Abstract
Ficus benghalensis (Moraceae) is widely found in Asian Countries and has been traditionally prescribed owing to its analgesic, anti-inflammatory, and muscle relaxation properties. Purposely, in this study, phytochemical composition and isolation of fixed oil from F. benghalensis were comprehended for the first time. The fatty acids were isolated from hexane (HO) and chloroform (CO) fractions and were characterized by gas chromatography-mass spectrometry (GC-MS) analysis. The fatty acid ester was identified by comparing the mass spectra with the equipment library. The major fatty acids identified from HO extract were oleic acid methyl ester (42.67), palmitic acid methyl ester (28.79), linoleic acid methyl ester (11.36), docosanoic acid methyl ester (8.83), and stearic acid methyl ester (3.21). Similarly, the major constituents of CO fraction were palmitic acid methyl ester (65.94), oleic acid methyl ester (10.18), stearic acid methyl ester (9.15), elaidic acid methyl ester (5.32), and linoleic acid methyl ester (3.94). Both of the isolated fixed oils were screened for antibacterial, antioxidant, analgesic, and muscle relaxant effects. Regarding the antibacterial effect, the maximum zone of inhibition against Bacillus subtilis was 26.32 and 25.09 mm by HO and CO fractions, respectively. Both HO and CO demonstrated significant antioxidant effects, i.e., 70.23 and 72.09 µg/mL at a higher dose (100 µg/mL). Similarly, both experimented fractions demonstrated significant analgesic effects while the muscle relaxant effect and sedative were non-significant. Results of the present study conclude that fixed oils are the significant antibacterial and analgesic.
1 Introduction
Ficus benghalensis L. is a member of the family Moraceae [1]. F. benghalensis is distributed abundantly in Asian countries, including Pakistan, Malaysia, China, India, and Nepal. In the traditional system, various parts of F. benghalensis, including, barks, roots, stems, buds, leaves, fruits, and latex, have been used in neurological disorders, such as insomnia, anxiety, and seizure [2,3]. Various researchers documented several neuropharmacological potentials of F. benghalensis [4]. The bark aqueous extract has been reported for excellent cognitive-enhancing potential [5]. Various parts of F. benghalensis have been reported for antimicrobial [6], analgesic [6], anti-inflammatory [7], antistress [8], anti-plasmodial [9], antiallergenic [8], anti-ulcer [10], mosquito larvicidal [11], antilipidemic [12], anti-atherogenic [13], and antidiabetic potency [14]. F. benghalensis aerial roots have been documented and reported for potential anxiolytic, memory enhancing, and muscle relaxation effects [15]. Phytochemical investigation of Ficus species indicated the presence of various classes of active secondary metabolites, such as phenol, flavonoids, alkaloids tannins, steroids, saponins, sugars, protein, essential oil, and volatile oils [16]. The ethyl acetate extracts of F. benghalensis have resulted in the isolation of O-α-l-rhamnopyranosyl-hexacosanoate-β-d-glucopyranosyl ester, mucusoside, carpachromene, and alpha amyrine acetate. The isolated compounds have shown excellent acetylcholinesterase and antioxidant activity [17]. The present study deals with the chemical composition and biological screening of fixed oil from F. benghalensis.
2 Materials and methods
2.1 Plant materials
F. benghalensis plant material was collected in March 2021 from District Swabi, KP, Pakistan (GPS coordinates: Latitude; 34.0613° North, 72.3927° East). The plant specimen was identified by Dr. Muhammad Ilyas, Department of Botany, University of Swabi, KPK, Pakistan. The voucher specimen number UOS-BOT/103 was deposited in the herbarium in the Department of Botany, University of Swabi, KPK, Pakistan.
2.2 Extraction and phytochemical screening
The extracts and fractions were assessed for various phytochemical screening as per the reported method [18,19]. Powdered F. benghalensis plant material (100 g) was extracted using the Soxhlet extraction method through n-hexane (HO) and chloroform (CO) separately. Afterward, the extracted oils were concentrated using a rotary evaporator. Phytochemical tests were conducted on CO and HO fractions of F. benghalensis extracts using defined procedures for the identification of respective constituents.
