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Assessment of antimicrobial and antioxidant potential of cytoplasmic male sterile lines of pepper

Assessment of antimicrobial and antioxidant potential of cytoplasmic male sterile lines of pepper In the present study eight Cytoplasmic Male Sterile (CMS) lines of pepper and one open pollinated commercial variety ‘California wonder’ were assessed for their antimicrobial and antioxidant potential. For antimicrobial activity, fresh fruits crushed in 10%, 20%, 30% and 40% methanol were analyzed. Thirty per cent and forty percent methanolic extracts were found to be best activator of metabolites exhibiting antibacterial activity. Highest antibacterial activity was shown by 40% methanoic extracts of KCS2A, KCS3A and AVPP9907-S of Capsicum CMS lines against different bacteria. The extracts of all lines found ee ff ctive against Yersinia pestia . Phenolic content of all CMS lines was determined by extracting fruit in abso- lute ethanol, it was found in the range of 594.99–1710.13 μg gallic acid/gfw. Phenolic content was highest in AVPP0710-S (1690.48 ± 19.65 μg gallic acid/gfw) and lowest in KCS 2A. FRAP, CUPRAC and DPPH assay were done to find out anti- oxidant potential of CMS lines under study. FRAP and CUPRAC were analyzed by crushing fruits in 80% ethanol. FRAP was found in the range of 0.9–3.12 µmol trolox/g, with AVPP0710-S exhibiting highest FRAP value (2.77 ± 0.3522 µmol trolox/g). CUPRAC was found in the range of 0.7–11.22 µmol trolox/g and AVPP9908-S showed highest CUPRAC value (8.55 ± 0.922 µmol trolox/g). Free radical scavenging DPPH assay was done by extraction in absolute methanol and was highest in AVPP9908-S (32.94 ± 3.61%) with 9.91–36.55% overall range. This study will pave the way for the development of genotypes with higher phenol, antioxidant and antimicrobial activity in pepper. Keywords Cytoplasmic male sterile · Pepper · Antimicrobial activity · Antioxidant · DPPH 1 Introduction unstable molecules that the body produces as a reaction to environmental and other pressures. Antioxidants can Peppers are an important group of vegetables which are protect against the cell damage that free radicals cause, grown on a large scale all over the world. It includes both, known as oxidative stress. They contain high concentra- mild or less pungent fruit types (sweet pepper) and pun- tion of polyphenols which includes flavonols, flavones, gent fruit types (hot pepper) [1]. A wide terminology is anthocyanins and hydroxycinnamates [4, 5]. These bio- used to designate peppers like chili, chile, chilli, aji, paprika compounds are known to enhance the antioxidant activ- and capsicum [2]. ity of peppers [4, 6, 7]. The antioxidant potential works Peppers are a rich source of various antioxidans like by preventing the oxidation of lipids by scavenging the capsaicinoids, ascorbic acid, carotenoids, Vitamin E, pro- free radicals. Vitamin E, C and beta carotene are of special vitamin A etc. [3]. Antioxidants are substances that can concern in increasing the antioxidant potential of peppers prevent or slow damage to cells caused by free radicals, [8]. Hot peppers are reported to show high antioxidant * Pooja Saklani, [email protected] | Department of Biotechnology, HNBGU (A Central University), Srinagar, Uttarakhand, India. Division of Vegetable Breeding, ICAR-IARI, Pusa Campus, New Delhi, India. SN Applied Sciences (2020) 2:1181 | https://doi.org/10.1007/s42452-020-2902-1 Vol.:(0123456789) | Research Article SN Applied Sciences (2020) 2:1181 https://doi.org/10.1007/s42452-020-2902-1 potential due to high levels of capsaicinoids in them which formulated to assess the antimicrobial and antioxidant also impart pharmacological properties to them [9]. Phe- potential of eight cytoplasmic male sterile lines of Capsi- nols reduce the risk of lipid autoxidation by scavenging cum annuum. The study would enforce the production of free radicals and thereby inhibit the initiation of oxidation good quality pepper hybrids, the consumption of which chain [10]. Flavonoids impart organoleptic properties such can act as robust nutraceutical. as taste to the fruits [11]. Anthocyanins are natural pig- ments which impart diverse colors to the plant life and flavonols are induced by sunlight which shields the plant 2 Materials and methods from harmful effects of ultraviolet rays [12]. In today’s scenario, microbial infections and resist- 2.1 Collection of plant material ance to antibiotic drugs have been the main causes that threaten the health of peoples. Over the decades, it has Eight CMS lines of pepper were used for conducting the been observed that the microbial infections are respon- present investigation. Out of eight CMS lines, five were sible for millions of deaths throughout the world. The procured from AVRDC, Taiwan (AVPP9908-S, AVPP9907-S, antimicrobial activity of plants is mainly attributed to two AVPP0710-S, AVPP9820-S, AVPP0516-S) and three (KCS1A, mechanisms, which include interfering chemically with KCS2A, KCS3A) and a non-CMS variety California wonder the synthesis or function of vital components of bacte - (CW ) which was used as a reference variety, were obtained ria, and/or circumventing the conventional mechanisms from ICAR-IARI, Regional Station, Katrain, Himachal of antibacterial resistance. These agents can act alone Pradesh, India. or in combination with