The antimicrobial and antioxidant property, GC–MS analysis of non-edible oil-seed cakes of neem, madhuca, and simarouba

Background: The antimicrobial activity against clinically and agriculturally important microorganisms, antioxidant property and chemical profiling of acetone crude extracts of non-edible oil-seed cakes of neem (Azadirachta indica A. Juss), madhuca (Madhuca longifolia syn. Madhuca indica Gmelin) and simarouba (Simarouba glauca DC) obtained by hot and cold extraction methods were tested in-vitro. Results: The hot neem and cold acetone extracts of madhuca and simarouba were inhibitory to Staphylococcus aureus. The enteric pathogens-Enterococcus faecalis and Salmonella enterica were inhibited by both hot and cold extracts of simarouba cake. Fusarium oxysporum and Colletotrichum capsici were sensitive to oil-seed cake extracts of madhuca and simarouba cake extracts followed by neem cake extract. The Aspergillus flavus was highly sensitive to neem followed by madhuca and simarouba extracts. The cyclic voltammetry of all extracts indicated the presence of oxidation peaks at different Epa values suggesting the presence of antioxidant ability. The GC–MS profile revealed the presence of pharmacologically important essential compounds. Conclusion: The study revealed the presence of bioactive compounds in non-edible oil-seed cakes that could be exploited for human welfare.


Background
India is a home to several non-edible oil-yielding plant species such as jatropha, castor, pongamia, neem, madhuca, and simarouba (Padhi and Singh 2011). Oil-seeds produced in such plant species are the potential source for biodiesel production (Gubitz et al. 1999;Sharmin et al. 2006;Martin et al. 2010). The biodiesel sourced from plants is better than the conventional diesel fuel in their physico-chemical properties and biodegradability (Knothe 2006;Scott et al. 2008). The above features of biodiesel prompted the increased utilization of nonedible oil-seeds to produce biodiesel. The oil seed-cake, the by-product of biodiesel industry from oil-seeds, can neither be directly utilized as an animal feed nor in agriculture due to the presence of certain phytotoxic compounds in them (Makkar et al. 1998;Akintayo 2004). However, the oil-seed cakes are rich in cellulose, hemicellulose, and lignin (Culcuoglu et al. 2002) besides high nitrogen, phosphorus, and potassium contents and hence cakes could be used as the source of organic fertilizers. Several toxic and non-toxic phytochemicals contained in oil-seed cakes could be isolated and utilized in various industries (Prasad et al. 2005;Govindaraju et al. 2009;Kumari et al. 2013).
The indiscriminate use of synthetic chemical compounds and antibiotics for the control of plant and Page 2 of 14 VasudhaUdupa et al. Bull Natl Res Cent (2021) 45:41 human pathogens poses severe problems to human and environmental health, resulting in the development of multi-drug-resistant pathogens and contamination of the food chain in the ecosystem (Crouse 1998). In contrast to this, the above plant-based metabolites are biodegradable and could become the best source alternative to synthetic chemicals and antibiotics (Joshi et al. 2011;Parimala and Shoba 2014).
The oil-yielding plant species such as neem, madhuca, and simarouba also find application in traditional medicinal preparations since they contain diverse phytochemicals with promising therapeutic value for treating several infectious diseases (Govindachari 1992;Mansi and Gaikwad 2011). Neem (Azadirachta indica A. Juss, family Meliaceae) is a predominant source of limonoids (Kavathekar 2003) like azadirachtin (0.2-0.6%, w/v) in the seed kernels (Govindachari 1992). Madhuca (Madhuca longifolia syn. Madhuca indica Gmelin, family Sapotaceae) is the primary source of semisolid fat along with other phytochemicals, such as saponins (Kumar et al. 2011). Simarouba (Simarouba glauca DC, family Simaroubaceae) also the rich source of oil (60%) contains the bitter terpenoids and quassinoids in the oil-cake (Severan 1953). The seeds, as well as seed-cakes of neem, madhuca, and simarouba, are being incorporated into the soil to control plant pathogenic nematodes and insects (Prasad et al. 2005;Saha et al. 2010;Ashraf and Khan 2010;Rizvi et al. 2012).
The utilization of non-edible oil-seed-cakes is mainly for their role in protecting plants from soil-borne phytopathogens, nematodes, and insect pests (Rameshchandran et al. 2007). Further, the literature survey indicated that the oil-seed cakes of the neem, madhuca, and simarouba had not been studied for their antimicrobial and antioxidant properties, as well as the phytotoxicity. The present study aims at determining the antibacterial, antifungal, and antioxidant activities in extracts of oilseed-cakes of neem, madhuca, and simarouba and the chemical profiling of oil-cakes by GC-MS analysis.

