Immunity-Enhancing Effect of Extracts Extracted from Leaves of Rubia hexaphylla, Cymbopogon citratus, and Dioscorea japonica for Sustainable Healthy Life

Abstract

The COVID-19 era requires research and development into eco-friendly immune technologies in human life. However, such research is limited because of a lack of interest. Therefore, in this study, sub-critical extracts extracted from Rubia hexaphylla leaves, Cymbopogon citratus, and Dioscorea japonica with physiological effects were tested for their ability to enhance immunitye and antioxidant activity in the body. Cell viability experiments, cytokine secretion induction experiments, nitrogen oxide induction experiments, and cytokine content measurements in mouse serum and NK activation experiments were conducted to verify the effects of improving immunitye and antioxidant activity in the body. First, the cell survival rate of all samples was found to be over 95%. Second, it was confirmed that the contents of IL-6 and TNF-α in the macrophages of all samples were increased, especially for RCD 3, at concentrations of 1000 μg/mL. Third, the nitrogen oxide content of all samples was increased in the nitrogen oxide induction evaluation, especially for RCD 3, at the highest concentration of 1000 μg/mL. Fourth, for RCD 3, IL-2 and TNF-γ were the highest measured in the measurement of the cytokine content in the mouse serum. Fifth, in the NK activation experiments, the positive control group of the samples was all found to have higher activity than the negative control group, with more than 60% cell activity in RCD 3.

1. Introduction

Inflammation reactions in the human body are related to a variety of biochemical phenomena, which are local biological defense reactions in response to pathological conditions such as physical trauma, harmful chemicals, or bacteria-, fungi-, and virus-induced infections. In particular, macrophages are known to play a key role in inflammatory reactions by producing nitrogen oxide and several inflammatory cytokines [1] according to chemical stimuli [2]. Natural killer cells (NK cells), one of these macrophages, are innate immune cells that are known to play a key role in immune response control by interacting directly with various macrophages, T cells, or by producing cytokines [3,4]. These NK cell activities are regulated by various activation and inhibitory receptor signaling balances, and normal cells are not recognized and attacked by inhibitory receptors in NK cells because MHC class I exists in the cells, while infected cells are removed by NK cells because of their reduced class I cells or ligands [5,6].

Meanwhile, Rubia hexaphylla is a herb of the family Rubiaceae, which is known for its young shoots that can be eaten as vegetables, and its roots that act as a haemorrhage cure, although little is known about the use of leaves. The leaves of the Rubia hexaphylla are long, oval and spiny, with six to ten at the original stem.

Dioscorea japonica is a member of the family Dioscoreaceae and has thick, long beetle roots [7]. It is native to China, and in Korea, it is native to various parts of the country, such as undergrowth or grasslands, and is widely cultivated in Gyeongsang-do. The main constituents of the Chinese yam are starch, amino acids, proteins, minerals, vitamin C and vitamin B1, and medicinal substances, such as saponine, arginine, choline, dioscin, batasin, mucin, amylase, etc. Dioscorea japonica contains a large amount of particularly sticky, viscous polysaccharides, which are mostly mannan dietary fibers [8]. The main physiological activity of Dioscorea japonica is the enhancement of coronary circulation [9], lipid improvement [10,11], blood flow improvement [12], blood pressure dropping [10,12], neurostabilization [13], and blood sugar improvement [14].

Additionally, Cymbopogon citratus is an herb in the family Gramineae. The prefix “lemon” is due to the typical lemon smell, caused by the presence of citral, a ring monoterpen. Cymbopogon citratus is native to southern India and Sri Lanka, and is now widely cultivated in tropical areas in the United States and Asia. Prior studies have shown that Cymbopogon citratus has a variety of effects such as antibacterial [15], antioxidant [16], anti-inflammatory [17], and antifungal [18] effects.

In particular, Rubia hexagonal leaves, Cymbopogon citratus, and Dioscorea japonica, which have proven pharmacological effects, have great academic and clinical value in sustainable combinations of mixtures, with the pharmacological effects of certain ingredients. However, studies on the function and use of these extracts are still insufficient. Therefore, the purpose of this study is to experimentally verify the immune enhancing effects that can be used in a sustainable environment, with extracts extracted from a mixture of Rubia hexapila leaves, Cymbopogon citratus, and Dioscorea japonica.

