism of the plant. Alkaloids, flavonoids, tannins, phenolic The aerial parts of P. farcta (Banks et Sol.) Eig. (Fabaceae) were collected from Alkharga Oasis, Alwady Algaded, Egypt on March, compounds, steroids, resins, fatty acids and gums are 2015. The plant was identified by Prof. Dr. Ibrahim A. Mashaly, the most important of these bioactive compounds of Professor of Plant Ecology and Flora, Botany Department, Faculty plants which are capable of producing specific of Science, Mansoura University, Egypt. physiological effect on body. Medicinal plants are used to treat about 80% of the world's population especially in India. The plant materials used in traditional medicine are Extraction and fractionation considered more effective and relatively cheaper than manufactured medicine (Iwu et al., 1999; Mann et al., The aerial parts were dried in an oven at 45°C for 24 h and been grinded to give 1.5 kg of dried powder. Then, powdered materials 2008). On the other hand, anticancers were isolated from were soaked in MeOH for 72 h, at room temperature. After filtration, different plant species. The first anticancer agents the solvent was evaporated using rotatory evaporator, resulting introduced in clinical use were vinca alkaloids, that is, crude extract that was undergo further solvent extraction using vinblastine (VLB) and vincristine (VCR) isolated from different organic solvents that is, n-hexane, methylene chloride, Catharanthus roseus (Parakash et al., 2013). ethyl acetate and n-butanol. The genus Prosopis L., is widely spread in different dry and semi-dry regions worldwide like; Africa, Australia, America and Asia (Perez-Garcia et al., 2012; Peña- Antimicrobial activity Avelino et al., 2014; Abdelmoteleb et al., 2017). The antimicrobial activity was evaluated by filter paper disc method Approximately, this genus comprises from 44 to 50 (Murray et al., 1998; Sardari et al., 1998). Briefly, filter paper discs, species (William and Jafri, 2015). Prosopis farcta (Banks 5 mm diameter, were saturated with 200 µg of tested extract. Stock et Sol.) Eig. (Family Fabaceae) is a little prickly spiny cultures of the test organisms were obtained from the shrub; it is native of United States, Kuwait, Turkey, Iraq, Microbiological Laboratory, Faculty of Medicine, Mansoura Iran, Northern Africa, and South Western Asia (Sharifi- University. Bacteria test microbes used were S. aureus, Rad et al., 2014). Different phytochemicals were reported Streptococcus pyogenes, Klebsiella pneumonia, Escherichia coli, Bacillus subtilis, Erwenia carotovora, Shigella sp., Erwinia sp., to be isolated and identified from the plant viz., volatile Enterobacter aerogenes, Pseudomonas aeruginosa, Proteus constituents (Harzallah-Skhiri et al., 2006), proteins and vulgaris and Staphylococcus epidermis. Whereas the fungus used unsaturated fatty acids (Lajnef et al., 2015), and was Candida albicans. The bacterial test microbes (106cells/ml)

*Corresponding author. E-mail: [email protected] Tel: +2 01012346834. Fax: +2 02 35 40 8125.

Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0 International License Saad et al. 69

were swapped on plates containing nutrient agar medium (DSMZ1) tested sample (El-Gazzar et al., 2009). whereas, the fungus test microbe (108 cell/ml) was swapped on plates containing Czapek-Dox medium (DSMZ130). The filter paper discs-containing the tested extracts were put on the surfaces of the GC/MS analysis inoculated plates. The plates were then incubated at 37 and 30°C, for bacteria and fungus test microbes respectively. The appearance GC/MS analysis was performed at the Central Laboratory of the of clear zones (mm diameter) was detected after 24 h of incubation. Ministry of Agriculture, Al-Bhooth Str., Cairo, on Agilent 6890 The activity index (%) is also measured as a correlation of the clear gas chromatograph equipped with an Agilent mass spectrometric zone of tested extract compared to standard antibiotics (Ampicillin, column PAS-5ms (30 m x 0.32 mm x0.25 um film thickness). The Streptomycin, Kanamycin, Tobarmycin and Clotrimazole). The plant extracts were injected under the following conditions. Helium activity index was measured according to the following equation: was used as carrier gas at approximately 1.0 ml /min, pulsed splitless mode. The solvent delay was 3 min, and the injection size

