Phytochemical and in-vitro biological study of Psidium guajava L ...

Wet OR F PHJournal ARMAof CY AND Pand HA R MACEUTICSciences AL SCIENCES Saber al.LD JOURNAL OWorld Pharmacy Pharmaceutical SJIF Impact Factor 5.210

Volume 4, Issue 05, 124-141.

Research Article

ISSN 2278 – 4357

Phytochemical and in-vitro biological study of Psidium guajava L. leaves cultivated in Egypt Fathy M. Soliman, Magda M. Fathy, Maha M. Salama, Engy A. Mahrous and *Fatema R. Saber Pharmacognosy Department, Faculty of Pharmacy, Cairo University, Kasr El-Aini Street, Cairo, 11562, Egypt.

Article Received on 28 Feb 2015, Revised on 22 March 2015, Accepted on 12 April 2015

ABSTRACT The Egyptian guava tree (Psidium guajava Linn.) is a member of the Myrtaceae family. In this study, seven compounds were isolated from chloroform: methanol (80:20) extract (CME) of Psidium guajava L. leaves; namely two flavonoids; Quercetin-3-O-α-arabinofuranoside

*Correspondence for

(Avicularin) and Quercetin-3-O-β-xylopyranoside (reynoutrin), two

Author

meroterpenoids; Psiguadial D and Psidial C in addition to β-sitosterol,

Dr. Fatema Saber Pharmacognosy

4-hydroxy phenyl ethanoid palmitate ester and Corosolic acid. The

Department, Faculty of

structures of the isolated compounds were elucidated on the basis of

Pharmacy, Cairo

their spectral data; MS, UV, 1D and 2D NMR analyses. The CME of

University, Kasr El-Aini

P. guajava leaves showed a moderate free radical scavenging activity

Street-11561, Cairo,

with IC50= 92.26 μg/mL, as compared to ascorbic acid (19.76 μg/mL)

Egypt.

in DPPH-based assay. Furthermore, the in vitro α- glucosidase inhibitory activity of the CME was measured using p-nitrophenyl-α- glucopyranose (pNPG) and a potent inhibitory activity was observed (IC50= 31.6±0.06 μg/mL) relative to acarbose (224±2.31 µg/mL), a reference antidiabetic drug. Moreover, the extract exhibited cytotoxic activity against HEPG2, MCF7 and HEP2 cell lines with (IC50= 19.3, 20.6, 17.5 μg/mL), respectively. The major isolated compounds; Psiguadial D, and Corosolic acid exhibited cytotoxic activity against MCF7 with IC50 = 22.1 & 10.4 μg/mL respectively. KEYWORDS: Psidium guajava, Myrtaceae, meroterpenoids, Corosolic acid, antidiabetic, cytotoxic.

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World Journal of Pharmacy and Pharmaceutical Sciences

INTRODUCTION Natural products once served humankind as the source of all drugs, and higher plants provided most of these therapeutic agents. Nowadays, natural products (and their derivatives and analogs) still represent over 50% of all drugs in clinical use, with higher plant-derived natural products Representing ca. 25% of the total

[1]

. The World Health Organization

estimates that 80% of the people in developing countries of the world rely on traditional medicine for their primary health care, and about 85% of traditional medicine involves the use of plant extracts. This means that about 3.5 to 4 billion people in the world rely on plants as sources of drugs.[2] Natural product functional activities are attributed to different classes of compounds, such as essential oils, alkaloids, flavonoids and carotenoids. The Myrtaceae (Myrtle, Eucalypts, Clove, or Guava family) is a large family of dicotyledonous woody plants placed within the order Myrtales containing over 5,650 species organized in 130 to 150 genera. Recognized as the eighth largest flowering plant family, it comprises several genera of outstanding ecological and economic relevance worldwide. The family occurs mainly in the Southern Hemisphere. It has centers of diversity in the wet tropics, particularly South America, Australia, and tropical Asia with occurrences in Africa and Europe.[3] The genus Psidium belongs to family Myrtaceae, which originated in tropical South America. It is now naturalized in tropical and subtropical countries. It comprises approximately 150 species of small trees and shrubs, in which only 20 species produce edible fruits. The most common cultivated species includes: the Common guava (Psidium guajava L.), the Cattely guava or Strawberry guava (P. cattleianum Sabine), the Brazilian guava (P.guineense Sw.) and Costa Rician guava or Chinese guava (P. friedrichsthalianum Ndz.).[4] The common guava leaf is used in many countries as traditional medicine for treating diarrhea, gastrointestinal and respiratory disturbances, hypertension, mellitus.[5,

