text:
Analysis of Capillary Water Migration Law of Compacted Loess in Ningxia

Capillary water is the main cause of freezing damage and collapsible damage of compacted loess. Therefore, researching capillary water migration law of compacted loess is the key to solve various damage in the infrastructure construction of loess. Different regions have different climatic conditions, which make the physical and mechanical properties of the loess is different. In order to reveal the capillary water migration law of compacted loess in Ningxia, the moisture migration law of compacted loess with different degrees of compaction was studied by using standpipe method. The results show that the migration rate of capillary water is rapid at the early stage and gradually became slower with the increase of time, which is negatively related to the degree of compaction and migration height. The maximum migration height of capillary water increases exponentially with the increase of compactness. Migration rate and migration height of capillary water are related to capillary water potential, gravitational potential, and pipe-resistance of capillary. In this study, the relational equation can predict capillary water distribution and the migration height of compacted loess was newly established. This equation can also provide a reference to reduce various damage and freeze-thaw effect in compacted

Introduction
regions [11][12][13][14]. It is cold and dry in winter, large areas of farmland are irrigated before winter, which makes the frost-heave of soil large and the rate of frost-heave reaches 13.0%, canals and subgrade projects are severely damaged by frost [15][16][17]. However, capillary moisture is the main source of water supply during the soil frost-heave process, which is an important factor affecting soil frost-heave deformation [18]. In order to reduce the freezing damage, satisfy the needs of production construction and theoretical research of compacted loess, investigating the capillary moisture migration law of compacted loess is necessary.
Previous studies have shown that the migration law of capillary water is related to temperature potential, gravitational potential, and matrix potential [19]. The temperature potential refers to the effect of temperature changes caused by the external environment on the migration direction, migration height and rate of capillary water. Gravity potential means that the selfgravity of capillary water has different effects on the migration law of capillary water in different directions. Matrix potential is related to the influence of different ionic and molecular attractive force on capillary water migration in different soils, which is caused by their different mineral components and physical and mechanical properties. Freeze-thaw cycles (FT) and dry-wet (TW) cycles are the main causes of damage to compacted loess. The freeze-thaw cycle is the main research direction of temperature potential on the migration law of capillary water, which is a seasonal process and exists widely in seasonal frozen ground regions. Meanwhile, the freeze-thaw cycle is usually accompanied by dry-wet cycle, both of which can decrease the mechanical properties of soils [20][21][22][23][24]. Capillary water migration rate in the compacted loess is inversely related to the number of dry-wet cycles, initial water content and compaction degree [25]. It is dry and windy in winter that capillary water migrates to ground surface due to strong evaporation. At the same time, the salt in the soil is also transported to the surface and accumulated under the action of capillary water [26]. When the temperature is lower than 0ºC, the capillary water in the soil will gradually be transformed into solid ice from top to bottom of the ground, which will attract moisture of soil migrate to the frozen zone too [27,28], the soil frosts heave and the volume will increase. As temperature increases, the water molecules between the soil particles are transformed from solid ice to liquid water. Meanwhile, the moisture content of soil increases, porosity increases. Accordingly, shear strength, compression strength, cementation between soil particles, compaction, and elastic modulus of the soil would be decreased during this process, all of which would cause collapsible deformation and uneven settlement of the soil [29].
The migration law of capillary water is also affected by gravitational potential. The migration rate of capillary water is negatively correlated with the migration height [30,31]. The higher the migration, the greater the gravitational potential energy of the water molecules which can decrease the migration rate of moisture. The distribution of capillary water is also related to the nature of the soil. Different mineral components and ionic content will cause differences in soil matrix suction. Capillary water migration equation and simplified equation based on particle size distribution can effectively describe the soil water retention curve and determine the soil hydraulic conductivity [32]. The study of geotechnical characteristics of loess shows that open pores in the soil are the main reason for loess collapsing [33]. Higher compaction degree and smaller water content can effectively reduce the rate of water migration and avoid the collapse of the loess [34].
Generally speaking, different soils have various physical and chemical properties [35,36], such as compaction, saturation, moisture content, dry density, thickness of combined water film, and so on. Different matrix potential can influence migration height and rate of capillary water too. In recent years, more and more scholars pay emphasis on the improvement of loess collapsible by adding additives loess and better drainage system, which can effectively improve the collapsible damage of the compacted loess [37][38][39][40].
Soil quality is an important factor in controlling capillary water migration. However, the interaction between the external environment and the soil will affect the soil quality. Accordingly, the hydro-geographic conditions in different regions will have different effects on the physical and mechanical properties of the soil, the physical and mechanical characteristics of loess in different regions are different. Ningxia loess has smaller content of hydrophilic minerals and smaller amount of clay, so it has a different capillary water migration law from other regions. However, the research on the capillary water migration law of Ningxia compacted loess is still in its infancy. In order to understand the capillary water migration law of compacted loess in Ningxia and provide a reliable theoretical basis for basic engineering construction, in this study a capillary water distribution equation based on saturation was established, and the effect of compaction on Capillary water saturation was analyzed. This study made a foundation of Ningxia compacted unsaturated loess in freeze-thaw cycles, and it provides a significant method for the evaluation and prevention of freezing damages in engineering.

Experimental Materials
According to the Specification of Soil Test (SL237-1999), the loess used in the test was taken from Tongxin County of Ningxia Hui Autonomous Region, the physical and mechanical indexes of the loess are shown Author Copy • Author Copy • Author Copy • Author Copy • Author Copy • Author Copy • Author Copy • Author Copy • Author Copy in Table 1. XRD analysis showed that the main minerals were quartz, calcite, Na 2 Al 2 Si 5 O 14 , and mica, as shown in Fig. 1. The particle size distribution is shown in Fig. 2. It is mainly silt-grained soil of 0.005 mm ~0.075 mm, accounting for 81.3%, and the clay-grained content is small, about 13.9%. The SEM micro-morphology of the loess particles is shown in Fig. 3. The morphology of loess particles is mainly granular, angular, elliptical, and surface is slightly convex. The surface of the coarse powder particles is adhered by small sticky particles or colloidal particles. There are few cohesive particles with a particle size smaller than 5 um.

Experimental Scheme
The physical and mechanical indexes of samples are shown in Table 2. the compaction degree was set at four levels of 0.86, 0.90, 0.94, and 0.98. At temperature of 20±1ºC, studied the capillary moisture migration height of loess with different compaction degrees. The capillary moisture migration rate is fast in the early period and slower in the later period, so the observation interval is set relatively short in the early days and long when it is stable or near the top of the tube. the early data observation interval was 1d, 3d, 7d, 12d respectively. when capillary action is stable or near the top of the tube, further data are observed at every 12d until 72d. In order to facilitate the preparation of samples and observe the position of the vertex about capillary moisture, the water content used in the sample preparation was w 0 = 8.0%.

Preparation of Samples
The loess was air-dried and crushed, and water was added according to the moisture content set in the plan. It was then mixed evenly and put in a sealed bag, and placed in a moisturizing container for not less than 24 hours to fully mixed the soil particles and water. According to the designed compaction and the thickness of each layer (5 cm), the quality of wet soil in each layer was calculated, and compacted each layer in an organic-glass sample tube. The sample is 10 cm in diameter and 200 cm in height.

Capillary Water Migration Test
The migration height of capillary moisture was measured by the standpipe method. The test device is shown in Fig. 4. The inner diameter of the standpipe  was 10 cm and the height was 200 cm. A ruler was attached upward from 10 cm at the bottom of the standpipe to locate the position of the vertex about capillary moisture. A 10 mm hole was drilled every 10 cm from 15 cm up from the bottom to determine the moisture content of the soil. The top of the standpipe was wrapped with a plastic film with small holes to prevent the water in the soil sample at the top of the pipe from evaporating too quickly. A 400-mesh gauze pad was placed on the bottom to prevent soil from escaping into the water. The bottom of the water storage tank was covered with fine-stones of 5 cm, and an overflow hole was opened at 15 cm. The thin stream continuously replenishes water, and the depth of the vertical pipe in the water was always maintained at 10 cm. The migration height of capillary water was observed by the direct-observation method under the guidance of technical code (SL237-1999), according to the color change of the soil sample and the ruler attached to the vertical pipe. The moisture content sensor can continuously monitor the dynamic changes of soil moisture, but there are many factors that affect the accuracy of the measurement, and there are certain measurement errors [41][42][43][44][45]. Especially for saline soils, the measurement results are significantly diverse due to different salt content. Although the quality-method cannot continuously monitor the soil moisture content, it has few influencing factors and high measurement accuracy.
The content of soluble salt in loess is more than others, and it is unevenly distributed due to migration of capillary moisture [46][47][48]. In the study of soil freeze-thaw effect, the most concerned is the steadystate moisture content when capillary moisture migrations to reach stability, so the quality-method was used to determine the moisture content. After the capillary moisture migration height was stable, samples were taken at about 2 cm and 5 cm from the tube wall, and the average value was used as the measurement result.