2.2.1 Alkaloids
Each extract (0.2 g) was mixed for 2 min with H2SO4 (2%). After filtration, Dragendroff’s reagent (a few drops) was further added. The presence of alkaloids was noticed due to the occurrence of orange-red precipitates.
2.2.2 Steroids
Each extract (0.5 g) having 2 mL of H2SO4 was mixed with acetic anhydride (2 mL). A change in color from violet to blue/green indicated the presence of steroids.
2.2.3 Tannins
Water was mixed with each extract and heated in a water bath followed by filtration. Afterward, ferric chloride (few drops) was mixed and filtered. The appearance of the dark green solution indicated the occurrence of tannins.
2.2.4 Glycosides
Plant extracts were hydrolyzed and neutralized with HCl and NaOH, respectively. Later, Fehling solutions A and B (a few drops) were mixed in the above solution. The appearance of red precipitates indicated the occurrence of glycosides.
2.2.5 Saponins
Extract (0.2 g) was mixed with distilled water and heated in a water bath. The presence of saponins was indicated owing to the appearance of creamy miss of small bubbles.
2.2.6 Reducing sugars
Extracts were mixed with distilled water and later filtered. Afterward, the filtrate was mixed with Fehling solutions A and B (a few drops) and boiled for few minutes. The appearance of orange-red precipitates indicated the occurrence of reducing sugars.
2.2.7 Protein
Extract (0.5 mg), NaOH solution (40%), and two drops of copper sulfate solution (1%) were mixed. The appearance of violet color indicated the occurrence of protein.
2.2.8 Amino acids
The extract (0.5 mg) was heated in the presence of a few drops of ninhydrin (0.2%). The appearance of pink/purple color indicated the occurrence of amino acids/proteins.
2.3 Isolation of fatty acids and preparation of fatty acid methyl esters (FAMEs)
The identified plant materials of stem (8.24 kg) were dried in shade and subjected to cold extraction with methanol for 2 weeks. The extract obtained was concentrated at low temperate and pressure, which yield 98 g of crude extract. The crude extract was fractionated to various fractions, such as HO (16 g), CO (27 g), and ethyl acetate (21 g). The HO fractions were subjected to column chromatography using HO and EtOAc as an eluting solvent, which afforded yellow color oil (1.65 g). Similarly, the CO fraction was subjected to chromatography analysis, which afforded yellowish color oil (1.03 g).
2.4 Methylation of fatty acids
The oils extracted from HO and CO fractions were methylated according to the published procedure. The oil was refluxed with hydrochloric acid and methanol for 2 min. The water was added to the reaction mixture medium, and then, the methylated fatty acids were extracted with HO. The methylated samples are used for the gas chromatography-mass spectrometry (GC-MS) analysis. The oil isolated from title plant was assessed by the GC-MS analysis. One microliter of the prepared sample was injected into GC-MS with the help of an auto-injector system.
2.5 Preparation of standard for GC-MS analysis
For GC-MS analysis, the internal standard was prepared by combining 1 mL of HO with 13.70 mg of tridecanoic acid methyl ester. The extremal standard was also prepared by adding 10 mL of dichloromethane with 10 mg of 37-compound FAMEs. This solution was used for the preparation of further standard solution.
2.6 GC-MS analysis
The constituents present in fixed oil were investigated on Shimadzu GC-MS according to our reported method [20]. This GC-MS comprised an auto-injector (AOC-20i) and auto-sampler (AOC-20S). Capillary column treated with polyethylene glycol had specifications as thickness (0.25 µm, id; 0.35 mm) and length (30 m). Chromatographic conditions were as follows: interface temperature: 240°C, EI-ion source temperature: 250°C, solvent cut time: 1.5 min, and pressure: 100 kPa. The injection temperature was set at 240°C, whereas injector has a split ratio of 1:50. In this chromatographic system, the elution time was 45 min. Scanning for MS was achieved from m/z 85 to m/z 380. The chemical constituents were identified by the comparison of the obtained mass spectra with the mass spectra of standard from the NIST Library.