antibiotics to enhance the anti- bacterial activity against a wide range of bacteria. In this 2.2 Bacterial strains regards, phytochemicals have revealed significant activi- ties while many researchers have used natural products The bacterial strains used for antimicrobial screening were to act against bacterial resistance [8–11]. Although syn- obtained from Department of Microbiology and Biotech- thetic antimicrobial agents have been already approved in nology, Himachal Pradesh University, Shimla, India. The many countries, yet the usage of natural compounds that following strains were used for the assay: are derived from microbial, animals, or plants attracts the Gram negative: Escherichia coli, Klebsiella pneumoniae, attention of many researchers [13, 14]. These compounds Pseudomonas aeruginosa, Salmonella typhi, Shigella dysen- have exhibited promising results in overcoming the emer- teriae, Yersinia pestis gence of antibiotic resistance in bacterial pathogens [15, Gram positive: Staphylococcus aureus, Bacillus subtilis. 16]. Among all of the available options, plant-derived Microbial strains were inoculated in nutrient broth at compounds have displayed more potential applications 37 °C for 8 h, strains were subcultured on nutrient agar in combating bacterial infections. Nowadays, people are medium at 37 °C and stored at 4 °C. getting more aware of the type of food they are having and explore the nutritional value of foods before consum- 2.3 Sample collection and extract preparation ing [17–19]. Numerous medicinal plants and many vegetable crops Fruits of pepper were collected, washed with distilled have been reported to possess antimicrobial activity. The water and wiped with 70% ethanol. Fruit of each line was antimicrobial potential is generally attributed to the pres- weighed 2 g and crushed in 30 ml of 10%, 20%, 30% and ence of polyphenols in them [20]. Peppers are bestowed to 40% methanol with the help of pestle and mortar. The inhibit the growth of many gram positive and gram nega- crude extract was transferred to the centrifuge tube and tive bacteria such as E. coli, Klebsiella pnuemoniae, Salmo- centrifuged at 7500 rpm for 10 min. The supernatant was nella typhi, Staphylococcus aureus, Streptococcus pyogenes, collected in a separate tube and was further concentrated Bacillus cereus etc. and fungal strains—Candida albicans, by reducing the volume to 15 ml in a water bath at 40 °C Aspergillus flavus [21, 22]. Many phytotherapy annuals for 1–2 h. The resultant extract was stored at − 20 °C for fur- have emphasized on use of botanicals for treating various ther use. Extraction was also done in distilled water (DW ) diseases of human body such as urinary and skin infection, which was used as negative control. Standard antibiotics, respiratory and gastrointestinal disorders [23]. streptomycin and bleomycin were used as positive control. In recent times, CMS character has been exploited to develop many hybrids in chilli. The analysis of the antioxi- 2.4 Determination of antibacterial activity dant and antimicrobial potential of CMS lines would help to select healthy lines for hybridization process. Keeping The antimicrobial activity was determined by agar well these viewpoints in mind, the present investigation was diffusion method. 100 µl of 8 h old inoculum was poured Vol:.(1234567890) | Research Article SN Applied Sciences (2020) 2:1181 https://doi.org/10.1007/s42452-020-2902-1 onto nutrient agar plates and evenly spread on the plate 2.8 Estimation of free radical scavenging by DPPH using a sterile spreader. Four wells of 6 mm diameter were made with the help of a sterile cork borer in each petri The antioxidant activity of fruit extracts was determined plate.  100  µl of 10%, 20%, 30% and 40% crude extract by free radical scavenging activity of 2,2-diphenyl-1-picryl was carefully added in each well and marked accordingly. hydrazyl (DPPH) as described by Bruits and Bucar [27]. 2 g Extract was allowed to diffuse through the well into the of sample fruits were crushed in 30 ml of absolute metha- gel by keeping the plates at the room temperature for nol and final volume made to 15 ml by concentrating in 2 h. Plates were then incubated at 37 °C for 18–24 h. Anti- the water bath. 20 µl of methanolic extract of fruits was microbial activity was measured as zone of inhibition of added to 2 ml of 0.004% freshly prepared DPPH solution bacterial growth. Standard antibiotics streptomycin and in methanol and allowed to stand for 30 min at room tem- bleomycin were used as positive controls and distilled perature. The absorbance was recorded at 517 nm and water as negative control. All the tests were performed compared to the control (DPPH solution without sample) in triplicates. and scavenging percentage was determined as follows Free radical scavenging %= A − A ∕A × 100 control sample control 2.5 Determination of total phenolic content The total phenolic content was determined spectrophoto- 2.9 Statistical analysis metrically following the procedure given by Muresan et al. [24] with slight modifications. In this 2 g of sample was All experiments were performed in triplicates. The data homogenized in 10 ml of 80% ethanol and centrifuged were analyzed by basic statistics and represented as at 5000 rpm for 10 min. Supernatant was stored at − 20 °C mean ± standard deviation. The data were analyzed using for further