Analysis of Water Migration
The relationship between the capillary moisture migration height h and time t of loess samples with different compaction degrees is shown in Fig. 5, and the moisture migration rate v of the corresponding period is shown in Fig. 6.
It can be seen from Fig. 5 and Fig. 6 that the early migration of capillary moisture in the compacted loess was fast, especially on the first day, the sample with a compaction degree of 0.86 had a capillary moisture migration rate of 60 cm/day. With the increase of time, the migration rate of capillary moisture decreased rapidly. The migration rate at the 2~3d was only 1/2.5~1/4.7 of the migration rate at the 1d, which had dropped by 60%~78%. After 36d, the rate of capillary moisture migration was less than 1.0 cm/day. The Capillary moisture migration rate decreased with the increase of compaction before 7d, and then increased with the increase of compaction. The capillary moisture migration height increased with time, and stabilized after a certain period which was related to the degree of compaction. The higher the degree of compaction, the longer the time for the capillary moisture migration height to reach stability. Capillary moisture potential, gravity potential, and resistance of capillary wall were the decisive factors at capillary moisture migration rate [49]. The potential energy of capillary moisture was related to capillary diameter, soil particle thickness, material composition, and water properties. For example, if the capillary diameter was small, the soil particles were fine, the constituents were highly hydrophilic, the degree of water mineralization and cations were small, the capillary moisture potential would be greater than other conditions. The resistance of capillary wall was related to capillary diameter and morphology, the property and thickness of water bound to the tube wall, the shape and surface state of soil particles, and the rate of capillary moisture migration [50,51]. If the capillary diameter was small, the longitudinal bend or planar shape was flat, the water binding on the wall of pipe was strongly bound-moisture, the combined water film was thick, the soil particles were angular, the surface was rough, the resistance of pipe-wall would greater than other conditions. Capillary moisture was driven by the surface tension of water vapor, overcomes gravity and the resistance of capillary wall to move upward. Early in the test, capillary moisture had a small migration height, low gravity potential, relatively thin water film combined with the tube wall, small resistance to the tube wall, and high capillary moisture migration rate. As the capillary moisture migrations, the gravity potential increases. At the same time, the tube wall in the capillary moisture infiltration area became thicker with the water film, the resistance of the pipe wall increased, and the capillary moisture migration rate decreased rapidly. Higher compactness of loess results in a reduction of the capillary while increasing the capillary moisture potential and the pipe wall resistance. when compaction degree is greater than 0.86, the capillary wall resistance of the loess increases faster than the capillary moisture potential with the increase of the compaction degree, the capillary moisture migration rate decreases, and the time to reach the maximum height was increased. Fig. 5 shows that the capillary moisture migration height h and time t of each sample show a clear hyperbolic relationship. In the Equation 1: a and h u are the fitting coefficients to be determined, h u is the extreme value of h when t tends to infinity, that is, the maximum height of capillary moisture, cm.
The fitting coefficients a and h u and the correlation coefficient R 2 of the capillary moisture migration equation of each sample are shown in Table 3, R 2 in Table 3 are all close to 1.0, and the relationship between capillary moisture migration height and time is fitted with a hyperbolic relationship, which has a high correlation of fit.
Using the least squares method to further fit the relationship among a, h u , compaction degree λ c and porosity e (Fig. 7), which can get: The correlation coefficient R 2 of fitting coefficient a and h u has an obvious correlation with compaction degree λ c and porosity e, which are all close to 1. From Equation 2 and Equation 3, it can be seen that the maximum migration height of capillary moisture increases exponentially with the degree of compaction, decreases with the exponential decreasing power ratio of porosity, and It is consistent with the Hazen formula h c = Ce -1 for estimating the maximum migration height of sandy capillary moisture [52]. When the radius of the pores between the particles is less than 2.75 μm, the capillary moisture phenomenon does not occur, and the corresponding particle size is 0.0024 mm [53]. The grain composition of Ningxia loess is mainly powder and less than 4% of particles smaller than 0.002 mm. The mineral composition is mainly quartz, calcite and mica with small hydrophilicity, the combined water film is thin, even in the most compact state, there are few pores with a radius less than 2.75 μm, and the path of capillary moisture migration will not be blocked. Similar to silt or coarse-grained soil, the maximum  predict the maximum height of capillary moisture in Ningxia loess. The maximum migration of capillary moisture in compacted loess is affected by porosity, but it is far less sensitive than in sand. In order to verify the reliability of the equation, the capillary moisture migration height of each group of samples was measured during the test. The measured values of the capillary moisture migration height of each group of samples for 28d, the equation predicted values, and the residuals and relative residuals of the measured and predicted values are listed in Table 5. It can be seen from Table 4 that the relative residuals between the predicted value and the measured value of the capillary moisture migration height equation are within 5%, and the equation prediction results are accurate and reliable. Fig. 8 shows the steady-state moisture content distribution of the loess samples with different degrees of compaction, and Fig. 8 is the saturation distribution. It is observed from Fig. 8 and Fig. 9 that the moisture content of each test point of the sample is significantly larger than the initial moisture content of the sample, this phenomenon indicated that moisture of the four samples had migration to the top of the standpipe. The water content and saturation decreased with the increase of height. Within the range of 65 cm from the water level, the water content and saturation changed slowly along with migration height, and the saturation can reach more than 80%, the other part above 65 cm decreased slightly with the increase of height. The moisture content of the sample at the same height decreased obviously with the increase of the compaction degree. The saturation increased with the increase of the compaction degree, but the change of saturation was not obvious with the increase of compaction in the regions with high saturation.

Analysis of Capillary Moisture Distribution
From Table 5, Table 6, Table 7 and Table 8, regression analysis and optimization were applied to obtain the distribution equations of moisture content and saturation along with the height of loess with different compaction degrees.
(4) (5) Note: because the correlation between saturation and compaction is very low, the average value of the four samples is taken as the result for regression analysis, the degree of freedom is 17. Fig. 8. Distribution of water content with height. Fig. 9. Distribution of saturation with height. The significance parameter F of the water content relationship equation is 841.911, the correlation coefficient R 2 is 0.96, and the critical value F 0.99 = 4.79. The F of the saturation relationship equation is 1031.0, the correlation coefficient is 0.98, and the critical value is F 0.99 = 8.53. It can be concluded from the above numerical analysis. Both of the water content relationship equation and the saturation relationship equation were highly significant and have a good correlation with factors. The saturation distribution equation has nothing to do with compaction, which indicated that the effect of compaction on saturation is not obvious.
The steady-state water content distribution curve of soil in the capillary moisture migration area is also the soil-water characteristic curve [54,55]. Equation 4 and Equation 5 are soil-water characteristic curves characterized respectively by mass water content and saturation. The soil-water characteristic curve characterized by saturation which had nothing to do with compaction. "In a wide range of matric suction, saturation is not affected by porosity" [56][57][58], which can be mutually proved by Equation 5.
In order to compare and analyze the degree of influence of compaction on water content and saturation, analysis of variance was performed respectively on water content and saturation. The distribution of significance parameter F along the height is shown in Fig. 10. Fig. 10 revealed that the F of the saturation was much smaller than the F of the water content in the range of moisture migration height, which indicated that the impact of the compaction on the saturation was less obvious than the effect of the water content. The F about water content was all greater than F 0.90 , and gradually increased from top to bottom. The effect of compaction on water content had increased rapidly from obvious to very obvious and then reach to the most obvious. The part of obvious and very obvious account for a large proportion, was about 85%. The F about saturation were all less than F 0.90 , and the impact of compaction on saturation was not obvious, mainly because saturation was a combination of water content and porosity. According to the variance theory, if the influence of a factor was not obvious, it can be seen that the factor has no effect. Therefore, when the degree of compaction of the loess is greater than 0.86, the effect of the compaction degree on the soil saturation in the capillary moisture migration area can be ignored. It can be inferred that loess has a limited increase or decrease in density after the compaction due to changes in load, temperature, humidity, and other factors, the steady-state water content in the capillary moisture migration zone increases and decreases, while the saturation remains unchanged. This inference provides a theoretical basis for the study of the effects of freezethaw cycles of unsaturated compacted loess on the hydration cycle.