2.7 Antibacterial activity
Antibacterial properties of HO extract and CO extract were determined against S. aureus, S. flexenari, Escherichia coli, Pseudomonas aeruginosa, and Bacillus subtilis by following the protocols adopted by Bawazeer et al. [21]. In this experiment, Staphylococcus aureus, Shigella flexenari, E. coli, P. aeruginosa, and B. subtilis were used. All the experimented bacterial strains were grown on a Müller–Hinton agar for 24 h at 37°C. Triplicate of cultures was incubated at 37°C for 24–72 h. At 37°C, incubation was achieved for 24 h and, later, the diameter of ZOI (zone of inhibition) for the growth of microbes was calculated in millimeters. This experiment was performed in triplicates.
2.8 Antioxidant activity
To determine the antioxidant effect of HO and CO fractions of F. benghalensis L., the methods described by Khalil et al. [22,23] were followed. Purposely, the antioxidant properties of oil extracts were measured in terms of the free radical scavenging activity through 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay. In cuvette, each extract (4 mL) was mixed with 1 mL of DPPH methanolic solution. This mixture was allowed to stand at 25°C for half an hour. By using the UV-Vis Spectro-photometer (CECIL CE7200), the absorbance of the prepared mixture was noticed at 520 nm. The following formula was used for the determination of percent inhibition:
A(b) is the absorbance of the blank sample (t = 0 min) and A(s) is the absorbance the of tested extract (t = 30 min).
2.9 Animals
Balb/c mice with having weight 24–27 g were used for in vivo biological screening. The animals used for this analysis were given the standard laboratory food and water ad libitum. The animals studied were approved by the Ethical Community of the Department of Pharmacy, University of Swabi (No. Pharm/298).
2.10 Muscle relaxant
The muscle coordination test was performed through the inclined plan test and the traction test. For both of these activities, animals were categorized into positive control, negative control, and tested groups. These groups were treated with Diazepam (0.5 mg/kg), normal saline (10 mL/kg), and fixed oil fractions (at the doses of 5, 10, 15, and 20 mg/kg). After 30 min of the above treatment, animals were subjected to the inclined plan and traction tests according to our published methods [24].
2.11 Analgesic activity
The acetic acid-induced writhing model was used for the evaluation of the fixed oil fraction for analgesic effect. The animals were classified into various groups: positive control, negative control, and tested groups. These groups were treated with Diclofenac sodium (2 mg/kg), normal saline (10 mL/kg), and samples (5–20 mg/kg) to be tested receptivity. After 30 min of this administration, all animals were injected with 1% acetic acid solution. After 5 min of acetic acid treatment, the number of writhing’s was counted for 10 min and, then, the percent effect was calculated according to our published method [24].
2.12 Sedative activity
The open-filed experimental procedure was adopted for the evolution of our tested sample. This procedure was according to our published paradigm [21]. The animals were categorized as above; the positive control was treated with Diazepam (0.5 mg/kg), the negative control was treated with normal saline (10 mL/kg), and the tested group was treated with fixed oil at the dose of 5, 10, 15, and 20 mg/kg. After 30 min of this administration, animals were placed at the center of the special box and the number of lines crossed was counted. Greater the number of lines crossed indicating lack of sedation and vice versa.
2.13 Statistical analysis
Results were expressed as mean ± standard deviation. One-way analysis of variance was used to evaluate the level of significance (p ≤ 0.05).
3 Results and discussion
3.1 Phytochemical analysis
The preliminary phytochemical study of various fractions is presented in Table 1. Different classes of phytoconstituents were identified in various fractions. The steroids, fatty acid, and carbohydrate were found in all tested samples. The methanolic and ethyl acetate fractions were the rich sources of various classes of phytochemicals. The HO fraction was not so much rich with tested classes of bioactive compounds.
