Effects of Sterilization on Bioactives of Jatropha dioica and Opuntia oligacantha Extracts, and on Antimicrobial Capacity against Streptococcus mutans

: Natural extracts have been used as antimicrobial agents, and extracts of the rhizomes of the dragon’s blood plant ( Jatropha dioica ) and of the fruit of xoconostle ‘Ulapa’ ( Opuntia oligacantha ) are among these agents. To be able to use the extracts, it is necessary to eliminate their natural microbiota by the means of sterilization methods; however, the effects of thermal sterilization on their properties have not been evaluated. The objective of the study was to evaluate the physico-chemical, antioxidant, and antimicrobial properties of extracts of the rhizomes of dragon’s blood and the fruit of xoconostle, when not sterilized, and when sterilized by autoclaving. The contents of phenolic compounds, ﬂavonoids, antioxidant, and antimicrobial activity against Streptococcus mutans were not affected in autoclave-sterilized extracts of dragon’s blood rhizomes, and naringenin, apigenin, galagina, vanillic, and caffeic acid were detected, while in autoclave-sterilized extracts of xoconostle fruits, the betalain content decreased, with galagin, gallic, vanillic, and p-coumaric acid being identiﬁed. The minimum inhibitory concentration for the extract sterilized by autoclaving dragon’s blood was 2 mg/mL, and for the extract sterilized by autoclaving xoconostle, it was 28 mg/mL. Both represent natural alternatives as antimicrobial agents against S. mutans. variance was carried out with a completely randomized design, where the effects of sterilization by autoclaving on the response variables was evaluated: total phenols, total ﬂavonoids, betalains, antioxidant activity, and antimicrobial activity against Streptococcus mutans . The analysis of means was performed by the Tukey test ( p < 0.05); this was carried out in the statistical package SAS version 9.4. After sterilization treatment by autoclaving, the antimicrobial properties of the dragon fruit rhizome extract were maintained, and its low concentration for the inhibition of S. mutans represents an alternative to synthetic antimicrobials as an adjuvant, or to prevent oral diseases such as caries. These results suggest that an extract of autoclave-sterilized dragon ’ s blood rhizomes could be an excellent natural source of antimicrobial compounds, as well as a safe ingredient for food or pharmaceutical products.


Introduction
Streptococcus mutans is a Gram-positive bacterial involved in oral health issues, as it is responsible for tooth decay, which triggers other illnesses such as gingivitis and periodontitis [1]. Some of the most commonly used chemical agents to control plaque and tooth decay related to S. mutans are chlorhexidine and triclosan; however, long-term use of these substances causes secondary effects such as brown staining in teeth, altered taste, and scaly lesions in the alveolar mucosa [2]. Currently, the use of natural antimicrobial agents with efficient performance in the inhibition of pathogenic Xoconostle fruits (Opuntia oligacantha CF Först var. 'Ulapa') were sampled during February 2017 at Ulapa Melchor Ocampo Hidalgo, in the Municipality of Tetepango, Hidalgo, Mexico, located at the coordinates 20 • 40 55 N and 99 • 04 59 W, at 2100 m a.s.l., with semi-dry temperate weather and alluvial soil. Xoconostle fruits were identified according to Hernández-Fuentes et al. 2015. Fruit at the stage of commercial maturity (completely colored) were selected and washed, and the glochids were removed. The fruits were stored at ultra-low temperature (−70 • C) (Thermo Scientific 703 Ultra-Low Freezer, Grand Island, NY, USA), lyophilized (Model 79480 LABCONCO, Kansas City, MO, USA), and stored (5 • C for one week before use).