6]

and

diabetes

Reports have suggested that guava leaves exhibited beneficial anti-oxidative,

anti-cancerous, anti-inflammatory, anti-coagulative, and anti-diabetic bioactivity.[7,

8]

been reported that guava leaves are rich in anti-oxidative components including

tannins,

It has

terpenoids, and flavonoids.[9] Recently, several novel sesquiterpenoid-based meroterpenoids with relative configurations had been reported from this plant.[10, 11] The skeletons of these compounds were characterized with a unique connection pattern between sesquiterpenoid and

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diphenylmethane moiety. It was assumed that meroterpenoids of guava may act as substrates for the efflux transporter P-glycoprotein pump.[12] The aim of this study is to evaluate the in-vitro biological activities of the leaf extract of P. guajava L. cultivated in Egypt, as well as to isolate the major active constituents which could justify these activities. MATERIAL AND METHODS Plant material Samples of P. guajava L. leaves were collected in 2012 from El-Behera Governorate, Egypt. The plant was kindly identified by Dr. Mohamed El-Gebaly (Senior Botanist). A voucher specimen (PG-12-12-2012) was kept in the herbarium of Department of Pharmacognosy, Faculty of Pharmacy, Cairo University. One kilogram of the powdered leaves was extracted using Soxhlet apparatus with chloroform:methanol (80:20), (CME) as the extracting solvent to yield 90 grams of the dried guava extract, adopting the method reported by Sidana, et al. 2010.[13] Chemicals and equipment Silica gel H (Merck, Darmstadt, Germany) for vacuum liquid chromatography (VLC), silica gel 60 (70–230 mesh ASTM, Fluka, Steinheim, Germany), silica gel (40-63µm, Fluka) and Sephadex LH-20 (Pharmacia, Stockholm, Sweden) were used for column chromatography. Thin-layer chromatography (TLC) was performed on silica gel GF254 precoated plates (EMerck) using the following solvent systems: S1: n-hexane: ethyl acetate (95:5 v/v), S2: nhexane: ethyl acetate (85:15 v/v), S3: chloroform: methanol (95:5 v/v) + few drops formic acid and S4: chloroform: methanol (85:15 v/v) + few drops formic acid. The chromatograms were visualized under UV (at 254 and 366 nm) before and after exposure to ammonia vapour and spraying with AlCl3 and NP/PEG, as well as after spraying with p-anisaldehyde/sulphuric acid reagent. α-Glucosidase enzyme from brewer’s yeast (EC 3.2.1.20), the substrate, p-NPG, and phosphate buffer (pH 6.8) were purchased from Sigma Chemical Co. (St Louis, MO, USA). Acarbose, was purchased from Bayer Pharmaceuticals Pty, Ltd (Montville, NJ, USA). Cell lines were obtained from National Cancer Institute, Kasr El Ainy, Cairo, Egypt. Melting points (uncorrected) were determined on an Electrothermal 9100 (Markham, Ontario, Canada). UV spectra were measured using a Shimadzu UV 240 (P/N 204-58000) spectrophotometer (Kyoto, Japan). 1H NMR (400MHz) and

13

C NMR (100 MHz) were

measured on a Bruker AVIIIHD400 FT–NMR Spectrometer (400/3) instrument (Japan). The www.wjpps.com