2. Experimental Methods

In this study, the sub-critical water of the mixture made with Rubia hexaphylla leaves, Cymbopogon citratus and Dioscorea japonica, which have these physiological effects, was extracted to confirm the in vivo immune and antioxidant performance.

For the experiments of this study, a mixture of Rubia hexaphylla leaves, Cymbopogon citratus and dioscorea japanica was produced, and extracts were produced using sub-critical water. Sub-critical water refers to water at a high temperature and high pressure below that of super-critical water at a critical point (374.2.1, 22.1 MPa) that is used to extract oil based on the strong hydrolysis and organic solubility of water when it is at as high temperature and high pressure.

2.1. Manufacturing of Experimental Samples

Rubia hexaphylla leaves, Cymbopogon citratus, and Dioscorea japanica supplied from Jibio Pharm Co., Ltd. (Goyang, Gyeonggi-do, Korea) were cleaned and dried at 70 °C for 48 h to filter and homogenize on 25 mesh bodies, each of which was mixed at 200 g:100 g:100 g weight. Three experimental samples were produced from the following processes with Rubia hexaphylla leaves, Cymbopogon citratus, and Dioscorea japanica mixtures (

Table 1. Test samples.

RCD-1	RCD-2	RCD-3
Hydrothermal extract	Ethanol Extract	Sub-critical Extract

).

• RCD-1
  Here, 20 g of the mixture and 400 mL of distilled water were placed in a 1 L Erlenmeyer flask, and the mixture was extracted while stirring in a magnetically stirring constant temperature water bath at 70 °C for 6 h. After extraction, the extract was cooled at room temperature for 1 h, and filtered with a 0.45 μm membrane filter. After filtration, RCD-1 was prepared by concentration in a vacuum and at room temperature for 3 h.
• RCD-2
  Here, 20 g of the mixture and 400 mL of 70% concentration ethanol were placed in a 1L round flask, and a reflux tube was connected. After connection, extraction was performed under reflux while stirring for 6 h in a magnetically stirring constant temperature water bath at 70 °C. After cooling the extract at room temperature for 1 h, the reflux tube was removed and filtered through a 0.45 μm membrane filter. After filtration, RCD-2 was prepared by concentration in vacuo and room temperature for 3 h.
• RCD-3
  Here, 20 g of the mixture was added to the accelerated solvent extractor, and 400 mL of distilled water for sub-critical treatment was added, followed by sub-critical extraction. According to the specific procedure, the temperature was raised to 100 °C using a thermostat, extracted under 3 MPa for 5 min, then 120 °C for 10 min under 3 MPa, and then extracted under 3 MPa for 30 min under 3 MPa. Then, the extract was collected in a vial and filtered through a 0.45 μm membrane filter. After filtration, RCD-3 was prepared by concentration in a vacuum and at room temperature for 3 h.

2.2. Cell Viability Experiment

The cell viability of the three experimental samples manufactured in this study were tested. Macrophages play a pivotal role in cell-mediated immune responses by transmitting external antigen information to T cells. To check the cytotoxicity of the samples, the cell survival rates of raw 246.7 cells, which are macrophages, were determined at concentrations of 100 μg/mL, 300 μg/mL, 500 μg/mL and 1000 μg/mL. Specifically, RAW 264.7 cells, a mouse-derived macrophage, were cultured in 100 IU/mL penicillin, 100 IU/mL streptomycin, and 10% bovine serum DMEM (Dulbecco’s modified egg’s medium) using a 5% carbon dioxide culture at 37 °C. Afterwards, RAW 264.7 cells were allocated at concentrations of 3 × 10⁴ cells/well into 96-well plates, incubated at 37 °C, 5% carbon dioxide incubators for 12 h, and the samples were diluted in phosphate-buffered saline (PBS) to treat with concentrations of 0, 100, 300, and 1000 μg/mL of MTT. Then, 100 μL of DMSO was treated, and an hour later, absorbance was measured at 517 nm using a micro-plate reader (Epoch^TM2, BioTECH, Miami, FL, USA), and cell survival rates were compared using these values.

