Effect of extraction temperature and solvent type on the bioactive potential of Ocimum gratissimum L. extracts

Ocimum gratissimum is a shrub that belongs to the Lamiaceae family of plants. Despite the known biological activities and ethnomedicinal applications, comparative evaluation of the effects of different extraction techniques on the chemical and bioactive properties of O. gratissimum extracts has not yet been performed. This study adopted different analytical techniques to determine the effect of extraction temperature and solvent type on the phytochemical and bioactive properties of O. gratissimum extracts. Chemical profiling showed increased concentrations of compounds for both the ethanolic and methanolic extracts compared to the water extracts. The results also revealed that the extraction temperature had an effect on the total phenolic content and radical-scavenging properties of the different extracts. The antioxidant kinetic modeling achieved the best fit when using the second-order kinetic model. Methanolic extracts had the highest levels of antibacterial activity against Escherichia coli, Bacillus cereus, Staphylococcus aureus, and Salmonella typhimurium. At high concentrations, all extracts lowered the viability of the breast cancer cell line MDA-MB-231. In conclusion, the chemical and bioactive properties of all extracts showed significant dependence on the extraction temperature and solvent type. With proper extraction methods, they boast a wide range of promising applications in the medical, pharmaceutical, and food industries.

Microbial pollution is a global problem that is primarily caused by pathogenic bacteria such as Bacillus cereus, Escherichia coli, Salmonella typhimurium, and Staphylococcus aureus 1 . Recent studies have focused on the extraction of polyphenols from plants due to the antimicrobial 2 and antioxidant 3 effects of these compounds, as well as their ability to decrease food spoilage resulting from lipid oxidation without affecting the quality, safety, and freshness of the food product 4,5 . Moreover, a study by Shahidi and Zhong 6 demonstrated the link between oxidative stress and the pathophysiology of several health conditions. Ocimum gratissimum L. often called clove basil or scent leaf, is indigenous to coastal, savannah, and tropical areas 7 , and is mostly found in tropical areas such as West Africa, Asia, Brazil, and India 8 . Ethnomedicinal applications have been reported for the plant due to its flowers and leaves being rich in polyphenols and other bioactive compounds 9 . Polyphenol isolation from plant sources can be achieved via several techniques. However, every technique has advantages and disadvantages. Hence, there is a need to ascertain which materials, techniques, and extraction conditions are optimal for polyphenol extraction, as the efficiency of the extraction process can affect the polyphenolic content and antioxidant capacity of the extract 10 . A universally acceptable protocol for extraction of polyphenols from plants would be difficult to establish due to the structural and compositional diversity of different plant phenolic compounds. Several variables, such as temperature and the nature of the solvent, may act independently or dependently to affect the extraction efficiency as well as the antioxidant capacity. Hence, there is a need to understand the mass transfer phenomenon for the extraction process using mathematical models. The use of kinetics and mathematical models facilitates the understanding of the mass transfer mechanism of the extraction process, which is essential for simulations, optimization, control, and design of the process and minimizes the number of experiments required 11 .
Although previous studies have reported the effects of different extraction solvents on the phytochemicals and bioactivity of plant extracts, none have developed extraction kinetic and phenomenological models for the effects of temperature variations and different common solvents (methanol, ethanol, and water) on the antioxidant and Gas chromatography-mass spectrometry analysis of O. gratissimum extracts. The chemical composition of the extracts was determined using gas chromatography-mass spectrometry (GC-MS), in a method modified from that reported by Kavaz et al. 5 . A fused-silica capillary column (film thickness: 30 × 0.25 HP-5Ms, 0.25 µm) was used for the determination of compounds, with helium acting as the carrier gas (1/mL flow rate). An oven temperature of 40 °C was set and held for 5 min, then increased by 3 °C/min up to 270 °C. The split ratio was set at 60:1. The connection parts and ion sources were set at a temperature of 180 °C, and an interface temperature of 240 °C was set for the mass spectrometer. For the ionization energy amount, 70 eV was adopted, while the electron impact (EI) mode was chosen to produce stable and reproducible mass spectra and a value range of 50-650 m/z was used for the running of samples. The MS delay time before scanning was 5 min. The identification of the extract components was performed by comparing each compound's mass spectra with records in the National Institute of Standards and Technology (Gaithersburg, MD, USA) 14 and Wiley (Hoboken, NJ, USA) MS libraries.
