Extraction and Fermentation of Schisandra | CCID

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Back to Journal »Clinical, Aesthetic and Research Dermatology» Volume 14

Study on Preparation of Effective Natural Substances by Extraction and Fermentation of Schisandra

Author Nguyen CT, Han JM, Tran VH, Jeong H, Kim ES

Published on November 2, 2021, Volume 2021: 14 pages, 1603-1612 pages

DOI https://doi.org/10.2147/CCID.S336280

Single anonymous peer review

Editor who approved for publication: Dr. Jeffrey Weinberg

Chi Thanh Nguyen,1 Jong-Man Han,2 Van Huong Tran,3 Hun Jeong,4 Eun Sook Kim5 1Department of Materials Technology, Faculty of Applied Science, Ho Chi Minh City University of Science and Technology, Ho Chi Minh City, 700,000, Vietnam; 2Department of Physical Therapy, Daegu College of Health , Daegu, 41453, Republic of Korea; 3 School of Mechanical Engineering, Hanoi University of Science and Technology, Hanoi, 100000, Vietnam; 4 Department of Nano Fusion Engineering, Polymer Materials Fusion Research Center, Chonbuk National University, Jeonju, Jeonbuk, 54896, Republic of Korea; 5 Seoul, South Korea 02713 Corresponding author: Hun Jeong; Eun Sook Kim [email protected]; [email protected] Purpose: In this study, a highly effective schisandra extract produced by effective microorganism (EM) fermentation (SCE) is used as an antioxidant material in the preparation of cosmetics. Subjects and methods: We conducted research on extracting S. chinensis through EM fermentation to improve efficiency. Determine the tyrosinase inhibitor analysis, pH value and thermal stability to verify the properties of the prepared product. Results: The efficacy and whitening effect of the prepared substances were verified by the analysis of tyrosinase inhibitors. It was found that both SCE and SCE fermentation (SCEF) showed high antioxidant capacity from natural sources. In addition, the pH value and thermal stability of the substance are also evaluated to optimize cosmetic manufacturing conditions. In this case, as the concentration of the added extract increases, the pH value decreases. The safety and stability evaluation showed that the substance contains effective chemical ingredients with anti-oxidation, inhibiting skin aging and whitening effects in the weak acid range of pH 6.25 to 2.98. In addition, the obtained product has no safety issues even if it is used after 60 days of storage. Conclusion: The SCE substance has proved to be a possible material for cosmetics. Keywords: Schisandra, effective microbial fermentation, antioxidant

In recent years, people's improvement and pursuit of living standards have caused many problems that affect people's health. Among them, cosmetics is a special case that requires careful study. The use of synthetic chemicals as the main ingredients of cosmetics can lead to many negative consequences, such as toxicity and high costs. This is why the research on natural products used in cosmetic applications has aroused great interest among many researchers. Schisandra (SC) is a medicinal and edible plant with five flavors (sweet, sour, bitter, salty and pungent)1,2, widely used in many food, beverage and herbal industries, etc. 3,4 SC contains many organisms Active compounds, such as gomisin, malic acid and citric acid, can be effectively used to treat cough and asthma. 5 In addition, due to its well-known antibacterial and antibacterial effects, it can be used in the food and cosmetic industries. Antioxidant capacity. In addition, it has excellent thermal stability and can be used in cosmetics and foods that do not affect human health. 6 Fermentation refers to the process of using microbial enzymes to decompose organic matter, derived from the Latin fervere. 7 It can be used in various ways and in different fields, such as food, medicine and cosmetics. 7 Food fermentation by the enzymatic action of microorganisms is traditionally used in the manufacturing process to improve flavor and destroy toxins, and it also has the effect of promoting biomolecules. 8,9 Effective microorganisms (EM) are useful microorganisms developed in 1982. 10 EM was originally developed for natural and organic agriculture. Subsequently, its scope of application gradually expanded, and it was widely used in Asian countries, Russia and the United States. 11 Initially, the EM solution was developed from 5 families, 10 genera and 80 species; however, this is a very complicated process. Therefore, EM was simply developed by some major organisms, such as photosynthetic bacteria, lactic acid bacteria, fungi, yeast, and actinomycetes. 12 Regarding fermentation applications, it is fermented under facultative anaerobic conditions, so its synthetic products, such as vitamins and carotene pigments, act as powerful antioxidants to prevent organic matter from decay. 13 The resulting amino acids and organic acids are converted into corresponding proteins and sugars, which are then immediately absorbed by plants. This greatly improves the efficiency of the synthesis and use of plant foods. EM was originally developed for natural and organic agriculture, but has now been used in various fields such as construction, medical and cosmetic industries. 14-16

In this study, SC was extracted with hot water, and the extract was made by fermentation with EM to improve efficiency. Tyrosinase inhibitor analysis is used to determine efficacy and whitening effect. In addition, the stability of the prepared product was verified by measuring the pH value and thermal stability, and then the effectiveness of the material in SC anti-oxidation and whitening effects was evaluated.