was 1.0 µl. The mass spectrophotometric detector was operated in electron impact ionization mode an ionizing energy of 70 eV scanning from m/z 50 to 500. The ion source temperature was 230°C and the quadrupole temperature was 150°C. The electron Anticancer activity (MTT assay) multiplier voltage (EM voltage) was maintained at 1250 V above auto tune. The instrument was manually tuned using The anticancer activity was evaluated according to the reported perfluorotributyl amine (PFTBA). The GC temperature program was procedure (Mauceri et al., 1998), using four human tumor cell lines started at 60°C then elevated to 280°C at rate of 8°C/min and 10 namely; hepatocellular carcinoma (HePG-2), mammary gland min hold at 280°C the detector and injector temperature were set at breast cancer (MCF-7), human prostate cancer (PC3), and 280 and 250°C, respectively. Wiley and Nist 05 mass spectral data Epitheliod carcinoma (Hela). The cell lines were obtained from base was used in the identification of the separated peaks. ATCC via Holding company for biological products and vaccines (VACSERA), Cairo, Egypt. 5-fluorouracil was used as a standard anticancer drug for comparison. Briefly, the different cell lines Chromatographic isolation of EtOAc and n-BuOH extracts mentioned above were used to determine the inhibitory effects of extracts/compounds on cell growth using the MTT assay. This The ethyl acetate extract (3.12 g) was subjected to polyamide colorimetric assay is based on the conversion of the yellow column chromatography, initially eluted with distilled water (H2O), tetrazolium bromide (MTT) to a purple formazan derivative by followed by gradient mix elution with methanol, acetic acid and mitochondrial succinate dehydrogenase in viable cells. The cells ammonia to afford one major fraction (I), further purification of (I) were cultured in RPMI-1640 medium with 10% fetal bovine serum. over Sephadex LH-20 column chromatography eluted with (CH2Cl2: Antibiotics added were 100 units/ml penicillin and 100 µg/ml MeOH; 1:9; v/v) followed by extra purification over preparative thin streptomycin at 37°C in a 5% CO2 incubator. The cells were seeds layer chromatography (PTLC) (EtOAc: MeOH; 3.1:1.9; v/v) led to in a 96-well plate at a density of 1.0x104 cells/well at 37°C for 48 h the isolation of compound 1. Moreover, the n-butanol extract (4.09 under 5% CO2. After incubation the cells were treated with different g) was subjected to polyamide column chromatography, initially concentration of compounds and incubated for 24 h. After 24 h of eluted with distilled water (H2O), followed by gradient mix elution drug treatment, 20 µl of MTT solution at 5 mg/ml was added and with methanol, acetic acid and ammonia to afford three major incubated for 4 h. Dimethyl sulfoxide (DMSO) in volume of 100 µl is fractions (II, III and IV).The first fraction (II) was eluted in MeOH and added into each well to dissolve the purple formazan formed. The resubmitted to further purification over Sephadex LH-20 column colorimetric assay is measured and recorded at absorbance of 570 chromatography eluted with (CH2Cl2: MeOH; 1:9; v/v) followed by nm using a plate reader (EXL 800, USA). The relative cell viability in extra purification over preparative thin layer chromatography percentage was calculated as (A570 of treated samples/A570 of (PTLC) (EtOAc: MeOH; 2.8:2.2; v/v) to afford compound 2. The untreated sample) X 100. second fraction (III) was eluted in MeOH+AcOH and undergoing further purification over Sephadex LH-20 column chromatography eluted with (CH2Cl2: MeOH; 1:9; v/v) followed by extra purification over preparative thin layer chromatography (PTLC) (EtOAc: MeOH; Antioxidant activity (ABTS assay) 3:2; v/v) to afford compound 3. Finally, the third fraction (IV) was eluted in AcOH and resubmitted to further purification over The antioxidant activity was evaluated via 2,2-azino-di-[3- Sephadex LH-20 column chromatography eluted with (CH2Cl2: ethylbenzo-thiazolin-sulphonate] (ABTS) method. Briefly, for each MeOH; 1:9; v/v) followed by extra purification over preparative thin of the investigated sample 2 mL of ABTS solution (60 mM) was layer chromatography (PTLC) (EtOAc: MeOH: H2O; 9:0.8:0.2; v/v/v) added to 3 M MnO2 solution (25 mg/ml) all prepared in phosphate to afford compound 4. The chemical structures of the four isolated buffer (pH 7, 0.1 M). The mixture was shaken, centrifuged, filtered, compounds are shown in Figure 1. and the absorbance (Acontrol) of the resulting green-blue solution (ABTS radical solution) was adjusted at ca. 0.5 at 734 nm. Then, 50 ml of (2 mM) solution of the test compound in spectroscopic grade MeOH/ phosphate buffer (1:1) was added. The absorbance (Atest) RESULTS AND DISCUSSION was measured and the reduction in color intensity was expressed as % inhibition. The % inhibition for each compound is calculated Identification and characterization of the isolated from the following equation: compounds (1-4)