Conclusions
This paper presents a study under the condition that the compaction degree is not less than 0.86, with a powder content of about 80.0% and the main mineral composition is mainly composed of Quartz, Calcite, Na 2 Al 2 Si 5 O 14 and Mica. The migration of capillary  Table 8. Regression parameters of saturation. (1) The capillary moisture migration height of Ningxia compacted loess is larger than 200cm. The maximum migration height increases exponentially with the increase of compactness.
(2) The effect of compactness on saturation is not obvious. Accordingly, it can be assumed that the saturation of the loess is almost unchanged after freezethaw cycles, when studying the effects of freeze-thaw cycles on capillary water migration laws of loess.
(3) The moisture content decreases with the increase of height, and the moisture content decrease rate increases with the increase of height. The moisture content of the sample at the same height decreases as the degree of compaction increases.
(4) The newly established equations can effectively reflect the relationship between saturation, compaction and capillary moisture migration height, and provide a theoretical basis for the study of freeze-thaw deformation of loess engineering.


extfieldsofstudy: Chemistry Medicine

provenance: AgriculturalAndFoodSciences,2019-2020-Agricultural And Food Sciences-2020.jsonl.gz.gz

text:
Chemical Composition, Antimicrobial, Antioxidant, and Antiproliferative Properties of Grapefruit Essential Oil Prepared by Molecular Distillation

Grapefruit essential oil has been proven to have wide range of bioactivities. However, bioactivity of its molecular distillate has not been well studied. In this study, a light phase oil was obtained by molecular distillation from cold-pressed grapefruit essential oil and GC-MS was used to identify its chemical composition. The antimicrobial activity of the light phase oil was tested by filter paper diffusion method, and the anticancer activity was determined by the Cell Counting Kit-8 (CCK-8) assay. Twenty-four components were detected with a total relative content of 99.74%, including 97.48% of terpenes and 1.66% of oxygenated terpenes. The light phase oil had the best antimicrobial effect on Bacillus subtilis, followed by Escherichia coli, Staphylococcus aureus and Salmonellaty phimurium. DPPH and ABTS assays demonstrated that the light phase oil had good antioxidant activity. The CCK-8 assay of cell proliferation showed that the light phase oil had a good inhibitory effect on the proliferation of HepG2 liver cancer cells and HCT116 colon cancer cells.

Introduction
With the frequent occurrence of food safety issues and the toxicity of synthetic chemicals, the demand for safe and natural alternatives is growing. Plant extracts have been used since ancient times, and now the focus is on their role in health promotion and their treatment and prevention properties for various diseases. In the past few decades, plant essential oils (EOs) have attracted a lot of interest due to their safety and pharmacological properties including bacteriostatic, free radical scavenging, anti-inflammatory, and inhibitory effects on malignant tumor cell proliferation [1][2][3][4]. Citrus Eos are the main aromatic by-products of the juice extraction industry and are widely used in food, cosmetics and pharmaceutical industry [5][6][7]. The annual global production of citrus EO is approximately 16,000 tons, and the cost is about $14,000/ton on the international market. Thus, citrus EO is of great demand and is one of the more promising market prospects [8]. Grapefruit (Citrus paradisi Macf.), one of the world s largest production citrus families [9], is famous for its taste and nutritional value. Grapefruit EO is extracted from grapefruit peel and has been used for a long time as a valuable ingredient for its characteristic aroma in flavor and fragrance [10,11]. Similar to most citrus EOs, its major components are terpenes and terpene oxides. Terpene oxides include alcohols, ethers, aldehydes, ketones, and esters [12][13][14], which are the main source of the aroma, whereas terpenes contribute less to the aroma. In spite of extensive studies on the aroma features of grapefruit EO, in recent years, more and more researchers have become interested in exploring their biological and pharmacological activities. Grapefruit EO has been reported to have a wide range of bioactivities. It was shown to inhibit the growth of food-borne spoilage bacteria and pathogenic strains [15][16][17]. Okunowo et al. (2013) found that grapefruit EO obtained from the peel by hydrodistillation exerted inhibitory effects against bacteria and fungi, andmay be further developed for the treatment of certain diseases [18]. Grapefruit EO has shown antioxidant activity, which was important for food preservation and disease prevention [19,20]. Ahmed et al. (2019) reported that grapefruit EO extracted by hydrodistillation had antioxidant activity by using DPPH and FRAP assays [21]. Grapefruit peel extracts have been shown to decrease the HL-60 cell viability in a concentration-dependent manner [22]. In fact, grapefruit extracts (a mixture of EO and other nonvolatile phytochemicals) could also inhibit the growth and proliferation of cancer cells such as neuroblastomas, leukemias, and prostate and lung cancer lines [23][24][25]. Cuthrell et al. (2006) reviewed the anticancer activities of phytochemicals found in grapefruit [26].
Most grapefruit EO samples used for bioactivity studies were made by cold-pressing, steam distillation, or hydrodistillation methods. Cold-pressing is the predominant method to extract most citrus peel EOs, including grapefruit EOs. In commercial practice, grapefruitis processed to obtain juice and other by-products. EO is one of the primary grapefruit by-products. Large-scale grapefruit EO is mainly prepared by a cold-pressing method based on John Bean FoodTech (JBT) juice extractors and its technology, which is used by 75% of the world's citrus juice production [27]. The juice and EO are extracted separately and simultaneously. The EO was extracted by mechanical rupturing of the oil sacs in the flavedo, expressing the oil as an aqueous emulsion from which it is separated by centrifuging. The EO recovery is a physical separation process and no heat is applied throughout the whole extraction procedure. The operation temperature is much lower than in the distillation procedure. Thus, the EOs will have characteristics that are closer to those of the essence present in the grapefruit matrix. Large scale production, low cost and the aroma characteristic remaining are the big advantages of the cold-pressing method. However, cold-pressed grapefruit oil contains waxes, pesticide residues, coumarins, carotenoids and other nonvolatile components [28][29][30], some of them also have good bioactivities that may cause bias in bioactivity research of EO. César et al. (2009) found that furanocoumarins isolated from grapefruit peel oil showed potent in vitro inhibitory activity against intestinal cytochrome P450 3A4, an enzyme involved in "grapefruit/drug" interactions in humans [28]. Steam distillation or hydrodistillation was carried out at relatively high temperature which may cause degradation of some thermal sensitive molecules [18,31]. To avoid such problems, and find a new way to use the commercial available cold-pressed grapefruit EO in biochemistry and pharmachutical fields, we used a molecular distillation method to prepare grapefruit oil samples for our bioactivity tests.
Molecular distillation is a special liquid-liquid separation technology under high vacuum, which is employed as a separation process in the food industry [32,33]. Molecular distillation can divide the EO mixture into two different phases according to the free path of different molecules at low temperature. Molecular distillation is prominent with the advantages of low temperature treatment and high vacuum application, which is very suitable for thermolabile compounds and is used for concentrating and refining EOs [34,35]. At present, there are seldom reports on the antibacterial and anticancer activity of Eos obtained by molecular distillation. In this study, the cold-pressed grapefruit EO was processed by molecular distillation technology and the light phase essential oil (LPEO) was collected. Its constituents were identified by GC-MS. The activities of LEPO were tested on microorganisms and malignant proliferating cells (HCT116 colon cancer cells and HepG2 liver cancer cells) were tested. We expect that this work can stimulate the development of new agents for food preservation and chemo-preventive anti-cancer treatments.

