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Objectives: Subcritical water extraction technique is considered as an environmentally extraction technique. The aim of this study was to compare the different characteristics of water extract and subcritical water extract of Helicteres isora L. Materials and Methods: Water extraction was performed under the following conditions: 25°C, 24 h, and solid-to-water ratio 1:30. Subcritical water extract was carried out under specific conditions (pressure = 10 bar, temperature = 160°C, solid-to-water ratio = 1: 30, time = 30 min). Chemical composition analysis was performed using GC–Mass chromatography. Anti-biofilm activity in the terms of anti-attach and removal of biofilm were assessed using the ELISA reader method and reading absorbance at 570 nm. Anti-microbial activity against Bacillus cereus, Staphylococcus aureus, Staphylococcus saprophyticus, and Bacillus subtilis was investigated by measurement of inhibitory zone diameter. Anti-enzymatic and antioxidant properties were also assessed. Results: The results of GC–Mass analysis showed some components extracted in subcritical method which were absent in water extract such as octadecanoic acid, hexadecanoic acid, and berberin. Antioxidant activity of the two tested extracts revealed that subcritical water extract had more antioxidant capacity than water extract (P ≤ 0.05). The two tested extracts exhibited anti-enzymatic activity against polyphenol oxidase enzyme with better performance of subcritical water extract. Anti-biofilm activity of the two extracts implies that, in the case of preventing biofilm formation, both extracts had similar efficiency but in the removal of biofilm, subcritical water extract showed better performance. Both extracts had anti-microbial activity against B. cereus, S. aureus, S. saprophyticus, and B. subtilis with better performance of subcritical water extract. Anti-enzymatic assay also showed similar results. Conclusions: Subcritical water extract of H. isora showed more antioxidant activity as well as anti- biofilm, anti-bacterial, and anti-enzymatic activity rather than ordinary water extract. Key words: Helicteres isora L.; subcritical water; anti-biofilm; antioxidant; anti-bacterial. © The Author 2019. Published by Oxford University Press on behalf of Zhejiang University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by- nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact firstname.lastname@example.org Downloaded from https://academic.oup.com/fqs/advance-article-abstract/doi/10.1093/fqsafe/fyz038/5679558 by guest on 19 February 2020 2 Didar Water extraction of H. isora Introduction For water extraction, the method outlined by Kumar et al. (2013) Plant materials possess various bio-molecule components with was adapted. One hundred grams of H. isora were soaked in 3-L beneficial biological activities including antioxidant, anti-microbial, distilled water. The extraction method was cold percolation (25°C, antifungal, antiviral, anti- inflammatory, and anticancer activities 24 h) (Kumar et al., 2013). (Cvetanović et al., 2018). Novel scientific trends application of nat- ural products (such as plants extract, pure compounds, or stand- Anti-microbial activity ardized extracts) in the different fields of science such as therapy, Microbial strains of S. aureus (PTCC 1112), S. saprophyticus prevention of numerous diseases, and food technology. Different (PTCC 1440), B. subtilis (PTCC1720), and B. cereus (PTCC researches affirmed beneficial of diet composed of plant in human 1015) were purchased from Iranian Research Organization for health and prevention of cancers, cardiovascular diseases and dia- Science and Technology (IROST) in a lyophilized form. Thereafter, betes, and osteoporosis and neurodegenerative diseases (Graf et al., lyophilized vials of bacteria were broken under sterile condition 2005). Accordingly, there is interest for investigation of characteris- and transferred to a suitable culture medium (according to the rec- tics of plant material sources and their beneficial impacts. Food in- ommendation of IROST) and incubated at 37°C for 24 h (Yolmeh dustry is one of the fields focused on application of natural products et al., 2015). Microbial cells were harvested by centrifugation at in food, especially for enhancement of the food functionality proper- 4000 g (ALC4232model). The MacFarland method was then ap- ties and replacement synthetic components with natural compounds. 