Scholarly article on topic 'In vitro evaluation of the total phenolic and flavonoid contents and the antimicrobial and cytotoxicity activities of crude fruit extracts with different polarities from Ficus sycomorus'

In vitro evaluation of the total phenolic and flavonoid contents and the antimicrobial and cytotoxicity activities of crude fruit extracts with different polarities from Ficus sycomorus Academic research paper on "Biological sciences"

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Abstract of research paper on Biological sciences, author of scientific article — Sheikha Khamis Al-matani, Ruqaiya Nasser Said Al-Wahaibi, Mohammed Amzad Hossain

Abstract Plants are a good source for discovering new drugs for human benefit. The objective of this study was to determine the total phenolic and flavonoid contents and to probe the antimicrobial and cytotoxic activities of fruit extracts of Ficus sycomorus (F. sycomorus), which is native to the Sultanate of Oman. A study was conducted to measure the total phenolic and flavonoid contents of the methanol extract of this fruit and its derived fractions [n-hexane, chloroform, ethyl acetate, butanol and residual aqueous fractions] by in vitro chemical analyses using the Folin-Ciocalteu reagent assay (FCR) and the aluminium chloride (AlCl3) method. The antimicrobial and cytotoxic activities were assessed using the slightly modified agar disc diffusion method and the brine shrimp lethality (BSL) method. The results of the analysis of the total phenolic content in different fruits extracts showed a significant amount of phenolic compounds, ranging from 1.48 to 213.79 mg of GAE/100 g of powder weight. However, the range of the total flavonoid content among the fruit extracts was found to be 0.100–0.527 mg QE/100 g of powder weight. The antimicrobial activity of the crude fruit extracts at different concentrations against four Gram-positive and Gram-negative bacterial strains, Staphylococcus aureus (S. aureus), Escherichia coli (E. coli), Haemophilus influenza (H. influenza), and Proteus spp (P. spp), showed strong activity in the range of 0–15 mm. The highest cytotoxic activity among the same extracts was that of the methanol extract, which had a LC50 value of 1.48 μg/mL, followed by hexane, chloroform, butanol, water and ethyl acetate, with LC50 values of 2.13, 3.16, 7.49, 133.04, and 213.79 μg/mL, respectively, in comparison to the control, DMSO. The findings of the present study suggest that the fruit extracts have significant amounts of total phenolic and flavonoid compounds with potential cytotoxic and antimicrobial activities.

Academic research paper on topic "In vitro evaluation of the total phenolic and flavonoid contents and the antimicrobial and cytotoxicity activities of crude fruit extracts with different polarities from Ficus sycomorus"

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Pacific Science Review A: Natural Science and Engineering

journal homepage: www.journals.elsevier.com/pacific-science-review-a-natural-science-and-engineering/

In vitro evaluation of the total phenolic and flavonoid contents and the antimicrobial and cytotoxicity activities of crude fruit extracts with different polarities from Ficus sycomorus

Sheikha Khamis Al-matani, Ruqaiya Nasser Said Al-Wahaibi, Mohammed Amzad Hossain*

School of Pharmacy, College of Pharmacy and Nursing, University ofNizwa, P.O. Box 33, Postal Code 616, Nizwa, Oman

ARTICLE INFO

ABSTRACT

Article history:

Available online 4 March 2016

Keywords: Ficus sycomorus Maceration method Total phenolics Total flavonoids Antimicrobial Cytotoxic activity