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NMR spectra were recorded in CDCl3 and MeOD and chemical shifts were given in δ (ppm) relative to TMS as internal standard. IN VITRO BIOLOGICAL ASSAYS 1. DPPH assay The antioxidant activity of the choloroform:methanol (80:20) extract was assessed using a modified quantitative 2,2-diphenyl-2-picrylhydrazyl hydrate (DPPH) assay,[14] using 0.004% solution of DPPH in methanol. The CME extract of P. guajava L. leaves was dissolved in 70% methanol at a concentration range 10-100 μg/mL and 0.3 mL of each concentration was added to 2 mL DPPH solution. Blank samples were run using only 70% methanol. After a 30 min incubation period at room temperature, the absorbance was measured at 492 nm. Ascorbic acid was used as a positive control at a concentration range 1-50 µg/mL. Antioxidant activity is expressed as IC50 value which is the concentration of the extract inhibiting DPPH-free radical formation by 50% relative to methanol (IC50 value). Inhibition of the DPPH free radical in percent (I%) was calculated according to the formula: "I%=" [(Ablank -Asample)/ Ablank]×100 Where Ablank is the absorbance of the control reaction (containing all reagents except the extract), and Asample is the absorbance of the tested sample. Each measurement was performed in triplicate. 2. α-glucosidase inhibitory activity The enzyme inhibition study was performed in a 96-well microplate.[15] A total of 100 μL reaction mixture containing 50 μL of 100 mM phosphate buffer (pH 6.8), 20 μL of 2.5 mM pNPG and 10 μL of the investigated extract in DMSO were added to each well, followed by 20 μL of 10 mM phosphate buffer (pH 6.8) containing 2.4 U/mL α-glucosidase enzyme. The plate was incubated at 37ºC for 15 min, and then 80 μL of 0.2 M sodium carbonate solution was added to stop the reaction. Following that, the absorbance was recorded at 405 nm. Control contained the same reaction mixture except that the same volume of phosphate buffer was added instead of the inhibitor solution. Acarbose was dissolved in water and used as a positive control. Inhibition% = [(AB-AA)/AB] ×100% Where AB is the absorbance of the control sample and AA is the absorbance of tested sample. www.wjpps.com

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3. IN VITRO CYTOTOXIC ACTIVITY The cytotoxicity of the (CME) of P. guajava L. leaves was tested against 4 human cancer cell lines: HEPG2 (hepatocellular carcinoma), MCF7 (breast carcinoma cell line), HEP2 (laryngeal carcinoma) and CAco-2 (colonic adenocarcinoma), In addition to HFB4 (normal melanocytes). While the major isolated compounds were tested against MCF7 (breast carcinoma cell line). This was performed by Sulphorhodamine B assay.[16] Briefly, cells were plated in a 96 well plate (104cells/well) for 24 hours before treatment with the tested sample to form monolayer. Different concentrations of the extract/or compounds (0, 5, 12.5, 25 and 50 μg/mL) were added to the cell monolayer and incubated for 48 hours at 37ºC in atmosphere of 5% CO2. The cells were then washed and stained with Sulphorhodamine B stain. Excess stain was washed with acetic acid and attached stain was recovered with Tris EDTA buffer. The color intensity was then measured at 564 nm. All testing was done in triplicate. The cell viability was expressed as the relation between surviving fraction and the concentration of the extract/ or compounds then plotted to get the survival curve of each tumor cell line for the specified tested compound. The curves were fitted using linear equation and IC50 (dose of the drug which reduces survival to 50%) was calculated. The IC50 was calculated for each cell line together with the reference drug, Doxorubicin. Extraction and Isolation The chloroform-methanol extract (80:20), (50g) was chromatographed on a VLC column (10 cm X 15 cm) of Silica gel H (300g). Gradient elution was carried out starting with 200 mL nhexane (100%) followed by 5% increments of EtOAc, up to 100% EtOAc, then by CHCl3/MeOH (5% increments), to a final concentration of 20% MeOH. Fractions, each of 200 mL, were collected and monitored by TLC. Similar fractions were pooled together to yield 3 collective fractions (Fr. I - Fr. III). For isolation and purification of individual compounds these fractions were further subjected to different chromatographic techniques as follows: Fr. I (2 g): showed a major spot, Rf = 0.56 in S1, In addition to several minor spots. It was chromatographed on a silica gel 60 column (27×2.5cm). Isocratic elution was performed with n-hexane:ethyl acetate (98:2). Fractions (10 mL each) were collected and monitored by TLC. Subfraction (Ia) was further purified on a silica gel column (40-63 μm) using 5% ethyl