2.3. Cytokine Secretion Induction Experiment

The cytokine secretion ability of the three experimental samples was tested. Cytokine is a signaling substance that is released when macrophages are activated by external antigens. To verify the cytokine secretion of samples (RCD-1~3), we identified the cytokine (IL-6, TNF-α) of raw 246.7 cells at concentrations of 100 μg/mL, 300 μg/mL, 500 μg/mL, and 1000 μg/mL. To this end, RAW 264.7 cells were deposited into 96-well plates at concentrations of 5 × 10⁴ cells/well, then incubated in 37 °C, 5% carbon dioxide incubators for 12 h to completely attach the cells, dilute the samples to PBS, and treat them at concentrations of 0, 100, 300, 500, and 1000 μg/mL for 24 h. After incubation, the supernatant was separated and the cytokine content in the supernatant was measured. The cytokine content was determined using an ELISA kit, and the concentration was calculated as the standard curve obtained from the standard solution contained in the kit.

2.4. Nitrogen Oxide Induction Experiment

The nitrogen oxide induction ability of the three experimental samples was tested. Nitrogen oxide is a signaling substance in the immune system that is caused by nitrogen oxide synthase, which can confirm the activation of the immune system. To verify the nitrogen oxide-inducing capability of the samples, the nitrogen oxide-inducement of raw 246.7 cells, identified through cell survival experiments, was verified at 100 μg/mL, 300 μg/mL, 500 μg/mL, and 1000 μg/mL concentrations. For this, RAW 264.7 cells were deposited into 96-well plates at concentrations of 5 × 10⁴ cells/well, incubated in 37 °C, 5% carbon dioxide incubators for 12 h to completely attach the cells, and diluted with PBS to incubate at concentrations of 0, 100, 300, 500, and 1000 μg/mL for 24 h. After incubation, 100 μL of supernatant was taken and reacted with 100 μL of Griess reagent for 10 min, absorbance was measured at 517 nm using micro plate reader, and NO concentration was calculated by comparing it with the standard straight line obtained using sodium nitrite (iNtRON Biotechnology, Seongnam, Korea).

2.5. Measurement of Cytokine Content in Mouse Serum

To determine the cytokine secretion of samples identified in macrophages in mice, an immuno-degradable mouse model was produced, and the negative and positive control samples produced in these processes were administered, and the mice later sacrificed. BALB mice (6 weeks old) were purchased from Orient Bio Company (Iksan, Korea), acclimatized for 1 week while supplying them with sufficient feed and water, and then used in the experiment. The breeding environment had a day and night cycle of 12 h each, and the temperature (20–22 °C) and humidity (50–60%) were kept constant.

After oral administration of the test sample obtained in the above process, (though not to be, negative control group (NC) and the positive control group (PC)), the mice were sacrificed and then cytokines (IL-2, TNF-γ) in the mouse serum were analyzed. After purification, the immuno-suppressant cyclophosphamide (CPA) was orally administration, at 150 mg/kg three days before sacrifice and 100 mg/kg a day earlier to create an immunosuppressive model.

A negative control group (water), test group (RCD-1 or RCD-3, 100 mg/kg), and positive control group (β-glucan, 50 mg/kg) were orally administered their respective substance once a day for 10 days. After 10 days of oral administration, they stopped eating for 12 h, was anesthetized with CO₂ and sacrificed. Afterwards, blood from the victimized mouse was collected and centrifuged at 2000 rpm for 15 min at 4 °C. The cytokine content was measured with an ELISA kit, and 50 μL of serum was added to a 96-well plate with capture antibody, incubated at room temperature for 1 h, and washed. After that, 100 μL of a TMB solution was added to react for 10 min, and then 100 μL of a reaction stop solution was added to stop the reaction. The absorbance was then measured at 450 nm using the micro-plate reader, the cytokine (IL-2, TNF-γ) content was measured using the ELISA kit, and the concentration was calculated as the standard curve obtained from the standard solution contained in the kit.

2.6. NK Activation Experiment

NK cell activation tests were conducted to verify the immunity of the three experimental samples. NK cells are key components of the immune system that directly remove cancer cells and infected cells, or secrete water-soluble factors, including cytokines, which activate other predatory cells, such as mononuclear or dendritic cells. Immunity was confirmed by analyzing spleen NK cell activity in mice administered with RCD-1, RCD-2, and RCD-3 samples, the negative control group (NC), and the positive control group (PC).