Plants are a good source for discovering new drugs for human benefit. The objective of this study was to determine the total phenolic and flavonoid contents and to probe the antimicrobial and cytotoxic activities of fruit extracts of Ficus sycomorus (F. sycomorus), which is native to the Sultanate of Oman. A study was conducted to measure the total phenolic and flavonoid contents of the methanol extract of this fruit and its derived fractions [n-hexane, chloroform, ethyl acetate, butanol and residual aqueous fractions] by in vitro chemical analyses using the Folin-Ciocalteu reagent assay (FCR) and the aluminium chloride (AlCl3) method. The antimicrobial and cytotoxic activities were assessed using the slightly modified agar disc diffusion method and the brine shrimp lethality (BSL) method. The results of the analysis of the total phenolic content in different fruits extracts showed a significant amount of phenolic compounds, ranging from 1.48 to 213.79 mg of GAE/100 g of powder weight. However, the range of the total flavonoid content among the fruit extracts was found to be 0.100—0.527 mg QE/100 g of powder weight. The antimicrobial activity of the crude fruit extracts at different concentrations against four Gram-positive and Gram-negative bacterial strains, Staphylococcus aureus (S. aureus), Escherichia coli (E. coli), Haemophilus influenza (H. influenza), and Proteus spp (P. spp), showed strong activity in the range of 0—15 mm. The highest cytotoxic activity among the same extracts was that of the methanol extract, which had a LC50 value of 1.48 mg/mL, followed by hexane, chloroform, butanol, water and ethyl acetate, with LC50 values of 2.13, 3.16, 7.49,133.04, and 213.79 mg/mL, respectively, in comparison to the control, DMSO. The findings of the present study suggest that the fruit extracts have significant amounts of total phenolic and flavonoid compounds with potential cytotoxic and antimicrobial activities. Copyright © 2015, Far Eastern Federal University, Kangnam University, Dalian University of Technology, Kokushikan University. Production and hosting by Elsevier B.V. This is an open access article under the CC

BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

1. Introduction

Since time immemorial, Omanis have been using plants as a medicine for the treatment of chronic diseases because of their abundant chemical properties. According to a report by the World Health Organization (WHO), more than 80% of the world's population depends on traditional medicine for their primary healthcare needs [1]. The demand for more drugs from plant sources is continuously increasing. All drugs currently used for clinical purposes are derived from two fundamental sources. The first group of

* Corresponding author. Tel.: +968 99708496; fax: +968 25446236. E-mail address: hossainabi@gail.com (M.A. Hossain).

Peer review under responsibility of Far Eastern Federal University, Kangnam University, Dalian University of Technology, Kokushikan University.

drug compounds is of synthetic origin and is not based on naturally occurring templates. The other large group is secondary metabolites, commonly called natural products, isolated from medicinal plants or cultures of microorganisms [1,2]. Most of the drugs that are currently available on the market for the treatment of various serious diseases have limited potential because they are expensive and produce detectable side effects. Therefore, it necessary to find effective treatments for various disorders excluding the above mentioned limitations of the marketed drugs. Fruits, vegetables, and herbal plants have been shown to be rich sources of chemicals with the potential to prevent incurable diseases. The medicinal value of plants lies in chemical substances that produce a definite physiological action in the human body [3]. There is now an urgent need to search for novel and effective medicines in plants that may be the remedy for incurable diseases. Scientists are now engaged in

http://dx.doi.org/10.1016/j.psra.2016.02.002

2405-8823/Copyright © 2015, Far Eastern Federal University, Kangnam University, Dalian University of Technology, Kokushikan University. Production and hosting by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

the search to find potent remedies for cancer and other infectious diseases through the discovery of new and effective drugs from plants, microbes and other suitable natural sources.

F. sycomorus is a short tree that belongs to the Moraceae family. It is native to Africa. Its fruit is called figs, which have been used as food and medicine. Locally, it is called subula, and it is widely distributed in the Mediterranean basin of Egypt. Among the important medicinal plants are species that belong to Moraceae, often called the mulberry family or fig family. This is a family of flowering plants comprising approximately 40 genera and over 1000 species. Most are widespread in tropical and subtropical regions [4,5]. The height of F. sycomorus is approximately 20 m and 6 m wide. The fruit is large, approximately 2—3 cm in diameter, maturing from buff-green to yellow or red. The bark is a green—yellow to orange colour. The leaves are heart-shaped with a cycle apex. They are 14 cm tall and 10 cm broad. Also, their margin is entire. The flower is cyclic, greenish, and unisexual. It contains latex, like all other figs [6,7]. Fig is the most important plant mentioned in the Holy Quran. The plant grows spontaneously and propagates from its seeds. It grows mostly in the plains and at the riverside, where the soil remains humid or wet, even during the hot and dry summer [8].