Phytochemical analysis of various extracts of F. benghalensis
| Secondary metabolites | Hexane | Chloroform | EtOAc | Methanol |
|---|---|---|---|---|
| Steroids | + | + | + | + |
| Alkaloids | − | + | + | + |
| Saponins | − | − | + | + |
| Glucosides | − | + | + | + |
| Phenolic compounds | − | − | + | + |
| Tannins | − | − | − | + |
| Amino acids | − | + | + | + |
| Fatty acids | + | + | − | + |
| Reducing sugars | + | + | + | + |
3.2 GC-MS analysis of fatty acids
Figure 1 and Table 2 show the identified fatty acids methyl ester and its relative concentration with the help of external standard methods. The experiment was performed in triplicate and values of concentration and area are listed in (Tables 2 and 3). Fatty acid methyl ester quantification was done with the help of a three-point calibration curve having an R value of less than 0.99 in every experiment. The GC-MS chromatogram of oil extracted from F. benghalensis has properly labeled signals of identified constituents. The results of extracted oils indicated the presence of saturated and unsaturated fatty acid methyl ester. The oil extracted from the HO fraction comprises the highest concentration of oleic acid methyl ester (42.67), palmitic acid methyl ester (28.79), linoleic acid methyl ester (11.36), decanoic acid methyl ester (8.83), and stearic acid methyl ester (3.21).
GC-MS analysis of oil extracted from HO fraction of F. benghalensis.
GC-MS analysis of oil extracted from CO fraction of F. benghalensis.
Quantitative results of oil extracted from HO fraction of F. benghalensis
| ID # | Name | R time | Area | Conc. (%) |
|---|---|---|---|---|
| 1 | C6:0:caproic acid methyl ester | 4.820 | 3,261 | 0.03 |
| 2 | C8:0:caprylic acid methyl ester | 9.997 | 5,920 | 0.05 |
| 3 | C10:0:capric acid methyl ester | 15.464 | 17,356 | 0.14 |
| 5 | C12:0:lauric acid methyl ester | 20.506 | 58,377 | 0.47 |
| 8 | C14:0:myristic acid methyl ester | 25.981 | 169,246 | 1.37 |
| 10 | C15:0:pentadecanoic acid methyl ester | 29.382 | 46,449 | 0.38 |
| 12 | C16:0:palmatic acid methyl ester | 33.131 | 3,546,214 | 28.79 |
| 14 | C17:1:heptadecanoic acid methyl ester | 37.604 | 98,458 | 0.80 |
| 16 | C18:1c:oleic acid methyl ester | 40.691 | 5,256,370 | 42.67 |
| 17 | C18:2c:linoleic acid methyl ester | 40.658 | 1,399,857 | 11.36 |
| 18 | C18:1n9T:elaidic acid methyl ester | 41.266 | 141,775 | 1.15 |
| 19 | C18:0:stearic acid methyl ester | 41.458 | 395,513 | 3.21 |
| 20 | Docosanic acid methyl ester | 42.515 | 1,087,130 | 8.83 |
| 25 | C20:1:eicosanic acid methyl ester | 45.424 | 91,392 | 0.74 |
Similarly, the oil isolated from CO extract indicated the presence of major constituent’s palmitic acid methyl ester (65.94), oleic acid methyl ester (10.18), stearic acid methyl ester (9.15), elaidic acid methyl ester (5.32), and linoleic acid methyl ester (3.94). Both saturated and unsaturated fatty acids were identified in the HO and CO fractions (Table 2 and 3).