use. 100 μl of sample was added to 2.9 ml dis- one way analysis of variance (ANOVA) using SPSS version tilled water and 0.5 ml Folin–Ciocalteu reagent. Reaction was neutralized after 3 min by adding 2 ml of 20% sodium carbonate and OD was recorded at 750 nm. Total phenols were calculated as μg gallic acid/gfw. 3 Results and discussion 2.6 Reducing ability using FRAP assay 3.1 Determination of antibacterial activity FRAP (Ferric ion reducing ability of plasma) assay was A significant zone of inhibition of methanolic extracts of conducted to determine the reducing power of peppers all eight CMS lines and a non CMS variety CW as observed as described by Benzie and Strain [25] with few modifica- ( Table 1). Among four methanolic extracts (10% to 40%), tions. Briefly, the FRAP reagent was prepared by mixing 30% and 40% were observed to be most effective in 300 mM acetate buffer, 10 mM TPTZ (prepared in 40 mM activating the metabolites against different pathogens. HCL) and 20 mM ferric chloride in the ratio of 10:1:1. Etha- Extracts of three CMS lines i.e. KCS 2A, KCS 3A and AVPP nolic extract was prepared by homogenizing 2 g of pepper 9907-S were found most effective in combating the bac - fruits in 10 ml of absolute ethanol. 100 µl of this extract terial growth of Staphylococcus aureus followed by Yers- was mixed with 3 ml FRAP reagent and kept in dark for inia pestis and Bacillus subtilis. On the other hand, 30% 30 min. Absorbance was recorded at 593 nm in UV–Vis methanolic extract of AVPP0710-S were found more spectrophotometer and reducing capacity was deter- effective than 40% counterparts against Bacillus subtilis. mined as µmol trolox/g. 10% and 20% methanolic extracts were also observed to show some inhibition against few bacterial strains such as Staphylococcus aureus, Bacillus subtilis and Yersinia but 2.7 CUPRAC assay in majority there was no inhibition at all. California won- der was also seen to inhibit Salmonella typhi, Staphylococ- CUPRAC (Cupric ion reducing antioxidant capacity) was cus aureus, Bacillus subtilis and Yersinia pestis at different determined using protocol of Cekic et al. [26]. 100 µl of methanolic concentrations. In the present study, Yersinia sample extract prepared previously for FRAP was mixed pestis was inhibited by almost all the fruit extracts whereas with 1  ml neucuproine, 1  ml ammonium acetate, 1  ml Shigella was found most resistant against many CMS lines copper chloride and 1 ml distilled water and absorbance of the pepper. Similar results have also been observed by was taken immediately at 450 nm and CUPRAC was deter- many other researchers using extracts of die ff rent types mined as µmol trolox/g. Vol.:(0123456789) | Research Article SN Applied Sciences (2020) 2:1181 https://doi.org/10.1007/s42452-020-2902-1 Table 1 Table showing zone of inhibition of fruit extracts against Escherichia coli (EC), Salmonella typhi (ST ), Staphylococcus aureus (SA), Pseu- domonas aerugenosa (PA), Shigella dysentriae (SD), Bacillus subtilis (BC), Klebsiella pnumoniae (KP), Yersinia pestis (YP) and distilled water (DW ) Line Extract Zone of Inhibition (mm) EC ST SA PA SD B S KP YP AVPP9908-S Methanol% 10 0 0 0 0 0 0.4 ± 0 0 0.65 ± 0.21 20 0.65 ± 0.7 1.45 ± 0.7 1 ± 0.42 0.35 ± 0.49 0 0.5 ± 0.14 0.75 ± .21 1.1 ± 0.28 30 0.4 ± .14 0.9 ± 0.14 0.9 ± 0.42 0.25 ± 0.35 0 0.95 ± 0.07 0.5 ± .14 0.8 ± 0.28 40 0 0 0.25 ± 0.07 0 0 0.45 ± 0.07 0 0.3 ± 0.28 DW 0 0 0 0 0 0 0 0 AVPP 0710-S Methanol% 10 0 0 0 0 0.55 ± 0.35 0.5 ± 0.14 0 0.55 ± 0.07 20 0 0 0 0 0 0.8 ± 0.14 0 0.7 ± 0.14 30 0.45 ± 0.07 0.75 ± 0.35 0.8 ± 0.28 0.4 ± 0 0 1.2 ± 0.28 0.4 ± 0.14 1.15 ± 0.07 40 0.85 ± 0.07 1.2 ± 0.42 1.35 ± 0.21 0.65 ± 0.07 0 1.15 ± 0.63 0.65 ± .07 1.2 ± 0 DW 0 0 0 0 0 0 0 0 AVPPO 516-S Methanol% 10 0 0 0.05 ± 0.07 0 0 0.2 ± 0.28 0 0.45 ± 0.07 20 0 0 0.1 ± 0.14 0 0 0.2 ± 0.28 0 0.6 ± 0.14 30 0.45 ± 0.07 1.15 ± 0.07 1.1 ± 0.14 0.2 ± 0.28 0 0.65 ± 0.21 0.4 ± 0 1 ± .14 40 0.85 ± 0.07 1.45 ± 0.07 1.3 ± 0.14 0.75 ± 0.21 0 1 ± 0.14 0.9 ± 0.14 1.25 ± 0.07 DW 0 0 0 0 0 0 0 0 AVPP 9907-S Methanol% 10 0 0 0.85 ± 0.35 0 0 0.2 ± 0.28 0.05 ± 0.07 0.3 ± 0.42 20 0 0 1.35 ± 0.07 0.25 ± 0.21 0 1 ± 0.14 0.1 ± 0 1 ± 0 30 0.4 ± 0 0.6 ± 0 1.45 ± 0.07 0.45 ± 0.21 0 1.15 ± 0.21 0.35 ± 0.07 1.15 ± 0.07 40 0.7 ± 0 1.05 ± 0.07 1.45 ± 0.07 0.7 ± 0.05 0 1.35 ± 0.21 0.75 ± 0.07 1.4 ± 0 DW 0 0 0 0 0 0 0 0 AVPP 9820-S Methanol% 10 0 0 0.1 ± 0.14 0 0 0.55 ± 0.07 0 0 20 0 0 0.5 ± 0.70 0 0 0.8 ± 0.28 0 0.6 ± 0.28 30 0.45 ± 0.07 1.25 ± 0.07 0.8 ± 0.42 0.35 ± 0.07 0 1.1 ± 0.28 0.45 ± 0.07 0.9 ± 0.14 40 0.6 ± 0 1.5 ± 0 1.05 ± 0.35 0.65 ± 0.07 0 1.15 ± 0.35 0.65 ± 0.07 1.1 ± 0.14 DW 0 0 0 0 0 0 0 0 KCS-1A Methanol% 10 0 0 0.2 ± 0.28 0 0 0.45 ± 0.21 0 0.6 ± 0.14 20 0 1 ± 0.07 0.5 ± 0.70 0.05 ± 0.07 0 0.95 ± 0.07 0 0.8 ± 0.14 30 0.3 ± 0.14 1 ± 0.70 0.5 ± 0.70 0.3 ± 0.28 0 0.95 ± 0.07 0.3 ± 0.14 1.15 ± 0.07 40 0.75 ± 0.07 1.3 ± 0.70 0.85 ± 0.49 0.55 ± 0.21 0 1.2 ± 0.28 0.6 ± 0.14 1.25 ± 0.07 DW 0 0 0 0 0 0 0 0 KCS-2A Methanol% 10 0 0 0 0 0.25 ± 0.35 0.25 ± 0.35 0 0.35 ± 0.49 20 0 0 0 0 0 0.3 ± 0.28 0 0 30 0.3 ± 0.14 1.15 ± 0.21 0.9 ± 0.56 0.35 ± 0.07 0.8 ± 0.28 0.75 ± 0.63 0.35 ± 0.21 1.1 ± 0.14 40 0.6 ± .14 1.5 ± 0.14 0.95 ± 0.35 0.65 ± 0.07 1.1 ± 0.14 1.4 ± 0.42 0.65 ± 0.07 1.35 ± 0.07 DW 0 0 0 0 0 0 0 0 KCS-3A Methanol% 10 0 0 0 0 0.35 ± 0.07 0.5 ± 0.05 0 0.5 ± 0.11 20 0 0.45 ± 0.07 1 ± 0.12 0 0.75 ± 0.07 0.95 ± 0.07 0.1 ± 0.05 0.85 ± 0.21 30 0.2 ± 0.07 0.85 ± 0.07 1.1 ± 0.14 0.2 ± 0.28 0.85 ± 0.07 1.1 ± 0.14 0.35 ± 0.07 1.05 ± 0.21 40 0.65 ± 0.07 1.15 ± 0.07 1.2 ± 0.28 0.35 ± 0.21 1.25 ± 0.07 1.3 ± 0.28 0.65 ± 0.07 1.25 ± 0.21 DW 0 0 0 0 0 0 0 0 CW Methanol% 10 0 0 0 0 0 0.3 ± 0.42 0 0.3 ± 0.42 20 0 0 0 0 0 0 0 0.4 ± 0.56 30 0 0 0 0 0 0 0 0.46 ± 0.12 40 0 0.65 ± 0.21 1.1 ± 0 0 0 0 0 0.55 ± 0.21 DW 0 0 0 0 0 0 0 0 Streptomycin 1.1 1.2 1.2 1.2 1.3 1.4 1.1 1.2 Bleomycin 1.2 1.1 1.3 1.3 1.2 1.1 1.1 1.3 Vol:.