Evaluation of chemical composition of seed oil and oil cake of Ailanthus excelsa (Roxb.) and its application ☆

– The purpose of this investigation was to examine the antibacterial activity of oil derived from Ailanthus excelsa (Roxb) as well as the chemical composition of seed oil and the proximate analysis of oil cake. The oil content of the seeds is ∼ 17%. The seed oil was analyzed using GC-MS/FID, and the results showed that it contained a variety of fatty acids, such as linoleic acid, oleic acid, and palmitic acid. When employed with 100 m L, the oil did not demonstrate any antibacterial activity against the bacteria Staphylococcus aureus , Salmonella typhi , Escherichia coli , Pseudomonas aeruginosa , and Bacillus subtilis . The oil does not possess any antifungal action against Candida albicans and Aspergillus ﬂ avus . The oil cake is rich in protein and minerals. These ﬁ ndings imply that A. excelsa seed oil and oil cake have the potential to be used in the food and pharmaceutical industries after ascertaining its non-toxic nature and absence of antinutrients. The oil is not having antibacterial activity hence it can be used as a part of nutrient media for bacterial cultures.


Introduction
A huge population (80%) of the world depends on traditional medicine for primary healthcare (World Health Organization, March 2022). About 70-90% of people in developing countries still use medicines made from plants and plant extracts (Chin et al., 2006). The plants have a lot of phytochemicals, which have anti-aging, anti-inflammatory, and antimicrobial properties (Cos et al., 2006) and are very interesting to the pharmaceutical industry. Antibiotics are generally target-specific as they affect cell wall synthesis, DNA replication, and the translational machinery of the bacterial cell (Krebs et al., 2017). Despite antibiotics' target functions, bacteria have evolved resistance mechanisms. Over the last decades, the exhaustive over prescription and selfmedication of clinically available antibiotics and long-term exposure of pathogenic microorganisms to these antibiotics have led to the development of antibiotic resistance (Harbottle et al., 2006). Currently, more than 70% of pathogenic bacteria are reported to have acquired resistance against antibiotic therapies (Harvey et al., 2006;Anand et al., 2019). Plants have been extensively used as a source of antibiotic, antineoplastic, analgesic, cardioprotective agents, etc. Natural products and their derivatives contribute to more than half of Food and Drug Administration (FDA)-approved drugs (Chavan et al., 2018). The development of novel, efficient, cost-effective, and noncross-resistant antibiotics has become the only alternative to treat bacterial diseases and remains a great challenge for the pharmaceutical industry (Chouhan et al., 2017). The oils have been reported to possess significant antiseptic, antibacterial, antiviral, antioxidant, anti-parasitic, anti-fungal, and insecticidal activities as these plant products contain bactericidal as well as bacteriostatic agents (Benjilali et al., 1986).
The seed oils have a variety of phytochemicals that have medicinal and nutraceutical properties. Many seed oils have been reported to have antibacterial properties. When bacteria were treated with 100 mg/mL neem oil, it created 11.7 mm and 13.0 mm of zone of inhibition for Escherichia coli and Staphylococcus aureus respectively (Sandanasamy et al., 2013). When Carnobacterium maltaromaticum, Brochothrix thermosphacta, Escherichia coli, Pseudomonas fluorescens, Lactobacillus curvatus, and Lactobacillus sakei were treated with 10 mL (by disc diffusion test) neem oil, it resulted in a minimum 89% growth reduction in each case (Del-Serrone et al., 2015). According to Khoobchandani et al. (2010) the seed oil of Eruca sativa has antimicrobial activity against Gram-negative (Shigella flexneri, Escherichia coli, and Pseudomoms aeruginosa) and Gram-positive (Bacillus subtilis and Staphylococcus aureus) bacteria. Seed oil presented the maximum zone of inhibition (74-97%) for Gram-negative bacteria and 97% for Gram-positive bacteria.
The seed oil cake is a by-product of the oil extraction process. Several studies have shown that it is a rich source of nitrogen, phosphorous, and potassium. Some seed oil cakes are rich sources of protein, minerals, and crude fibers. Jatropha curcus seed oil cake can be used as fertilizer in the tuber, leafy vegetable, and fruit crops as green manure (Kumar and Sharma, 2008). The seed cake of Blighia sapida is rich in starch (44.2%), protein (22.4%), and fibre (15.6%) (Djenontin et al., 2009). The oil cake also has a good amount of minerals like K, Ca, and Mg.
Ailanthus excelsa (Roxb.) is a multipurpose tree that belongs to the family Simaroubaceae. It is distributed in semiarid and subtropical regions. Traditionally, it has been used in the treatment of bacterial and fungal diseases. Various extracts of the root, stem, bark, and leaf of Ailanthus excelsa were analysed for their phytochemicals and used against various human pathogens (Lavhale and Mishra, 2007). The bark of A. excelsa may be recommended as a potential antimicrobial agent (Malviya and Dwivedi, 2019). The seed oil of A. excelsa has been studied for its chemical composition and its application in biodiesel (Devi et al., 1984;Kundu and Laskar, 2007;Anjaneyulu et al., 2017). Devi et al. (1984) reported 18% fat in kernel weight in Ailanthus excelsa from Andhra Pradesh (India). Similarly, the seed oil has been reported to be 32.3% in Ailanthus excelsa from the same state (Anjaneyulu et al., 2017). Kundu and Laskar (2007) reported seed oil to be 65 g/kg from West Bengal (India). The fatty acid composition of the seed oil has also been worked out, and it was found that the oil is rich in oleic acid (Kundu and Laskar, 2007;Anjaneyulu et al., 2017). However, to the best of our knowledge, there has been no report on the antimicrobial activity of the seed oil of A. excelsa. Similarly, the seed oil cake of A. excelsa has yet to be explored.
In this study, oil from A. excelsa seeds has been extracted, and its fatty acid and phytochemical composition have been analyzed using gas chromatography with a flame ionization detector (GC-FID) and gas chromatography coupled with mass spectrometry (GC-MS) respectively. The antimicrobial activity of the oil has been explored against selected bacterial and fungal strains. In this study, we have also carried out a proximate analysis of seed oil cake and tried to present its potential applications.