Extraction Procedure
Extracts of dragon's blood rhizomes were obtained following the methodology suggested by Wong-Paz et al. [12], with subtle modifications. The experimental conditions allowed for higher yields and the contents of the bioactive compounds to be obtained, and modifications were made in the drying of the rhizomes; they were not dried at 60 • C, and lyophilization was used instead. Extraction was performed using 70% ethanol at a rhizome/solvent ratio of 1:5. The liquid fraction was filtered using Whatman paper n • 5, and the solvent was brought to dryness in a rotary evaporator (Büchi R-215, Flawil, Switzerland) at 50 • C under vacuum conditions. The extract was recovered from the rotavapor flask and weighed, redissolved with distilled water at a concentration of 0.2-5 mg of dry extract/mL, and sonicated in a bath sonicator (Ultrasonic Cleaner, Mod. 32V118A, Freeport, IL, USA) for complete dissolution at 30 • C for 20 min.
Xoconostle extracts were obtained from lyophilized whole fruit (peel, pulp, and seeds). The lyophilized fruit was ground, resuspended in water, and centrifuged (Thermo Scientific, Mod. ST 16R, Waltham, MA, USA) at 17,500× g for 10 min at 5 • C. The supernatant was separated, and the procedure was repeated with the precipitate until there was total loss of color in the liquid phase. The resulting supernatants were collected and lyophilized. The dry samples were ground, weighed, and redissolved with distilled water at a concentration of 0.2-80 mg dry extract/mL, and sonicated for complete dissolution.

Non-Sterilized or Autoclave-Sterilized Extracts
Previous microbiological analyses of the extracts showed the presence of microorganisms due to contamination during their extraction; for this reason, the extracts were subjected to a sterilization process by autoclaving. The conditions of the autoclave sterilization were 121 • C for 15 min at 15 psi (Tuttnaver, 354SE, Hauppauge, NY, USA). On the other hand, the non-sterilized extracts were filtered through 0.20 µm acrodiscs (syringe filter). The nomenclature used to identify the non-sterilized extracts of dragon's blood plant rhizomes and xoconostle fruit were S A and X A , respectively; autoclave-sterilized extracts of dragon's blood rhizomes and xoconostle fruit were S B and X B , respectively.

Determination of Physicochemical Properties
Physical-chemical properties were determined for the non-sterilized and autoclave-sterilized extracts. pH was measured according to the AOAC 920.151 method [16], using a digital potentiometer (Hanna Instruments Woonsocket, RI, USA). The total sugar content was determined by spectrophotometry, following the method described by Dimler et al. [17] (antrone-H 2 SO 4 assay), measuring its absorbance at 640 nm (model 6715 UV/Vis, Jenway, Techne Inc., Staffordshire, UK). The concentration of the reducing sugars was detected by spectrophotometry, according to the method suggested by Miller [18] with 3,5-dinitrosalicylic acid (DNS) at 540 nm. Glucose was employed as a standard for the latter two estimations.
Ascorbic acid content was determined by spectrophotometry following the methodology developed by Jagota and Dani [19]. Briefly, 0.1 mL of the extract was used, which was diluted in 10 mL of trichloroacetic acid (10% w/v), and allowed to react for 10 min, and its absorbance was subsequently measured at 760 nm. The color attributes of the extracts (values of L*, a*, and b*) were measured using a Hunter Lab colorimeter (Minolta, CM508d, Minolta Camera Co., Ltd., Osaka, Japan) total color difference (∆E) was obtained with Equation (1): where ∆L* is the difference in lightness, ∆a* is the difference in red, and ∆b* is the difference in yellow detected in extracts that were non-sterilized and sterilized by autoclaving.

Total Phenolic Analysis
Phenolic compounds content was determined for non-sterilized and sterilized extracts of dragon's blood rhizomes and xoconostle fruit. The total phenolic content was estimated using the methodology proposed by Singleton and Rossi [20]. Extracts dissolved in distilled water (0.5 mL) were mixed with 2.5 mL Folin-Ciocalteu reagent and 2 mL of Na 2 CO 3 (0.7 N), and maintained under darkness conditions for 2 h. The absorbance was measured at 760 nm using a spectrophotometer (model 6715 UV/Vis, Jenway, Techne Inc., Staffordshire, UK). The total phenolic content was expressed as mg GAE/g.

Flavonoid Content
Flavonoid content was estimated according to the methodology adapted by Arvouet-Grand et al. [21]. A solution containing extracts dissolved in distilled water (2 mL) and AlCl 3 (2%) in pure methanol was prepared. The absorbance was measured at a 415 nm using a spectrophotometer. Results were expressed as mg QE/g.

Quantification of Betalain
Betalains were quantified exclusively for non-sterilized and sterilized extracts of xoconostle fruit, following the methodology suggested by Nilsson [22] for estimating the concentration of betanins and vulgaxanthins (dragon's blood plant has anthocyanins, thus excluding the presence of betalains). Xoconostle extracts were dissolved in distilled water. Measurements were recorded at 476, 538, and 600 nm using a spectrophotometer (A 476 , A 538 , and A 600 were the absorbance values obtained at these wavelengths, respectively). Betanins and vulgaxanthins were determined using Equations (2) and (3), modified by Nilsson. The content is expressed as mg/100 g dry extract (DE).