Quantitative results of oil extracted from CO fraction of F. benghalensis
| ID # | Name | R time | Area | Conc. (%) |
|---|---|---|---|---|
| 1 | C6:0:caproic acid methyl ester | 4.820 | 2,605 | 0.04 |
| 2 | C8:0:caprylic acid methyl ester | 9.998 | 8,261 | 0.12 |
| 3 | C10:0:capric acid methyl ester | 15.466 | 10,331 | 0.14 |
| 5 | C12:0:lauric acid methyl ester | 20.507 | 727,774 | 1.02 |
| 8 | C14:0:myristic acid methyl ester | 25.984 | 127,025 | 1.78 |
| 10 | C15:0:pentadecanoic acid methyl ester | 29.384 | 12,944 | 0.18 |
| 12 | C16:0:palmatic acid methyl ester | 33.137 | 4,716,935 | 65.94 |
| 14 | C17:1:heptadecanoic acid methyl ester | 37.602 | 17,127 | 0.24 |
| 16 | C181c:oleic acid methyl ester | 40.700 | 728,552 | 10.18 |
| 17 | C18:2c:linoleic acid methyl ester | 40.465 | 281,985 | 3.94 |
| 18 | C18:1n9T:elaidic acid methyl ester | 40.915 | 380,555 | 5.32 |
| 19 | C18:0:stearic acid methyl ester | 41.462 | 654,550 | 9.15 |
| 20 | Docosanic acid methyl ester | 42.500 | 95,084 | 1.33 |
| 25 | C20:1:eicosanic acid methyl ester | 45.425 | 44,958 | 0.63 |
3.3 Antibacterial effect
The antibacterial effect of HO and CO is presented in Table 4. The fractions were tested against various pathogenic bacteria. The maximum effect shown by HO and CO against B. subtilis was 26.32 and 25.09 mm, respectively. The growth of P. aeruginosa and E. coli was also inhibited by both of the tested extracts in a significant manner. The standard antibacterial samples demonstrated maximum effect against all tested bacteria.
Antibacterial effects of oil extracts from HO and CO fractions of F. benghalensis
| Bacterial strain | Zone of inhibition (mm) | ||
|---|---|---|---|
| HO | CO | Streptomycin | |
| S. aureus | 15.98 ± 2.65 | 12.65 ± 2.76 | 30.87 ± 0.99 |
| S. flexenari | 4.87 ± 3.86 | 3.09 ± 3.01 | 32.87 ± 0.87 |
| E. coli | 18.76 ± 2.85 | 16.98 ± 2.09 | 31.87 ± 0.46 |
| P. aeruginosa | 22.98 ± 1.87 | 20.76 ± 3.89 | 35.87 ± 0.76 |
| B. subtilis | 26.32 ± 2.00 | 25.09 ± 2.09 | 30.66 ± 0.89 |
3.4 Antioxidant effect
A dose-dependent antioxidant effect was observed against HO and CO as shown in Table 5. The maximum free radical scavenging effect was noted against at higher doses, i.e., 70.23 and 72.09 µg/mL, by HO and CO, respectively. The outstanding effect of the standard drug was also recorded.
Antioxidant effects of oil extracts from HO and CO fractions of F. benghalensis
| Samples | Concentrations | |||||
|---|---|---|---|---|---|---|
| 10 µg/mL | 20 µg/mL | 40 µg/mL | 60 µg/mL | 80 µg/mL | 100 µg/mL | |
| HO | 6.54 ± 2.00 | 14.98 ± 1.23 | 30.76 ± 1.98 | 45.65 ± 1.23 | 58.65 ± 1.65 | 70.23 ± 1.87 |
| CO | 8.32 ± 1.87 | 16.43 ± 1.43 | 32.98 ± 1.77 | 49.09 ± 1.65 | 60.23 ± 1.44 | 72.09 ± 1.98 |
| BHA | 90.22 ± 1.70 | 91.09 ± 1.87 | 92.65 ± 1.87 | 92.99 ± 1.64 | 95.00 ± 1.00 | 96.98 ± 1.23 |
3.5 In vivo screening of fixed oil
3.5.1 Analgesic effect
The analgesic effect of HO and CO is presented in Table 6. A dose-dependent effect was noticed against both of the tested samples. The maximum percent effect was 63.09 and 68.32 against HO and CO. The positive control (Diclofenac sodium) demonstrated a maximum analgesic effect (85.00%).