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acetate in n-hexane to yield 80 mg of yellow oil (compound G1). Fr. II (9 g): showed two major spots, Rf = 0.44, 0.27 in S2, in addition to other minors. It was rechromatographed on a silica gel column (50×5cm). Isocratic elution was carried out using n-hexane:ethyl acetate (90:10). Fractions, 20 mL each, were collected and monitored by TLC. Sub-fraction (IIb), was washed with acetone followed by methanol to give 26 mg of white needle crystals (compound G2), while subfraction (IIc), yielded 125 mg of white powder (compound G3). Fr. III (2.7 g): showed four major spots, Rf = 0.58, 0.47 in S3 and 0.56, 0.37 in S4, together with some impurities. It was first purified on a silica gel column (27×3cm). Gradient elution was adopted starting with 100% chloroform then reaching chloroform:methanol (95:5) and yielded 3 main sub-fractions. Sub-fraction (IIId): was rechromatographed on Sephadex LH20 column (28×1.5cm) using chloroform:methanol (3:1) followed by several Sephadex columns (100% methanol) to give 10 mg of yellow powder (compound G4). Subfraction (IIIe) was purified on a silica gel (40-63μm) column (27×1.5cm). Elution was acheived using 5-15% ethylacetate in chloroform, and yielded 120 mg of white needles, compound G5. Subfraction (III f): this was purified on successive Sephadex LH-20 = columns (100% methanol) to give 30 mg of yellow powder (compound G6) and 39 mg of yellow powder (compound G7). RESULTS AND DISCUSSION The phytochemical study of CME of Psidium guajava L. leaves resulted in the isolation of seven compounds (Fig. 1) designated as; two meroterpenoids: Psiguadial D (G1) and Psidial C (G4), two flavonoids: Quercetin-3-O-α-arabinofuranoside (Avicularin, G6 ) and Quercetin3-O-β-xylopyranoside (reynoutrin, G7), Corosolic acid (G5), in addition to, β- sitosterol (G2), 4-hydroxy phenyl ethanoid palmitate ester (G3). The isolated compounds were identified by comparing their UV, 1H and 13C NMR spectroscopic data with those in the literature [17-23] Spectral data of the isolated compounds Compound G1: Yellow oil. Rf: 0.56 in S1. UV (MeOH), λmax: 276. ESI-MS (-ve mode): 473 [M-H]-. 1H-NMR (CDCl3 , 400 MHz): 0.66 (1H, m, H-7), 0.75 (3H, s, H-15), 0.94 (1H, m, H-6), 0.96 (1H, m, H-8b), 1.16 (3H, s, H-13), 1.22 (3H, s, H-12), 1.59 (2H, m, H-3), 1.74 (3H, s, H-14), 1.92 (1H, m, H-8a), 2.1 (1H, m, H-2b), 2.11 (2H, m, H-9), 2.76 (1H, m, H-2a), 3.7 (1H, d, J =8, H-5), 4.41 (1H, s, H-1’), 5.30 (1H, dd, J =12, 4, H-1), 6.81 (1H, d, J =8, H9’), 7.18 (1H, H-10’), 7.23 (1H, H-11’), 7.36 (1H, H-12’), 10.13 (2H, s, H-14’, 15’), 13.17 (s, 7’-OH), 13.64 (s-5’-OH). 13C-NMR (CDCl3 , 100 MHz): 17.48 (C-14), 18.61 (C-15), 19.35 www.wjpps.com