% Inhibition = Acontrol-Atest/Acontrol x 100 Dihydrokaempferol-3-O-α-L-rhamnoside (1): Was

obtained as white powder, m.p. 282°C, Rf 0.56 in (15% Ascorbic acid (vitamin C) was used as standard antioxidant (positive control). Blank sample was run without ABTS and using AcOH; PC) and 0.34 in (EtOAc: MeOH; 3.1:1.9; TLC). It MeOH/phosphate buffer (1:1) instead of sample. Negative control showed a dark purple spot upon paper chromatography 1 sample was run with MeOH/phosphate buffer (1:1) instead of under UV light. H-NMR (DMSO-d6, 400 MHz) δ in ppm: 70 J. Pharmacognosy Phytother.

Figure 1. Chemical structures of pure compounds were isolated from the EtOAc and n-BuOH extracts of P. farcta.

7.73 (2H, d, J= 8.8 Hz, H-2',6'), 6.94 (2H, d, J= 8.8 Hz, H- authentic sample (identical Rf and spot colour). Moreover, 3',5'), 6.49 (1H, d, J= 2.0 Hz, H-6), 6.18 (1H, d, J= 2.0 Hz, spectral data of compound 2 were in agreement with the H-8), 4.73 (1H, d, J= 10.8 Hz, H-2), 4.60 (1H, d, J= 10.8 reported data (Fujiwara et al., 2011). Hz, H-3), 4.14 (1H, d, J= 2.0 Hz, Rha-1''), 0.90 (3H, d, J= 7.2 Hz, CH3) and 3.31-3.79 ppm (m, rest of sugar 4′-methoxyquercetin (tamarixetin) (3): Was obtained protons). Complete acid hydrolysis of compound 1 as yellow powder, m.p. 216°C, Rf 0.09 in (15% AcOH; revealed the presence of dihydrokaempferol as aglycone PC) and 0.41 in (EtOAc: MeOH; 3:2; TLC). It showed (Co-PC) and rhamnose as sugar moiety (Co-TLC). yellow spot upon paper chromatography under UV light. 1 Moreover, all protons signal (chemical shifts and coupling H-NMR (DMSO-d6, 400 MHz) δ in ppm revealed the constants) were matched with the reported in the presence of three characteristic signals for three aromatic literature (Fujiwara et al., 2011; Xiao et al., 2011). protons at 7.80 (1H, d, J= 8.6 Hz, H-2'), 7.50 (1H, dd, J= 8.6, 2.4 Hz, H-6'), and 6.90 ppm (1H, d, J= 8.6 Hz, H-5') Apigenin (2): Was obtained as faint yellow powder, m.p. for ring-B. Also, two meta coupled protons at 6.65 (1H, d, 358°C, Rf 0.05 in (15% AcOH; PC) and 0.29 in (EtOAc: J= 2.4 Hz, H-8), and 6.32 ppm (1H, d, J= 2.4 Hz, H-6) for MeOH; 2.8:2.2; TLC).It showed a dark purple spot upon ring-A. The appearance of one signal for methoxyl group paper chromatography under UV light without any at 3.80 (3H, -OCH3), spectral data of compound 3 were in 1 change after spraying with AlCl3. H-NMR (DMSO-d6, 400 agreement with the reported data (Awaad et al., 2012). MHz) δ in ppm revealed the presence of two doublet of doublets at 7.91 (2H, d, J= 8.4 Hz, H-2',6') and 6.94 ppm Acacetin-7-O-α-L-rhamnoside (4): Was obtained as (2H, d, J= 8.4 Hz, H-3',5'), a singlet signal at 6.75 ppm yellow powder, m.p. 260°C, Rf 0.48 in (15% AcOH; PC) (1H, s, H-3), two meta coupled aromatic doublets and 0.52 in (EtOAc: MeOH: H2O; 9:0.8:0.2; TLC). It characteristic for ring-A at 5.97 (1H, d, J= 2.0 Hz, H-6) showed dark purple spot upon paper chromatography 1 and 6.49 ppm (1H, d, J= 2.0 Hz, H-8), these spectral data under UV light. H-NMR (DMSO-d6, 400 MHz) δ in ppm were characteristic for a 5,7,4'-trisubstituted flavone revealed the presence of two doublet signals at 7.93 (2H, skeleton which was confirmed via Co-paper with apigenin d, J= 8.6 Hz, H2',6') and 6.94 ppm (2H, d, J= 8.6 Hz, Saad et al. 71