Plant material
Mature fruits of A. excelsa were collected from the Amity University Rajasthan campus located in Jaipur, India. The seeds were separated from the fruits by a mechanical method. To find out the moisture content in seeds, 10 g seeds were kept in a hot air oven at 65°C. After 24 h, the seed weight was recorded. This experiment was replicated thrice. The average moisture content was found to be 18.67% (w/w).

Oil extraction from seeds
The seeds of A. excelsa were crushed with a mortar and pestle into a fine powder. The oil was then extracted using the soxhlet apparatus using n-Hexane as a solvent (boiling point: 67-68°C) (Saini et al., 2019). The oil was then kept at room temperature in a sealed glass vial until it was used to study its chemical composition and determine its antimicrobial activities.

Phytochemicals in seed oil
A. excelsa seed oil was analyzed by GC-MS (Agilent 8890/ 5977B series Agilent 5977B EI/CIMSD) at a pressure range of 0.001 to 13.886 psi with 0.01 to 100 psi resolution, with a 30 m Â 250 mm Â 0.25 mm DB 5 MS column front SS inlet with nitrogen as the carrier gas and the same in the back SS inlet with helium as the carrier gas, which flowed 1 mL/min for 50 minutes, the temperature range was set at 160-300°C. Oil was diluted in hexane at 0.1 mg/mL with a molecular weight of less than 500 g/mol, and 1 mL solution was injected. The initial average velocity at 160°C was 38.194 cm/s, and the hold-up time was 1.30 minutes. At the rear SS, a triple-axis detector with a high-energy dynode and electron multiplier autosampler had a temperature range of 114.3-300°C and an electron mass of 236.3 Hz, which was connected to the TIC, MS library (NIST 20 L), and Agilent Mass Hunter.

Fatty acids composition study
For fatty acid composition, gas chromatography was used. FID No Trace 1300 with analytical column ZB FAME 30 mm Â 0.25 mm MID Â 0.24 m was used for fatty acid analysis. The oil was converted to fatty acid methyl ester (FAME), and 1 mL of the sample was injected into the inlet column with the help of a syringe. Hydrogen was used as a carrier gas, and the flow rate was 1.2 mL/min, the initial temperature of the inlet column was 100°C, the rise in temperature was 10°C/min; the final temperature was 240°C and the FID detector temperature was 260°C with 2 minutes of hold time.

Preparation of test sample
The seed oil was used directly (1X) and at 50% concentration by dissolving it into n-Hexane (0.5X).