Antimicrobial Activity
Grapefruit EOs prepared by cold-pressing or hydrodistillation using a Clevenger-type apparatus have shown a wide spectrum of antimicrobial activity in vitro [18,37]. However, antimicrobial activity of grapefruit EO prepared by molecular distillation has not been well studied. We tested LPEO on five microorganisms and the results obtained are shown in Table 2. The filter paper diffusion method was used to test the antibacterial activity of LPEO against different bacteria, and the activity of LPEO was evaluated according to the diameter of the inhibition zone and the minimum inhibitory concentration (MIC) values. LPEO exhibited strong antibacterial effects on the four bacteria tested. From the scale of the inhibition zone, LPEO had the strongest inhibitory effect on B. subtilis with a maximum diameter of 35.59 mm, followed by E. coli, S. aureus, and S. typhimurium. LPEO had no inhibitory activity against P. aeruginosa with an inhibition zone of 8.57 mm. P. aeruginosa belongs with E. coli and S. typhimurium to the group of Gram-negative bacteria but exhibits quite a different response to LPEO. This phenomenon may be partly due to its relatively low outer membrane permeability. LPEO molecules enter the periplasm by diffusion through the channels of nonspecific porins in the outer membrane, and this pathway in P. aeruginosa is 10-to 100-fold less efficient than that in E. coli [38]. Regarding MIC values, LPEO showed the best antimicrobial activity against Bacillus subtilis with a MIC value of 0.78 µL/mL. Based on the inhibition zone and MIC values, the order of sensitivity of the different bacteria was: B. subtilis > E. coli > S. aureus > S. typhimurium > P. aeruginosa. Since cold-pressed grapefruit oil (Citrus paradisi Macf.) has been evaluated as "generally recognized as safe" (GRAS) by the Expert Panel of the Flavor and Extract Manufacturers Association (FEMA) [39], and LPEO, a distillate from cold-pressed EO, showed strong sensitivity to most tested microorganisms, it appeared to be suitable to food applications. These results demonstrate that molecular distillation technology can provide a grapefruit EO fraction with good antimicrobial activity. Disk diameter is 6.0 mm. Zone of growth inhibition values are presented as mean± standard deviation for at least three experiments. Different superscript letters represent the significant differences at P < 0.05 according to Tukey's multiple range test. The scale of zone of inhibition measurement was the following (disk diameter included): ≥20 mm is strongly inhibitory; <20-16 mm is moderately/mildly inhibitory; <15-10 mm is weak inhibitory; <9-7 mm is not inhibitory.
Uysal et al. (2011) evaluated the antibacterial activities of grapefruit Eos from solvent-free microwave extraction (SFME) and hydrodistillation (HD) by the disc-diffusion method [17]. The Eos obtained from SFME and HD showed the highest activity against S. aureus with inhibition zones of 53 and 41 mm, respectively, higher than LPEO (24.34 mm). The activity against E. coli (30 mm and 28 mm) was close to our result (26.86 mm). Both of their samples and LPEO showed no obvious activity against P. aeruginosa. LPEO showed better activity against S. typhimurium (21.70 mm) than their samples (15 mm and 13 mm). Although a lot of plant EOs have shown antimicrobial activity, the reason of this capacity is not well known. It could be provoked by the major components of the EOs or due to a synergistic effect among the major components and the minor ones. Different preparation methods yield EO samples with differences in chemical composition and relative content, and cause differences in antimicrobial activity.

Antioxidant Activity
A lot of EOs have been reported to scavenge the free radicals that cause damage to the body and reduce the risk of many diseases originating from oxidative stress. In order to measure the effect of LPEO and determine its potential application in food, cosmetic or pharmaceutical industries, we evaluated its antioxidant activity using two different assays: The 2,2-diphenyl-1-picrylhydrazyl (DPPH)and 2,2 -azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) radical (ABTS) assays. Butylated hydroxytoluene (BHT) was used as positive control. The IC 50 values of BHT in DPPH and ABTS were 0.03 mg/mL and 0.01 mg/mL, which was consistent with the literature [40]. The DPPH and ABTS activities of LPEO were obtained with IC 50 values of 22.06 ± 0.92 mg/mL and 15.72 ± 0.32 mg/mL, respectively. LPEO had better antioxidant activity than cold-pressed grapefruit EO in the DPPH assay (EC 50 > 40 mg/mL) and hydrodistilled grapefruit EO in the ABTS assay (EC 50 = 27.5 mg/mL) [35]. Compared with cold-pressed orange oil, LPEO had much lower antioxidant activity in the DPPH assay (IC 50 = 3.01 ± 0.20 mg/mL) and better activity in the ABTS assay (IC 50 = 23.25 ± 0.84 mg/mL) [41].

Antiproliferative Activity of LPEO in HepG2 and HCT116 Cancer Cells
The effects of different concentrations of LPEO on the proliferation of HepG2 liver cancer and HCT116 colon cancer cells were tested by the Cell Counting Kit-8 (CCK-8) method [42,43]. The results are shown in Figure 2. The viability rate of both cell types decreased with increasing LPEO concentration. When the concentration of LPEO was less than 0.1 µL/mL, no obvious change of viability of HepG2 cells was observed. However, when the concentration of LPEO was higher than 0.1 µL/mL, the viability of HepG2 cells significantly decreased; at the LPEO concentration of 0.3 µL/mL, the viability was 7.4%only. LPEO also had a good inhibitory effect on the growth of HCT116 colon cancer cells. At the concentration of0.05 µL/mL or higher, the viability of HCT116 cells significantly decreased. It was as low as 7.43% when the concentration of LPEO was 0.5 µL/mL. GraphPad Prism™ (Version 5.00) software (GraphPad Software, San Diego, CA, USA) was used to calculate IC 50 values. IC 50 value of HepG2 and HCT116 was 0.24 and 0.20 µL/mL, respectively. These results indicate that LPEO has a significant inhibitory effect on the proliferation of HepG2 hepatoma cells and HCT116 colon cancer cells in vitro.  [22]. The extract showed antiproliferative activity in a dose-dependent manner with the median effective dose (EC50) value of 130.09 mg/mL. However, they did not identify the specific phytochemicals which were responsible for antiproliferative activity. Manassero et al. (2013) studied the antiproliferative activity of cold-pressed EO from mandarin peel and its principal component limonene [44]. Mandarin EO and limonene exhibitedIC50 of 0.063 µL/mL and 0.150 µL/mL against HepG2 cells, respectively. The much higher activity of mandarin EO than LPEO (0.24 µL/mL) may attributed to other high potent phytochemicals in cold-pressed EO. We have reported antiproliferative activity of the 'Gannanzao' orange EO (GOEO) prepared by hydrodistillation, which exhibited IC50 of 0.29 µL/mL and 0.35 µL/mL against HepG2 cells and HCT116 colon cancer cells, respectively [43]. LPEO showed a slightly higher activity than GOEO, which may be attributed to its higher limonene content (LPEO 93.33%, GOEO 88.07%).
The discussion about anticancer activity of some EO components has been made by Mukhtar et al. [45]. Our study preliminarily tested the inhibitory effect of LPEO on the proliferation of HepG2 liver cancer cells and HCT116 colon cancer cells. The anticancer activity of LPEO and its components on cancer cells and their mode of action deserve further study.

Preparation of Grapefruit Light Phase EO Sample
Grapefruit light phase EO(LPEO) was obtained by molecular distillation from cold-pressed grapefruit EO (Citrus paradisi Macf., Lakeland, FL, USA) using a wiped-film molecular distillation Significant decreases in cell viability of cancer cells are seen at increasing LPEO concentrations compared to untreated controls (control group was set to 100%). **-Very significant at p < 0.01, ***-Highly significant at p < 0.001. Sun et al. (2002) studied antiproliferative activity of grapefruit fruit extract on the growth of HepG2 human liver cancer cells in vitro [22]. The extract showed antiproliferative activity in a dose-dependent manner with the median effective dose (EC 50 ) value of 130.09 mg/mL. However, they did not identify the specific phytochemicals which were responsible for antiproliferative activity. Manassero et al. (2013) studied the antiproliferative activity of cold-pressed EO from mandarin peel and its principal component limonene [44]. Mandarin EO and limonene exhibited IC 50 of 0.063 µL/mL and 0.150 µL/mL against HepG2 cells, respectively. The much higher activity of mandarin EO than LPEO (0.24 µL/mL) may attributed to other high potent phytochemicals in cold-pressed EO. We have reported antiproliferative activity of the 'Gannanzao' orange EO (GOEO) prepared by hydrodistillation, which exhibited IC 50 of 0.29 µL/mL and 0.35 µL/mL against HepG2 cells and HCT116 colon cancer cells, respectively [43]. LPEO showed a slightly higher activity than GOEO, which may be attributed to its higher limonene content (LPEO 93.33%, GOEO 88.07%).
The discussion about anticancer activity of some EO components has been made by Mukhtar et al. [45]. Our study preliminarily tested the inhibitory effect of LPEO on the proliferation of HepG2 liver cancer cells and HCT116 colon cancer cells. The anticancer activity of LPEO and its components on cancer cells and their mode of action deserve further study.

Preparation of Grapefruit Light Phase EO Sample
Grapefruit light phase EO(LPEO) was obtained by molecular distillation from cold-pressed grapefruit EO (Citrus paradisi Macf., Lakeland, FL, USA) using a wiped-film molecular distillation apparatus (Pope Two Inch Laboratory Scale Wiped-Film Molecular Still & Evaporator, Pope Scientific Inc., Saukville, WI, USA). The evaporation temperature and operation pressure were 55 • C and 6.0 Torr, respectively. Cold-pressed grapefruit EO was fed at room temperature and the feeding rate was 3.0 mL/min. The rotational speed of the roller wiper (Pope Scientific Inc., Saukville, WI, USA) was 325 rpm, and the condenser temperature was 0 • C. The final grapefruit EO sample (LPEO) was obtained from the light phase outlet with the yield of 86%.