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(C-13), 19.82 (C-11), 22.44 (C-8), 23.41 (C-2), 26.9 (C-6), 30.41 (C-12), 31.58 (C-7), 35.36 (C-3), 38.17 (C-9), 41.39 (C-4), 43.92 (C-1’) 85.27 (C-5), 104.23 (C-6’), 104.47 (C-4’), 105.21 (C-2’), 126.31 (C-11’), 127.48 (C-10’), 127.50 (C-9’), 127.55 (C-1), 127.79 (C-13’), 130.25 (C-12’), 130.71 (C-10), 140.41 (C-8’), 170.6 (C-7’), 166.40 (C-3’), 168.27 (C-5’), 191.54 (C-15’), 192.18 (C-14’). Compound G2: White needle crystals. m.p.: 140-141°C. Rf: 0.44 in S2. EI-MS m/z (70 ev, rel. int.): 414 (M+, 86.8%), 396 (45.7%), 329 (46.15%), 303 (42%), 273 (23.3%), 255 (31%), 55 (100%). Compound G3: Whit e powder. Rf: 0.27 in S2. 1H-NMR (CDCl3 , 400 MHz): 0.851 (3H, t, J= 6.8 Hz, H-16'), 1.23–1.38 (24H, m, H- 4'-15'), 1.58 (2H, m, H-3'), 2.52 (2H, m, H2'), 2.81 (2H, t, J= 6.4 Hz, H-7), 4.19 (2H, t, J= 6.8 Hz, H-8), 6.74 (2H, d, J= 8 Hz, H-3, 5), 7.03 (2H, d, J= 8 Hz, H-2, 6).13C-NMR (CDCl3 , 100 MHz): 14.05 (C-16'), 22.65 (C-15'), 24.87 (C-3'), 29.09-29.66 (C-4'-13'), 33.97 (C-14'), 34.23 (C-7), 34.34 (C-2'), 65.12 (C-8), 115.25 (C-3, 5), 128.95 (C-1), 129.87 (C-2, 6), 155.16 (C-4), 174.17 (C-1’). Compound G4: Yellow powder. Rf: 0.58 in S3. 1H-NMR (CD 3 OD, 400 MHz): 0.82 (3H, s, H-13), 0.85 (3H, s, H-12), 0.98 (3H, d, J=7.6 Hz, H-15), 1.17 (3H, s, H-14), 2.4 (1H, m, H4), 2.6 (1H, m, H-1), 4.11 (1H, d, J=5.6 Hz, H-9’), 5.12 (1H, d, J=4.4 Hz, H-6), 6.92 (1H, t, J=7.2 Hz, H-13'), 7.04 (2H, t, J=7.6 Hz, H-12', 14'), 7.33 (2H, d, J=7.2 Hz, H-11', 15'), 9.72 (2H, s, H-7',8').

13

C-NMR (CD 3 OD, 100 MHz): 20.82 (C-8), 21.67 (C-15), 22.44 (C-14),

23.52 (C-2), 27.55 (C-13), 27.73 (C-12), 33.17 (C-3), 35.21 (C-9), 39.59 (C-10), 39.65 (C-4), 40.47 (C-9'), 45.57 (C-7), 45.75 (C-1), 72.09 (C-11), 104.3 (C-3', 5'), 110.44 (C-1'), 123.44 (C-6), 124.7 (C-13'), 127.12 (C-12', 14'), 128.45 (C-11', 15'), 144.08 (C-10'), 150.54 (C-5), 167.95 (C-2', 4', 6'). Compound G5: White needle crystals. m.p.: 252-254˚C. 1H-NMR (CD 3 OD, 400 MHz): 0.83 (3H, s, H-24), 0.87 (3H, s, H-26), 0.88 (1H, m, H-5), 0.89 (1H, m, H-1b), 0.91 (3H, d, J= 6.4 Hz, H-29), 0.98 (1H, m, H-20), 0.99 (3H, br. s, H-30), 1.03 (3H, s, H-23), 1.03 (3H, s, H-25), 1.13 (1H, m, H-15b), 1.14 (3H, s, H-27), 1.36 (1H, m, H-21b), 1.39 (1H, m, H-7b), 1.4 (1H, m, H-19), 1.46 (1H, m, H-6b), 1.51 (1H, m, H-21a), 1.56 (1H, m, H-6a), 1.58 (1H, m, H-7a), 1.6 (1H, m, H-9), 1.68 (2H, m, H-16), 1.7 (2H, m, H-22), 1.9 (2H, m, H-11), 1.94 (1H, m, H-15a), 1.97 (1H, m, H-1a), 2.1 (1H, d, J= 11.2 Hz, H-18), 2.9 (1H, d, J= 9.6 Hz, H3), 3.64 (1H, m, H-2), 5.25 (1H, t, J= 3.2 Hz, H-12). 13C-NMR (CD 3 OD, 100 MHz): 15.7 (C-25), 16.12 (C-24), 16.23 (C-26), 16.39 (C-29), 18.14 (C-6), 20.16 (C-30), 22.6 (C27), 23.1 (C-11), 23.8 (C-16), 27.5 (C-15), 27.92 (C-23), 30.33 (C-21), 32.6 (C-7), 36.63 (C-22), 37.78 (C-10), ), 38.65 (C-20), 39.08 (C-19), 39.2 (C-4), 39.4 (C-8), www.wjpps.com