Determination of total phenolic content. The total phenolic content (TPC) of the extracts was evaluated using the Folin-Ciocâlteu technique as described by Shahidi and Zhong 6 , with a few modifications. Briefly, 100 µL (0.5 mg/mL) of the OGE, OGM, and OGW extracts was placed in separate tubes. Next, 500 µL (1% v/v) of Folin-Ciocâlteu reagent was added to each tube and the tubes were gently agitated for 5 min. Subsequently, 400 µL (20% w/w) of sodium carbonate was added to the aliquots, which were then incubated in the dark at room temperature for 20 min. The absorbance of each mixture was evaluated with an ultraviolet-visible spectrophotometer at 765 nm. Sodium carbonate solution without any addition of Folin-Ciocâlteu reagent were used as blanks. For the calibration curve, gallic acid standards were used. TPCs of the samples were determined from the linear regression of the gallic acid standards. The results were represented as the gallic acid equivalent (GAE) per gram of dry weight of O. gratissimum extract (mg GAE/g). The procedure was conducted in triplicate (n = 3). DPPH free radical scavenging assay. The free radical scavenging activity of each extract was determined according to the methods described by Rakmai et al. 12 , with slight modifications. In brief, 2 mL of DPPHmethanol solution (180 µmol/L) was mixed with the different extracts (0.1 mg/mL). The aliquots were incubated in the dark at 25 °C. The absorbance of each sample was determined using a spectrophotometer at 517 nm at different time intervals (0-60 min). Aliquots of the extracts without addition of DPPH-methanol solution were used as blanks. Trolox, a synthetic analog of vitamin E, was used as a positive control. Equation (1) was used to evaluate the scavenging properties of the extracts. The procedure was carried out in triplicate (n = 3) where A sample is the extract + DPPH, A blank is the extract only, A control is the absorbance of the control solution (containing only DPPH).

Mathematical modeling of antioxidant activity.
To generate a description of the process, some assumptions were made, including: (1) The particles are solid, with a uniform distribution of bioactive components within the sphere.
(2) Absolute miscibility of solvent, with negligible liquid phase transfer resistance. To determine the antioxidant kinetic pattern of the O. gratissimum extracts, the general kinetic models of zero-, first-, and second-order reactions were adopted, as presented in Eqs. (2)-(4), respectively 13 where t is the reaction time (day); k (/day) represents the reaction constant; [C 0 ] and [C] are the initial and final amounts of compounds, respectively, at the different times t and temperatures (°C).
Calculation of phenolic content coefficient and relative antioxidant activity index. The relative antioxidant capacity (RACI) was calculated to further compare the antioxidant activity of the different extracts according to methods by Gorjanovic et al. 14 . RACI was calculated by subtracting the antioxidant mean values of the extracts from the raw data divided by the standard deviation. The formula for calculating the RACI is presented in Eq. (5) where σ represents the standard deviation, y raw data, and x the mean. The phenolic antioxidant coefficient (PAC) were calculated as the ratio between the total phenolic content and the antioxidant capacity of the extracts 15 .
Evaluation of antibacterial activity of O. gratissimum extracts. Two strains of Gram-negative bacteria, S. typhimurium (ATCC 13311) and E. coli (ATCC 8739), and two strains of Gram-positive bacteria, B. cereus (ATCC I4579) and S. aureus (ATCC 25923), were obtained in suspension from the American Type Culture Collection (Manassas, VA, USA). All were adjusted to 1.5 × 10 8 CFU/mL which is the McFarland standard, and examined to discern their minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) relative to OGE, OGM, and OGW extracts using the microdilution method described by Devrnja et al. 16 , with some modifications. Briefly, extracts were diluted to concentrations of 500-2 mg/mL and added to the micro-well plates containing MHB. Next, 10 µL of the McFarland standardized microbial suspension inoculums were added to all of the wells for each type of bacteria strain, and the plates were then incubated for 24 h at 37 °C. The MIC of the samples were established following the addition of 40 µL of iodonitrotetrazolium chloride (INT; 0.02% m/v) and incubation for 30 min at 37 °C. The lowest concentration having no form of microbial growth (P ≤ 0.005) in comparison with the positive control (culture medium containing Tween 80, microbial suspension, and ethanol) was established as the MIC. Culture medium (MHB) containing only the different types of bacteria strain served as the negative control. To further establish the MIC, the bacteria in the culture medium showing no microbial growth were transferred to an MHA plate and then incubated at 37 °C for 24 h. The lowest concentrations of the O. gratissimum extracts (OGE, OGM, and OGW) that inhibited the proliferation of the test microorganism after 24 h of incubation at 37 °C were reported as the respective MBCs. All experimental procedures were conducted in triplicate (n = 3).