(1234567890) | Research Article SN Applied Sciences (2020) 2:1181 https://doi.org/10.1007/s42452-020-2902-1 of pepper and other medicinal plants [28–31]. Plant sec- [38–40]. In previous studies, the antibacterial activity of ondary metabolites such as phenols act as antimicrobial Scutellaria baicalensis extract has been reported [41]. The agents and inhibit the growth of gram positive and gram antimicrobial activities of different extracts of S. litwin- negative bacteria. The efficiency of inhibition depends on owii have been screened against the standard strains of S. the chemical composition, concentration and structure of aureus, Bacillus cereus, P. aeruginosa, E. coli and C. albicans. antimicrobial agents [7]. Methanol extract has been found The results of this investigation indicated that extracts to be effective in solubilising or activating the antibacte - obtained from the aerial parts of S. litwinowii possessed rial agents from fruits or various plant parts [28, 32]. Some antioxidant and antimicrobial properties [42]. Many other researchers have reported antimicrobial activity in sterile researchers have also reported good amount of phenolic water extracts but the activity is quite lower as compared acids in capsicum cultivars [17–19]. to the methanol extracts [21, 22]. The role of solvents in extraction of various metabolites have been well docu- 3.3 Reducing ability using FRAP assay mented by other researchers also [19, 33]. Ferric reducing ability of plasma (FRAP) ranged from 3.2 Determination of total phenolic content 0.9–3.12 µmol trolox/g among CMS cultivars. It was found highest in AVPP0710-S with 2.77 ± 0.35 µmol trolox/g as A significant amount of total phenolic content and anti- genotype mean and lowest in AVPP9908-S with genotype oxidant capacities was observed among different CMS mean of 1.06 ± 0.16 µmol trolox/g. Bogusz et al. [18] have lines and a non CMS variety (California wonder) of Capsi- also reported high FRAP activity in C. frutescens, C. chinense cum annum (Table 2). The total phenolic content ranged and C. Baccatum. This assay is based on the reduction of 3+ 2+ from 594.99 to 1710.13 µg gallic acid/gfw in all studied ferric ions (F e ) to ferrous ions (Fe ) by donation of elec- lines. Highest phenolic content was found in AVPP0710-S trons from antioxidants. The antioxidant potential of plant (1690.48 ± 19.65 13 µg gallic acid/gfw) and lowest in KCS extract depends upon their reducing power [43] which in 2A (621.63 ± 26.64 µg gallic acid/gfw). They are the most turn depends on presence of various metabolites [44]. bountiful antioxidants available in the human food, [34, The phenolic acids have been advocated to offer resist - 35]. Saha et al. [36] have also reported phenols in Capsi- ance against various oxidative stresses by scavenging free cum annum in the range of 1012–4135 µg gallic acid/g FW. radicals, inhibiting lipid peroxidation etc. [45–47]. Phenols present in the fruit extract donate hydrogen ion to ferric ions converting them to stable, less reactive ferrous 3.4 CUPRAC assay form [37]. This action helps the human body in fighting against oxidative stress which leads to various degenera- Copper reducing antioxidant capacity (CUPRAC) ranged tive processes [17]. from 0.7–11.22 µmol trolox/g with highest in AVPP9820- Phenolic compounds include a wide range of bioactive S (11.06 ± 0.16) and lowest in KCS 1A (0.81 ± 0.11). This 2+ natural compounds that are extensively utilized for medi- activity is based on the reduction of cupric ions (Cu ) to 1+ cal purposes. These compounds, as bioactive molecules, cuprous ions (Cu ) by the antioxidant agents present in play an important role in enhancing antibiotic activity the extracts. A chelating agent, neocuproine, aids in the against resistant pathogens through various mechanisms above reaction [48]. CUPRAC assay has been employed Table 2 Mean values and Pepper line Phenolic content FRAP (µmol trolox/g) CUPRAC (µmol trolox/g DPPH (%) range of phenolic content, (μg gallic acid/gfw) CUPRAC, FRAP and DPPH activity of various CMS lines of AVPP9908-S 725.02 ± 14.19 1.06 ± 0.16 8.55 ± 0.9 32.94 ± 3.61 pepper AVPP 0516-S 952.99 ± 15.77 2.14 ± 0.28 8.41 ± 0.17 14.24 ± 2.71 AVPP 0710-S 1690.48 ± 19.65 2.77 ± 0.35 1.15 ± 0.09 21.28 ± 2.10 AVPP 9907-S 1534.50 ± 20.18 1.53 ± 0.21 1.78 ± 0.19 16.15 ± 2.44 AVPP 9820-S 1499.65 ± 50.76 1.35 ± 0.19 11.06 ± 0.16 11.64 ± 1.73 KCS 1A 1062.61 ± 31.09 1.49 ± 0.24 0.81 ± 0.11 21.80 ± 2.27 KCS 2A 621.63 ± 26.64 1.42 ± 0.28 1.09 ± 0.16 30.27 ± 3.27 KCS 3A 1478.23 ± 46.11 1.31 ± 0.27 4.08 ± 0.15 27.66 ± 3.44 California won- 1159.18 ± 38.72 1.47 ± 0.19 1.25 ± 0.09 11.11 ± 1.52 der (variety) Range 594.99–1710.13 0.9–3.12 0.7–11.22 9.91–36.55 Vol.:(0123456789) | Research Article SN Applied Sciences (2020) 2:1181 https://doi.org/10.1007/s42452-020-2902-1 for antioxidant detection in human serum, food and References plant extracts [49–52]. 1. 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Assessment of antimicrobial and antioxidant potential of cytoplasmic male sterile lines of pepper