Analgesic effect of oil extracts from HO and CO fraction of F. benghalensis
| Treatment | Dose | % inhibition of writhing |
|---|---|---|
| Saline | 10 mL/kg | — |
| Diclofenac sodium | 2 mg/kg | 85.00 ± 1.24 |
| HO | 5 mg/kg | 20.09 ± 2.09 |
| 10 mg/kg | 35.07 ± 2.00 | |
| 15 mg/kg | 49.32 ± 1.88 | |
| 20 mg/kg | 63.09 ± 1.98 | |
| CO | 5 mg/kg | 23.54 ± 2.87 |
| 10 mg/kg | 38.23 ± 2.65 | |
| 15 mg/kg | 52.11 ± 2.54 | |
| 20 mg/kg | 68.32 ± 2.00 |
3.5.2 Muscle relaxant effect
Both of the tested samples were tested for muscle relaxant effect. The muscle relaxant effect was tested in two models as presented in Table 7. The muscle coordination potential was not found in both of the tested samples.
Muscle relaxant effect of oil extracts from HO and CO fractions of F. benghalensis
| Groups | Dose | Inclined plan (% effect) | Traction (% effect) | ||||
|---|---|---|---|---|---|---|---|
| 30 min | 60 min | 90 min | 30 min | 60 min | 90 min | ||
| Distilled water | 10 mL | 0.00 ± 0 | 0.00 ± 0 | 0.00 ± 0 | 0.00 ± 0 | 0.00 ± 0 | 0.00 ± 0 |
| Diazepam | 1 mg/kg | 100 ± 0.00 | 100 ± 0.00 | 100 ± 0.00 | 100 ± 0.00 | 100 ± 0.00 | 100 ± 0.00 |
| HO | 5 mg/kg | 17.20 | 22.55 | 23.65 | 17.43 | 23.54 | 24.98 |
| 10 mg/kg | 22.70 | 27.43 | 28.04 | 23.45 | 28.54 | 29.65 | |
| 15 mg/kg | 28.65 | 32.54 | 33.54 | 29.54 | 33.54 | 34.87 | |
| 20 mg/kg | 32.09 | 37.54 | 36.33 | 33.45 | 38.09 | 37.65 | |
| CO | 5 mg/kg | 21.43 | 26.43 | 37.09 | 22.43 | 27.00 | 38.09 |
| 10 mg/kg | 25.54 | 31.20 | 37.43 | 26.32 | 32.00 | 39.01 | |
| 15 mg/kg | 32.43 | 36.43 | 42.33 | 33.98 | 37.87 | 43.09 | |
| 20 mg/kg | 38.87 | 42.98 | 43.00 | 49.09 | 43.09 | 51.00 | |
3.5.3 Sedative effect
The sedative activity of HO and CO is given in Table 8. The isolated oil samples (HO and CO) exhibited a moderate sedative effect as compared to the standard.
Sedative activity of oil extracts from HO and CO fractions of F. benghalensis
| Treatment | Dose | Lines crossed |
|---|---|---|
| Normal saline | 10 mL | 128.90 ± 0.23 |
| Diazepam | 0.5 mg/kg | 6.32 ± 0.98 |
| HO | 5 mg/kg | 115.43 ± 0.54 |
| 10 mg/kg | 110.22 ± 0.43 | |
| 15 mg/kg | 104.76 ± 0.65 | |
| 20 mg/kg | 97.98 ± 0.76 | |
| CO | 5 mg/kg | 118.98 ± 0.99 |
| 10 mg/kg | 112.87 ± 0.76 | |
| 15 mg/kg | 106.98 ± 0.54 | |
| 20 mg/kg | 98.867 ± 0.54 |
4 Discussion
Natural products gaining popularity with the passage of time due to their safety and easy availability. It is a general perception that natural products are free of side effects, but these natural remedies having actions as well as reactions are not possibly free of side effects. Actually, natural products accumulate various chemical constituents. These constituents might have agonistic and antagonistic effects. These agonistic and antagonistic effects are overcome by other antagonist chemicals. There is need to screen natural products rich in phytoconstituents to assess their various biological activities. In tropical and subtropical regions globally, the genus Ficus is characterized as one of the prime genera of angiosperms encompassing over 800 species of shrubs, trees, vines, and epiphytes [25,26]. These plants are most commonly known as fig trees or figs. Asian and Australasian regions are reported to be predominant regions as they contain nearly 500 species of Ficus; however, about 100–130 species have been found in Neotropics and Africa [27]. Since ancient times, F. benghalensis L. is renowned in Asian cultures owing to religious, medicinal, and mythological views. Reference of genus Ficus is present in various Holy Scriptures of different religions, such as Islam, Buddhism, and Hinduism. On the other hand, their medicinal usage has been significantly indicated in diverse traditional medicinal systems, such as Unani and Ayurveda [28].