SC fruit was purchased from a farm in South Korea. The SC is treated with deionized (DI) water and then naturally dried in a well-ventilated and cool place to produce substrates. Diethylene glycol (DEG, (HOCH2CH2)2O), folin-Ciocalteu phenol reagent, sodium hydroxide (NaOH) and trisodium citrate dihydrate (Na3C6H5O7) were purchased from Sigma Chemical Co. (USA). Purified deionized water was used in all experiments. The EM active solution was used as a reagent for EM fermentation and was purchased from the EM Center of Jeonju University (South Korea).

After titration, the dried SC was extracted 10 times with a reflux condenser in hot water. The extraction temperature is 80°C, which is consistent with the extraction time of 24 h. Impurities are removed from the SC solution through a filtration process. The filtered solution was evaporated in a rotary evaporator (N-1000SW), and then freeze-dried for one day to completely remove the solvent, leaving a solid product. The resulting product was stored under vacuum during the experiment.

In order to compare and analyze the extracts from common SC and EM fermented SC, different concentrations of 1, 5, 10, 20 and 40 mg·mL-1 were produced. For the extract from EM fermentation SC, each part of EM fermentation broth and sugar is kept at 6% and 5%. The EM fermented SC extract was incubated at 37°C for 7 days before use.

According to the Codex Alimentarius method,17 1.0 g sample was dissolved in 100 mL nitric acid and 100°C deionized water. Then, an element analyzer (Vario EL, Germany) was used to measure and analyze the content of trace elements in the sample.

The polyphenol content per gram of sample is measured by the Folin-Denis method. 18 Therefore, 100 μL of the extract and 2 wt% Na2CO3 were mixed in the EP tube. The EP tube is kept at room temperature for 2 minutes for reaction. After that, add 50% Folin-Ciocalteu phenol reagent to the tube. Place the sample in a vortex mixer for 30 minutes at room temperature, and then analyze it with an ultraviolet-visible spectrophotometer at 750 nm.

The total content of flavonoids per gram of extract was measured by the diethylene glycol colorimetric method. 19 Therefore, 100 μL of extract and 100 μL of 1.0 N NaOH were added to the EP tube and mixed using a vortex mixer. After mixing, the solution was kept at 30°C for 1.0 hour for reaction. The yield of the reaction was analyzed by an ultraviolet-visible spectrophotometer at 420 nm.

The improved Blois method is used to measure the ability of 1,1-diphenyl-2-picrylhydrazine (DPPH) to scavenge free radicals. 20 In this case, 0.1 M Trizma base-HCl buffer (Tris buffer, pH 7.4) and 500 mM DPPH were initially prepared with methanol. Select butylated hydroxytoluene (BHT) and butylated hydroxyanisole (BHA) as the standard products for the control experiment. Then, mix 100 μL of the extract sample and 400 μL of Tris buffer in the EP tube, and then add 500 μL of DPPH solution. The mixture was kept in a dark room for 20 minutes and then analyzed by an ultraviolet-visible spectrophotometer at 517 nm. In the control experiment, 100 μL of BHT and BHA were added instead of the extract sample. In the non-additive group, 100 μL of Tris buffer was added to the EP tube instead of the extract sample. The measurement of electronic power is as follows: 21 (1)

An improved method developed by Kim et al. was used to measure nitrite scavenging activity. 22,23 Specifically, 0.3 mL of the extract sample was mixed with 0.1 mL of 1.0 mM NaNO2 solution and 0.2 M citrate buffer-HCl (pH 2.5) to obtain a final volume of 1.0 mL. The mixture was then reacted at 37°C for 1.0 hour. Subsequently, it was mixed with 0.4 mL Griess reagent (30% CH3COOH solution containing p-aminobenzenesulfonic acid (1.0 wt%): naphthylamine (1 wt%)) and 3.0 mL CH3COOH solution (2.0 wt%). Then, the reaction was carried out at room temperature for 15 minutes. (2)

Where A is the absorbance at 520 nm measured with the test sample, B is the absorbance at 520 nm measured with H2O instead of NaNO2, and C is the absorbance measured at 520 nm with H2O instead of the test sample, using an ultraviolet-visible spectrophotometer.