GC-MS Analyses
The constituents of LPEO were analyzed using an Agilent 7890B gas chromatograph coupled with an Agilent mass spectrometer detector (Agilent Technologies, Santa Clara, CA, USA). The GC was equipped with a HP-5 column (30.00 m × 0.25 mm × 0.25 µm). Mass spectra were obtained by electron ionization (EI) at 70 eV. The injector and detector were operated at 250 • C and 300 • C, respectively. The temperature program was 80 • C for 4 min, and then increased at 5 • C/min to 250 • C and held constant for 10 min. The constituents were identified by comparing their mass spectra with the National Institute of Standards and Technology (NIST, version 2010, U.S. Department of Commerce, Gaithersburg, MD, USA) data reference. The retention indices (RI) of the constituents were determined by adding a C8-C20 n-alkanes mixture to the essential oil before injecting in the GC-MS equipment and analyzing it under the same conditions described above.

Microbial Growth Conditions
The microbial strains were maintained in nutrient agar media at 37 • C. Subsequently, one colony from each culture was inoculated in liquid medium for 18-24 h with shaking (200 rpm) to obtain freshly cultured microbial suspensions (>10 8 CFU mL −1 ) for test.

Determination of Diameter of the Inhibition Zone
LPEO was tested on five microbial strains, using filter paper diffusion method [46]. Briefly, a suspension of the tested microorganism (10 6 CFUmL −1 ) was spread on the solid media plates. The paper discs (Whatman No. 1 filter paper, 6 mm diameter) were impregnated with 20 µL LPEO and placed on the inoculated agar. The plates inoculated with bacterial strains were incubated for 24 h at 37 • C. After incubation, diameter of the inhibition zone was measured in millimeters. Each test was performed in triplicates on at least three separate experiments.

Determination of Minimum Inhibitory Concentration (MIC)
MIC values of LPEO against microorganisms were determined by disc-diffusion method [46,47] Sterile filter paper discs were placed on the surface of Petri dishes and impregnated with 20 µL of EO at different concentrations (100.00, 50.00, 25.00, 12.50, 6.25, 3.125, 1.56, 0.78, 0.39, and 0.195 mg/mL) in dimethyl sulfoxide (DMSO). DMSO alone was used as negative control. After staying at 4 • C for 2 h, all Petri dishes were incubated at 37 • C for 24 h. All determinations were performed in triplicates. The minimum inhibitory concentration (MIC) values were determined as the lowest concentration of EOs that inhibited visible growth of the tested microorganism.

DPPH Radical-Scavenging Assay
The free radical-scavenging activity of LPEO was measured using the stable radical 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay [48]. DPPH was dissolved in ethanol at concentration of 0.1 mmol L −1 . The absorbance of 2.7 mL DPPH solution and 0.3 mL ethanol was measured as the negative control. A different concentration of the sample solution in ethanol (0.3 mL) was pipetted into a cuvette with 2.7 mL DPPH solution. The resultant solution was incubated for 30 min at room temperature in the dark, and then monitored at 517 nm. The DPPH scavenging activity was expressed according to the following equation: where A C is the absorbance of the negative control, and A S is the absorbance containing 0.3 mL sample and 2.7 mL DPPH solution. All samples were analyzed in triplicates, and the results are expressed as the mean ± standard deviation. The scavenging activity was expressed as the 50% inhibitory concentration (IC 50 ), which was defined as the sample concentration necessary to inhibit DPPH radical activity by 50% after incubation.

ABTS Radical-Scavenging Assay
This method was performed as described by Teles et al. [49], based on the capacity of LPEO to inhibit the 2,2 -azinobis (3-ethylbenzthiazoline-6-sulfonic acid) radical (ABTS). Twenty-five mL of ABTS (7 mM) were added to 440 µL of potassium persulfate (K 2 S 2 O 8 , 140 mM), and the solution was kept in darkness for 12 h at room temperature in order to form the radical. An accurate volume of the solution was diluted in ethanol until an absorbance of 0.70 at 734 nm. Once the radical was formed, 2 mL of ABTS solution were mixed with 100µL of LPEO and the absorbance measured at734 nm. ABTS scavenging effect was calculated using the following equation: ABTS scavenging activity (%) = (A C − A S )/A C × 100 (2) where A S is the absorbance of the solution when the sample has been added and A C is the absorbance of the ABTS solution as control. The IC 50 was calculated from the graph of scavenging percentage against LPEO concentration. The results are expressed as the mean ± standard deviation.

Cancer Cell Culture
HCT116 colon cancer cells and HepG2 liver cancer cells were purchased from Library of Typical Culture of Chinese Academy of Sciences (Shanghai, China). HCT116 cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Hyclone, UT, USA), supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (Hyclone, UT, USA). HepG2 cells were cultured in MEM containing 10% FBS and 1% penicillin/streptomycin (Hyclone, UT, USA). The above-mentioned cells were maintained in 25 cm 2 cell culture flasks in a humidified atmosphere containing 5% CO 2 at 37 • C. Cells were fed until 90% confluence and the confluent cells were washed twice with phosphate buffered saline (PBS), treated with 0.25% trypsin (Invitrogen, MA, USA) for about 1 min, and incubated at 37 • C. When the cells were contracted and rounded under the microscope, FBS (Hyclone, UT, USA) containing medium was added, centrifuged at 200× g for 3 min, and subcultured at a split ratio of 1:3.

Antiproliferative Activity Test of LPEO
The cell proliferation inhibition rate of LPEO was evaluated by CCK-8 assay [42,43]. LPEO (50 µL) was added to the medium and mixed well. The mixture was diluted in DMSO to prepare solutions at a concentration of 0.5, 0.4, 0.3, 0.2, 0.1, 0.05, and 0.0 µL/mL, respectively. The cells were placed into 96-well plates (3 × 10 3 cells/well). After 24 h, 100 µL of LPEO at different concentrations was added and continued to incubate for 48 h at 37 • C in a CO 2 incubator, after which the medium in the 96-well plate was disposed. A 100 µL of CCK-8 test solution (DojinDo, Tokyo, Japan) was added and incubated for 2 h at 37 • C. The optical density (OD) for each well was measured at 450 nm using a microplate reader (BioTek, Winooski, VT, USA). The cell viability rate at different concentrations of LPEO treatment was calculated according to the formula: Viability rate (%) = (OD sample − OD blank )/(OD control − OD blank ) × 100% (3)

Statistical Analysis
The mean and standard deviation of three experiments were determined. Statistical analyses of the differences between mean values obtained for experimental groups were calculated using IBM SPSS Statistics 23.0. (IBM Corp. Released 2015. IBM SPSS Statistics for Windows, Version 23.0. Armonk, NY, USA). p values < 0.05 were regarded as significant, p values < 0.01 as very significant and p values < 0.001 as highly significant.

Conclusions
Essential oils are valuable plant extracts used in food, medicine and complementary treatment strategies [41]. The beneficial role of grapefruit EO has been widely reported. However, the bioactivities of grapefruit EO prepared by molecular distillation has not been well studied. Molecular distillation is a very useful technique to separate thermally-sensitive EOs. In our study, molecular distillation was used to remove undesired components from the cold-pressed grapefruit EO to provide light phase EO (LPEO). The chemical composition and antimicrobial activity of LPEO were studied. LPEO showed a wide spectrum of antimicrobial activity against some Gram-positive and Gram-negative microorganisms, with MIC values ranging from 0.78 to 12.50 µL/mL. LPEO might be used as a novel antimicrobial agent in the food industry. The antioxidant activity of LPEO by DPPH and ABTS was obtained with IC 50 values of 22.06 ± 0.92 mg/mL and 15.72 ± 0.32 mg/mL, respectively. An in vitro test showed a dose-dependent antiproliferative activity of LPEO on HepG2 and HCT116 cancer cells. Thus, LPEO may potentially be used as a new complementary anticancer agent. However, this still needs further studies.
Author Contributions: J.C. and B.Z. contributed to the conception and design of the study and data analysis; W.D., K.L., J.S., and J.C. collected the plant material and performed distillation and GC-MS analysis; K.L. and S.C. carried out bioactivity test; W.D. and J.C. wrote the manuscript. All authors have read and agreed to the published version of the manuscript.