6 −1 plied for adjustment microbial population equal to 10 CFU ml Extraction approach is one of the main factors affecting the efficiency (Moradian Eivari et al., 2015). of resulted plant extract. For this purpose, various techniques are Anti-bacterial activity was evaluated using the disk diffusion available in which most of them are in the basis of extraction via method. For this reason, bacterial population equal to the popu- organic solvents. Organic solvents have a negative impact on human 6 −1 lation of 10 CFU ml was transferred to culture media. Then, health as well as environment. Some environmentally benign tech- 20 μl of extracts of H. isora poured on the disks and incubated at nologies are introduced by some researchers which imply on water 37°C for 24 h. Inhibition zone was measured by the Guanglu 25-0 usage as solvent. A disadvantage of these methods includes low solu- Digital Caliper and considered as the susceptibility of the bacteria bility of many organic components in water and high energy need for (Mohammadi et al., 2015). solvent removal (Cvjetko Bubalo et al., 2015). Subcritical is a tech- nique in which water acts as an excellent solvent and dissolves all the components and could be used for the extraction of non-polar and Anti-attach activity of H. isora extracts against moderately polar compounds under the controlled test conditions. biofilms of bacterial strains Helicteres isora L. is a plant that belongs to the Sterculiaceae Preparation of bacterial strains was carried out according to the fol- family. Various beneficial properties of H. isora were reported that lowing steps. To determine the biofilm formation, 200 μl of broth include anticancer, antioxidant, antiperoxidative potency, and anti- 6 media containing bacterial strains (in population equal to 10 bacterial potency (Kumar & Kumar Singh, 2014). −1 CFU ml ) were transferred to polystyrene microplates. Two hun- Subcritical water extraction has been applied to extract value-added dred microliters of H. isora extracts were added to each well of a by-products (e.g. bioactive phenolic compounds) from plants such as microplate and then, incubated at 37°C for 24 h. The wells con- xanthone from mangosteen pericarps (Machmudaha et al., 2018), taining sterile broth alone were used as control. After incubation, flavanones from defatted orange peel (Lachos-Pereza et al., 2018), the broth culture medium was drained and each well was washed antioxidants from mountain germander (Nastić et al., 2018), and three times with 200 μl of phosphate-buffered saline (pH = 7.7) and Nannochloropsis salina oil (Eikani et al., 2019). Cvetanovic et al. reversed to dry, then washed with ethanol 95%, and stained with (2017) pointed out subcritical water at specific conditions that re- 100 μl of 1% Crystal Violet for 5 min. The remaining colour was sulted in suitable performance in extraction of apigenin (Cvetanovic washed three times with sterile distilled water. The microplate was et al., 2017). Gabaston et al. (2018) concluded that applying subcrit- dried for 30 min, and then the optical density at 570 nm was meas- ical water extraction method resulted in more extraction of stilbenes ured by ELISA Reader (AWARNESS model). The biofilm formation from grapevine by-products (Gabaston et al., 2018). was classified as follows: OD > 1, high levels of biofilm formation; The aim of this research was to compare water extract of 0.1 ˂ OD > 1, average biofilm formation; and OD ˂ 0.1, no biofilm H. isora, in terms of chemical composition, antioxidant property, formation (Noumi et al., 2017). anti-enzymatic, anti-microbial, and anti-biofilm activities using water and subcritical water extraction methods. Biofilm removal activity of extracts of H. isora To investigate the effect of extracts of H. isora on the removal of 8 −1 formed bacterial biofilms, a specific population (10 CFU ml ) of Materials and Methods each bacterial strain was cultured in microplate and incubated at Plant material 37°C, 24 h. Then, extracts of H. isora were added to each well and Helicteres isora was purchased from local market and its variety incubated at 37°C for 150 min. Then the contents of each well were was confirmed in Systematic Biology of Islamic Azad University, drained and the other steps of washing and staining of the microplate Neyshabur branch. were similar to those described above (Todorov et al., 2018). Preparation of subcritical water extract Anti-enzymatic activity of water extracts of H. isora The extraction of H. isora was performed under recommended con- The anti-enzymatic activity of extracts against polyphenol oxidase of ditions by Mohammadi et al. (2014): 160°C, 30 min, pressure = 10 potato was determined as follows: first, potato samples were subjected bar, sample-to-solvent ratio = 1:30. The obtained extracts were fil- to water and subcritical water extracts for 72 h and dried at room tered and stored in a refrigerator for further analysis. temperature (Fasih et al., 2016). Thereafter, to determine the activity Downloaded from https://academic.oup.com/fqs/advance-article-abstract/doi/10.1093/fqsafe/fyz038/5679558 by guest on 19 February 2020 A study on Helicteres isora extracts 3 0.05 mM (300 µl) was blended to 40-ml extract with 1000 µg/ml concentrations. After 5 min, absorbance was read at 517 nm. The radical scavenging activity of the plant