The superoxide dismutase (SOD)-like activity (SODA) was measured using the method according to Marklund and Marklund. SODA shows the degree of oxidation of pyrogallol that catalyzes the conversion of hydrogen peroxide (H2O2). EDTA, pH 8.5) Add 2.6 mL to 0.2 mL of 7.2 mM pyrogallol and mix with a vortex mixer. After 10 minutes, the reaction was stopped by adding 1.0 N HCl solution. Use an ultraviolet-visible spectrophotometer to measure at 420 nm. SODA shows the percentage difference in absorbance between the sample addition group and the non-addition group. (3)

In the formula, A is the absorbance of the standard solution without sample, and B is the absorbance of the standard solution with sample.

The tyrosinase inhibitory activity was measured by an improved version of the method proposed by Masamoto et al. 25 In order to measure the inactivation characteristics of mushroom tyrosinase under in vitro conditions, 0.3 mL of 2.5 mM 3.4 dihydroxyphenylalanine (L-DOPA), 0.05 mL of extract was used to mix the sample with 0.1 M in a vortex mixer. Mix the phosphate buffer solution (pH 6.8, total volume 1.5 mL), and then pre-incubate at 25°C. Then, add 0.05 mL of 1380 unit·mL-1 (Sigma Co., USA) mushroom tyrosinase, and mix with a vortex mixer. After that, the reaction was carried out at 25°C for 2.0 minutes. (4)

Where A is the absorbance value of the reaction solution with no sample in 0.5-1 min, measured at 475 nm with an ultraviolet-visible spectrophotometer; B is the absorbance value of the reaction solution in 0.5-1.0 min, with UV-Vis spectrophotometer. Visible spectrophotometer measured at 475 nm.

The formula of the cream based on distilled water, extracted oil and additives was prepared as listed in Table 1. Weigh the water, additives and oil, and then heat it in a water bath at 80°C. In a small mixer (DS-1800; Korea) slowly add water and mix vigorously with oil. Cream A was prepared without extracting oil. Creams B, C, D, E, and F contained 1, 5, 10, 20, and 40 mg·mL-1 of SC extract (SCE), respectively. Creams G, H, I, J and K contain 1, 5, 10, 20 and 40 mg·mL−1 SCE fermentation (SCEF). Table 1 Different basic cream formulas containing SCE and SCEF

Table 1 Different basic cream formulas containing SCE and SCEF

The pH value is measured by a pH meter (professional meter pp-15; Germany) at 25°C for 10 minutes. Before measuring the pH value, immerse the glass electrode in an alkaline buffer solution or deionized water.

In order to evaluate the stability based on the ambient temperature, the stability of creams containing SCE or SCEF was evaluated at 4, 25 and 40°C.

The inductively coupled plasma mass spectrometry (ICP) analysis results of SCE are shown in Table 2, which shows that 1.0 mg·mL−1 SCE contains 23.71, 0.42, and 0.03 mg·kg−1 of K, Fe and respectively. When the amount of SCE increases, the content of these trace elements increases. In this regard, the increase in K is dominant, while the increase in Mn, Fe, Cu and Zn is not significant. Regardless of the concentration of SCE, the content of Se remains the same. These trace elements contribute to the action of many physiologically active substances inside and outside the human body, and play important roles, including antioxidant and immune activities. Table 2 ICP analysis of SCE

Table 2 ICP analysis of SCE

The content of SCE in 100 g SC is 27.91 wt%. When the procedure was performed in water and ethanol, the extraction provided the same yield. However, the yield of the extract is lower than in previous papers. 26,27 This is due to the place where SC grows, as well as the difference in culture conditions and extraction methods. 26 The content of polyphenols is shown in Table 3. 1.0 mg·mL-1 ordinary SCE extract provides 1.53±0.02 mg·g-1 polyphenols, while the same amount of EM SCEF provides a higher polyphenol content (20.84±0.04 mg·g-1) than Non-fermented extract. The extraction results of EM SCEF at 5, 10, 20 and 40 mg⋅mL-1 were 25.82±0.04, 29.13±0.05, 42.07±0.05 and 59.22±0.09 mg⋅g-1, respectively. Table 3 The content of polyphenols in SCE and SCEF