The fruits of F. sycomorus are an important herbal medicine and food that is used to treat fungal infections, jaundice and dysentery [9]. In addition, it is used for the treatment of cough, diarrhea, skin infection, stomach disorders, liver disease, epilepsy, tuberculosis, lactation disorders, helminthiasis, infertility and sterility [10—12]. The extracts of fruits of F. sycomorus are also used for the treatment of various diseases, such as cough, diarrhea, stomach disorder, skin infections, epilepsy, liver disease, tuberculosis, lactation disorder, infertility and sterility and helminthiasis [13—15]. The target plant contains several bioactive chemical constituents. The most important bioactive constituents of this plant are alkaloids, tannins, fla-vonoids and phenolic compounds [2,10]. Literature searches reveal that several studies have been performed on this plant species by several authors [5—12]. However, no works have been performed on the Omani species. Hence, there is need to screen medicinal plants for promising biological activity. Therefore, in the present study, we describe the preparation of crude extracts with different polarities of fruits of locally grown F. sycomorus and determine their total phenolic and flavonoid contents as well as their antimicrobial and cytotoxic activities.

2. Materials & methods

2.1. Chemical reagents

Methanol, acetone, ethyl acetate, butanol, chloroform, hexane were purchased from Sigma-Aldrich Company (St. Louis, MO, USA). Dimethyl sulfoxide (DMSO) and amoxicillin were obtained from Fisher Scientific Company, Mexico. Gallic acid, sodium carbonate, Folin-Ciocalteu reagent and aluminium chloride were purchased from New Jersey, USA. The rotary evaporator (Yamato, Rotary Evaporator, and Model- RE801) and UV—visible spectrophotometer were from Japan.

2.2. Collection of microorganism

Gram-positive and Gram-negative bacterial strains Staphylococcus aureus (S. aureus), Escherichia coli (E. coli), Haemophilus influenza (H. influenza), and Proteus spp (Proteus spp) were collected from Nizwa Hospital, Nizwa during the month of January, 2015.

2.3. Plant samples

Selected fruit samples for our study were collected in the Al Sharqea region and the Al Batinah region of Oman. The fruit samples were harvested on November 1, 2014, at 5 pm.

2.4. Extracts preparation

The fruit samples were washed with water and dried at room temperature under shade for 3 days. The dried samples were coarsely powdered using a Wiley Mill to a 60 mesh size and used for solvent extraction. The powdered samples (114.08 g) were macerated in methanol (500 mL) for 3 days. The whole mixture was filtered under pressure. Then, the filtrate was dried by a rotary evaporator under reduced pressure at 22 °C to yield the crude methanolic extract (11.43 g). The crude residue (10 g) was suspended in distilled water (150 mL) and transferred into a separation funnel. Thirty millilitres of hexane was added to it and shaken by hand for 20 min. After 30 min, the mixture was separated into two layers. The whole process was repeated with 20 mL of hexane. The two hexane fractions were combined and evaporated to obtain the hexane extract (1.88 g). Similarly, chloroform, ethyl acetate and butanol were used to prepare the chloroform (1.79 g), ethyl acetate (2.16 g) and butanol extracts (1.2 g). The remaining water fraction was evaporated to yield the water extract (1.57 g) [16].

2.5. Estimation of the total phenolic content

The fruit extracts were used for the determination of the total phenolic content by using a modified Folin-Ciocalteu reagent assay [17]. Each extract (1 mL) or a gallic acid standard (12.5, 25, 50,100 and 200 mg/L) was added to a volumetric flask (25 mL) containing methanol (9 mL). Two-hundred microlitres of each sample was added to a test tube, and 10% FCR (1.5 mL) was added and shaken. After 5 min in the dark, 1.5 mL of 6% Na2CO3 was added to the mixture. The mixture was diluted with water to 25 mL and incubated for 90 min at room temperature. Then, it was incubated for 2 h in a dark place. The absorbance of the different concentrations of each extract was measured against a reagent blank at 760 nm by using a UV—Vis spectrophotometer. The total phenolic content of each extract was determined using a conventional method and external calibration with gallic acid GAE/100 g powder weight [17]. All of the crude samples and the standards were analysed in triplicate.