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Abstract

In the present study eight Cytoplasmic Male Sterile (CMS) lines of pepper and one open pollinated commercial variety ‘California wonder’ were assessed for their antimicrobial and antioxidant potential. For antimicrobial activity, fresh fruits crushed in 10%, 20%, 30% and 40% methanol were analyzed. Thirty per cent and forty percent methanolic extracts were found to be best activator of metabolites exhibiting antibacterial activity. Highest antibacterial activity was shown by 40% methanoic extracts of KCS2A, KCS3A and AVPP9907-S of Capsicum CMS lines against different bacteria. The extracts of all lines found ee ff ctive against Yersinia pestia . Phenolic content of all CMS lines was determined by extracting fruit in abso- lute ethanol, it was found in the range of 594.99–1710.13 μg gallic acid/gfw. Phenolic content was highest in AVPP0710-S (1690.48 ± 19.65 μg gallic acid/gfw) and lowest in KCS 2A. FRAP, CUPRAC and DPPH assay were done to find out anti- oxidant potential of CMS lines under study. FRAP and CUPRAC were analyzed by crushing fruits in 80% ethanol. FRAP was found in the range of 0.9–3.12 µmol trolox/g, with AVPP0710-S exhibiting highest FRAP value (2.77 ± 0.3522 µmol trolox/g). CUPRAC was found in the range of 0.7–11.22 µmol trolox/g and AVPP9908-S showed highest CUPRAC value (8.55 ± 0.922 µmol trolox/g). Free radical scavenging DPPH assay was done by extraction in absolute methanol and was highest in AVPP9908-S (32.94 ± 3.61%) with 9.91–36.55% overall range. This study will pave the way for the development of genotypes with higher phenol, antioxidant and antimicrobial activity in pepper. Keywords Cytoplasmic male sterile · Pepper · Antimicrobial activity · Antioxidant · DPPH 1 Introduction unstable molecules that the body produces as a reaction to environmental and other pressures. Antioxidants can Peppers are an important group of vegetables which are protect against the cell damage that free radicals cause, grown on a large scale all over the world. It includes both, known as oxidative stress. They contain high concentra- mild or less pungent fruit types (sweet pepper) and pun- tion of polyphenols which includes flavonols, flavones, gent fruit types (hot pepper) [1]. A wide terminology is anthocyanins and hydroxycinnamates [4, 5]. These bio- used to designate peppers like chili, chile, chilli, aji, paprika compounds are known to enhance the antioxidant activ- and capsicum [2]. ity of peppers [4, 6, 7]. The antioxidant potential works Peppers are a rich source of various antioxidans like by preventing the oxidation of lipids by scavenging the capsaicinoids, ascorbic acid, carotenoids, Vitamin E, pro- free radicals. Vitamin E, C and beta carotene are of special vitamin A etc. [3]. Antioxidants are substances that can concern in increasing the antioxidant potential of peppers prevent or slow damage to cells caused by free radicals, [8]. Hot peppers are reported to show high antioxidant * Pooja Saklani, [email protected] | Department of Biotechnology, HNBGU (A Central University), Srinagar, Uttarakhand, India. Division of Vegetable Breeding, ICAR-IARI, Pusa Campus, New Delhi, India. SN Applied Sciences (2020) 2:1181 | https://doi.org/10.1007/s42452-020-2902-1 Vol.:(0123456789) | Research Article SN Applied Sciences (2020) 2:1181 https://doi.org/10.1007/s42452-020-2902-1 potential due to high levels of capsaicinoids in them which formulated to assess the antimicrobial and antioxidant also impart pharmacological properties to them [9]. Phe- potential of eight cytoplasmic male sterile lines of Capsi- nols reduce the risk of lipid autoxidation by scavenging cum annuum. The study would enforce the production of free radicals and thereby inhibit the initiation of oxidation good quality pepper hybrids, the consumption of which chain [10]. Flavonoids impart organoleptic properties such can act as robust nutraceutical. as taste to the fruits [11]. Anthocyanins are natural pig- ments which impart diverse colors to the plant life and flavonols are induced by sunlight which shields the plant 2 Materials and methods from harmful effects of ultraviolet rays [12]. In today’s scenario, microbial infections and resist- 2.1 Collection of plant material ance to antibiotic drugs have been the main causes that threaten the health of peoples. Over the decades, it has Eight CMS lines of pepper were used for conducting the been observed that the microbial infections are respon- present investigation. Out of eight CMS lines, five were sible for millions of deaths throughout the world. The procured from AVRDC, Taiwan (AVPP9908-S, AVPP9907-S, antimicrobial activity of plants is mainly attributed to two AVPP0710-S, AVPP9820-S, AVPP0516-S) and three (KCS1A, mechanisms, which include interfering chemically with KCS2A, KCS3A) and a non-CMS variety California wonder the synthesis or function of vital components of bacte - (CW ) which was used as a reference variety, were obtained ria, and/or circumventing the conventional mechanisms from ICAR-IARI, Regional Station, Katrain, Himachal of antibacterial resistance. These agents can act alone Pradesh, India. or in combination with antibiotics to enhance the anti- bacterial