Table 3 The content of polyphenols in SCE and SCEF

Taking common SC extract as an example, the corresponding results were 6.07±0.01, 11.87±0.01, 20.57±0.03 and 40.95±0.02 mg·g-1. Therefore, the polyphenol content of EM SCEF extract was higher than that in the SCE group. As the concentration of the schisandra extract increases, the polyphenol content in the extract also increases. In comparison, the extract of the EM SCEF group showed a higher content than the SC group. This means that SCE reacts with the EM active solution and causes the proliferation of polyphenols, which is a very useful physiological reaction substance for the human body. According to many reports by other researchers, there is a proportional relationship between antioxidant capacity and the content of flavonoids. 28-30 proves that the content of flavonoids depends on the concentration of EM SCEF and SCE (Table 4). For low SCE and SCEF content, such as 1, 5, and 10 mg⋅mL−1, no flavonoid values ​​were found. When the concentration of EM SCEF group and SCE group was 40 mg·mL-1, the flavonoid content was 2.79±0.02 and 1.59±0.08 mg·g-1, respectively. The results show that EM SCEF has high antioxidant capacity. Table 4 The content of flavonoids in SCE and SCEF

Table 4 The content of flavonoids in SCE and SCEF

Free radicals in the body can promote biological aging by reacting with lipids and proteins. In order to eliminate this phenomenon, many studies have conducted research on natural products. 31 The DPPH free radical scavenging test method is used in many natural products, using the electron donating ability of antioxidants to perform antioxidant measurements. The 32-34 SCE and EM SCEF groups are shown in Figure 1. In the case of SCE's DPPH free radical scavenging ability, as the concentration changes from 1.0, 10 to 40 mg·mL-1, the antioxidant capacity increases from 37%, 72% to 74%. In the EM SCEF group, the antioxidant capacity was 63%, 67%, and 79% at concentrations of 1.0, 10, and 40 mg·mL-1, respectively. In the EM SCEF group, the slight change in antioxidant capacity with concentration seems to be due to the reaction of EM SCEF with microorganisms to produce antioxidants. The antioxidant capacity of SC is compared with some well-known antioxidants such as BHT (89%) and BHA (88%). It is found that the free radical scavenging ability of SC is not much different from them. In addition, even at low concentrations, SCEF has a higher free radical scavenging ability than SCE. This means that when the SC and EM active solutions react with each other, the microorganisms produce physiologically active substances with antioxidant capacity. Therefore, it is possible to use less EM SCEF than SCE to produce materials with higher levels of antioxidants. Our conclusion is that this can solve the dosage problem in the manufacture of cosmetics, while improving the functional aspects of cosmetics containing plant natural substances. Figure 1 DPPH free radical scavenging ability of SCE and SCEF.

Figure 1 DPPH free radical scavenging ability of SCE and SCEF.

Nitrite reacts with secondary amines (compounds in which the two hydrogen atoms of ammonia are replaced by the hydrocarbon functional group R) to form nitrosamines, which is a notorious carcinogen; in other words, nitrites are nitrosamines Precursor. Therefore, the formation of nitrosamines can be effectively inhibited by removing nitrite. 35 If the reactivity between the analytical sample and nitrite is high, nitrite will be removed because it reacts in an ionized state, thereby inhibiting the formation of nitrosamines. The same applies to other substances in the form of ions or electrons. The high reactivity of the sample is equivalent to or evaluated as high activity in nitrite scavenging and anti-oxidation. The greater the amount of total phenolic compounds in the sample, the stronger the nitrite removal reaction that occurs in the lower pH range. On the contrary, the removal effect will be reduced in the higher pH range. 36 Table 5 shows that SCE is 15% at 1 mg⋅mL-1, 40% at 10 mg⋅mL-1, and 89% at 40 mg⋅mL-1. On the other hand, SCEF showed 51% nitrite scavenging activity at 1 mg⋅mL-1, 69% at 10 mg⋅mL-1, and 98% at 40 mg⋅mL-1. From this test, it was observed that as the concentrations of the two groups increased, the scavenging activity also increased. In addition, SCEF has better nitrite scavenging activity than SC, which is similar to the results of other experiments. At a concentration of 1.0 mg·mL-1, the difference in scavenging activity is 40 mg·mL-1, which is the largest among different concentrations and shrinks as the concentration increases. From this comparative experiment, through the EM fermentation process, the scavenging effect of nitrite, which is evaluated as quite good, is further improved. This phenomenon can be attributed to the production of more biologically active substances during the fermentation process, which in turn leads to increased inhibition of the formation of nitrosamines, and many phenols, as crude plant components, also contribute to effective nitrites Scavenging reaction. Table 5 Nitrite scavenging ability of SCE and SCEF at pH 2.5