Table 1. The compounds identified in the n-hexane extract of P. farcta by GC/MS analyses.

Molecular ion peak, M+ R.T. No. Compound Name Area% M.F Base peak m/z %

12.31 1 Thiophene, tetrahydro-, 1,1-dioxide 0.76 C4H8O2S 120 60.24 41

17.10 2 Tetradecane 0.30 C14H30 198 3.21 57

19.57 3 Pentadecane 0.75 C15H32 212 3.03 57

21.62 4 Dodecanoic acid 0.36 C12H24O2 200 7.24 119

21.74 5 1-Tridecene 0.33 C13H26 182 4.65 55

23.00 6 4-methyl-14-pentadecenoic acid 3.46 C16H30O2 254 2.04 91

26.11 7 1-Icosene 0.58 C20H40 280 1.65 55

26.26 8 Octadecane 0.78 C18H38 254 3.65 57

28.26 9 Nonadecane 0.79 C19H40 268 2.84 57

28.83 10 Pentadecanoic acid 1.67 C15H30O2 242 2.97 74

30.05 11 Nonadecanoic acid 9.10 C19H38O2 298 1.95 73

32.02 12 Henicosane 1.06 C21H44 296 3.19 57

32.36 13 trans-Phytol 1.39 C20H40O 296 1.97 71

32.57 14 Methyl icosanoate 0.21 C21H41O2 326 2.47 74

33.25 15 (Z)9,17-Octadecadienal 10.60 C18H32O 264 15.67 55

33.54 16 stearic acid 0.47 C18H36O2 284 16.98 55

33.80 17 Docosane 0.25 C22H46 310 4.16 57

37.10 18 Tetracosane 0.73 C24H50 338 2.12 57

38.66 19 Pentacosane 0.29 C25H52 352 1.94 57

40.16 20 Hexacosane 0.26 C26H54 366 1.08 57

41.61 21 Heptacosane 0.15 C27H56 380 2.87 57

43.47 22 Supraene 0.25 C30H50 410 1.69 69

47.58 23 dl-α-Tocopherol 6.23 C29H50O2 430 100 430

49.81 24 γ-Sitosterol 0.51 C29H50O 414 100 414

50.80 25 Taraxasterol 0.81 C30H50O 426 50.62 207

50.93 26 Urs-20-en-16β-Ol. 0.53 C30H50O 426 12.97 189 Total % 42.62%

H3',5'), a singlet at 6.50 ppm (1H, s, H-3) and two meta extract composition. (Z)9,17-octadecadienal (10.60%), coupled protons at 6.41 (1H, d, J= 2.0 Hz, H-8) and 6.37 nonadecanoic acid (9.10%), dl-α-tocopherol (6.23%) and ppm (1H, d, J= 2.0 Hz, H-6), anomeric proton of 4-methyl-14-pentadecenoic acid (3.46%) were the major rhamnosyl moiety at 5.35 ppm (1H, brd, Rha-1''), components (Figure 2). On the other hand, the chemical methoxy group at 4.04 ppm (3H, -OCH3), 3.83-3.25 (m, constituents of the methylene chloride extract are shown rest of sugar protons) and methyl protons of the in (Table 2), which revealed the presence of thirty two rhamnosyl moiety were appeared at 1.23 ppm (3H, d, J= compounds representing 85.34% of the total extract 6.4, Rha-CH3). Moreover, complete acid hydrolysis of composition. Tricosanoic acid (9.24%), (Z,Z)-9,12- compound 4 revealed the presence of acacetin as octadecadienoic acid (9.10%), 3-hydroxy-β-damascone aglycone (Co-PC) and rhamnose as sugar moiety (Co- (6.53%), 1,2-dihydrophenanthrene-4-carboxylic acid TLC). All protons resonances of compound 4 were in (5.54%), and trans-piperitone oxide (5.11) were the major agreement with those reported by Rashed et al. (2015). components (Figure 3). Reviewing the literature revealed that there is no any reported data about the GC/MS analyses of P. farcta extracts, but the available data were GC/MS analyses of the n-hexane and methylene reported on the volatile constituents of roots, leaves, chloride extracts pods, flowers and branches parts of P. farcta growing in Tunisia, the major compounds were identified in the root The chemical constituents of the n-hexane extract are part are octadecanal (10.5%), hexadecanal (9.0%), shown in (Table 1). These constituents were identified heptadeca-1, 11,13-triene (8.3%) and octadeca-1-ene using GC/MS analysis which revealed the presence of (7.0%); while for the branch part are 6,10,14- twenty six compounds representing 42.62% of the total trimethylpentadecan-2-one (8.6%), D-limonene (5.4%) 72 J. Pharmacognosy Phytother.