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41.89 (C-14), 46.88 (C-1), 47.46 (C-17), 47.53 (C-9), 52.92 (C-18), 55.31 (C-5), 68.09 (C-2), 83.05 (C-3), 125.31 (C-12), 138.34 (C-13), 180.18 (C-28). Compound G6: Yellow powder. Rf: 0.56 in S4 . UV λmax (MeOH): 261, 269 sh., 358; CH 3 ONa: 269, 328, 405; AlC l 3 : 274, 302 sh, 428; AlCl 3 /HCl: 269, 298 sh, 364, 401; CH 3 COONa: 273, 322 sh, 390; CH 3 COONa/H 3 BO 3 : 263, 298 sh, 370. 1 H-NMR (CD 3 OD, 400 MHz): 7.43 (1H, d, J= 2 Hz, H-2'), 7.39 (1H, dd, J= 2, 8.36 Hz, H-6'), 6.79 (1H, d, J= 8.36 Hz, H-5'), 6.29 (1H, d, J= 2 Hz, H-8), 6.10 (1H, d, J= 2 Hz, H-6), 5.37 (1H, br. s, H-1''), 3.76-4.23 (5H, H-2''-5''). 13 C-NMR (CD 3 OD, 100 MHz): The sugar moiety: 61.13 (C-5''), 77.28 (C-3''), 81.90 (C- 2''), 86.62 (C-4''), 108.11 (C-1''). The aglycone moiety: 93.46 (C-8), 98.62 (C-6), 104.09 (C-10), 115.04 (C-5'), 115.41 (C-2'), 121.55 (C-6'), 121.69 (C-1'), 133.47 (C-3), 144.97 (C-3'), 148.47 (C- 4'), 157.2 (C-9), 157.89 (C-2), 161.65 (C-5), 165.07 (C-7), 178.54 (C-4). Compound G7: Yellow powder. Rf: 0.37 in S4. UV λmax (MeOH): 258, 356; CH 3 ONa: 270, 409; 273, 302 sh., 450; AlCl 3 / HCl: 269, 298 sh, 364, 402; CH 3 COONa: 273, 325 sh., 394; CH 3 COONa/H 3 BO 3 : 263, 292 sh., 372. 1 H-NMR (CD 3 OD, 400 MHz): 7.51 (1H, d, J= 2 Hz, H-2'), 7.48 (1H, dd, J= 2, 8.4 Hz, H-6'), 6.75 (1H, d, J= 8.32 Hz, H-5'), 6.27 (1H, d, J= 2 Hz, H-8), 6.09 (1H, d, J= 2 Hz, H-6), 5.06 (1H, d, J= 7.12 Hz, H-1''), 2.98-3.66 (5H, m, , H-2''-5’’). 13 C-NMR (CD 3 OD, 100 MHz): The suga r mo iet y: 65.81 (C-5''), 69.60 ( C-4''), 73.87 (C- 2''), 76.16 ( C-3''), 103.26 (C1''). T he aglyco ne mo iet y: 93.46 (C -8), 98.68 (C-6), 104.08 (C-10), 114.61 (C-5'), 115.81 (C-2'), 121.61 (C-6'), 121. 89 (C-1'), 133.99 (C-3), 144.65 (C3'), 148.50 (C-4'), 157.04 (C-9), 157.44 (C-2), 161.60 (C-5), 165.14 (C-7), 177.92 (C-4). Concerning meroterpenoids, Psiguadial D (G1) showed a molecular ion peak at m/z 473 [MH]-, which was established to be C30H34O5. While Psidial C (G4) gave a molecular ion peak at m/z 491[M-H]- corresponding to molecular formula C30H36O6. The NMR spectra of the two compounds revealed that both contain the basic structural unit of a 3,5- diformyl phloroglucinol as evident by presence of two formyl signals which appeared as singlet protons at δ 9.7 as well as carbon signals at δ 191.5 and 192.1 in 1H and respectively. This was consistent with published data.