Solvent extraction of oil-seed cakes
The oil-seed-cakes of neem, madhuca, and simarouba were collected from the Biofuel Production Unit, Hassan, Karnataka. The above oil-seed cakes were air-dried under ambient conditions in the shade for 5-7 days and stored in air-tight zip-lock covers at 5º C. The oil-seed-cake (500 g) of each plant species was subjected to sequential hot extraction with hexane, ethyl acetate, acetone, and water for 24 h using the Soxhlet apparatus (Soxhlet 1879). Another set consisting of the above cake samples was also extracted with the hexane, ethyl acetate, acetone, and water solvents, but in the cold for 24 h, using a rotary shaker (120 rpm). The crude extracts were recovered under vacuum and stored at 4ºC in air-tight vials, until use.

Phytochemical assay
The acetone extracts of oil-seed cakes obtained from both methods were subjected to qualitative phytochemical analysis (Sasidharan et al. 2011). The stock solution of each extract was prepared by dissolving the crude residue of plant extract (100 mg) separately in DMSO and stored in glass bottles. The stock solutions were then subjected to the assay of alkaloids, flavonoids, flavones, phenols, sterols, triterpenoids, coumarins, glycosides, tannins, lignin, quinones, anthraquinones, plobatannins, volatile oil and saponins, and primary metabolites, as well.

Antimicrobial assay
The in vitro antimicrobial activity of acetone extracts of oil-seed-cakes was determined by the well diffusion method (Bauer et al. 1966 All the bacterial isolates were cultured in the nutrient broth for 16 h, and 100 µL of each culture was spread on a nutrient agar medium. One loop-full of three-day-old sporulating fungal culture was suspended in sterile water (1 mL), and 100 µL of each mixture spread separately on potato dextrose agar. The antibacterial standards (ampicillin, chloramphenicol, or ciprofloxacin) and antifungal standards (fluconazole, griseofulvin), or bavistin (5 mg each) taken separately in DMSO (1 mL) were considered as positive controls. At the same time, only DMSO was recognized as the negative control. The stock solution of each extract in DMSO (200 mg mL −1 ) was prepared and diluted to obtain the concentration of 200, 100, 50, or 25%. The individual extract was placed into 5 mm dia. wells in the agar medium and incubated at 37ºC for 24 h in case of test bacteria and 30ºC for 72 h in case of fungal isolates. The clear zone of inhibition (ZI, mm) around the well indicated the antimicrobial activity of the extract.

Assay of the electrochemical potential of extracts
The electrochemical potential of the acetone extract of oil-seed cakes was determined by the cyclic voltammetry (CV model CHI660c potentiostat) (Arulpriya et al. 2010). The analytical system consisted of a two-compartment Pyrex cell with a conventional three-electrode configuration, a carbon paste working electrode (Teflon tube 3 mm dia) activated by mixing 70% graphite powder and 30% silicon oil, a platinum wire counter electrode and a saturated calomel reference electrode. About 10 mL of phosphate buffer (pH) (supporting solution) was dispensed into an electrochemical cell to record the voltammogram of the blank. The appropriate volume (2 mM) of acetone extract was added to the supporting solution, and CV measurements were recorded at positive potential (− 0.2 to + 1.0) at the scan rate of 50 to 300mVs −1 at room temperature.

GC-MS analysis
The acetone extracts of neem, madhuca or simarouba oil-seed cakes were analyzed for their chemical constituents by a combined Gas Chromatography (GC) (Thermo Scientific Trace 1310) and triple quad Mass Spectrometer (MS) (Thermo Scientific TSQ 8000) using DB 5MS (30 cm length, 0.250 mm ID) silica column and helium as a carrier gas. One microliter of the sample was injected into the column at 250˚C. A column temperature initiated the GC program at 40˚C for 2 min. and increased to 240˚C at 5˚C m −1 rate.
Further, the temperature was increased to 300˚C at a scan range of 50 and 600 Da. The total period of 47 min. was required to complete the entire run. The data were analyzed by the software Calibur 4.0, and chromatograms obtained were analyzed and identified by matching their mass spectral fragmentation patterns with a database deposited at the National Institute of Standards and Technology Mass spectral database (NIST 2.2) library.

Statistical analysis
Each assay was performed in three replicates with a completely randomized design, and the Zone of Inhibition (ZI) was expressed as the mean ± standard error.