2.6. Estimation of total flavonoid content

A total of six extracts from the fruit of F. sycomorus were used in this present study. The extracts and a quercetin standard were used for the determination of the total flavonoid content by the AlCl3 method with modification [18]. The crude extract samples or quercetin standard (12.5, 25, 50,100 and 200 mg/L) was added to a 10-mL volumetric flask containing 4 mL of methanol. A portion of each extract (0.25 mL) was added to a separate test tube, and 1.25 mL of water and 75 ml of a sodium nitrate solution were added to the samples. The mixture solution was kept for 6 min in a dark place; 150 ml of aluminium chloride was added to each test tube and kept in a dark place for 5 min. Finally, 0.5 mL of sodium hydroxide (NaOH) and 0.275 mL of water were added to the mixture. The mixture was mixed well, and the absorbance was measured against a prepared blank reagent using UV—Vis spectroscopy at a fixed wavelength of 510 nm. The total flavonoid content of the fruit extracts was determined using the conventional method and an external calibration with the quercetin standard QE/100 g of

powder weight [18]. All of the crude fruit samples and the standard were analysed in triplicate.

2.7. Antimicrobial activity

The antibacterial activities of the selected plant extracts were determined using the disc diffusion method as described by Amzad et al. [19]. One Gram (+) bacteria, S. aureus, and three Gram (—) bacteria, E. coli, Proteus spp, and H. influenza, were used in the present study. All of the test bacteria were collected from Nizwa Hospital, Nizwa. Different known concentrations of plant extracts, such as 2, 1, 0.5 and 0.25 mg/mL, were prepared using dimethyl sulfoxide (DMSO). Amoxicillin was used as a positive control. Sterile filter paper discs (6-mm diameter) were impregnated with each concentration of the extracts. Finally, all of the plates were incubated micro aerobically at 37 °C for 24 h. Evaluation of the antibacterial activity of the tested material was measured by measuring the diameter of the zone of inhibition in millimetres against the employed cultured bacteria strains [19].

= 0.0032x + 0.312 R2 = 0.9763

Concentration (mg/L)

Fig. 1. Gallic acid standard calibration curve.

3. Results

2.8. Cytotoxic activity assay

The cytotoxic activities of fruit extracts with different polarities from the selected plant were determined by the brine shrimp lethality method [20]. Brine shrimp eggs were hatched in the covered chamber of a duo compartment plastic container with sea water for 24 h. After hatching, the active nauplii were separated from the eggs and used for the cytotoxic activity assay. Four milligrams of different plant extract samples were placed in a vial and dissolved with 4 mL of dimethyl sulfoxide (DMSO). Seven concentrations, 1,000, 500, 250, 125, 62.50, 31.25 and 15.75 mg/mL, of each extract were prepared with DMSO. Fifty microlitres of each of these test solutions were added to pre-marked test tubes containing 5 mL of sea water and 10 nauplii. After 24 h, the number of surviving nauplii in each test tube was counted using a magnifying glass and recorded. The percentage of the mortality of the brine shrimp was calculated for each concentration of sample. The LC50 values were calculated using Microsoft Excel (log concentration and ant log).

2.9. Statistical analyses

Experimental results were analysed by SPSS 10 (SPSS Inc. Chicago, IL). The data represent the mean ± S.D. of three parallel measurements. The differences in the means were calculated using Tukey multiple comparisons and the least significant difference (LSD) test. Correlations were determined using the Pearson correlation coefficient in bivariate correlations. P values <0.05 were regarded to be significant.

3.1. Yield of extracts

The extractive yields of the hexane, chloroform, ethyl acetate, butanol and water extracts of the fruit of F. sycomorus were 18.8%, 17.9%, 21.6%, 12% and 15.7%, respectively (Table 1). The highest amount of total extractable compounds was in the ethyl acetate extract, and the lowest was in butanol in descending order: hexane > chloroform > ethyl acetate > butanol > water extract [16].