Identification of Extract Compounds by HPLC
Compounds of non-sterilized and autoclave-sterilized extracts were identified by high-performance liquid chromatography (HPLC). Extracts S A , S B , X A , and X B were prepared at a concentration of 5 mg/mL in distilled water, centrifuged at 17,500× g for 15 min, and filtered (0.2 µm). The extract content and type of metabolites were identified by HPLC, according to the methodology described by Aguiñiga-Sánchez et al. [23]. Flavonoids were determined in a HP Hypersil ODS column (125 × 40 mm) under gradient conditions of (A) H 2 O at pH 2.5 with trifluoroacetic acid (TFA), and (B) acetonitrile (ACN) with the following parameters: injection volume was 20 µL, flow rate was fixed at 1 mL/min, reaction temperature was 30 • C, and analysis period was 25 min. Standards were rutin, phlorizidin, myricetin, quercetin, naringenin, phloretin, apigenin, and galagin (Sigma Aldrich, St. Louis, MO, USA). Phenolic acids were determined with a Nucleosil (Macherey-Nagel) column (125 × 4.0 mm) under gradient conditions of (A) H 2 O at pH 2.5 with TFA and (B) ACN. The experimental parameters were the same as for the flavonoids, while the standards were caffeic, gallic, chlorogenic, vanillic, p-hydroxybenzoic, p-coumaric, ferulic, and syringic acids (Sigma Aldrich).

Determination of Antioxidant Activity by the DPPH Method
The antioxidant activities were determined for the non-sterilized and autoclave-sterilized extracts of dragon's blood rhizomes and xoconostle fruit. The 2,20-diphenyl-1-picrylhydrazyl (DPPH) method suggested by Brand Williams et al. [24], was employed for the estimations. A total of 300 µL of extract prepared in distilled water with 2.7 mL DPPH solution and maintained under darkness for 60 min, was used. The absorbance was measured at 517 nm using a spectrophotometer; the antioxidant activity was expressed as mg TE/g.

Determination of Antioxidant Activity by the ABTS Method
The 2,2 -azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) method was carried out according to Re et al. [25]. A total of 100 µL of extracts prepared with distilled water and mixed with 3.9 mL of ABTS solution (previously prepared with K 2 S 2 O 8 (2.45 mM) was used. The absorbance was measured in a spectrophotometer at 754 nm. The results were expressed as mg TE/g.

Antimicrobial Activity
The antimicrobial activities of the non-sterilized and autoclave-sterilized extracts of dragon's blood rhizomes and xoconostle fruit were determined according to the methodology developed by Domínguez et al. [26]. A mixture was prepared by combining 10 mL of Brain Heart Infusion (BHI) broth, 5 mg/mL non-sterilized or autoclave-sterilized extract, and 100 µL of liquid medium containing Streptococcus mutans (ATCC 25175, lot number: 62284317) at a concentration of 103 colony-forming units (CFU) per mL. Samples were incubated at 37 • C for 80 h. Changes in turbidity were measured every 4 h in the spectrophotometer at 600 nm, using a quartz cell (1000 µL).
Each extract was evaluated with the following: a blank containing BHI broth and the extract without Streptococcus mutans, a positive control containing 10 mL of BHI broth and 103 CFU/mL of S. mutans, and a negative control containing 10 mL of BHI broth, 103 CFU/mL of S. mutans, and 0.012 µL of chlorhexidine/mL (GUM Paroex, Sunstar Suisse S.A., Étoy, Switzerland). The concentration of chlorhexidine was established according to research developed by Aguilera et al. [27]. Results were expressed as log CFU/mL. The effect of each extract over the growth rate (µ) of S. mutans was assessed.
In addition, the minimum inhibitory concentration (MIC) was determined, which was defined as the lowest extract concentration at which the assayed bacteria did not exhibit visible growth. The BHI-broth macrodilution method and a slightly modified spectrophotometric method [28] were employed to determine the MIC. In this experiment, a strain of S. mutans (at a concentration of 103 log CFU/mL) was used, as well as two controls: negative (the strain of S. mutans and chlorhexidine) and positive (containing only the strain of S. mutans). The absorbance was read at 600 nm at 96 h. The controls were prepared with BHI broth and each extract. Turbidity was defined and measured by spectrophotometry and used as a growth parameter.