activity against a wide range of bacteria. In this 2.2 Bacterial strains regards, phytochemicals have revealed significant activi- ties while many researchers have used natural products The bacterial strains used for antimicrobial screening were to act against bacterial resistance [8–11]. Although syn- obtained from Department of Microbiology and Biotech- thetic antimicrobial agents have been already approved in nology, Himachal Pradesh University, Shimla, India. The many countries, yet the usage of natural compounds that following strains were used for the assay: are derived from microbial, animals, or plants attracts the Gram negative: Escherichia coli, Klebsiella pneumoniae, attention of many researchers [13, 14]. These compounds Pseudomonas aeruginosa, Salmonella typhi, Shigella dysen- have exhibited promising results in overcoming the emer- teriae, Yersinia pestis gence of antibiotic resistance in bacterial pathogens [15, Gram positive: Staphylococcus aureus, Bacillus subtilis. 16]. Among all of the available options, plant-derived Microbial strains were inoculated in nutrient broth at compounds have displayed more potential applications 37 °C for 8 h, strains were subcultured on nutrient agar in combating bacterial infections. Nowadays, people are medium at 37 °C and stored at 4 °C. getting more aware of the type of food they are having and explore the nutritional value of foods before consum- 2.3 Sample collection and extract preparation ing [17–19]. Numerous medicinal plants and many vegetable crops Fruits of pepper were collected, washed with distilled have been reported to possess antimicrobial activity. The water and wiped with 70% ethanol. Fruit of each line was antimicrobial potential is generally attributed to the pres- weighed 2 g and crushed in 30 ml of 10%, 20%, 30% and ence of polyphenols in them [20]. Peppers are bestowed to 40% methanol with the help of pestle and mortar. The inhibit the growth of many gram positive and gram nega- crude extract was transferred to the centrifuge tube and tive bacteria such as E. coli, Klebsiella pnuemoniae, Salmo- centrifuged at 7500 rpm for 10 min. The supernatant was nella typhi, Staphylococcus aureus, Streptococcus pyogenes, collected in a separate tube and was further concentrated Bacillus cereus etc. and fungal strains—Candida albicans, by reducing the volume to 15 ml in a water bath at 40 °C Aspergillus flavus [21, 22]. Many phytotherapy annuals for 1–2 h. The resultant extract was stored at − 20 °C for fur- have emphasized on use of botanicals for treating various ther use. Extraction was also done in distilled water (DW ) diseases of human body such as urinary and skin infection, which was used as negative control. Standard antibiotics, respiratory and gastrointestinal disorders [23]. streptomycin and bleomycin were used as positive control. In recent times, CMS character has been exploited to develop many hybrids in chilli. The analysis of the antioxi- 2.4 Determination of antibacterial activity dant and antimicrobial potential of CMS lines would help to select healthy lines for hybridization process. Keeping The antimicrobial activity was determined by agar well these viewpoints in mind, the present investigation was diffusion method. 100 µl of 8 h old inoculum was poured Vol:.(1234567890) | Research Article SN Applied Sciences (2020) 2:1181 https://doi.org/10.1007/s42452-020-2902-1 onto nutrient agar plates and evenly spread on the plate 2.8 Estimation of free radical scavenging by DPPH using a sterile spreader. Four wells of 6 mm diameter were made with the help of a sterile cork borer in each petri The antioxidant activity of fruit extracts was determined plate.  100  µl of 10%, 20%, 30% and 40% crude extract by free radical scavenging activity of 2,2-diphenyl-1-picryl was carefully added in each well and marked accordingly. hydrazyl (DPPH) as described by Bruits and Bucar [27]. 2 g Extract was allowed to diffuse through the well into the of sample fruits were crushed in 30 ml of absolute metha- gel by keeping the plates at the room temperature for nol and final volume made to 15 ml by concentrating in 2 h. Plates were then incubated at 37 °C for 18–24 h. Anti- the water bath. 20 µl of methanolic extract of fruits was microbial activity was measured as zone of inhibition of added to 2 ml of 0.004% freshly prepared DPPH solution bacterial growth. Standard antibiotics streptomycin and in methanol and allowed to stand for 30 min at room tem- bleomycin were used as positive controls and distilled perature. The absorbance was recorded at 517 nm and water as negative control. All the tests were performed compared to the control (DPPH solution without sample) in triplicates. and scavenging percentage was determined as follows Free radical scavenging %= A − A ∕A × 100 control sample control 2.5 Determination of total phenolic content The total phenolic content was determined spectrophoto- 2.9 Statistical analysis metrically following the procedure given by Muresan et al. [24] with slight modifications. In this 2 g of sample was All experiments were performed in triplicates. The data homogenized in 10 ml of 80% ethanol and centrifuged were analyzed by basic statistics and represented as at 5000 rpm for 10 min. Supernatant was stored at − 20 °C mean ± standard deviation. The data were analyzed using for further use. 100 μl of sample was added to 2.9 ml