Conflicts of Interest:
The authors declare no conflict of interest.


text:
Influence of Acetylated Annealed Starch on the Release of β-Escin from the Anionic and Non-Ionic Hydrophilic Gels

Naturally sourced products introduced to human nutrition and rediscovered for therapy include polysaccharides from potatoes. The starch may obtain unique properties via acetylation with acetic anhydride at 13 cm3/100 g of starch as the basic dose of reagent used in industrial conditions. The hydrogel formulation was applied as a carrier for escin included in the dry extract of Aesculus hippocastanum. Six hydrogels were evaluated (methylcellulose, polyacrylic acid-Carbopol 980 NF and polyacrylate crosspolymer 11—Aristoflex Velvet) with various concentrations of the modified starch. The kinetic studies of in vitro β-escin release were carried out in purified water at 37 ± 0.5 °C using a paddle apparatus at 50 rpm and a time period of 7 h. The criterion for the most suitable model was based on a high correlation coefficient of evaluated release profiles. The addition of modified annealed acetylated potato starch resulted in prolongation of β-escin release.

Introduction
Modern pharmaceutical technology is based, among others, on different carriers of medicinal substances, including synthetic and natural polymers. Among the latter, the high availability in plant sources should be considered an advantage, but the disadvantage is the variability of the composition, depending on the origin of the raw material. Starch and cellulose are known as very common natural polymers [1,2]. Previously used starch formulations, based on the pharmacopoeial rules, have been described as powders with different grain diameters. An alternative is semisynthetic polymers, e.g., modified cellulose or starch. Enzymatic, physical and chemical complex methods are used to modify starch molecules. The improvement of functional properties or the imparting of new, specific properties required for the desired application has been achieved. Modified starch (MS) is used, among others, in the food, textile, paper, construction and pharmaceutical industries. Adhesives, superabsorbents, biodegradable materials or heavy metal-absorbing beds are produced from MS [3]. Starch acetate is used in the food industry as a food additive. This modification is obtained on an industrial scale by esterifying starch in an aqueous suspension with acetic anhydride in an alkaline medium. It is characterized by a lower gelatinization temperature and higher viscosity of prepared pastes compared to those of natural starch and forms stable gels resistant to retrogradation.
An example of physical modification of starch is hydrothermal treatment, which includes annealing, i.e., native starch incubation with an excess of water at a temperature between the glass transition temperature and the initial gelatinization temperature. The purpose of this transformation is to receive Pharmaceutics 2020, 12 such a modification temperature that stimulates the molecular mobility of starch molecules without transforming them to the gelatinized form. This process leads to an increase in the homogeneity and stability of the crystal structure of hardened starch, thereby protecting the structure of the final products [4,5]. The proposed technology allows for empty capsules to be prepared with interesting application features. Polymeric materials can affect the release kinetics of drug substances and provide controlled release of drugs in the appropriate area of the body. Therefore, research has been conducted on the effect of modified polymers of natural origin on the release kinetics of selected, model therapeutic substances [6,7]. Escin is the main active pharmaceutical ingredient in a horse chestnut seed (EH). It occurs as a mixture of triterpenoid saponins, existing in two forms, α-escin and β-escin (βE). They are characterized by different melting points, specific optical rotation, solubility in water and hemolytic index. βE is the major active component, and may be transformed into α-escin at elevated temperatures [8]. The application of EH in the form of hydrogel applied topically on the skin surface is an interesting alternative to peroral delivery of EH in the form of tablets used in the varicose veins [9].
The aim of the study was to evaluate the influence of the modified acetylated potato starch polymer (MSA) addition on the release profile of βE, depending on the amount of MSA added, and the type of basic hydrophilic gel, preformulated for the topical application.
A previous study on release kinetics from hydrogel formulations has been used, and the same cellulose and acryl derivatives polymers have been explored [9]. The previous study compared the release kinetics of βE from a variety of hydrogels prepared with commercially available polymers: Methylcellulose (MC), polyacrylic acid (Carbopol 980 NF, PA1), and polyacrylate crosspolymer 11 (PC-11), and from commercially available medicinal gel preparation with polyacrylic acid (Carbopol 5984). Formulations with MC presented the release profile comparable to those obtained from the marketed gel, whereas, the application of PA1, and PC-11 resulted in the prolonged release of βE [9]. Due to the present research, the addition of starch derivative-MSA-resulted in a stronger marked prolongation of the release.

Hydrogel Preparation
The composition of the prepared gels is shown in Table 1. A concentration of 2.0% MC and 1.5% PC-11 and PA1 was prepared in gel formulations: F0, G0 and H0, respectively. The mixture with MC was achieved via adding distilled water of 80 • C and vigorously stirring in a mortar. The formulation with PA1 was completed by casting with 50.0 mg of 50% NaOH. The gels containing MSA were made with 20% and 40% of the starch derivative, respectively, by adding appropriate amounts of MSA instead of water: These were formulations F20 and F40, G20 and G40, as well as H20 and H40, respectively. The gels with MSA were gently mixed with a glass spatula to avoid foaming. The gels were conditioned in a refrigerator at 8 • C for 48 h. However, the temperature was brought to 25 • C before subsequent experiments.

Starch Preparation
A large starch knob fraction with an average volume diameter D(4,3) equal to 61.7 µm was isolated from natural potato starch with an average volume diameter D(4,3) of 39.1 µm, determined using a Malvern laser particle size analyser [4]. The large fraction of starch knobs was acetylated with acetic anhydride at 13 cm 3 /100 g of starch as the basic dose of reagent used in industrial conditions. After drying the acetylated starch, an initial gelatinization temperature of 49.17 • C using a Mettler differential scanning calorimeter was determined [5]. Two litres of a 10% starch suspension was prepared from acetylated starch and kept under constant stirring for 24 h at 48 • C. The starch was then washed three times with five-litre portions of distilled water. The starch pellet was separated from the suspension in each case using a Contifuge Stratos flow centrifuge (Heraeus, Germany) and then dried for 24 h in an air dryer at 30 • C. A photo of sample starch knobs was taken under a Stereo Zoom Microscope SMZ-171-TLED (Motic, Hong Kong, China) at fifty times magnification ( Figure 1).

Starch Preparation
A large starch knob fraction with an average volume diameter D(4,3) equal to 61.7 µm was isolated from natural potato starch with an average volume diameter D(4,3) of 39.1 µm, determined using a Malvern laser particle size analyser [4]. The large fraction of starch knobs was acetylated with acetic anhydride at 13 cm 3 /100 g of starch as the basic dose of reagent used in industrial conditions. After drying the acetylated starch, an initial gelatinization temperature of 49.17 °C using a Mettler differential scanning calorimeter was determined [5]. Two litres of a 10% starch suspension was prepared from acetylated starch and kept under constant stirring for 24 h at 48 °C. The starch was then washed three times with five-litre portions of distilled water. The starch pellet was separated from the suspension in each case using a Contifuge Stratos flow centrifuge (Heraeus, Germany) and then dried for 24 h in an air dryer at 30 °C. A photo of sample starch knobs was taken under a Stereo Zoom Microscope SMZ-171-TLED (Motic, Hong Kong, China) at fifty times magnification ( Figure 1).

Kinetics Study
The release kinetics study was performed in a drug dissolution Tester Erweka GmbH DT 700 (Heusenstamm, Germany) paddle-over-disc apparatus using the pharmacopoeial method at a stirring rate maintained at 50 rpm. A dialysis tubing cellulose membrane 43 mm × 27 mm in size with