3.2. Total phenolic content

The total phenolic contents of the fruit extracts were evaluated using a modified Folin-Ciocalteu reagent method reported by Amzad et al. [17]. The total phenolic content of fractions with different polarities that were extracted using solvents such as hexane, chloroform, methanol, water, ethyl acetate and butanol from the fruits of F. sycomorus are presented in Table 1. The butanol extract exhibited the highest total phenolic content (81.56 ± 0.43 mg of GAE/100 g powder weight), followed by water (32.81 ± 0.20), ethyl acetate (26.56 ± 0.29), hexane (10.62 ± 0.12), methanol (8.75 ± 0.23) and chloroform (3.43 ± 0.14). Gallic acid was used as a standard for the calculation of the total phenolic content (Table 1 and Fig. 1).

3.3. Total flavonoid content

The determination of the total flavonoid content of the fruit extracts was estimated using a modified AlCl3 method reported by Amzad et al. [18]. The total flavonoid content of different extracts of the fruits of F. sycomorus are also presented in Table 1. The

Table 1

Total phenolics and flavonoids contents, ratio and extraction yield of fruits of F. sycomorus.

Crude Total phenolics content (mg of GAE/100 gm powder weight) Total flavonoids content (mg QE/100 gm powder weight) Flavonoids/phenolics Yield (%)

extracts ratio

Methanol 8.75±0.23 0.527+0.15 0.06 9.6

Hexane 10.62+0.12 0.100+0.09 0.009 18.8

Chloroform 3.43±0.14 0.477+0.52 0.14 17.9

Butanol 81.56+0.43 0.410+0.56 0.005 12

Ethyl acetate 26.56+0.29 0.364+0.87 0.014 21.6

Water 32.81+0.20 0.135+0.12 0.004 15

Absorbance

0.05 0

0 50 100 150 200 250

Concentration (mg/L)

Fig. 2. Qurecetin standard calibration curve.

methanolic extract exhibited the highest total flavonoid content (0.527 ± 0.15 mg QE/100 g powder weight), and the lowest was that of the hexane extract (Table 1). Quercetin (QE) was used as a standard for the calculation of the total flavonoid content (Table 1 and Fig. 2).

3.4. Antimicrobial activity

Fruit extracts of different polarities from the selected plant showed moderate activity against all three Gram (—) bacteria, but poor activity was found against Gram (+) bacteria [19]. The zone of inhibition against S. aureus, E. coli, H. influenza and Proteus spp bacteria at the concentrations of 2 mg/mL, 1 mg/mL, 0.5 mg/mL and 0.25 mg/mL was in the range of 0—15 mm (Table 2), which indicated its antimicrobial activity compared to the zone of inhibition

of the standard amoxicillin, which gave values of 6—13 mm. Almost all of the fruit extracts showed significant antimicrobial activity against E. coli and S. aureus at different concentrations. However, all of the extracts of the fruit of F. sycomorus did not show any activity against H. influenza, except the butanol extract. The butanol extract showed the highest antimicrobial activity against H. influenza at all employed concentrations (Table 2). The hexane extract also did not show any antimicrobial activity against Proteus spp at all employed concentrations (Table 2).

3.5. Cytotoxic activity

The cytotoxic assay was performed using the brine shrimp lethality bioassay method [20]. Among the six extract fractions, the ethyl acetate extract exhibited the highest cytotoxic activity, with an LC50 value of 26.82 mg/mL, followed by chloroform, methanol, hexane, water and butanol, which had LC50 values of 86.09,138.96, 281.83, 341.19 and 463.44 mg/mL, respectively, in comparison with the control, DMSO (Table 3 and Fig. 3).