Figure 2. Gas ion chromatogram of the n-hexane extract of P. farcta aerial part.

and 2-methyl-1-tertiobutilprop-1,3-yl- diisobutylate (5.4%); Streptomycin and Ampicillin as standard antibiotics with for leaves part are phytol (39.65%) and benzyl benzoate inhibition zones of 35, 24 mm respectively (Table 3). On (6.8%), for flower part are 6,10,14-trimethylpentadecan-2- the other hand, the ethyl acetate extract exhibited strong one (4.8%) and tetracosane (4.4%); and finally for pod antimicrobial effect against four tested strains namely; part are phytol (15.9%), 3-hydroxy-beta damascone Shigella spp., E. coli, P. vulgaris and C. albicans with (4.8%) and methyl hexadecanoate (3.1%) (Harzallah- inhibition zones of 7.3, 11, 6 and 7.3 mm, respectively; Skhiri et al., 2006). compared to Streptomycin, Ampicillin and Clotrimazole with inhibition zones of 14, 24, 18 and 20 mm, respectively. Moreover, the n-butanol extract exhibited Antimicrobial activity strong antimicrobial effect against six tested strains namely; Shigella spp., Erwinia spp., E. coli, P. vulgaris, S. The antimicrobial activity of the different solvent extracts, epidermis and C. albicans with inhibition zones of 11, 9, that is, n-hexane, methylene chloride, ethyl acetate and 17, 12.4, 9.7 and 11 mm, respectively; compared to n-butanol of P. farcta was evaluated using disk agar Streptomycin, Ampicillin and Clotrimazole with inhibition assay against twelve pathogenic microbial strains. The zones 14, 35, 24, 18, 24 and 20 mm respectively (Table results revealed that the n-hexane extract showed 4). The antibacterial activity of the 85% methanol extract moderate antimicrobial activity (relative to the used of different parts of P. farcta growing in Iran against standard antibiotic) against only three tested strains that methicillin-resistant S. aureus (MRSA) was evaluated is, Shigella spp., E. coli and P. vulgaris with inhibition with inhibition zones of 5, 6, 8 and 12 mm, respectively zones of 4.7, 8.3 and 6.3 mm, respectively; compared to for roots, leaf, pods and seeds extracts (Sharifi-Rad et Streptomycin, Ampicillin as standard antibiotics with al., 2014). Also, Miri et al. (2015) reported on the inhibition zones of 14, 24 and 18 mm respectively. Also, antibacterial activity of silver nanoparticles (Ag-NPs) from the methylene chloride extract showed moderate the crude extract of P. farcta growing in Iran against four antimicrobial activity against three tested strains namely; human pathogenic bacteria namely; S. aureus, B. subtilis, Erwinia spp., E. coli and S. epidermis with inhibition E. coli and P. aeruginosa with inhibition zone of 9.5, 9, zones of 6.2, 7.2 and 8.4 mm respectively; compared to 9.5 and 9.5 mm respectively (Miri et al., 2015). In, Saad et al. 73