Trypan blue exclusion assay. The determination of the cytotoxic properties of O. gratissimum extracts on MDA-MB-231 cells was carried out using a trypan blue dye exclusion assay. Cells (3 × 10 4 /mL) were plated in 35 mm dishes and allowed to incubate overnight before treatment with several concentrations of extract (0-100 µg/mL) according to the respective OGE-40, OGE-50, OGE-60, OGM-40, OGM-50, OGM-60, OGW-90, OGW-100, and OGW-110 protocols. The control group was treated with 1 mL of Dulbecco's modified eagle medium. After a 48-h incubation period, trypan blue dye (4%) was dropped into all culture plates, which were further incubation for 40 min. The viability of the cells was determined using an inverted microscope (DFC295; Leica Camera, Wetzlar, Germany), choosing cells from 30 randomly selected sites within the dishes.

Statistical analyses.
All experiments were conducted a minimum of three times (n ≥ 3). One-way analysis of variance (ANOVA) and Student's two-tailed t test were used for statistical comparisons. Differences in mean values were regarded as very significant at P ≤ 0.0001, significant at P ≤ 0.05, and nonsignificant at P > 0.

Results and discussion
Gas chromatographic chemical composition analysis of O. gratissimum extracts. The extraction procedure is one of the most important steps in the use of natural resources, as it can affect the chemical makeup as well as the biological properties of extracts obtained. Several methods of extraction rely on different www.nature.com/scientificreports/ analyte mechanisms of isolation from matrices, and the technique used may rely on the nature of the plant material. For this study, the chemical profiles of three different extracts (OGE, OGM, and OGW) were revealed using GC-MS. The results obtained for the OGE are presented in Table 1, which shows that the lowest concentration (% wt/wt) of observed components was found in extracts prepared at 50 °C (OGE-50), while the concentrations were higher in extracts prepared at 40 °C (OGE-40) and at 60 °C (OGE-60). Samples obtained using ethanolic and methanolic extracts contained a higher amount of bioactive compounds, including sabinene, terpenene, thymol, copaene, caryophyllene, humulene, selinene, caryphylene oxides, and phytol, compared to OGW extracts. The concentration of constituent compounds in OGE extracts varies at different temperatures, with OGE-60 > OGE-40 > OGE-50. This might be due to altered dissolving abilities of the solvents at different temperatures during the process of extraction 17 . A study by Cvetanović et al. 18 on extractions of Aronia melanocarpa M. stem reported more flavonoids and phenolic content in extractions with ethanol compared to extractions with methanol. In contrast, our results revealed that a high number of compounds was observed in OGM extracts in all cases (Table 2). Hence, it can be generalized that extraction with methanol is the preferred technique for isolation of several bioactive compounds from the O. gratissimum plant. As reported by Gharaati 19 , methanol can easily infiltrate into plant tissue and increase the process of extraction. The concentration of compounds in the methanolic extracts was highest in OGM-40, followed by OGM-60 and OGM-50. For OGW extracts, those prepared at 110 °C showed the highest number and concentration of identified compounds, as reported in Table 3.
The differences in concentration of compounds could be a consequence of the extraction temperature resulting in rupture of the plant cell walls, leading to diffusion of the plant constituents into the water medium. Moreover, the lower number of bioactive compounds in OGW extracts compared to OGE and OGM extracts might be due to reduced compound solubility in water, as well as the extraction conditions. Nevertheless, an increase in temperature causes a decrease in water's dielectric constant, and as a result, fewer polar compounds will be dissolved in it. Chemical and physical properties of water can change drastically under supercritical conditions. In addition, under certain operational conditions, the polarity of water can be fine-tuned 18 . Antioxidant activities of O. gratissimum extracts. In a biological system, free radicals can cause damage in vivo and are consequently considered a cause of numerous diseases. Hence, the scavenging of free radicals is an essential task for antioxidant compounds to protect living systems. The results of our experiments testing antioxidant activity revealed that the extracts investigated showed notable potential for oxidation inhibition. As shown in Fig. 1, the activity can be ranked as follows: OGM-40 > OGE-60 > OGW-110 > OGM-50 > OGE-40 > OGE-50 > OGW-90 > OGW-100. The free radical-scavenging activity of compounds, as outlined by Kfoury et al. 24 , is examined after 30 min contact of DPPH solution with the compound. However, Kamimura et al. 25 proposed that the antioxidant activity of compounds is likely to last much longer, depending on the oxidation kinetics over time. OGM antioxidant ability was higher from the period of 1-5 h compared to the rest of the extracts. The high antioxidant activity of OGM-40 is attributed to the high level of phenolics in this extract compared to the others. The findings of this study are in agreement with those of the study by Ngo et al. 26 on antioxidant activity of methanolic extracts of Salacia chinensis L. root. Among the OGW extracts, the scavenging power was the greatest at 100 °C, less at 90 °C, and even more reduced at 110 °C. This shows that antioxidant properties can be greatly reduced at extraction temperatures above the optimum/boiling temperature of the solvent, as this can lead to the denaturation of phenolics, as was the case with OGW-110.