extract was expressed as per- centage of inhibition against control (Braca et al., 2002). Statistical analysis All analyses were performed in triplicate and are expressed as means ± standard deviation (SD). Mean values were considered as signifi- cantly different at P < 0.05 confidence level, after the ANOVA ana- lysis. Comparison of means performed by Duncan. Results and Discussion Chemical composition of water extracts of H. isora The obtained results of chemical composition analysis by GC– Mass chromatoraphy revealed that various chemical components including aldehydes, alchohols, alkaloids, fatty acids, and esters are present in extracts of H. isora (Figure 1). Some differences in chem- ical composition of the two examined extracts were observed and some chemicals were present only in subcritical extracted extract (including hexadecanoic acid, Octadecnoic acid, and berberine). The main reason of these observations might be attributed to the specific characteristics of the subcritical method in extracting com- pounds. Subcritical water can be an alternative approach for the extraction of components with low polarity. With the aid of this method, several components with low polarity could be select- ively extracted at various temperatures (100–374°C) and pressure. Under subcritical water conditions, increasing temperature resulted in weakening of hydrogen bonding of water and enhancement of dielectric constant of molecules of water (Teo et al., 2010). As the temperature of water reaches 225°C, the dielectric constant of water reaches close tor the dielectric constant of methanol and ethanol at ambient temperature (Miller et al., 1998). Other re- ports pointed out more efficiency of subcritical water extraction method in extracting different chemical components. Nastić et al. (2018) reported that extraction of bioactive components such as gallic acid, catechin, chlorogenic acid, vanillic acid, caffeic acid, and epicatechin through subcritical water extraction from trad- itional Serbian medicinal plants had better performance (Nastić et al., 2018). Figure 1. GC–Mass of Helicteres isora L. extracts. (A) Water. (B) Subcritical Antioxidant activity of water extract of H. isora water. According to the results, antioxidant capacity of ordinary water extract and subcritical water extract of H. isora was 21.2% and of polyphenol oxidase enzyme, 2.3 ml of phosphate buffer (pH = 7) 35.5%, respectively. This result approved the antioxidant capacity −1 and 0.6 ml of pyrocatechol 100 mmol ml were placed in a water bath of two tested extract as well as higher antioxidant capacity of sub- at 25°C. The reaction was started by adding 0.1 ml of enzyme extract. critical water extract, which could be attributed to the presence Absorption was measured by a spectrophotometer (Jenway 6305, UK) of chemical compounds with antioxidant characteristics such as at 420 nm in a 10-min period. In the control sample, distilled water hexadecanoic acid, octadecanoic acid, and berberine in subcritical was used instead of the enzyme extract (Lante et al., 2015). water extract (Table 1). According to statistical analysis, antioxi- dant activity of the two extracts of H. isora significantly differed (P ≤ 0.05). Chemical composition This result is in accordance with the results obtained from GC– GC–MS analysis was performed by using Agilent 7890 A, injector Mass analysis of the two tested extracts. The subcritical method 7683B, capillary column HP with the length of 30 m, ID 0.25 µm, shows more extraction of chemical compounds with antioxidant and film thickness of 0.25 µm (Barupal et al., 2019). potency resulted in more antioxidant activity of the extract. Some researchers also reported similar results. Higher antioxidant activity DPPH radical scavenging assay against 1,1-Diphenyl-2-picrylhydrazyl radicals was observed in sub- For the assessment of antioxidant activity of H. isora extracts, DPPH critical water extracts of tested plant materials (G. macrorrhizum, radical scavenging assay was applied. Ethanolic solution of DPPH T. chamaedrys) (Nastić et al., 2018). Downloaded from https://academic.oup.com/fqs/advance-article-abstract/doi/10.1093/fqsafe/fyz038/5679558 by guest on 19 February 2020 4 Didar Anti-attach activity of water extracts of H. isora The anti-attach property of H. isora extracts might be attributed to the existence of chemical components with bioactive characteristics. Biofilm formation causes remarkable issues in food industry in the Some researchers reported the efficiency of plant extracts view point of safety. This is more considerable in relation to the for- against bacterial biofilms. Mohammadi et al. (2019) confirmed anti- mation of biofilm from pathogenic bacteria. biofilm activity of cold extract of Carum copticum against biofilm Anti-biofilm activity included two different parts: prevention of of B. cereus, S. aureus, Pseudomonas aeruginosa, Escherichia coli, biofilm