Kinetics Study
The release kinetics study was performed in a drug dissolution Tester Erweka GmbH DT 700 (Heusenstamm, Germany) paddle-over-disc apparatus using the pharmacopoeial method at a stirring rate maintained at 50 rpm. A dialysis tubing cellulose membrane 43 mm × 27 mm in size with pores of 14 kDa (Sigma-Aldrich, Saint Louis, MO, USA) was applied to the extraction cells with an area accessible to the dissolution of 28.26 cm 2 . Purified water was used as a dissolution medium in a volume of 900 mL at 37 ± 0.5 • C [10][11][12][13] without returning the medium. Six parallel measurements were performed in extraction cells by sampling 3 mL of the acceptor fluid every 5 min for 7 h. Analysis of the samples was performed by spectrophotometry using a UV/VIS spectrophotometer Jasco V-530 (Tokyo, Japan) at 265 nm, according to the available bibliography and comparing to the absorption spectrum of β-escin (βE) in an aqueous solution [14]. A standard curve based on three series of measurements with six concentration points from 59.0 to 236.0 µg/mL was generated. The results were examined according to zero-order kinetics, first-order kinetics, and second-order kinetics models [11,15,16], according to Higuchi [17]. The Weibull model was used as a preference statistic model for the hydrogel formulations, and Statistica software was used to predict the plot of the release kinetics [18].
The models applied in the study ( Table 2) come from the Fick's first law, which may be interpreted in the context of the diffusion process through the semi-permeable membrane. The general equation is presented as below (Equation (1)), where J is the amount of solute passing through a unit area perpendicular to the surface per unit time, D is the diffusion coefficient, and dC/dx is the concentration gradient, which represents a driving force for diffusion [19]. There are conditions which must be met to apply zero-order model: Dissolved drug form does not aggregate, dosage form surface does not change, and no equilibrium state is achieved. This type of model usually concerns the prolonged release process [17].
The first-order model shows the connection between the dissolution of solid particles and surface action [19,20]. In second-order kinetics model release, process depends on the time units and concentration both. Higuchi model is usually applied to the release of water-soluble and poorly water-soluble drugs included in semi-solid and/or solid matrices [19]. The Weibull model is frequently used in all kinds of dissolution curves [17]. Table 2. Kinetic models applied to estimate obtained results. Q 0 is initial percentage of the released drug; Q t is percentage of the released drug after time t; K is rate constant, for zero-order kinetics (K 0 ), 1st-order kinetics (K I ), 2nd-order kinetics (K II ), and Higuchi model (K H ), respectively; Td is time after release of 69.3% of drug from the formulation; t 0.5 is half release time; β is a shape parameter in the Weibull model; a, b, are slope and intersection of the graph representing the release process, respectively.

Models Applied General Equation Parameters
Zero-order

Zero-Order Kinetics Model
The initial amount of EH in the formulations is presented in Table 1. The plateau phase was established after 420 min. The largest released amount of βE in the equilibrium state was 44.43% and belonged to MC formulation (Figure 2a), and the smallest was 19.98% for PA1 hydrogel (Figure 2b). Generally, when more MSA was added to the formulation, less βE was released ( Figure 2). In the case of the formulations with MC and PA1, 40% MSA addition caused the released amount to decrease by almost half (Figure 2a,b). In the hydrogels with PC-11, MSA supplementation exhibited an approximate Generally, when more MSA was added to the formulation, less βE was released ( Figure 2). In the case of the formulations with MC and PA1, 40% MSA addition caused the released amount to decrease by almost half (Figure 2a,b). In the hydrogels with PC-11, MSA supplementation exhibited an approximate 30% decrease (Figure 2c). The release processes did not follow the linear course of the zero-order kinetics function ( Figure 2).

First-Order Kinetics Model
The flow of first-order kinetics model function indicated a departure of linearity, especially applied to formulations with PC-11 ( Figure 3).

First-Order Kinetics Model
The flow of first-order kinetics model function indicated a departure of linearity, especially applied to formulations with PC-11 ( Figure 3). Generally, when more MSA was added to the formulation, less βE was released ( Figure 2). In the case of the formulations with MC and PA1, 40% MSA addition caused the released amount to decrease by almost half (Figure 2a,b). In the hydrogels with PC-11, MSA supplementation exhibited an approximate 30% decrease (Figure 2c). The release processes did not follow the linear course of the zero-order kinetics function ( Figure 2).

First-Order Kinetics Model
The flow of first-order kinetics model function indicated a departure of linearity, especially applied to formulations with PC-11 ( Figure 3).

Second-Order Kinetics Model
The charts for the second-order kinetics model were well suited to the linear course of the function, especially used to formulations with MC ( Figure 4).

Second-Order Kinetics Model
The charts for the second-order kinetics model were well suited to the linear course of the function, especially used to formulations with MC ( Figure 4).

Second-Order Kinetics Model
The charts for the second-order kinetics model were well suited to the linear course of the function, especially used to formulations with MC ( Figure 4).

The Weibull Model
The logarithmic equation of the Weibull model introduced a good fit for the rectilinear course of graphs ( Figure 6).

Kinetics Models Parameters
The range of the release rate constants of the formulations was from 4.37 × 10 −2 %·min −1 to 9.50 × 10 −2 %·min −1 ( Table 3). The best match belonged to the second-order kinetics and Higuchi model, as shown in Table 3. The release rate constants for the second-order kinetics were in the range of 5.59 × 10 −6 % −1 ·min −1 to 1.80 × 10 −5 % −1 ·min −1 . The Higuchi model showed a minimum release rate constant of 1.05 %·min −1/2 and a maximum release rate of 2.31 %·min −1/2 ( Table 3). The calculated values of the shape parameter β in the Weibull model were between 0.598 and 0.769 in preparations F20 and F40, respectively. The constants and regression coefficients are shown in Table 3. The range of the regression coefficients of the kinetics models was from 0.9522 to 0.9992, and the highest was observed for the H20 formulation. The Weibull model showed regression coefficients between 0.9889 and 0.9993 (Table 3).

Kinetics Models Parameters
The range of the release rate constants of the formulations was from 4.37 × 10 −2 %·min −1 to 9.50 × 10 −2 %·min −1 ( Table 3). The best match belonged to the second-order kinetics and Higuchi model, as shown in Table 3. The release rate constants for the second-order kinetics were in the range of 5.59 × 10 −6 % −1 ·min −1 to 1.80 × 10 −5 % −1 ·min −1 . The Higuchi model showed a minimum release rate constant of 1.05 %·min −1/2 and a maximum release rate of 2.31 %·min −1/2 ( Table 3). The calculated values of the shape parameter β in the Weibull model were between 0.598 and 0.769 in preparations F20 and F40, respectively. The constants and regression coefficients are shown in Table 3. The range of the regression coefficients of the kinetics models was from 0.9522 to 0.9992, and the highest was observed for the H20 formulation. The Weibull model showed regression coefficients between 0.9889 and 0.9993 (Table 3). The minimal values were exhibited for the F0 and the maximal for the G40 formulations according to the kinetics and Weibull models. Table 3. The parameters determined in the course of release kinetics evaluation of gel formulations F, G, H, where K-release rates for respective model: zero-order kinetics (K (0) ), 1st-order kinetics (K (I) ), 2nd-order kinetics (K (II) ) and the Higuchi model (K (H) ), β-a shape parameter in the Weibull model, r 2 -correlation coefficient for the regression, SD-standard deviation, MC-methylcellulose, PA1-polyacrylic acid derivative (Carbopol 980 NF), PC-11-modified polyacrylic acid (Aristoflex Velvet).

Discussion
The best kinetics model match was chosen for the studied hydrogel formulations according to the highest correlation coefficients, as presented in Table 3. The formulations with MC hydrogel showed the best match to the Higuchi model (F0) and the second-order kinetics model (F20, F40). The PA1 and PC-11 formulations released the active substance in accordance with the Higuchi model.