For the OGE and OGM extracts, the effect of temperature on the oxidant-scavenging properties of these extracts was in line with the findings of Molaveisi, Beigbabaei, Akbari, Noghabi, and Mohamadi 27 . www.nature.com/scientificreports/ The DPPH scavenging capacity of the extracts was also evaluated by calculating their IC 50 values, which correlates to the amount of extract that is capable of scavenging 50% of the free radicals contained in the reaction mixture. A low IC 50 value indicates high free radical-scavenging activity, and vice versa. The IC 50 values of the extracts are within the range of 0.75-2.84 mg/mL (Table 5). Based on the IC 50 values, the order of free radicalscavenging activity of the extracts is as follows: OGE-40 > OGE-50 > OGE-60 > OGW-90 > OGM-50 > OGM-40 > OGM-60 > OGW-100 > OGW-110 ( Table 5). The high antioxidant capacity of O. gratissimum is often considered a function of its high phenolic content. However, our results showed that extracts with low TPC also exhibited high antioxidant activity, suggesting that in addition to phenolics, other factors, such as extraction temperature and solvent type, can also contribute to an extract's antioxidant activity.
Phenolic antioxidant coefficient and relative capacity index. In order to further compare the TPC and antioxidant capacity of the extracts, two additional parameters, namely, PAC, which is the ratio between TPC, and particular antioxidant capacity; and RACI, which determines the total reducing capacity, were introduced. In Fig. 2, it can be seen that the highest RACI value is ascribed to OGE-40 (1.  (Fig. 2). A study by Wojdylo et al. 28 suggest that with high PAC values are not a reflection of high antioxidant activity in plants; however, plants with substantial PAC and RACI values is an indication of their rich antioxidant potential. The findings of this study are similar to those from a study by Petrovic et al. 29 .  www.nature.com/scientificreports/ Kinetic models for the study of the effect of solvent composition and temperature on the antioxidant activity of O. gratissimum extracts. As shown in Fig. 3, varying the temperature and type of solvent had a significant effect on the antioxidant kinetics of O. gratissimum extracts. Data analysis using several kinetic models showed that the highest coefficient of determination (r 2 ) value for both the zero-and first-order kinetic models was achieved with OGE-50, with r 2 values of 0.694 and 0.793, respectively. In contrast, for the second-order model, the highest adjusted r 2 value was achieved with OGE-60 (r 2 = 0.436, K = 0.314; Table 6). The linear relationship between lnK for the antioxidant activity for all the extracts over time had the best fit to the second-order kinetics model. These results are in agreement with previous studies, such as a study that reported the kinetics of the effect of temperature on antioxidant activity of Iranian jujube honey 27 (Fig. S1). For the cancer cells treated with OGM-40, membrane blebs were also observed after 48 h of incubation. This is an indication of cell apoptosis, as suggested by Onyebuchi and Kavaz 7 . The high antiproliferative activity of OGM-40 compared to the rest of the extracts is likely a result of oxidative stress generation within the cell membrane.

Conclusion
The chemical profile and bioactive properties of O. gratissimum extracts prepared using different extraction temperatures and solvent types were examined in this study. The results obtained herein suggest that both extraction temperature and solvent type have a significant effect on the phenolic content yield, antioxidant capacity, www.nature.com/scientificreports/ antimicrobial activity, and cancer cell cytotoxic properties of O. gratissimum extracts. For extracts prepared with methanol, extraction performed at 40 °C (OGM-40) was the optimal protocol for obtaining high TPC and desirable chemical properties. The extracts showed strong temperature dependence and best fit with the second-order kinetic model, as the rate of antioxidant activity tends to increase with an increase in extraction temperature. The findings of this study further establish the potential use of the extracts as alternative natural food antioxidants over synthetic preservatives. The antimicrobial and anticancer properties of the extracts also demonstrate their potential applications in the pharmaceutical and food industries.