Statistical Analysis
All trials were carried out in triplicate, and the averages were expressed. An analysis of variance was carried out with a completely randomized design, where the effects of sterilization by autoclaving on the response variables was evaluated: total phenols, total flavonoids, betalains, antioxidant activity, and antimicrobial activity against Streptococcus mutans. The analysis of means was performed by the Tukey test (p < 0.05); this was carried out in the statistical package SAS version 9.4. Table 1 shows the physicochemical parameters of the extracts obtained for the natural sources used. The sterilization of the extracts caused an increase in the pH, the S B value was 16.9% higher than the S A values, while X B presented an increase of 21.87% with respect to X A . This was related to the reduction of organic acids, such as ascorbic acid. Additionally, in the autoclave-sterilized extracts, the concentration of ascorbic acid due was reduced, due to the thermolabile character of the molecule, which could explain the increase of the pH in the sterilized samples. [29]. The contents of the total sugars during the sterilization process in sample S B increased by 13.4% with respect to S A , and the reducing sugars content increased by 33.1%; however, they were not statistically different according to the Tukey test (p < 0.05). Meanwhile, in the X B samples, the content of total sugars increased by 13.0%, and the reducing sugars by 10.98%; their increase may have been due to thermal hydrolysis processes from heating during the autoclave sterilization, generating a break in the bonds of the glycosylated compounds, and increasing the concentration the concentrations of simpler sugars [30]. Letters in each column indicate statistically significant differences; pH was measured at extract concentrations of 10 mg/mL at 25.5 • C; TSC: total sugar content; RS: reducing sugars; ASC: ascorbic acid; S A : unsterilized dragon's blood rhizome extract; S B : autoclave-sterilized dragon's blood rhizome extract; X A : unsterilized xoconostle fruit extract; X B : autoclave-sterilized xoconostle fruit extract; GluE: Glucose equivalents; DW: dry weight of the sample; ASCE: ascorbic acid equivalents.

Physicochemical Analyses
Regarding the color of the extracts, it was observed that sterilized extracts of dragon's blood became darker with an amber hue with respect to the non-sterilized extracts; this was due to the reduction of luminosity and the increase of the b * -value, which tended to the color yellow ( Table 2). The color change in the dragon's blood rhizome extracts could be linked to caramelization or Maillard reactions, due to the presence of sugars and the conditions of elevated temperature during the process of autoclave-sterilization [15]. The non-sterilized xoconostle extracts had a reddish coloration (greater a* value and lower luminosity), compared to the sterilized extracts. In the latter case, this may be due to the loss of betalains that are responsible for attributing a red coloration to the fruits of xoconostle, as also occurs in other Cactaceae fruits [8,31].