Phytochemical screening of oil-seed cakes
The oil-seed-cakes of neem, madhuca, and simarouba upon extraction by cold and hot solvents yielded primary and secondary metabolites. The hot extraction method produced more volume of extracts when compared to the cold extraction method. The acetone extracts yielded polar aprotic phytochemicals. The oil-seed-cakes of selected species tested positive for alkaloids, flavonoids, glycosides, lignins, phenols, sterols, tannins, and triterpenoids. Coumarins were documented only in neem oilseed cake. Flavones and lignins were present in the cold and hot acetone extract, respectively, in all seed-cake extracts. Quinones and anthraquinones in madhuca and simarouba oil-seed cakes were extractable only by the hot acetone.

Antibacterial activities of oil-seed cakes
Hot acetone fraction of neem oil-seed-cake produced prominent ZI to S. aureus. In contrast, the cold acetone fraction strongly inhibited E. faecalis, which was comparable to that of the standard antibiotic amoxicillin. However, cold acetone fraction was less active for S. aureus and E. faecalis, respectively. This observation pointed out that compounds extracted into cold acetone could be acting with a different mode of action depending on the test isolate. Alternatively, a high extract concentration of 200% could be required to produce a high ZI (Table 1).
Cold acetone fraction of madhuca oil-seed-cake exhibited the ZI of 8.1 mm to S. aureus, which is slightly less effective than amoxicillin (11 mm). On the other hand, hot acetone fraction failed to inhibit test isolates, except P. fluorescence. In the case of simarouba oil-seed-cake, cold acetone extract produced a good ZI comparable to that of amoxicillin. In contrast, the hot acetone extract at 100% caused high ZI (12.2 mm) to S. enteritica, which is similar to that of the amoxicillin. A further increase to a concentration of up to 200% increased inhibition to 18.9 mm. On the other hand, cold acetone extract was the least effective for E. coli and P. fluorescence even at an elevated concentration.
The cold acetone fraction of simaouba oil-seed cake at high concentration also inhibited the colony growth of S.aureus, E. faecalis, and S. enterica, comparable to amoxicillin control (Table 1). The above observations indicated that inhibition of colony culture of test organisms increased with an increase in the concentration of the extract. Among the three antibiotic standards used, ciprofloxacin followed by chloramphenicol was most effective by producing the high zone of inhibition.

Antifungal activity of oil-seed cakes
The oil-seed cakes of selected plant species also showed antifungal activity to certain test fungal species. Both the hot and cold extractions were active for at least three fungal species. Fusarium. oxysporum, C. capsici, and A. flavus as well as C. albicans, were sensitive to all the oil-seed-cake extracts. The colony growth of F. oxysporum and A. flavus was inhibited by cold and hot acetone extracts of neem cake, much like that exhibited by standards-fluconazole and bavistin (Table 2). While hot acetone extract was efficacious for C. capsici, it was ineffective for C. albicans. The madhuca oil-seed-cake extract was comparatively similar to standards fluconazole and bavistin for F. oxysporum. Cold acetone extracts inhibited the growth of C. albicans. (Table 2). The electrochemical potential of extracts An irreversible anodic peak was produced due to hot acetone extract of neem at 0.42 V, but no peaks were recorded in the cold extract, which could be due to the absence of compounds with observable redox potential (Fig. 1). On the other hand, the voltammogram of a hot extract of simarouba showed two peaks at 0.19 V and 0.68 V, while one peak at 0.22 was observed in the cold extract at 300 Vs −1 scan rates. Both the hot and cold acetone extracts of madhuca oil-seed cake produced similar peaks at 0.19 V (Fig. 1).