Antibacterial assay
Bacterial cultures were resurrected using appropriate nutrient media. Staphylococcus aureus ATCC 25923, Salmonella typhi ATCC 733, Escherichia coli ATCC 25922, and Pseudomonas aeruginosa ATCC 27853 were streaked and incubated at 37°C for 24 h. Cultured bacterial strains were activated and diluted aseptically with sterile peptone water to obtain 0.5 McFarland turbidity for working standard inoculum and then cultured on Muller-Hinton agar (MH) media. Similarly, an antibacterial study against Bacillus subtilis, E. coli, and Pseudomonas aeruginosa was carried out where the activated bacterial cultures with more than 0.6 optical density (600 nm) have been spread on Muller-Hinton agar (MH) media.
A hole with 8 mm internal diameter was created to add 100 mL sample (1X oil, 0.5X oil, n-Hexane, standard antibiotics) with the bacterial lawn (Tab. 1A). The oil was diluted to 0.5X using n-Hexane. A suspension of streptomycin (0.3 mg) was used as a positive control for Staphylococcus aureus ATCC 25923 and Escherichia coli ATCC 25922. A positive control for Salmonella typhi ATCC 733 was chloramphenicol disc (0.3 mg), and a positive control for Pseudomonas aeruginosa ATCC 27853 was ceftazidime disc (0.3 mg). As a negative control, n-Hexane was used.

Antifungal activity
Fungal cultures were revived on Sabouraud dextrose agar plates. Candida albicans ATCC 14053 and Aspergillus flavus (clinical isolate: 46047918) were streaked and incubated at 27°C for 2-3 days. For the antifungal activity study well diffusion method was used with Sabouraud dextrose agar medium. The standard 0.5 McFarland turbidity value for each culture was obtained as mentioned in the earlier method. The culture was swabbed on a Sabouraud dextrose agar surface, and wells were created with the help of sterile tips. 100 mL of each test sample (1X and 0.5X oil) was loaded into the respective well. A suspension of itraconazole in DMSO (5 mg/mL) was used as a positive control and n-Hexane as a negative control for both fungal strains. The plates were incubated at 27°C for 2-3 days for fungal growth, and then the diameter of the inhibition zone was recorded in mm.

Proximate analysis of seed oil cake
Proximate analysis refers to the quantitative analysis of macromolecules, a combination of different techniques such as extraction, Kjeldahl, and NIR (near infrared) are used to determine protein, fat, moisture, ash, carbohydrate, and mineral levels in a sample. The fat content of seed oil cake was determined using the BIOSOX automatic solvent extraction system IS 4684 (1975). Crude fibre, ash, and moisture content were determined by using the IS 7874, Part I, (1975). Phosphorous was determined using the ISO 6491 (1998) animal feed phosphorous determination method. The protein content of the sample was estimated using the BIOKJEL nitrogen estimation system IS 7219 (1996) and the FSSAI Lab Manual (2016). Potassium and zinc were determined according to the protocol for metals in food established by the AOAC in 2015 (Gill et al., 2015).  3 Results and discussion

Seed oil and chemical composition
The oil content in the seeds of Ailanthus excelsa has been found to be 16.67% (w/v) when the seed moisture was 18.67% (w/w). These findings are a bit different from earlier reports of oil/fat variation from 6.5% to 32.3% (Devi et al., 1984;Kundu and Laskar 2007;Anjaneyulu et al., 2017). This variation could be due to the seed material used, the extraction methods used, or a regional or climate effect.
This light-yellow oil was used to study chemical composition using GCMS, and the results are mentioned in Table 2. Based on the results, the oil is mostly consisting of oleic acid, n-Hexadecanoic acid, and octadecenoic acid. The oil is rich in oleic acid. The oil also contains 9-Octadecenoic acid, 2-Hydroxyethyl ester, and 1-Ethynycyclododecanol in addition to fatty acids. Our findings are in agreement with earlier reports where the oil has been found to be rich in oleic acid (Kundu and Laskar, 2007;Anjaneyulu et al., 2017).

Antibacterial activity
The antimicrobial activity of Ailanthus excelsa seed oil against Staphylococcus aureus ATCC 25923, Escherichia coli ATCC 25922, Salmonella typhi ATCC, and Pseudomonas aeruginosa ATCC 27853 was investigated, and the results are shown in Table 1A and in Figures 1 and 2. The results indicate that the oil has no antibacterial activity against Staphylococcus aureus ATCC 25923, Escherichia coli ATCC 25922, and Salmonella typhi ATCC 733. Similarly, no antibacterial activity of the seed oil has been recorded against Bacillus subtilis, E. coli, and Pseudomonas aeruginosaw. However, low-level antimicrobial activity against Pseudomonas aeruginosa ATCC 27853 has been found with 1X seed oil with a very poor zone of inhibition, while the standard antibiotic Ceftazidime could create a zone of inhibition with a diameter of 26 mm (Tabs. 1A and Figs. 1 and 2). The antibacterial property of the seed oil depends on its chemical composition. The seed oil of A. excelsa seems to have no such phytochemicals that can inhibit bacterial growth, or the concentration of such compound(s) is too low to be toxic for  the bacterial strains under study. However, seed oils obtained from Leuconia leucocephala, Callophyllum inophyllum, Moringa oleifera, Balanites aegyptiaca, Prosopis spp., Eruca sativa, and Azadirechta indica have been found to have significant antibacterial activities (Khoobchandani et al., 2010;Aderibigbe et al., 2011;Chothani and Vaghasiya, 2011;Saadabi and Zaid, 2011;Sandanasamy et al., 2013;Adewuyi et al., 2014;Imam et al., 2019).