13

CNMR,

[17, 18]

Corosolic acid (CA) was obtained as white amorphous powder and its (-)-ESIMS spectrum showed the molecular ion peak at m/z 471 [M-H]-, accounting for the molecular formula

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C30H48O4. Comparing thoroughly the NMR data of Corosolic acid with that of ursolic acid, showed very close similarities between them. The major difference in the NMR spectra, was the presence of an additional oxygenated methine signal at δH 3.64 (1H, m) and δC 68.0 (C2), suggesting that CA is a 2, 3-dihydroxy-ursane type triterpenoid. The 2α, 3β- configuration of the two secondary hydroxyl groups were suggested by the coupling constant (J = 9.6 Hz) between H-2 and H-3. Thus, compound G5 was assumed to be Corosolic acid (2α, 3βdihydroxy-urs-12-en-28-oic acid) and this was further confirmed by comparison of its spectral data with literature.[21, 22] Antioxidant drugs that help in the control or elimination of free radicals can reduce cellular oxidation in the body providing an important defense against degenerative diseases caused by oxidative stress as diabetes and cancer. Experimental evidences suggest the involvement of free radicals in the pathogenesis of diabetes [24], and more importantly in the development of diabetic complications. [25-27] Many recent studies reveal that antioxidants capable of neutralizing free radicals are effective in preventing experimentally induced diabetes in animal models. [28,

29]

Our findings

evidenced moderate free radical scavenging activity of CME of P. guajava leaves, using DPPH method (Fig. 2) with IC50 of 92.26 μg/mL while that of ascorbic acid was 19.76 μg/mL. α-Glucosidase is a key enzyme for carbohydrate digestion, located in the brush border of the small intestine. This enzyme has been recognised as a therapeutic target for the modulation of postprandial hyperglycaemia (PPHG), which is the earliest metabolic abnormality to occur in type 2 diabetes mellitus. Furthermore, a highly potent α-glucosidase inhibitory activity was observed with IC50= 31.6 ± 0.06 μg/mL for CME of P. guajava leaves relative to acarbose, a known inhibitor α-glucosidase enzyme, which exhibited IC50 at 224 ± 2.31μg/mL in the same assay (Fig. 3). On the other hand, CME exhibited cytotoxic activity against HEPG2, MCF7 and HEP2 cell lines with IC50=19.3, 20.6, 17.5 μg/mL, respectively, while it was inactive against colonic adenocarcinoma (CAco-2 human cell lines). Table (1).

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Table (1): Results of the cytotoxic activity of CME of guava leaves and major isolated compounds on human cancer cell lines: Tested extract CME of guava Psiguadial D Corosolic acid Doxorubicin

HFB4 IC50 µg/mL 25.3 4

HEPG2 IC50 µg/mL 19.3 4.2

HEP2 IC50 µg/mL 17.5 4.35

CAco-2 IC50 µg/mL 45.5 4.88

MCF7 IC50 µg/mL 20.6 22.1 10.4 4.2

The presence of high amount of Corsolic acid may explain the antidiabetic and anticancer activities of the tested extract. Corosolic acid (10mg/kg) was found to significantly reduce the level of blood glucose of KK-Ay mice through increasing glucose transporter isoform 4 translocation in muscle, improving glucose metabolism through reducing insulin resistance, [30]

inhibiting hydrolysis of sucrose [31] and inhibiting alpha-glycosidase activity [32, 33].