Table 2. The compounds identified in the methylene chloride extract of P. farcta by GC/MS analyses.

Compound Name Molecular ion peak, M+ Base R.T Area% M.F No. m/z % peak

17.85 1 Vanillin 2.84 C8H8O3 152 100 151

20.15 2 1,2-dihydrophenanthrene-4-carboxylic acid 5.54 C15H12O2 224 2.97 191 20.51 3 Dihydroactinidiolide 1.24 C11H16O2 180 43.97 111 (1S,4aS,8aS)-2,5,5,8a-tetramethyl-1,4,4a,5,6,7,8,8a- 22.26 4 1.91 C15H24O2 236 2.67 43 octahydronaphthalene-1-carboxylic acid

22.49 5 3-Hydroxy-β-damascone 6.53 C13H20O2 208 66.34 69

22.69 6 trans-piperitone oxide 5.11 C10H16O2 168 9.35 71

22.90 7 2-Hydroxypinan-3-one 2.78 C10H16O2 168 2.35 71 23.27 8 3a,7a-dimethylhexahydro-4,7-epoxyisobenzofuran-1,3-dione 0.17 C10H12O2 196 1.65 43

23.55 9 Ar-turmerone 1.20 C15H20O 216 30.75 83

23.78 10 Benzenemethanol, 3,4,5-trimethoxy- 3.09 C10H14O4 198 100 198

24.21 11 Phenethyl carbamic acid, ethyl ester 3.06 C11H15NO2 193 100 193 Spiro[2,4,5,6,7,7a-hexahydro-2-oxo4,4,7a-trimethylbenzofuran]-7,2'- 24.89 12 1.16 C12H16O3 208 6.29 43 (oxirane)

25.65 13 Phenol, 4-(3-hydroxy-1-propenyl)-2-methoxy 3.48 C10H12O3 180 90.38 137

26.02 14 Loliolide 3.62 C10H16O3 196 10.61 111

26.46 15 Myristicin 1.90 C11H12O3 192 1.96 124

26.63 16 Cedrelanol 1.07 C15H26O 222 1.60 43

26.83 17 7-Oxabicyclo[4.1.0]heptan-2-one, 1-methyl-4-(2-methyloxiranyl) 1.95 C10H14O3 182 2.68 111

27.20 18 6,10,14-Trimethylpentadecan-2-one 1.26 C18H36O 268 1.61 43 (1R,4S,5R,6R,7S,10R)-7-isopropyl-4,10-dimethyl- 27.77 19 0.97 C15H26O 222 3.97 149 tricyclo[4.4.0.0(1,5)]decan-4-ol