Total Phenolics and Flavonoids
Autoclave-sterilization significantly affected the concentration of phenolic compounds in dragon's blood extracts, but not of those in xoconostle fruit extracts (Table 3). It was observed that the concentrations of total phenols and flavonoids in S B increased by 95.7% and 92%, respectively, almost double the amount with respect to the S A extracts. Randhir et al. [32] mention that heat treatment promotes the release of phenolic compounds, due to the breakdown of cell walls and cellular components such as vacuoles, and they also mention that thermal treatment dissociates conjugated polyphenols, such as tannins, to simpler phenolic compounds. In some studies where thermal treatment by autoclaving is used, samples of grain sprouts, seedlings, and legumes have also presented an increase in the total phenolic content [33,34]. Phenolic contents in extracts can be affected by extrinsic factors such as temperature, pH, light, and oxygen content, among others [35]. In this study, the ultrasound bath that was used for rehydration of the extracts may also have had an effect on increasing the total phenol content of dragon's blood rhizome extracts. This is because ultrasound treatment causes micro-ruptures in the suspended particles via cavitation, increasing the surface area and enhancing the solubilization of phenolic compounds [36]. On the other hand, some flavonoids are stable at high temperatures. Elhamirad and Zamanipoor [37] reported that when the temperature is increased, thermal hydrolysis of glycosylated compounds occurs, increasing their solubility and modifying the diffusion coefficient, causing an increase in flavonoid content. In this study, autoclave-sterilization did not significantly affect the content of phenolic compounds in xoconostle extracts. In contrast, the flavonoids in sterilized extracts only increased by 16.8%, compared to the non-sterilized extracts. Other compounds, such as mucilage and soluble polysaccharides (15% found in xoconostle), may have protected the bioactive compounds under high temperatures [38,39]; this may be related to the small variation of the content of phenolic compounds in the X A extracts compared to the X B extracts. Also, other researchers state that high temperatures can increase or decrease the concentration of the phenolic compounds; these results are related to factors such as temperature, exposure time, pH, water content, and raw material composition [40]. On the other hand, a slight increase in flavonoid content in X B compared to X A extracts may be due to the thermal stability that these present, depending on the media, chemical structures, and pH [37]. Also, Elhamirad and Zamanipoor [37] reported that when the temperature is increased, thermal hydrolysis of glycosylated compounds occurs, which causes an increase in the flavonoid content.
Unsterilized extracts of dragon's blood showed 75% higher contents of phenolic compounds than non-sterile extracts of xoconostle, while sterilized extracts of dragon's blood were 87.7% higher in phenolic content than xoconostle extracts that had been sterilized by autoclaving. This may be due to the fact that in the dragon's blood plant, the roots are the first tissues that detect a water deficit condition and induce stress signals, which translate into a higher concentration of secondary metabolites in which the phenolic compounds are included [13]. Another reason could be due to the Maillard reaction (non-enzymatic browning), caramelization, and the chemical oxidation of phenols, since the rhizomes of dragon's blood extracts become an amber coloration (Table 2) when subjected to autoclave sterilization [32,41]. Martínez et al. [42] reported free amino acids and amines for dragon's blood rhizomes under thermal conditions; these compounds react with reducing sugars, producing other compounds with chelating and reducing properties [43]. All of the above could have an effect on increasing the quantification of total phenols in SA.

Betalains in Xoconostle Extracts
Measurement of the content of betalains in the dragon's blood rhizome extracts was not made, due to its absence in this species. For this reason, betalain content was only taken in xoconostle fruit extracts, where a decrease of 85.19 % in the content of betanins was observed in the X B extracts with respect to the X A extracts (Table 3), while the vulgaxathin content was 23% higher in the X B extracts compared to X A extracts. The typical color loss found in the xoconostle fruit extracts subjected to high temperatures was related to a decrease in betalain and isobetanin contents (these give the red and orange coloration in xoconostle). This reduction is due to a dehydrogenation reaction in betanins and isobetanins during thermal treatments, forming compounds such as neobetanins [44]. The loss of the reddish coloration of xoconostle fruits extracts and its change to a yellow color during the process of sterilization by autoclaving may be due to the increase of neobetatins. Neobetanins have a yellow coloration, absorbing at wavelengths that are similar to vulgaxathins; therefore, their presence could be related to an observed increase of vulgaxathin content [45].

Identification of Extracted Compounds by HPLC
Some phenolic acids and flavonoids were detected and confirmed by HPLC in rhizomes of dragon's blood plant extracts and xoconostle fruits extracts. In the S B extracts, an increase of phenolic compounds was detected with respect to the S A extracts; this result has a behavior similar to that found in the quantification of the total content of phenols by the spectrophotometric method, where the sterilization process increased the content of the phenolic compounds. In the present study, the phenolic acids detected in S A and S B were vanillic and caffeic acids, while the flavonoids were naringenin, apigenin, and galagin (Tables 4 and 5). Wong-Paz et al. [12] analyzed ethanolic-aqueous dragon's blood rhizome extracts by HPLC; in their study, phenolic compounds were not detected due to their low concentrations and the poor purification of the extracts.
The phenolic compounds quantified in the xoconostle fruits extracts were gallic acid, vanillic acid, and p-coumaric acid, and for the flavonoids, it was only galagina. In this sense, the extract with the highest content of phenols was the xoconostle fruit extract, while the extract with the highest concentration of flavonoid compounds was the dragon's blood rhizome extract. On the other hand, in the extracts of the xoconostle fruit, the variation of the phenolic compounds was lower than in the dragon's blood rhizome extracts; X B extracts presented concentrations of gallic, vanillic, and p-couramic acid that were similar to that of X A extracts, while the flavonoid content increased in X B extracts (increase the content of galagin) during autoclave-sterilization treatment (Tables 4 and 5).
Some reports suggest that the presence of compounds such as caffeic, ferulic, and p-coumaric acids inhibit Gram-negative and Gram-positive microorganisms such as E. coli, S. aureus, and B. cereus [46]; the presence of these compounds could maintain the properties of antimicrobial extracts after being subjected to sterilization by autoclaving.