Although Corosolic acid has been semi-synthesized,

[34]

the process is too complicated and

guava leaves could be considered a potential resource for its production.[35] According to the latest report of The International Agency for Research on Cancer (GLOBOCAN 2012), breast cancer (BC) is by far the world’s most common cancer among women, and the most likely cause that a woman will die from cancer worldwide. In Egypt, the percentage of breast cancer among cancer cases among women accounts to 37.5 %. [36] Natural products from plants used in traditional medicines are currently one of the main sources in cancer chemo-preventive drug discovery. [37] In that view, the major isolated compounds from CME viz., Psiguadial D and Corosolic acid were tested for their cytotoxic activity against human cancer cell lines (MCF7). Corosolic acid evidenced significant cytotoxic activity (IC50=10.4 µg/mL) against MCF7 while Psiguadial D showed less activity with IC50= 22.1 μg/mL (Fig. 4) and (Table 1). Synergistic antitumor effects of Corosolic acid (CA) and chemotherapeutic agents was previously reported. [38] The combined effect of CA and chemotherapeutic agents was measured on tumor cell proliferation using tumor cell lines. CA was used at a concentration of 20 𝜇M, as this dose has been shown to suppress STAT3 activation but not inhibit tumor cell viability. Consequently, CA significantly increased the antitumor effects of adriamycin and cisplatin in ovarian cancer cells. These data suggest that CA suppresses tumor proliferation and is a potential candidate agent for enhancing anticancer chemotherapeutic agents in several types of tumor cells.

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Fig. (2): DPPH anti-oxidant activity of P. guajava leaves’ extract against ascorbic acid.

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Fig. (3): Antidiabetic effect of P. guajava leaves’ extract against acarbose.

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CME,

CME,

IC50= 45.5 µg/mL

IC50= 19.3 µg/mL

CME,

CME,

IC50= 20.6 µg/mL

IC50= 17.5 µg/mL

Psiguadial D,

Corosolic acid,

IC50= 22.1 µg/mL

IC50= 10.4 µg/mL

Fig. (4): Cytotoxic activity of P. guajava L. leaves’ extract and major compounds on human cancer cell lines.

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CONCLUSION This work identifies the isolation of seven compounds from the bioactive CME of P. guajava leaves. It also highlights on the α-glucosidase inhibitory activity to rationalize the mechanism of action of its antidiabetic activity. Moreover, Corosolic acid and Psiguadial D evidenced cytotoxic activity against human breast cancer cell lines which is a major problem in Egypt. Our study may provide validation for some of guava leaves’ medicinal uses, but further in vivo studies are still necessary to confirm its effectiveness as an adjuvant therapy that may aid in the treatment of different human ailments. REFERENCES 1. Balandrin, N.F.; Kinghorn, A.D., and Farnsworth, N.R., Human Medicinal Agents from Plants; Kinghorn, A. D.; Balandrin, N. F., Eds. ACS Symposium Series, 1993. 534: p. 212. 2. Farnsworth, N.R., Screening plants for new medicines. Biodiversity, 1988(Part II): p. 8397. 3. Govaerts, R.; Sobral, M.; Ashton, P.; Barrie, F.; Holst, B.; Landrum, L.; Matsumoto, K.; Mazine, F.; Lughadha, E.N., and Proneça, C., World checklist of Myrtaceae. 2008: Royal Botanic Gardens. 4. Thomas, A.M. and Mishra, R., Elucidation of diversity among Psidium species using morphological and SPAR methods. Journal of Phytology, 2011. 3(8). 5. Ojewole, J., Hypoglycemic and hypotensive effects of Psidium guajava Linn.(Myrtaceae) leaf aqueous extract. Methods and findings in experimental and clinical pharmacology, 2005. 27(10): p. 689-696. 6. Gutiérrez, R.M.P.; Mitchell, S., and Solis, R.V., Psidium guajava: a review of its traditional uses, phytochemistry and pharmacology. Journal of Ethnopharmacology, 2008. 117(1): p. 1-27. 7. Oh, W.K.; Lee, C.H.; Lee, M.S.; Bae, E.Y.; Sohn, C.B.; Oh, H.; Kim, B.Y., and Ahn, J.S.,

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