For this, as much as 5 mL of 4% FBS-RPMI 1640 was injected into the abdominal cavity of the mouse, which was sacrificed in the above experiment, and the cells inside the abdominal cavity were collected by massaging them for 5 min to collect NK cells. After centrifuging the collected cells at a temperature of 4 °C for 10 min at 3000 rpm, the cell pellets were suspended in a lysing buffer to remove and clean the red blood cells. Afterwards, the cells were diluted to 1 × 10⁶ cells/mL with 10% FBS-RPMI 1640 and 1000 μL of cells were deposited in a 24-well plate, left at 37 °C in a CO₂ incubator for 2 h, and experimented upon to test for their attached cells. NK cells were then separated into 96-well plates at 1 × 10⁵ cells in each, and target Yac-1 cells (KCLB40160) were deposited at 1 × 10⁴ cells (effector-to-target cell ratio 10:1) per well. After 16 h of incubation at 37 °C, in 5% CO₂ incubator, MTS was added and reacted for 2 h. Finally, absorbance values were measured at 490 nm with the micro-plate reader.

3. Results

3.1. Cell Viability

The comparison of cytotoxicity in macrophages on the basis of cell viability (100%) for this study showed that the cell viability of all samples at a concentration of 0 to 1000 μg/mL was greater than 95%. Therefore, all samples in this study were found to have no influence on cytotoxicity in macrophages, and subsequent experiments on cytokine and nitrogen oxide production were conducted at concentrations of 0 to 1 μg/mL (see Figure 1).

3.2. Cytokine Secretion Induction

Cytokines are proteins secreted by immune cells and are very important mediators that regulate immune cell activity and the immune system. Therefore, cytokines are very important for identifying immune enhancement, and studies on IL-2, IFN-γ, TNF-α, etc. whose functions have already been identified are in progress [19,20].

As a result of this study, for the macrophages, all samples were found to have increased IL-6 and TNF-α content as concentrations increased, while RCD 3 samples showed relatively higher IL-6 and TNF-α content compared to RCD 1 and RCD 2 samples. In particular, for RCD 3 samples, both the IL-6 content and the TNF-α content were found to have the highest concentration at 1000 μg/mL. In addition, the content of IL-6 and TNF-α were found to be higher at all of the concentrations in RCD 2, an ethanol extract sample, than the hydrothermal extract sample, RCD 1. Therefore, it has been confirmed that the cytokine secretion induction ability is in the order of RCD 3 > RCD 2 > RCD 1 (see Figure 2a,b). TNF-α and IL-6 are mainly produced in T cells or activated macrophages, and act on endothelial cells and leukocytes to control the initial inflammatory response to infection with pathogenic microorganisms [21]. In addition, IL-6 is a cytokine that functions in both innate immunity and adaptive immunity, and is secreted from T cells to activate an immune response to infection, and promotes the proliferation and differentiation of T cells and B cells [22,23]. As a result of the experiment, RCD 3 was found to induce an increase in TNF-α and IL-6, and thus it can be seen that it is a very useful extract for enhancing immunity in the body.

3.3. Nitrogen Oxide Induction

Nitric oxide (NO) is an important signal transmitter that acts as a defense against foreign substances in the immune system and is produced by L-arginine due to the action of nitric oxide synthase (NOS). Nitric oxide is known as a neurotransmitter and a defense material of the immune system because it is involved in various biological processes, such as the immune response, cytotoxicity, and the neurotransmitter system [24]. Therefore, an increase in nitric oxide production at a concentration without cytotoxicity can be seen to activate the immune system in the body.

In macrophages, all samples were found to have increased nitrogen oxide content as concentrations increased, while the RCD 3 samples showed relatively higher nitrogen oxide content compared to RCD 1 and RCD 2 samples. In particular, for RCD 3 samples, the nitrogen oxide content was highest at the concentration of 1000 μg/mL. In addition, the nitrogen oxide content was found to be higher at all of the concentrations in RCD 2, an ethanol extract sample, than the hydrothermal extract sample, RCD 1. Therefore, it has been confirmed that the nitrogen oxide induction ability is in the order of RCD 3 > RCD 2 > RCD 1 (see Figure 3).

3.4. Cytokine Content in Mouse Serum

The immune initiation response depends on the immediate response of immune-related cells, such as macrophages, natural killer cells (NK), T cells, and B cells, to foreign antigens. In addition, mutual cooperation between immune cells and tissue cells such as fibroblasts, chondrocytes, and osteoblasts, is very essential for the inflammatory response, and the mutual cooperation between these cells is mediated by a protein called cytokine. Therefore, the generation and secretion of cytokines generated from immune cells occupies a very important position in regulating the immune response [25].