Table 5 Nitrite scavenging ability of SCE and SCEF at pH 2.5

SOD is an enzymatic antioxidant that can detoxify and inhibit the toxicity of O2, H2O2, peroxide, and OH free radicals. 37,38 Table 6 shows SODA ⋅mL−1 with SCE (or SCEF) concentrations of 1.0, 10, and 40 mg. When the concentration was increased from 1 to 40 mg·mL−1, the SODA of the SCE group was 6%, 18%, and 41%, while the SODA of the EM SCEF group was 28%, 32%, and 43%. In order to analyze the difference in activity between the two groups, the difference in SODA value is smaller at low concentration (1.0 mg·mL-1) and high concentration (40 mg·mL-1) of SCE and SCEF. In this test, SODA levels in both groups were very high. 26,39 Therefore, it can be judged that both SCE and SCEF have high natural antioxidant capacity. Table 6 SOD-like activities of SCE and SCEF

Table 6 SOD-like activities of SCE and SCEF

The mechanism of tyrosinase inhibitory activity is very important in the cosmetics industry and can be used as a measure of skin whitening effect. 40 In the SCE group, the tyrosinase inhibitory activity increased from 35% to 36%, 37%, 38%, and 39% with increasing extract concentration (Table 7). In the EM SCEF group, when the concentration increased, the tyrosinase inhibitory activity increased from 38% to 39%, 40%, 41%, and 42%. EM SCEF has more effective tyrosinase inhibitory activity than normal extracts, but the difference is not significant. However, it is believed that both of these extracts have skin whitening effects when used in the manufacture of cosmetics. 41 Table 7 Inhibition of SCE and SCEF on melanin synthesis by tyrosinase in vitro

Table 7 Inhibition of SCE and SCEF on melanin synthesis by tyrosinase in vitro

The cosmetic formulations of different concentrations of SCE and EM SCEF, namely 0.0, 1.0, 5.0, 10, 20 and 40 mg·mL-1, are shown in Figure 2. The finished cosmetics are W/O formulations by adding the water phase to the oil phase. The pH value of the human skin surface is generally between 4.5 and 6.5, which is slightly acidic or neutral. 42 If the pH becomes alkaline, the skin's resistance will be weakened, causing bacteria to multiply and eventually leading to skin diseases. Therefore, it is strongly recommended to use neutral or slightly acidic cosmetics. The change of pH value with storage time is shown in Figure 3. Using SCE-free cream, the pH slightly increased from its initial value of 6.25 to 6.23 after 60 days. The initial pH values ​​of cream products with SCE concentrations of 1.0, 5.0, 10, 20, and 40 mg·mL-1 were 5.53, 3.87, 3.43, 3.15, and 3.03, respectively. These pH values ​​did not change after 60 days. The initial pH values ​​of EM SCEF-based creams with EM SCEF concentrations of 1.0, 5.0, 10, 20, and 40 mg·mL-1 were 4.12, 3.46, 3.37, 3.15, and 2.98, respectively. Similar pH values ​​were observed after 60 days. These results mean that there is no significant difference in pH changes between the two groups, and when the extract concentration increases, the pH value decreases. These results mean that there are no safety issues when using these cosmetics. Figure 2 A photo of W/O emulsion containing SCE and SCEF. Figure 3 Changes in pH of emulsions containing (A) SCE and (B) SCEF.

Figure 2 A photo of W/O emulsion containing SCE and SCEF.

Figure 3 Changes in pH of emulsions containing (A) SCE and (B) SCEF.

The evaluation of the influence of temperature on the stability of cosmetics helps us understand the chemical and physical changes of cosmetics. It was found that after 60 days, cosmetics appeared acidification, discoloration, evaporation, flotation, precipitation, turbidity, separation and other phenomena at different temperatures (Table 8). Table 8 Stability test of emulsions containing SCE and SCEF at different temperatures

Table 8 Stability test of emulsions containing SCE and SCEF at different temperatures

In this research, we successfully synthesized SCE and SCEF, which provide a source of biologically active substances and have potential application value in the design of innovative natural cosmetics with a wide range of activities. In the antioxidant activity test of SCE, the SODA measurements of DPPH, free radical scavenging, nitrite scavenging and EM fermentation SC were the highest in the test group. There is little difference in skin whitening effects between SCE and SCEF. The stability and safety evaluation of the determination show that the obtained substances contain effective chemical components, which have anti-oxidation, inhibit skin aging and whitening effects in the weak acid range of pH 6.25 to 2.98; therefore, these substances can be nominated for future cosmetic applications s material.

The research was supported by Shiqing University in 2020.

The authors report no conflicts of interest in this work.

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