dis- one way analysis of variance (ANOVA) using SPSS version tilled water and 0.5 ml Folin–Ciocalteu reagent. Reaction was neutralized after 3 min by adding 2 ml of 20% sodium carbonate and OD was recorded at 750 nm. Total phenols were calculated as μg gallic acid/gfw. 3 Results and discussion 2.6 Reducing ability using FRAP assay 3.1 Determination of antibacterial activity FRAP (Ferric ion reducing ability of plasma) assay was A significant zone of inhibition of methanolic extracts of conducted to determine the reducing power of peppers all eight CMS lines and a non CMS variety CW as observed as described by Benzie and Strain [25] with few modifica- ( Table 1). Among four methanolic extracts (10% to 40%), tions. Briefly, the FRAP reagent was prepared by mixing 30% and 40% were observed to be most effective in 300 mM acetate buffer, 10 mM TPTZ (prepared in 40 mM activating the metabolites against different pathogens. HCL) and 20 mM ferric chloride in the ratio of 10:1:1. Etha- Extracts of three CMS lines i.e. KCS 2A, KCS 3A and AVPP nolic extract was prepared by homogenizing 2 g of pepper 9907-S were found most effective in combating the bac - fruits in 10 ml of absolute ethanol. 100 µl of this extract terial growth of Staphylococcus aureus followed by Yers- was mixed with 3 ml FRAP reagent and kept in dark for inia pestis and Bacillus subtilis. On the other hand, 30% 30 min. Absorbance was recorded at 593 nm in UV–Vis methanolic extract of AVPP0710-S were found more spectrophotometer and reducing capacity was deter- effective than 40% counterparts against Bacillus subtilis. mined as µmol trolox/g. 10% and 20% methanolic extracts were also observed to show some inhibition against few bacterial strains such as Staphylococcus aureus, Bacillus subtilis and Yersinia but 2.7 CUPRAC assay in majority there was no inhibition at all. California won- der was also seen to inhibit Salmonella typhi, Staphylococ- CUPRAC (Cupric ion reducing antioxidant capacity) was cus aureus, Bacillus subtilis and Yersinia pestis at different determined using protocol of Cekic et al. [26]. 100 µl of methanolic concentrations. In the present study, Yersinia sample extract prepared previously for FRAP was mixed pestis was inhibited by almost all the fruit extracts whereas with 1  ml neucuproine, 1  ml ammonium acetate, 1  ml Shigella was found most resistant against many CMS lines copper chloride and 1 ml distilled water and absorbance of the pepper. Similar results have also been observed by was taken immediately at 450 nm and CUPRAC was deter- many other researchers using extracts of die ff rent types mined as µmol trolox/g. Vol.:(0123456789) | Research Article SN Applied Sciences (2020) 2:1181 https://doi.org/10.1007/s42452-020-2902-1 Table 1 Table showing zone of inhibition of fruit extracts against Escherichia coli (EC), Salmonella typhi (ST ), Staphylococcus aureus (SA), Pseu- domonas aerugenosa (PA), Shigella dysentriae (SD), Bacillus subtilis (BC), Klebsiella pnumoniae (KP), Yersinia pestis (YP) and distilled water (DW ) Line Extract Zone of Inhibition (mm) EC ST SA PA SD B S KP YP AVPP9908-S Methanol% 10 0 0 0 0 0 0.4 ± 0 0 0.65 ± 0.21 20 0.65 ± 0.7 1.45 ± 0.7 1 ± 0.42 0.35 ± 0.49 0 0.5 ± 0.14 0.75 ± .21 1.1 ± 0.28 30 0.4 ± .14 0.9 ± 0.14 0.9 ± 0.42 0.25 ± 0.35 0 0.95 ± 0.07 0.5 ± .14 0.8 ± 0.28 40 0 0 0.25 ± 0.07 0 0 0.45 ± 0.07 0 0.3 ± 0.28 DW 0 0 0 0 0 0 0 0 AVPP 0710-S Methanol% 10 0 0 0 0 0.55 ± 0.35 0.5 ± 0.14 0 0.55 ± 0.07 20 0 0 0 0 0 0.8 ± 0.14 0 0.7 ± 0.14 30 0.45 ± 0.07 0.75 ± 0.35 0.8 ± 0.28 0.4 ± 0 0 1.2 ± 0.28 0.4 ± 0.14 1.15 ± 0.07 40 0.85 ± 0.07 1.2 ± 0.42 1.35 ± 0.21 0.65 ± 0.07 0 1.15 ± 0.63 0.65 ± .07 1.2 ± 0 DW 0 0 0 0 0 0 0 0 AVPPO 516-S Methanol% 10 0 0 0.05 ± 0.07 0 0 0.2 ± 0.28 0 0.45 ± 0.07 20 0 0 0.1 ± 0.14 0 0 0.2 ± 0.28 0 0.6 ± 0.14 30 0.45 ± 0.07 1.15 ± 0.07 1.1 ± 0.14 0.2 ± 0.28 0 0.65 ± 0.21 0.4 ± 0 1 ± .14 40 0.85 ± 0.07 1.45 ± 0.07 1.3 ± 0.14 0.75 ± 0.21 0 1 ± 0.14 0.9 ± 0.14 1.25 ± 0.07 DW 0 0 0 0 0 0 0 0 AVPP 9907-S Methanol% 10 0 0 0.85 ± 0.35 0 0 0.2 ± 0.28 0.05 ± 0.07 0.3 ± 0.42 20 0 0 1.35 ± 0.07 0.25 ± 0.21 0 1 ± 0.14 0.1 ± 0 1 ± 0 30 0.4 ± 0 0.6 ± 0 1.45 ± 0.07 0.45 ± 0.21 0 1.15 ± 0.21 0.35 ± 0.07 1.15 ± 0.07 40 0.7 ± 0 1.05 ± 0.07 1.45 ± 0.07 0.7 ± 0.05 0 1.35 ± 0.21 0.75 ± 0.07 1.4 ± 0 DW 0 0 0 0 0 0 0 0 AVPP 9820-S Methanol% 10 0 0 0.1 ± 0.14 0 0 0.55 ± 0.07 0 0 20 0 0 0.5 ± 0.70 0 0 0.8 ± 0.28 0 0.6 ± 0.28 30 0.45 ± 0.07 1.25 ± 0.07 0.8 ± 0.42 0.35 ± 0.07 0 1.1 ± 0.28 0.45 ± 0.07 0.9 ± 0.14 40 0.6 ± 0 1.5 ± 0 1.05 ± 0.35 0.65 ± 0.07 0 1.15 ± 0.35 0.65 ± 0.07 1.1 ± 0.14 DW 0 0 0 0 0 0 0 0 KCS-1A Methanol% 10 0 0 0.2 ± 0.28 0 0 0.45 ± 0.21 0 0.6 ± 0.14 20 0 1 ± 0.07 0.5 ± 0.70 0.05 ± 0.07 0 0.95 ± 0.07 0 0.8 ± 0.14 30 0.3 ± 0.14 1 ± 0.70 0.5 ± 0.70 0.3 ± 0.28 0 0.95 ± 0.07 0.3 ± 0.14 1.15 ± 0.07 40 0.75 ± 0.07 1.3 ± 0.70 0.85 ± 0.49 0.55 ± 0.21 0 1.2 ± 0.28 0.6 ± 0.14 1.25 ± 0.07 DW 0 0 0 0 0 0 0 0 KCS-2A Methanol% 10 0 0 0 0 0.25 ± 0.35 0.25 ± 0.35 0 0.35 ± 0.49 20 0 0 0 0 0 0.3 ± 0.28 0 0 30 0.3 ± 0.14 1.15 ± 0.21 0.9 ± 0.56 0.35 ± 0.07 0.8 ± 0.28 0.75 ± 0.63 0.35 ± 0.21 1.1 ± 0.14 40 0.6 ± .14 1.5 ± 0.14 0.95 ± 0.35 0.65 ± 0.07 1.1 ± 0.14 1.4 ± 0.42 0.65 ± 0.07 1.35 ± 0.07 DW 0 0 0 0 0 0 0 0 KCS-3A Methanol% 10 0 0 0 0 0.35 ± 0.07 0.5 ± 0.05 0 0.5 ± 0.11 20 0 0.45 ± 0.07 1 ± 0.12 0 0.75 ± 0.07 0.95 ± 0.07 0.1 ± 0.05 0.85 ± 0.21 30 0.2 ± 0.07 0.85 ± 0.07 1.1 ± 0.14 0.2 ± 0.28 0.85 ± 0.07 1.1 ± 0.14 0.35 ± 0.07 1.05 ± 0.21 40 0.65 ± 0.07 1.15 ± 0.07 1.2 ± 0.28 0.35 ± 0.21 1.25 ± 0.07 1.3 ± 0.28 0.65 ± 0.07 1.25 ± 0.21 DW 0 0 0 0 0 0 0 0 CW Methanol% 10 0 0 0 0 0 0.3 ± 0.42 0 0.3 ± 0.42 20 0 0 0 0 0 0 0 0.4 ± 0.56 30 0 0 0 0 0 0 0 0.46 ± 0.12 40 0 0.65 ± 0.21 1.1 ± 0 0 0 0 0 0.55 ± 0.21 DW 0 0 0 0 0 0 0 0 Streptomycin 1.1 1.2 1.2 1.2 1.3 1.4 1.1 1.2 Bleomycin 1.2 1.1 1.3 1.3 1.2 1.1 1.1 1.3 Vol:.