Free Radical Scavenging Ability as Estimated by DPPH and ABTS
The potential benefits that phenolic compounds have for health are attributed mainly to their antioxidant activities by donating a hydrogen atom from the aromatic hydroxyl group to free radicals [31]. In the present investigation, the in vitro antioxidant activities of rhizomes of dragon's blood plant and xoconostle fruit extracts were tested through the DPPH and ABTS assays. Table 3 shows the effect of thermal treatment by autoclaving on antioxidant activity. The antioxidant activity, according to the DPPH method, was higher in dragon's blood rhizome extracts than in xoconostle fruit extracts (Table 3). In the S A extracts, no significant difference was observed in the activity with respect to the S B extracts, despite the increase in the content of phenolic compounds in the S B samples. This was not reflected in an increase in antioxidant capacity; however, it was maintained through the autoclave-sterilization process, probably due to the loss of other antioxidant compounds such as nitrogen compounds, carotenoids, or ascorbic acid (Table 1), which contributed to the total sum of the antioxidant capacity of the extract [29,40,47,48]. On the other hand, some studies indicate that the reaction mechanisms of the antioxidant and DPPH depend on the structural conformation of the antioxidants [49], and these could be altered during the autoclave-sterilization process [30].
Nevertheless, a decrease of 25.7% was observed for antioxidant activity in X B when compared to X A , and this could be related to the decrease in betanin content caused by the thermal effect (Table 3). Other thermolabile compounds with antioxidant properties, such as ascorbic acid, can be affected during sterilization by autoclaving, influencing the final activity of the extracts [29,50].
The results obtained by the ABTS method indicated that S B showed a slight increase in antioxidant activity of 11% over S A (Table 3). This increase could be associated with an increase of phenols and flavonoids, as well as some compounds derived from color change reactions (caramelization and Maillard reactions) with the ability to chelate metals and to provide antioxidant activities [51].
The difference between the results observed by the ABTS method with respect to DPPH could be due to the affinity of both techniques; quantification by the ABTS method tends towards hydrophilic and lipophilic compounds, while the DPPH method tends towards slightly polar compounds [52].
With regard to the antioxidant activity by the ABTS method on xoconostle extracts, there was a decrease of 18.3% in X B with respect to X A ( Table 3). The decrease in antioxidant activity in the autoclaved extracts could be attributed to the loss of thermolabile compounds such as betalain and ascorbic acid, which are present in xoconostle fruit extracts [7].
The behaviors of compounds from extracts with complex compositions cannot be predicted when they are affected by severe thermal treatments, given that the antioxidant properties can decrease or increase, due to factors such as exposure time to the thermal treatment, the temperature, the pH of the medium, the water activity, or the composition of the extract and its interactions, when its structure is modified [40].