28.82 20 Hexadecanoic acid, methyl ester 3.91 C17H34O2 270 13.83 74

29.81 21 Tricosanoic acid 9.24 C23H46O2 354 1.64 73

30.06 22 1-Docosene 2.04 C22H44 308 1.76 97

32.11 23 11-Octadecenoic acid, methyl ester 2.26 C19H36O2 296 4.37 55

32.33 24 Cis-Phytol 2.82 C20H40O 296 2.94 71

33.03 25 (Z,Z)-9,12-Octadecadienoic acid 9.10 C18H32O2 280 19.64 67

33.68 26 10-Heneicosene 1.23 C21H42 294 2.67 97 34.36 27 1-Heptacosanol 0.88 C27H56O 396 1.37 55

35.45 28 Cyclopropaneoctanal,2-octyl 0.51 C19H36O 280 2.35 57

36.44 29 2(3H)-Furanone, 5-butyldihydro-4-methyl 1.73 C9H16O2 156 1.87 99

37.01 30 Cyclotetracosane 1.07 C24H48 336 1.96 83 47.55 31 3β- Cholest-5-en-3-ol 0.59 C31H50O2 454 2.39 165

49.82 32 D:C-Friedoolean-8-en-29-oic acid 1.08 C30H48O3 456 1.98 207 Total % 85.34%

conclusion these extracts may be used as a source of (PC3) and Epitheliod carcinoma (Hela). According to naturally occurring antimicrobial agents and alternative to National Cancer Institute (NCI) criteria, the plant extract the synthetic antibiotics to overcome the microbial with IC50 (µg/ml): 1-10 was considered very strong; 11-20 infections. (strong); 21-50 (moderate); 51-100 (weak) and above 100 (non-cytotoxic). Therefore, the ethyl acetate extract showed very strong anticancer activity against Hela and Anticancer activity MCF-7 cell lines with IC50 of 6.9 and 8.8 µg/ml compared to 5-fluorouracil as standard with IC50 of 4.8 and 5.4 The in vitro anticancer activity of different solvent extracts µg/ml respectively. On the other hand the n-butanol of P. farcta was evaluated against four human tumor cell extract showed very strong anticancer activity against lines viz., hepatocellular carcinoma (HePG-2), mammary MCF-7, HePG-2 and PC3 cell lines with IC50 of 5.6, 8.4 gland breast cancer (MCF-7), human prostate cancer and 9.6 µg/ml respectively (Table 5). To the best of our 74 J. Pharmacognosy Phytother.

Figure 3. Gas ion chromatogram of the methylene chloride extract of P. farcta aerial part.

Table 3. The inhibition zones in mm and activity index% of the n-hexane and methylene chloride extracts of P. farcta compared to standard antibiotics.

Standard antibiotic/ n-hexane Methylene chloride Microorganism Inhibition zone (mm) Inhibition zone (mm) Activity index% Inhibition zone (mm) Activity index% Klebsiella pneumoniae Ampicillin/ 25 0 0 0 0 Shigella sp. Streptomycin/ 14 4.7 33.5 0 0 Erwinia sp. Streptomycin/ 35 0 0 6.2 17.7 Escherichia coli Ampicillin/ 24 8.3 34.5 7.2 30 Enterobacter aerogenes Kanamycin/ 20 0 0 0 0 Pseudomonas aeruginosa Tobramycin/ 15 0 0 0 0 Proteus vulgaris Ampicillin/ 18 6.3 35 0 0 Staphylococcus epidermis Ampicillin/ 24 0 0 8.4 35 Streptococcus pyogenes Ampicillin/ 20 0 0 0 0 Staphylococcus aureus Ampicillin/ 24 0 0 0 0 Bacillus subtilis Kanamycin/ 20 0 0 0 0 Candida albicans Clotrimazole/ 20 0 0 0 0

knowledge, there is very little information about the Free radical scavenging antioxidant activity (ABTS anticancer activity reported about the plant. The assay) anticancer activity of the hydroalcoholic extract of P. farcta was evaluated against MKN45 (human gastric The free radical antioxidant activity of different solvent cancer) cell line (Dejamfekr and Khaleghian, 2016). The extracts of P. farcta was evaluated via ABTS assay which above mentioned results suggest that P. farcta plant may based on their abilities to reduce the radical cation be used as a source of naturally occurring anticancer ABTS+°. The results in (Table 6) revealed that the % agents. inhibition of ABTS radical was 83.1, 82.0, 87.2 and 87.0% Saad et al. 75

Table 4. The inhibition zones in mm and activity index% of the ethyl acetate and n-butanol extracts of P. farcta compared to standard antibiotics.