Discussion
The potential of oil-seed cake extracts of neem, madhuca, and simarouba as antibacterial, antifungal and antioxidant, along with GC-MS profile was studied. The acetone extract of all three oil-seed cakes had high bioactivities. Acetone extracts both hydrophilic and lipophilic components of plants and is less toxic to bioassays (Das et al. 2010). The antibacterial activity depended on the chemical constituents of oil-seed-cake and the method of extraction. According to Eloff (1998), acetone is the most suitable solvent for the removal of both polar and nonpolar phytochemicals and microbial inhibitors. The hot neem cake extract was more efficient in inhibiting the S.
aureus and E. faecalis than the cold extract. The observed antibacterial activity of neem cake was evidenced with the presence of several biologically active limonoids such as azadirachtin A, and triterpenoids like salanin, nimbin, nimbidin, and gedunnin (Govindachari 1992). Salanin, the primary compound in neem cake, was shown with excellent antibacterial activity to meat-borne bacteria and hence is known as the natural preservative for meat storage (Sarone and Nicholetti 2013). Cold acetone fraction of madhuca oil-seed-cake was inhibitory to S. aureus, but the hot extract was not effective to the bacterial pathogen. The reduced antibacterial activity in hot extracts as compared to the cold ones could probably hint at the denaturation of certain thermolabile compounds with antibacterial activity (Adeshina et al. 2011;Rubila and Ranghanathan 2014). The madhuca cake contains compounds like saponins,  flavonoids, and glycosides that have been known for their antibacterial activity (Yadav and Singh 2012). However, certain extracts extracted either by hot or cold methods had a relative activity like that of amoxicillin. Amoxicillin, being bactericidal is shown to inhibit bacterial cell wall synthesis (Harold 1974). Some of the oil seed-cake extracts showing good inhibition ability might be having the same mode of action similar to that of amoxicillin. Previous reports have shown that specific metabolites containing alkaloids, flavonoids, and phenols inhibited the clinical pathogen S. aureus (Shinde and Mulay 2015). The optimal effectiveness of these secondary metabolites and their antimicrobial activity and exhibited mechanisms are not fully understood, although specific studies attempted to decipher the mechanism(s) involved. While alkaloids are microbicidal (Ghoshal et al. 1996), phenolic compounds such as flavones were inhibitory to the growth of A. niger and C. albicans and B. subtilis, E. coli,  et al. Bull Natl Res Cent (2021) 45:41 and S. epidermidis (Rahua et al. 2000). The mode(s) of action of flavonoids is related to the formation of complexes with extracellular soluble protein and bacterial cell walls (Gorniak et al. 2019) and inhibition of DNA gyrase and β-hydroxy-acyl carrier with protein dehydrogenase activity (Tsuchiya et al. 1996;Cushine and Lamb 2005). On the other hand, tannins and terpenes are shown to act by disturbing the cell membrane integrity by binding with polysaccharides and proline-rich proteins. At the same time, coumarins affected the respiration rate of the cell (Chung et al. 1998;Shimada 2006). Previous studies also demonstrated the antibacterial activity in leaf extracts of neem, madhuca, and simarouba (Vinoth et al. 2012;Kathuria and Singh 2015;Valdes et al. 2008), and the findings of this study that oil-seed-cakes also exhibited antibacterial activity might throw light on the presence of similar compounds with antibacterial activity. An ethnobotanical investigation of simarouba indicated that leaf and bark extracts could be used to treat dysentery and malaria (Mansi and Gaikwad 2011). The findings of the present investigation also suggested that the oil-seed cake of simarouba inhibited enteric bacterial species used in the study (Table 2). This indicated the requirement of more detailed reviews to exploit the benefits of simarouba oil-seed cake, which is otherwise disposed-off unutilized. The active compound in simarouba, the quassinoids, is bitter triterpenoid with a polycyclic skeleton. The most known quassinoids in simarouba are alanthinone, glaucarubinone, and holacanthone along with benzoquinone, glaucarubine, melianone, simaroubidin, simaroubin, and sitosterol (Valdes et al. 2008). These quassinoids are shown with  et al. Bull Natl Res Cent (2021) 45:41 antimicrobial, antiamoebic, and antiprotozoal activities (Write 2005;Nurhannan et al. 2005), and the compound inhibited the synthesis of protein and nucleic acid in the malarial pathogen (Kirby et al. 1989). The present study confirmed that the oil-seed-cakes of plant species were inhibitory to colony growth of F. oxysporum, C. capsci and C. albicans. The hot acetone simarouba oil-seed cake extract moderately inhibited A. flavus. This fungus is a common saprophyte capable of causing aspergillosis involving paranasal sinuses (Kameswaran and Raghunandan 2009) and aflatoxicosis in humans following the consumption of cereal grains and pulses contaminated with toxigenic strains of A.flavus (Abd Ek-Raheem et