In the current study, the fixed oil was screened against various biological effects. The natural fixed oil is a potential source of various non-polar constituents. The GC-MS study of our tested samples indicates the presence of different constituents, which might be responsible for various biological actions. The antibacterial effect represents that the plant is a good remedy against various pathogenic health issues. In this study, the antibacterial properties of oil extracts from HO and CO fractions of F. benghalensis revealed significant inhibition of S. aureus, S. flexenari, E. coli, P. aeruginosa, and B. subtilis. The result of our study is in accordance with an earlier study conducted by Murti and Kumar [29]. They compared antibacterial properties of two different species, i.e., Ficus racemosa and F. benghalensis, and were of the view that ethanolic extract of F. benghalensis showed a zone of inhibition of 30 and 22 mm against S. aureus and E. coli, respectively. Similarly, methanolic extract of F. benghalensis showed the highest inhibitory activity against Pseudomonas aeruginosa MTCC 424 [30].
In this current study, HO and CO showed the maximum free radical scavenging effect, i.e., 70.23 and 72.09 µg/mL at higher doses (100 µg/mL), respectively. The outcomes of our study were in harmony with the results of a previous study conducted by Bhaskara Rao et al. [31]. They reported that methanol extract of F. benghalensis showed significant antioxidative properties as determined by the DPPH assay. This antioxidant property of F. benghalensis might be owing to the occurrence of the high content of polyphenols in respective extracts. Moreover, Mohan et al. [32] and Ahmed et al. [33] have reported similar results.
Oil extracts from HO and CO fractions of F. benghalensis demonstrated momentously (p < 0.001) percent inhibition (63.09 and 68.32%) of writhing at a dose of 20 mg/kg. These results reveal the analgesic properties of experimented fractions. The analgesis effect of fixed oil found in this study validates the traditional usage of this plant. Results of our study regarding analgesic properties of F. benghalensis are in harmony with earlier finding of Deraniyagala et al. [34] as they documented significant analgesic effects of aqueous leaf extract. Likewise, Panday and Rauniar [35] concluded that aqueous extracts of F. benghalensis roots showed significant analgesic properties at 200 mg/kg concentration. In addition to analgesic effect, the F. benghalensis is muscle relaxant. Results of our study revealed non-significant muscle relaxant property of experimented plant. It may be possible that fixed oil being a non-polar fraction not acted as muscle relaxant while other fractions showed this property. The injection of extracted oil samples produced a slight reduction in the locomotive effect at a higher dose. The difference in pharmacological actions among the fractions attributes to the difference in chemical composition. The current study suggests further research for designing the topical pharmaceutical dosage and then testing further against various painful conditions and wounds.
5 Conclusion
The fixed oil of F. benghalensis is a significant antimicrobial and analgesic. These experimental reports support the folklore of this plant. It is further suggested to formulate the topical dosage of fixed oil.
Acknowledgment
The authors are thankful to the University of Swabi, KP, Pakistan, for providing an opportunity to complete this project.
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Funding information: None.
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Author contributions: Writing original draft preparation, Muhammad Ibrahim and Abdur Rauf; software, data curation, Naveed Muhammad; conceptualization, Anees Ahmed Khalil; methodology, formal analysis, Muhammad Akram and Qasim Aziz; writing and editing and English corrections, Zia Ullah, Yahya S. Al-Awthan, Omar Bahattab, and Mohammed A. Al-Duais. All authors read and approve this article for publication.
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Conflict of interest: The authors declare no potential conflict of interest.
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Ethical approval: The animals studied were approved by the Ethical Community of the Department of Pharmacy, University of Swabi (No. Pharm/298).
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Data availability statement: The data associated with this article are mentioned in the main text of this article.
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