Antifungal activity
The observations of the antifungal activities of the seed oil of Ailanthus excelsa against Candida albicans ATCC 14053 and Aspergillus flavus (clinical isolate: 46047918) are presented in Table 1B and Figure 3. The seed oil of some plants had antifungal properties. Aderibigbe et al. (2011) reported that the seed oil of Leuconia leucocephala had antifungal activity against Aspergillus niger, Rhizopus stolon, Penicillum notatum, and Candida albicans. Antifungal activity of Hyptis suaveolens (Poit.) seed oil from Kalagarh region (Uttarakhand State, India) was tested against fungal strains of Candida albicans MTCC 227 and Candida tropicalis MTCC 227, which show minimal inhibitory concentrations of 0.125 mg/mL and 0.25 mg/mL, respectively (Bachheti et al., 2015). This variation in antifungal activity in the seed oils can be due to variations in the chemical composition of the seed oil, which is determined by the genus of the plant species and climatic conditions.

Potential applications of the seed oil of A. excelsa
The phytochemicals present in A. excelsa seed oil can be used to treat health problems. Aparna et al. (2012) and Ravi and Krishnan (2017) reported that n-Hexadecanoic acid has anti-inflammatory and anti-cancer properties. According to their molecular docking analysis, n-Hexadecanoic acid interacts with topoisomerase I (a DNA replication and repair enzyme). They observed significant cytotoxicity against human colorectal carcinoma cells (HCT-116) with an IC 50 value of 0.8 mg/mL. The seed oil of A. excelsa is also rich in n-Hexadecanoic acid. Hence, it may be used for antiinflammatory and anti-cancer purposes. Oleic acid may be utilized for anti-inflammatory, anti-androgenic, anti-cancer, preservative, and hypocholesterolemic properties (Sreekumar et al., 2014). The seed oil of A. excelsa can also serve the same purpose due to the presence of oleic acid in it. Other seed oil constituents (octadecanoic acid, 9-Octadecenoic acid 2-Hydroxyethyl ester) have antioxidant, anti-inflammatory, antimicrobial, and diuretic activity Hussein et al. (2016), Osuntokun (2021) and Burt (2004) reported that some of the oil constituents can be used for the cosmetic, sanitary, food industry, and antimicrobial activity. These properties of oil components indicate potential applications of the seed oil of A. excelsa for various purposes. Table 4 shows the approximate composition of A. excelsa seed oil cake. The proximate analysis of A. excelsa seed oil cake shows that it is a good source of protein (51.38%), fibre (7.22%), and ash (10.87%), which is higher than the oil cake of Balanites aegyptiaca where protein, crude fibre, and ash contents are 17.7%, 5.95%, and 9.1% respectively (Ogori et al., 2017(Ogori et al., , 2018. The oil cake of A. excelsa can be used as feed for livestock and as a protein source for humans after assuring the absence of any toxic substance(s) or antinutrient(s). Swietenia mahagoni seed oil cake had 8.76% protein and 19.60% crude fibre (Mostafa et al., 2011). Protein quantity varies in seed oil cakes in different species, like Cucurbita pepo, Cannabis sativa, and Linum usitatissimum, which were reported to have 38.27%, 24.77%, and 32.83% of protein, respectively (Budzaki et al., 2018). The high ash content (10.87%) makes it a good source of minerals for animals and biofertilizers.

Conclusion
The oil from the seeds of Ailanthus excelsa was analyzed using GC-MS, and it was found to contain the following compounds: n-Hexadecanoic acid, oleic acid, octadecanoic acid (stearic acid), 9-Octadecenoic acid, 2-Hydroxyethyl ester, and 1-Ethynycyclododecanol. The fatty acid composition study reveals the predominance of oleic acid followed by linolenic acid in the oil. The oil has antibacterial activity against Pseudomonas aeruginosa. However, the oil has been found to have no cidal activity against Staphylococcus aureus ATCC 25923, Salmonella typhi ATCC 733, Escherichia coli ATCC 25922, Candida albicans ATCC 14053, and Aspergillus flavus. The seed oil cake has a high protein content (> 51%) and is also rich in ash and fibres. So, it may be used as a feed or food after assuring its nontoxic nature and absence of antinutrients.