(1234567890) | Research Article SN Applied Sciences (2020) 2:1181 https://doi.org/10.1007/s42452-020-2902-1 of pepper and other medicinal plants [28–31]. Plant sec- [38–40]. In previous studies, the antibacterial activity of ondary metabolites such as phenols act as antimicrobial Scutellaria baicalensis extract has been reported [41]. The agents and inhibit the growth of gram positive and gram antimicrobial activities of different extracts of S. litwin- negative bacteria. The efficiency of inhibition depends on owii have been screened against the standard strains of S. the chemical composition, concentration and structure of aureus, Bacillus cereus, P. aeruginosa, E. coli and C. albicans. antimicrobial agents [7]. Methanol extract has been found The results of this investigation indicated that extracts to be effective in solubilising or activating the antibacte - obtained from the aerial parts of S. litwinowii possessed rial agents from fruits or various plant parts [28, 32]. Some antioxidant and antimicrobial properties [42]. Many other researchers have reported antimicrobial activity in sterile researchers have also reported good amount of phenolic water extracts but the activity is quite lower as compared acids in capsicum cultivars [17–19]. to the methanol extracts [21, 22]. The role of solvents in extraction of various metabolites have been well docu- 3.3 Reducing ability using FRAP assay mented by other researchers also [19, 33]. Ferric reducing ability of plasma (FRAP) ranged from 3.2 Determination of total phenolic content 0.9–3.12 µmol trolox/g among CMS cultivars. It was found highest in AVPP0710-S with 2.77 ± 0.35 µmol trolox/g as A significant amount of total phenolic content and anti- genotype mean and lowest in AVPP9908-S with genotype oxidant capacities was observed among different CMS mean of 1.06 ± 0.16 µmol trolox/g. Bogusz et al. [18] have lines and a non CMS variety (California wonder) of Capsi- also reported high FRAP activity in C. frutescens, C. chinense cum annum (Table 2). The total phenolic content ranged and C. Baccatum. This assay is based on the reduction of 3+ 2+ from 594.99 to 1710.13 µg gallic acid/gfw in all studied ferric ions (F e ) to ferrous ions (Fe ) by donation of elec- lines. Highest phenolic content was found in AVPP0710-S trons from antioxidants. The antioxidant potential of plant (1690.48 ± 19.65 13 µg gallic acid/gfw) and lowest in KCS extract depends upon their reducing power [43] which in 2A (621.63 ± 26.64 µg gallic acid/gfw). They are the most turn depends on presence of various metabolites [44]. bountiful antioxidants available in the human food, [34, The phenolic acids have been advocated to offer resist - 35]. Saha et al. [36] have also reported phenols in Capsi- ance against various oxidative stresses by scavenging free cum annum in the range of 1012–4135 µg gallic acid/g FW. radicals, inhibiting lipid peroxidation etc. [45–47]. Phenols present in the fruit extract donate hydrogen ion to ferric ions converting them to stable, less reactive ferrous 3.4 CUPRAC assay form [37]. This action helps the human body in fighting against oxidative stress which leads to various degenera- Copper reducing antioxidant capacity (CUPRAC) ranged tive processes [17]. from 0.7–11.22 µmol trolox/g with highest in AVPP9820- Phenolic compounds include a wide range of bioactive S (11.06 ± 0.16) and lowest in KCS 1A (0.81 ± 0.11). This 2+ natural compounds that are extensively utilized for medi- activity is based on the reduction of cupric ions (Cu ) to 1+ cal purposes. These compounds, as bioactive molecules, cuprous ions (Cu ) by the antioxidant agents present in play an important role in enhancing antibiotic activity the extracts. A chelating agent, neocuproine, aids in the against resistant pathogens through various mechanisms above reaction [48]. CUPRAC assay has been employed Table 2 Mean values and Pepper line Phenolic content FRAP (µmol trolox/g) CUPRAC (µmol trolox/g DPPH (%) range of phenolic content, (μg gallic acid/gfw) CUPRAC, FRAP and DPPH activity of various CMS lines of AVPP9908-S 725.02 ± 14.19 1.06 ± 0.16 8.55 ± 0.9 32.94 ± 3.61 pepper AVPP 0516-S 952.99 ± 15.77 2.14 ± 0.28 8.41 ± 0.17 14.24 ± 2.71 AVPP 0710-S 1690.48 ± 19.65 2.77 ± 0.35 1.15 ± 0.09 21.28 ± 2.10 AVPP 9907-S 1534.50 ± 20.18 1.53 ± 0.21 1.78 ± 0.19 16.15 ± 2.44 AVPP 9820-S 1499.65 ± 50.76 1.35 ± 0.19 11.06 ± 0.16 11.64 ± 1.73 KCS 1A 1062.61 ± 31.09 1.49 ± 0.24 0.81 ± 0.11 21.80 ± 2.27 KCS 2A 621.63 ± 26.64 1.42 ± 0.28 1.09 ± 0.16 30.27 ± 3.27 KCS 3A 1478.23 ± 46.11 1.31 ± 0.27 4.08 ± 0.15 27.66 ± 3.44 California won- 1159.18 ± 38.72 1.47 ± 0.19 1.25 ± 0.09 11.11 ± 1.52 der (variety) Range 594.99–1710.13 0.9–3.12 0.7–11.22 9.91–36.55 Vol.:(0123456789) | Research Article SN Applied Sciences (2020) 2:1181 https://doi.org/10.1007/s42452-020-2902-1 for antioxidant detection in human serum, food and References plant extracts [49–52]. 1. 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