al 2014). The most notable view of the present study is the very high antifungal activity of the cold acetone extracts of madhuca against F. oxysporum, which had an inhibitory effect by nearly two folds to that of the systemic fungicide bavistin. Bavistin being a systemic fungicide is very useful to control Fusarium species, which caused wilt and other symptoms of disease in crop plants (Wani et al. 1982). The oil-seedcake extracts of neem, madhuca, or simarouba could be considered the promising sources of compounds to manage A.flavus contamination of grains (Kavitha et al. 2014). Except for the cold acetone extract from madhuca oil-seed cake, most oil-seed cake extracts of the above plant species were not highly effective for C. albicans. Hence, Candida albicans is a clinical pathogen capable of causing infections that persist for a long time in humans (Calderone and Clancy 2012).   , antifungal, antioxidant, antiseptic, antitumor, anti-inflammatory, anti-hyperlipidaemia (Marchese et al. 2016) 7-Methyl-Z-tetradecen-1-ol acetate Madhuca (cold) 0.52 Anticancer, anti-inflammatory, hepatoprotective (Hameed et al. 2015) Chloramben, methyl ester Madhuca (cold) 0.12 Herbicide (Wehtje et al. 1992) Nonanal Simarouba (cold) 0.99 Antibacterial (Bisignano et al. 2001), Antifungal (Zhang et al. 2017) Antidiarrheal (Miguel et al. 2008) VasudhaUdupa et al. Bull Natl Res Cent (2021 The cyclic voltammetry is a powerful electrochemical technique employed to investigate the reduction and oxidation potential of molecular species (Suliborska et al. 2019). This method correlated with the antioxidant assays by chemical and spectrometric means (Martinez et al. 2006;Keffous et al. 2016). Firuzi et al. (2005) observed that there was a correlation between electrochemical activity and redox potential of molecules/compounds with antioxidant properties. In the present study, maximum peaks were observed at relatively low potential (Epa < 0.45 V). It is found that compounds with strong scavenging capacities are oxidized at relatively low potential (Sousa et al. 2004). Simic et al. (2007) demonstrated that compounds with little Epa (< 0.45 V) act as high antioxidants and with high Epa (> 0.45 V) act as prooxidants. The observed peaks in the oil-seed cake extracts indicated the presence of low molecular weight antioxidants. According to Born et al. (1996), compounds with catechol group oxidized below 0.4 V and compounds having one or two phenolic groups showed antioxidant potential between 0.45 and 0.8 V.
Based on the above studies, some of the constituents revealed by GC-MS are biologically active compounds. They were proven to possess pharmacologic activities that may contribute to the healing potential of the cake extracts. The major chemicals include glycerine in madhuca, and neem cake has antibacterial activity (Nalawade et al. 2015). In the case of simarouba cake extract, diacetone and hydroxyl ketone were documented as an antioxidant and anticancer agent (Zhang et al. 2015;Lyantagaye 2013;Kawsar et al. 2015). The GC-MS profile of the extracts revealed the presence of potential antioxidants ( Table 3). The hot neem extract contained previously known antioxidants that includes methyl gallate (Lubis et al. 2018), vinyl sulphide (Ianiski et al. 2018), 5, hydroxymethylfurfural (Zhao et al. 2013), 6-hydroxyl-3-phenyl coumarin (Matos et al. 2015), cyclic octa atomic sulfur (Ojinnaka et al. 2015). The anti-HIV metabolites include methyl gallate (0.39%), canthin-6-one (2.02%) from neem hot and simarouba cold acetone extracts, respectively (Guvensen et al. 2019;Wang et al. 2014;Ahmed et al. 2017), while 2,4-di-tert-butylphenol and vanillic acid are also anticancer agents besides its antimicrobial, antioxidants activity (Gazoni et al. 2018;Yemis et al. 2011;Dehpour et al. 2012). On the other hand, angelic acid is identified as the potent antioxidants, spasmolytic, besides its usefulness to treat nervous problems (Varsha et al. 2015). Several compounds listed are also reported with pharmaceutical applications (Table 3).

Conclusion
This study demonstrated that extracts from neem, madhuca, and simarouba oil-seed cakes had antimicrobial activities against significant bacterial and fungal isolates with clinical or agricultural importance in the highest concentration (200%). The oil-seed cakes contained a variety of phytochemicals with biological significance. The oil-seed cakes could be exploited for chemicals that find application as antimicrobial agents in new drugs for the treatment of infectious diseases caused by pathogens and for the remedy of phytopathogenic diseases responsible for yield reduction. The isolation and utilization of herbicidal compounds (Halim et al. 2017) from oil cakes of simarouba and madhuca could be a promising strategy in organic farming practices. Further studies are required to enhance the activity of oil-seed extracts and study the mode of action of the bioactive compounds individually and in combination. The present investigation highlights the utilization of bio-waste for the development of new environmentally safe antimicrobial and antioxidant agents of plant origin.