4. Discussion

The total phenolic content of the extracts was solvent-dependent and expressed as milligrams of gallic acid equivalents. The total content of phenolic compounds in different extracts was determined from the regression equation of the calibration curve (y = 0.00032x + 0.0312, R2 = 0.976) and expressed in gallic acid equivalents (GAE). In our present study, the total phenolic content in fractions of different polarities varied, ranging from 3.43 ± 0.14 to 81.56 ± 0.43 mg of GAE/100 g powder weight (Table 1). The butanol extract exhibited the highest total phenolic content (81.56 ± 0.43 mg of GAE/100 g powder weight), followed by water (32.81 ± 0.20), ethyl acetate (26.56 ± 0.29), hexane (10.62 ± 0.12),

Table 2

Antimicrobial activity of different polarities fruits crude extracts of F. sycomorus.

Fruits crude extracts Conc mg/ml E. coli nm Proteus SPP nm S. aureus nm H. infleunza nm

Hexane 2 7±0.18 nd 7±0.13 nd

1 9 ±0.10 nd 8±0.76 nd

0.5 11±0.15 nd 15 ±0.43 nd

0.25 12±0.22 nd 8±0.12 nd

Control 7±0.16 12±0.13 11±0.17 nd

Chloroform 2 8±0.21 9±0.23 7±0.51 nd

1 12 ±0.35 9±0.17 9±0.11 nd

0.5 11±0.13 nd 11±0.23 nd

0.25 8±0.10 nd 7±0.57 nd

Control 12±0.25 9±0.10 10±0.19 nd

Ethyl acetate 2 7±0.14 11±0.19 10±0.23 nd

1 7 ±0.19 10±0.17 11±0.15 12±0.12

0.5 6±0.09 9±0.11 8±0.09 nd

0.25 6±0.31 9±0.23 7±0.19 nd

Control 9±0.21 10±0.23 10±0.12 6±0.10

Butanol 2 8±0.355 12±0.15 10±0.55 15±0.14

1 8 ±0.18 10±0.11 9±0.67 15±0.10

0.5 13±0.43 10±0.15 nd 14±0.34

0.25 6±0.63 9±0.19 nd 13±0.23

Control 12±0.17 13±0.29 10±0.12 9±0.10

Methanol 2 9±0.18 10±0.24 6±0.12 nd

1 9 ±0.15 10±0.13 8±0.19 nd

0.5 7±0.72 9±0.95 7±0.97 nd

0.25 nd 9±0.20 8±0.30 nd

Control 9±0.17 10±0.55 7±0.16 nd

Water 2 11±0.13 14±0.17 11±0.10 nd

1 9 ±0.10 12±0.09 11±0.13 nd

0.5 8±0.09 10±0.75 11±0.20 nd

0.25 7±0.32 9±0.10 10±0.14 nd

Control 8±0.49 10±0.11 13±0.16 nd

nd= not detected.

Table 3

Cytotoxic activity of different polarities fruits crude extracts of F. sycomorus.

Fruits crude extract Conc mg/ml Mortality (%) LC50 (mg/ml)

Hexane 1000 100

500 100

250 80 281.83

125 50

62.5 50

31.25 50

15.75 40

Control 0

Ethyl acetate 1000 100

500 100

250 80 26.82

125 80

62.5 80

31.25 50

15.75 40

Control 0

Chloroform 1000 100

500 90

250 90 86.09

125 80

62.5 70

31.25 50

15.75 30

Control 100

Butanol 1000 100

500 90

250 80 463.44

125 60

62.5 60

31.25 40

15.75 30

Control 0

Methanol 1000 100

500 90

250 80 138.96

125 60

62.5 60

31.25 50

15.75 40

Control 0

Water 1000 100

500 90

250 70 341.19

125 70

62.5 50

31.25 40

15.75 40

Control 0

(0.410 ± 0.56 mg QE/100 g powder weight), ethyl acetate (0.364 ± 0.87 mg QE/100 g powder weight), water (0.135 ± 0.12 mg QE/100 g powder weight) and hexane (0.100 ± 0.09 mg QE/100 g powder weight). The results for the total phenolic and flavonoid contents and their ratios in the selected plant material are shown in Table . Almost similar results were obtained for the fruits extract of F. sycomorus that have been previously reported [22].