Antimicrobial Activity
With regard to the growth kinetics of Streptococcus mutans in S A and X A , growth was not observed in the latter extract ( Figure 1). A similar result was obtained with the negative control (chlorhexidine). Some studies state that the contents of flavonoids, tannins, and condensed phenols of high molecular weight in dragon's blood rhizomes are responsible for the antimicrobial properties found within the extracts [9,53]. Other research suggests that the antimicrobial activity of dragon's blood rhizome extracts is due to the presence of terpenes such as β-sitosterol and citlalitrione [11]. The synergy among these compounds could play an important role in the antimicrobial properties found in the extracts. The results observed for the negative control (chlorhexidine) are mainly due to its cationic nature, which allows its adherence to the bacterial cell wall; at low concentrations, it increases permeability, filtrating intracellular components (bacteriostatic effect), while at high concentrations, it precipitates the bacterial cytoplasm and causes cell death (bactericidal effect) [2].   Exponential growth of S. mutans was observed after 20 h in the adaptation phase (Lag) in the positive control; the growth rate at the logarithmic phase was µ = 0.487 h −1 (r 2 = 0.976) (Figures 1  and 2). The non-sterilized xoconostle fruit extract (5 mg/mL) only delayed S. mutans growth until 4 h, compared to the positive control during the adaptation phase (Lag); the growth rate at the logarithmic phase was µ = 0.588 h −1 (r 2 = 0.970). The antimicrobial properties of xoconostle extracts were reported by Hayek and Ibrahim [6]; however, the concentration at which xoconostle fruit extracts reported activities similar to the negative control (chlorhexidine) ranged between 28 and 80 mg/mL, which is relatively high compared to the antiseptic compounds that are contained in commercial mouth rinses, considering that the concentrations of the active compounds in these commercial products range between 1 and 2 mg/mL [54].     The growth kinetics of S. mutans in S B and X B are shown in Figure 2. S B and S A extracts presented similar behaviors; the growth values (log CFU/mL) did not vary, and thus, the temperature during the sterilization treatment did not affect the antimicrobial activity in the dragon's blood rhizome extracts. On the other hand, the growth was delayed by 12 h in the X B extracts when compared with the X A extracts during the adaptation phase (Lag), while the growth rate at the logarithmic phase in the X B extracts was µ = 0.289 h −1 (r 2 = 0.991). Therefore, the antimicrobial properties of the non-sterilized and autoclave-sterilized extracts of dragon's blood plant rhizomes did not change their behavior; meanwhile, the extracts of autoclave-sterilized xoconostle fruit delayed the appearance of the logarithmic phase and decreasing growth rate values (µ); however, they did not show antimicrobial activity at a concentration of 5 mg/mL.
After comparisons of growth kinetics between non-sterilized and autoclave-sterilized extracts were performed, we decided to determine the MIC exclusively for the autoclave sterilized extracts (the antioxidant properties did not vary, the antimicrobial activity against S. mutans was maintained, and native microbiota were deleted from the extracts). Although filtration of the extracts through a 20 µm filter is enough to remove microorganisms, sterilization by thermal treatments is usually easier to perform, in order to consider these extracts for industrial applications as antimicrobials.
In that sense, only autoclave-sterilized extracts were used to calculate the MIC. The MIC value for S B against S. mutans was 2 mg/mL ( Figure 3A), while for X B , the MIC value was 28 mg/mL ( Figure 3B). The antimicrobial extract of the autoclave-sterilized dragon's blood plant rhizomes showed antimicrobial activity with a lower amount of extract (92% less), compared to the xoconostle fruit extract; in the literature, there is no information on the antimicrobial properties of these extracts against pathogenic strains of the oral cavity, like Porphyromonas gingivalis, that are related to periodontal diseases.

Conclusions
After the autoclave-sterilization of extracts from dragon's blood plant rhizomes and xoconostle fruit, the native microbiota were eliminated, allowing microbiological studies to be carried out without interference with the fact that the bioactive compounds were maintained after the thermal treatment. According to the HPLC method, five compounds were identified from the autoclaved dragon blood rhizome extracts, and two phenolic acids, and three flavonoids were related to antioxidant and antimicrobial properties. After sterilization treatment by autoclaving, the antimicrobial properties of the dragon fruit rhizome extract were maintained, and its low concentration for the inhibition of S. mutans represents an alternative to synthetic antimicrobials as an adjuvant, or to prevent oral diseases such as caries. These results suggest that an extract of autoclave-sterilized dragon's blood rhizomes could be an excellent natural source of antimicrobial compounds, as well as a safe ingredient for food or pharmaceutical products.

Conclusions
After the autoclave-sterilization of extracts from dragon's blood plant rhizomes and xoconostle fruit, the native microbiota were eliminated, allowing microbiological studies to be carried out without interference with the fact that the bioactive compounds were maintained after the thermal treatment. According to the HPLC method, five compounds were identified from the autoclaved dragon blood rhizome extracts, and two phenolic acids, and three flavonoids were related to antioxidant and antimicrobial properties. After sterilization treatment by autoclaving, the antimicrobial properties of the dragon fruit rhizome extract were maintained, and its low concentration for the inhibition of S. mutans represents an alternative to synthetic antimicrobials as an adjuvant, or to prevent oral diseases such as caries. These results suggest that an extract of autoclave-sterilized dragon's blood rhizomes could be an excellent natural source of antimicrobial compounds, as well as a safe ingredient for food or pharmaceutical products.