Discussion
The best kinetics model match was chosen for the studied hydrogel formulations according to the highest correlation coefficients, as presented in Table 3. The formulations with MC hydrogel showed the best match to the Higuchi model (F0) and the second-order kinetics model (F20, F40). The PA1 and PC-11 formulations released the active substance in accordance with the Higuchi model. The worst fitting kinetics were observed for the zero-order kinetics model considering all formulations. This suggests the course of release depended on substances concentration in the donor compartment. Moreover, in the case of first-order kinetics, we did not obtain the very high values of r 2 coefficient. Due to the known theory, the first-order process kinetics depends on the concentration of one molecular species engaged in the release. However, there should be another additional factor, as the r 2 coefficient are not very high. The evaluation of second-order process was motivated by our scientific interest and curiosity, if maybe another molecular species may influence the process parallelly with the active substance. Surprisingly we obtained very high values of r 2 coefficient in selected cases. This leads us to the concept which should be further studied, that two dynamic factors influence the release.
Our previous study results were compared, and similar results have been observed [9]; additionally, the Higuchi model has been complemented. The Higuchi model is based on a few parameters. Many factors may influence the release rate process, including following assumptions: The substance concentration exceeds its solubility, the matrix thickness is much larger than the size of the active substance, the swelling and dissolution of the matrix has no effect on the process, and there is a one-way diffusion and "sink conditions" are preserved, and the substance already present in the solution does not have a significant effect on the rate of release of its remaining amount [16]. In the case of second-order kinetics, a change in the concentration of two substrates influences the process rate [10]. The observed decreased rate of the release process might have been impacted by the interaction of MSA with the components of the hydrogels, as well as the active substance itself. This was more likely in the PA1 and PC-11 hydrogels than in MC because of the presence of reactive functional groups. The methyl groups in MC are substituted with hydroxyl groups at positions C2, C3 and/or C6 on glucose. This molecule is non-ionic and has an amphiphilic character. The structure of the MC makes it flat and stiff [21]. The heterogeneous distribution of the methyl groups and degrees of substitution larger than 1.3 enables hydrophobic interactions of the polymer [22]. As mentioned in our previous study [9], the anionic character of polyacrylic acid (PA1) may facilitate the production of complexes with cationic residues of other compounds [23]. In the case of the PC-11 hydrogel, cationic properties were observed. The amide groups may bond to the carboxyl groups of βE on the basis of hydrogen bonding. Navarro et al. investigated interactions between the carboxyl group of benzoic acid and the amide group of cyclophane. They found superiority of the O carboxyl -H· ·O=C amide bond over the C=O carboxyl · ·H-N amide bond as a result of a large negative charge density on the C=O oxygen [24]. The MSA was formed by means of acetic anhydride esterification, according to the method described by Zieba et al. [25]. The reaction proceeds as follows (Equation (2)) [26]: Considering the aqueous conditions of the reaction, there is a likelihood of hydrogen bonds between the carbonyl groups of MSA and βE (Figure 9). The most interesting parameter which reflects the expected release prolongation is the halfrelease time calculated on various equations according to available sources [9]. The release rate is inversely proportional to the half-release time. However, the half-release time units enable simple comparisons. The respective half-release times are presented in Figure 8. The release rates, as presented in Table 3, were between 4.37 × 10 −2 %·min −1 and 9.50 × 10 −2 %·min −1 for the zero-order kinetics and between 4.93 × 10 −4 ·min −1 and 1.29 × 10 −3 ·min −1 for the first-order kinetics. For the secondorder kinetics, they were between 5.59 × 10 −6 % −1 ·min −1 and 1.80 × 10 −5 % −1 ·min −1 , and for the Higuchi model, they were between 1.05 %·min −1/2 and 2.31 %·min −1/2 . The highest values of release rate were observed in the case of the formulations with MC, and the lower values were similar to each other in the case of the preparations with PA1 and PC-11. MC was applied in the formulations F0, F20, and F40. MC exhibits water-and organo-solubility and thermal gelation properties. Its wide range of viscosity ,000 mPa·s at 2%) corresponds to an average molecular weight range of 10,000-220,000 Da, making it useful as a carrier, a coating agent, and a disintegrant for tablets and matrix tablets [28]. MC 0512 used in our study had a viscosity of 4000 mPa·s, which is a medium-compact property, compared with that of the formulations with acrylic acid and modified acrylic acid (PA1 and PC-11), which show hydrophilic properties. The viscosity range of the PA1 hydrogel was approximately 60,000 mPa·s at 1.5%, and that of PC-11 was approximately 20,000 mPa·s at 1.5% [29]. Additionally, the viscosifying properties of polyacrylic acid polymers are strongly dependent on the molecular weight, degree of cross-linking, level of hydrophobic alkyl groups in their backbone, pH and concentration in the formulation. Additionally, the charge character of acrylate-based polymers is typically imparted by carboxylic acid functionality, and is, therefore, dependent upon the pH of the  Figure 9. The scheme of possible hydrogen bonding between the carboxyl groups of MSA and βE [27].
The most interesting parameter which reflects the expected release prolongation is the half-release time calculated on various equations according to available sources [9]. The release rate is inversely proportional to the half-release time. However, the half-release time units enable simple comparisons. The respective half-release times are presented in Figure 8. The release rates, as presented in Table 3, were between 4.37 × 10 −2 %·min −1 and 9.50 × 10 −2 %·min −1 for the zero-order kinetics and between 4.93 × 10 −4 ·min −1 and 1.29 × 10 −3 ·min −1 for the first-order kinetics. For the second-order kinetics, they were between 5.59 × 10 −6 % −1 ·min −1 and 1.80 × 10 −5 % −1 ·min −1 , and for the Higuchi model, they were between 1.05 %·min −1/2 and 2.31 %·min −1/2 . The highest values of release rate were observed in the case of the formulations with MC, and the lower values were similar to each other in the case of the preparations with PA1 and PC-11. MC was applied in the formulations F0, F20, and F40. MC exhibits water-and organo-solubility and thermal gelation properties. Its wide range of viscosity ,000 mPa·s at 2%) corresponds to an average molecular weight range of 10,000-220,000 Da, making it useful as a carrier, a coating agent, and a disintegrant for tablets and matrix tablets [28]. MC 0512 used in our study had a viscosity of 4000 mPa·s, which is a medium-compact property, compared with that of the formulations with acrylic acid and modified acrylic acid (PA1 and PC-11), which show hydrophilic properties. The viscosity range of the PA1 hydrogel was approximately 60,000 mPa·s at 1.5%, and that of PC-11 was approximately 20,000 mPa·s at 1.5% [29]. Additionally, the viscosifying properties of polyacrylic acid polymers are strongly dependent on the molecular weight, degree of cross-linking, level of hydrophobic alkyl groups in their backbone, pH and concentration in the formulation. Additionally, the charge character of acrylate-based polymers is typically imparted by carboxylic acid functionality, and is, therefore, dependent upon the pH of the formulation. At pH above 3.5, the carboxyl groups exhibit a negative charge and interact with polar solvents, such as water, as it is in the case of presented preparations. Moreover, the polymer strands uncoil into a swollen structure owing to internal crosslinks [29]. The ability of chitosan hydrogel, whose structure includes amino groups, to engage in ionic crosslinking with other chemical compounds has been indicated by many scientists, such as Muzzarelli [30], Berger [31] and Mahdavinia [32]. The above-presented factors may modify the βE release in specific hydrogels; however, the MSA addition tends to prolong the βE release in all the cases-the formulations: F20, F40, G20, G40, H20, H40.
According to the Weibull model, a β-factor has been established ( Table 3). The range of β was between 0.598 and 0.769, and the average value of β was 0.656. The lowest value was observed for F20, and the highest was observed in the case of F40. Several authors have treated the dependency of the β-value on the release mechanism of active substances. Papadopoulou et al. showed a dependence of the β-value up to 0.75 on the Fickian diffusion mechanism. In the β range between 0.75 and 1.0, Fickian diffusion is connected with case II transport. The presence of β-values greater than 1.0 highlights a complex release mechanism [33]. Peppas and coworkers [34] have interpreted a β-value of 0.5 as an indicator of Fickian law-dependent diffusion, where a β-value equal to 1.0, the drug release rate is independent of time (corresponding to zero-order release kinetics and case II transport). The values of β between 0.5 and 1.0 indicate the mixed mechanism of both (anomalous transport). Furthermore, Siepmann et al. [35] interpreted β = 0.5 as a diffusion-controlled drug release meter and β = 1.0 as an indicator of swelling-controlled drug release. Fickian law deviation is connected with the finite rate of the release reaction at which hydrophilic polymers may be relaxed to accommodate water [36]. Hopkinson et al. [37] studied the effect of water on amylose using stray field NMR. They demonstrated characteristics of case II transport for the ingress of liquid water. Similarly, Gümüşderelioglu and Kesgin [38] tested the release of bovine serum albumin from poly(vinyl ether)-based hydrogels and observed the absorption of water and the release of a drug via a swelling-controlled diffusion mechanism that occurred simultaneously. The range of β was 0.46 and 0.84, depending on the pH of the loaded gel. They recognized the existence of some molecular relaxation processes in addition to diffusion as responsible for the Fickian deviation.

Conclusions
The influence of MSA concentration in the evaluated hydrogel formulations on the release rate of βE was confirmed. The formulations of MSA with MC (F40) and PA1 (G40) had the highest impact on the release prolongation. The highest release prolongation was obtained in the formulation G40 containing PA1 and MSA. The PC-11 (H0) itself decreased βE release, whereas, the MSA addition slightly influenced the process (H40). Studies are still needed on the mechanism of binding the active substance to the PC-11 hydrogel, as there may exist strong interaction of βE with the polymer, presumably via the amide bonds. This may be ascribed to the presence of amide groups bonding to the carboxyl groups of βE and carboxylic groups interacting with polar solvents, such as water. The best fit of βE release pattern belonged to second-order kinetics and the Higuchi models. The Weibull model adapted to the experimental data. The appearance of β-value between 0.5 and 1.0 pointed to the mixed mechanism of the occurred processes.