The determination of the antimicrobial activity of the fruit extracts of the selected plant was performed using the agar disc diffusion method reported by Amzad et al. [19]. One Gram-positive and three Gram-negative bacterial strains were used in this study. In our study, all of the extracts showed moderate activity against three Gram (—) bacterial strains, but very poor activity was found against Gram (+) bacteria [19]. The zone of inhibition against S. aureus, E. coli, H. influenza and Proteus spp bacteria at concentrations of 2 mg/mL, 1 mg/mL, 0.5 mg/mL and 0.25 mg/mL of extract was in the range of 0—15 mm (Table 2). However, the zone of inhibition of all of the bacterial strains against the standard amoxi-cillin was in the range of 6—13 mm. Almost all of the extracts showed significant antimicrobial activity against E. coli and S. aureus at different concentrations. However, all of the extracts of the fruit of F. sycomorus did not show any activity against H. influenza, except the butanol extract. The butanol extract showed the highest antimicrobial activity against H. influenza at all employed concentrations (Table 2). The hexane extract also did not show any antimicrobial activity against Proteus spp at all employed concentrations (Table 2). This is the first report for the antimicrobial activity of the extracts of different polarities of locally available F. sycomorus against bacterial strains. Therefore, we are unable to compare our results with reported values.

The cytotoxic assay was determined using the brine shrimp lethality bioassay method reported by Weli et al. [20]. In our study, the highest activity was found for the ethyl acetate extract, and the lowest was that of the butanol extract (Table 3 and Fig. 3). Among the six extract fractions, the ethyl acetate extract exhibited the highest cytotoxic activity, with an LC5o value of 26.82 mg/mL, followed by chloroform, methanol, hexane, water and butanol, which had LC50 values of 86.09,138.96, 281.83, 341.19 and 463.44 mg/mL, respectively, in comparison with the control DMSO (Table 3 and Fig. 3). Similar results and a similar relationship related to the cytotoxic activity were previously reported for the crude extracts of the fruit of the Ficus species [23,24].

5. Conclusion

methanol (8.75 ± 0.23) and chloroform (3.43 ± 0.14). Gallic acid was used as a standard for the calculation of the total phenolic content (Table 1 and Fig. 1). In this way, the determination of the total phenolic content using the Folin-Ciocalteu reagent assay provides absolute measurements of the amounts of phenolic compounds in the extracts. In fact, the determination of the total phenolic contents is based upon their chemical reducing capacity relative to gallic acid [21]. Almost similar total phenolic content results were obtained for the fruit extracts of F. sycomorus that have been previously reported [22].

Determining the total flavonoid content using the aluminium chloride method is based on the formation of a stable complex between aluminium chloride and the keto and hydroxyl groups of flavones and flavonoids. The total content of flavonoids in different extracts was determined from the regression equation of the calibration curve (y = 0.0006x + 0.1093, R2 = 0.917) and expressed in quercetin equivalents (QE). In the present study, the methanol extract exhibited the highest total flavonoid content (0.527 ± 0.15 mg QE/100 g powder weight), followed by chloroform (0.477 ± 0.52 mg QE/100 g powder weight), butanol

The total phenolic and flavonoid contents and the antimicrobial and cytotoxic activities of the fruit extracts of F. sycomorus were

500 450 400 350 300 250 200 150 100 50 0

Chloroform Ethyl acetate Hexane

Butanol Methanol

Fig. 3. Comparison of LC50 values of different polarities fruits crude extracts of F. sycomorus.

estimated using well-established, conventional methods. Significant amounts of phenolic and flavonoid contents were found in all of the extracts. Similarly, the antimicrobial and cytotoxic activity results also showed significant activity for all of the extracts of the fruit. Therefore, further studies are in progress in our laboratory is to isolate the bioactive components from the fruit of F. sycomorus.

Acknowledgements

The authors are grateful to the Oman Research Council of the Sultanate of Oman for providing funding through the FURAP project to carry out this project. The authors are also grateful to the University of Nizwa, Nizwa, Sultanate of Oman for providing the laboratory and other facilities. The authors would like to thank the staff of the Pharmaceutical Chemistry Laboratory, Pharmacognosy Laboratory and Organic Chemistry Laboratory for their assistance to carry out the FURAP project successfully.

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