Standard antibiotic/ Ethyl acetate n-butanol Microorganism inhibition zone (mm) Inhibition zone (mm) Activity index% Inhibition zone (mm) Activity index% Klebsiella pneumoniae Ampicillin/ 25 0 0 0 0 Shigella spp. Streptomycin/ 14 7.3 52.1 11 78.5 Erwinia spp. Streptomycin/ 35 0 0 9 25.7 Escherichia coli Ampicillin/ 24 11 45.8 17 70.8 Enterobacter aerogenes Kanamycin/ 20 0 0 0 0 Pseudomonas aeruginosa Tobramycin/ 15 0 0 0 0 Proteus vulgaris Ampicillin/ 18 6 33.3 12.4 68.8 Staphylococcus epidermis Ampicillin/ 24 0 0 9.7 40.4 Streptococcus pyogenes Ampicillin/ 20 0 0 0 0 Staphylococcus aureus Ampicillin/ 24 0 0 0 0 Bacillus subtilis Kanamycin/ 20 0 0 0 0 Candida albicans Clotrimazole/ 20 7.3 52.1 11 78.5

Table 5. In vitro anticancer activity of different solvent extracts of P. farcta against four human tumor cell lines compared to 5-fluorouracil as standard.

1 IC50 (µg/ml) Sample HePG-2 Hela PC3 MCF-7 5-FU2 7.9±0.28 4.8±0.21 8.3±0.35 5.4±0.20 n-hexane 36.2±2.32 51.5±2.98 40.4±2.35 33.9±2.74 Methylene chloride 62.4±3.74 67.1±3.81 41.2±2.28 57.7±3.35 Ethyl acetate 14.6±1.06 6.9±0.36 12.4±1.24 8.8±0.86 n-butanol 8.4±0.41 13.5±0.98 9.6±0.87 5.6±0.53

1 2 IC50 (µg/ml): 1-10 (very strong); 11-20 (strong); 21-50 (moderate); 51-100 (weak) and above 100 (non-cytotoxic). 5- FU: 5-fluorouracil.

Table 6. In vitro ABTS free radical antioxidant activity of different solvent extracts of P. farcta compared to ascorbic acid.

ABTS Abs(control)-Abs(test)/Abs(control) × 100 Sample Absorbance of samples % inhibition Ascorbic acid 0.055 89.2 n-hexane 0.086 83.1 Methylene chloride 0.092 82.0 Ethyl acetate 0.065 87.2 n-butanol 0.066 87.0

respectively for the n-hexane, methylene chloride, ethyl the antioxidant activity of four solvent extracts from pod acetate and n-butanol extracts compared to ascorbic acid and seed of P. farcta was evaluated using DPPH assay, as standard (89.2%). The IC50 value of the ABTS free and the IC50 of the tested extracts from pod part were 1.0, radical activity of the methanol extract of P. farcta fruits 6.15, 3.55 and 7.75 µg/ml respectively for ethanol, growing in Tunisia was 270 μg/ml (Lajnef et al., 2015). To methanol, octanol and n-heptane extracts, while and the the best of our knowledge, there are limited reports about IC50 of the tested extracts from seed part were 2.0, 1.51, the antioxidant activity of P. farcta using the ABTS assay, 0.95 and 4.45 µg/ml respectively for ethanol, methanol, but there are available data about other assays that is, octanol and n-heptane extracts (Poudineh et al., 2015). 76 J. Pharmacognosy Phytother.

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