formation (anti-attach property) and biofilm removal. In the Acinetobacter baumannii, and Klebsiella pneumonia. According to present study, the effect of two tested extracts of H. isora was as- their report, susceptibility of bacterial biofilm structure was varied sessed in terms of anti-attach property and biofilm removal. The re- against plant extract (Mohammadi et al., 2019). sults of anti-attach activity of H. isora extracts are depicted in Table 2. Accordingly, all tested bacteria were capable of forming biofilm Biofilm removal activity of water extracts of H. isora and their biofilm formation ability was moderate (OD > 0.1). In the case of both forms of H. isora extracts, biofilm formation was pre- against biofilm of bacterial strains vented (OD < 0.1). So, it could be concluded that both extracts have The formation of biofilms, especially biofilms of pathogenic bacteria, similar efficiency in preventing bacterial biofilm formation (Table 2). causes important safety issues; so, various approaches can be used for removal of formed bacterial biofilms, including application of Table 1. GC–Mass analysis of subcritical water extract of Helicteres plant extracts and plant essential oils. Table 3 depicts the results bio- isora L. film removal activity of the two tested extracts. Accordingly, both extracts of H. isora were effective in removal of biofilms of B. cereus, Components Components Biological activity B. subtilis, S. aureus, and S. saprophyticus. Only in the case of water detected in the detected in the extract of H. isora, optical density more than 0.1 implies retention subcritical water water extract the biofilm of B. cereus in a moderate magnitude (OD = 0.167 ± extract 0.011). Formic acid, Formic acid, Preservative, anti-bacterial 1-methylethyl 1-methylethyl agent, treatment for warts Anti-bacterial activity of water extracts of H. isora ester ester (Tyagi & Agarwal, 2017). Anti-microbial activity of H. isora extracts was assessed by measure- 1-Butanol,2- 1-Butanol,2- – ment inhibition zone and the results are shown in Table 4. As it is ap- methyl methyl parent, both extracts have anti-microbial activity against B. cereus, Hexadecanoic – Anti-inflammatory S. aureus, S. saprophyticus, and B. subtilis. The highest inhibition acid property (Aparna et al., 2012). zone belongs to B. subtilis and this bacteria showed more suscep- 1-Octen-3-ol 1-Octen-3-ol Anti-microbial activity tibility against both types of H. isora extracts. The results revealed (Xiong et al., 2017). Heptadecen-(8) Heptadecen-(8) – that the subcritical extract had more impact on all tested bacteria. -carbonic acid-(1) -carbonic acid-(1) This could be ascending to more bioactive components in subcritical Octadecnoic acid – – water extract of H. isora than ordinary water extract, according to Berberine – Anti-inflammatory, stomachic, GC–Mass analysis. anticancer, analgesic, antibiotic, Inhibition growth of microbes due to the plant extracts is re- anticholera, antidysentric, anti- ported by other researches. Mohammadi et al. (2019) showed that bacterial (Deepak et al., 2014). efficiency of different extracts of C. copticum against planktonic Table 2. Anti-attach activity of water extracts of Helicteres isora L. against different bacterial strains biofilms Extract type Optical density at 570 nm Bacillus cereus Staphylococcus aureus Staphylococcus saprophyticus Bacillus subtilis aB aA aC aD Control 0.167 ± 0.011 0.254 ± 0.03 0.146 ± 0.017 0.138 ± 0.019 bA bA bC bB Ordinary water extract 0.014 ± 0.01 0.013 ± 0.01 0.007 ± 0.01 0.011 ± 0.01 cA cA cB bA Subcritical water extract 0.012 ± 0.01 0.011 ± 0.01 0.005 ± 0.01 0.010 ± 0.01 Means with different uppercase letters in the same row show significance (P ≤ 0.05). Means with different lowercase letters in the same column show signifi- cance (P ≤ 0.05). Table 3. Biofilm removal activity water extracts of H. isora against different bacterial strains Extract type Optical density at 570 nm Bacillus cereus Staphylococcus aureus Staphylococcus saprophyticus Bacillus subtilis aA aB aC aD Control 0.401 ± 0.02 0.254 ± 0.03 0.146 ± 0.017 0.138 ± 0.019 bA bB bC bD Water extract 0.167 ± 0.011 0.084 ± 0.01 0.066 ± 0.011 0.052 ± 0.01 cA cB cC cD Subcritical water extract 0.09 ± 0.011 0.075 ± 0.01 0.054 ± 0.011 0.041 ± 0.12 Means with different uppercase letters in the same row show significance (P ≤ 0.05). Means with different lowercase letters in the same column show signifi- cance (P ≤ 0.05). Downloaded from https://academic.oup.com/fqs/advance-article-abstract/doi/10.1093/fqsafe/fyz038/5679558 by guest on 19 February 2020 A study on Helicteres isora extracts 5 Table 4. Inhibitory zone of water extracts of H. isora against differ- Barupal, T., Meena, M., Sharma, K. (2019). 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Food Quality and Safety – Oxford University Press
Published: Jul 17, 2020
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