The content of cytokine in mice serum was measured and found to be consistent with the tendency of cytokine secretion identified by macrophages. For RCD 3, the IL-2 and TNF-γ contents were 45.2 mg/mL and 101.6 mg/mL, respectively, which were the highest among extracts, but the cytokine content was lower than that of the positive control, beta-glucan. On the other hand, cytokine content was found to be higher in RCD 2, an ethanol extract sample, than in RCD 1, a hydrothermal extract sample, and the cytokine content in all samples was higher than the negative control (see Figure 4). Therefore, it was confirmed that RCD 3 is a very useful extract for in vitro immunity enhancement.

3.5. NK Activation

NK cells play an important role as immune factors responsible for innate cellular immunity and secrete cytokines, such as IFN-γ, TGF-γ, and IL-10. NK cell activity modulates the immune system by stimulating macrophages or enhancing the cytotoxicity of CD8+ T lymphocytes [26]. Comparing the activity of spleen NK cells with the negative control, both samples and positive control (PC) showed higher activity than the negative control (NC). The cell activity of the sub-critical extract sample, RCD 3, was the highest with more than 60%, followed by the ethanol extract sample, RCD 2, and hydrothermal extract RCD 1. However, the positive control group (PC) had the highest cell activity and was found to have the same tendency as the cytokine experiment results (see Figure 5).

4. Discussion and Conclusions

Immunity in the human body is an essential physical function for eco-friendly, sustainable, and healthy human life. Immunity refers to the physical action of rejecting, eliminating, or discharging anything other than what should be there, and when these immune functions disappear, you are immediately attacked by viruses or bacteria and exposed to all diseases. In particular, in the COVID-19 era, the immune system is a very important area of life, and a good immune system must be built into human life in an eco-friendly and sustainable manner. In addition, the oxidation reaction in vivo is mainly carried out through a chain reaction in which radicals are involved. Active oxygen is involved in various diseases, such as arteriosclerosis, brain diseases, cardiovascular disorders, aging, and cancer, so antioxidant function, which is a body function that eliminates active oxygen, is essential for a sustainable and healthy life.

In this study, sub-critical extracts extracted from Rubia hexaphylla leaves, Cymbopogon citratus, and Dioscorea japonica with physiological effects were tested for immunity-enhancing activity. Rubia hexaphylla leaves, Cymbopogon citratus, and Dioscorea citratus were cleaned, dried at 70 °C for 48 h to homogenize, and then the mixture was prepared at 200 g:100 g:100 g weight, respectively. Cell viability experiments, cytokine secretion induction experiments, nitrogen oxide induction experiments, and cytokine content measurements in mouse serum and NK activation experiments were conducted to verify the effects of improving immune system in vivo.

The main experimental results of this study are as follows.

First, the cell survival rate of all samples was found to be over 95%. When the extract concentration was lower than 300 mg/L, the cell viability of the samples extracted with hot water (RCD-1) was measured to be slightly higher compared to other samples, but when the concentration was 500 mg/L, the cell viability of sub critical-water extracted RCD-3 sample was shown to be the highest. At a concentration of 1000 mg/L, all samples, RCD-1, RCD-2, and RCD-3, showed at least 95% cell viability. The decrease in cell viability can be seen as a result of damage to the intracellular nucleic acid materials or protein synthesis system, as with other toxic substances [27,28]. It was observed that cytotoxicity did not appear because the extracts used in this study showed at least 90% cell viability.

Second, it was confirmed that the contents of IL-6 and TNF-α in macrophages of all samples were increased, especially for RCD-3, at concentrations of 1000 μg/mL. Cytokines are proteins secreted by immune cells and are important mediators that regulate the activity between immune cells and the immune system [23]. The role of cytokines is very important for identifying the causes and treatment of certain diseases or factors for enhancing and suppressing immunity, and in the case of IL-6 and TNF-α, whose functions have already been identified, drugs that would increase or inhibit the secretion are already being developed [22]. TNF-α and IL-6 are mainly produced by T cells or activated macrophages, and are related to innate immunity as they act on endothelial cells and leukocytes to induce and control the initial inflammatory responses to infections by pathogenic microorganisms [21]. In particular, IL-6 is a cytokine that functions in both innate immunity and adaptive immunity, and it is known to be secreted from T cells to activate the immune response to trauma [20] and promote the proliferation and differentiation of T cells and B cells [22,23]. From these results, it can be seen that the administration of the extracts used in this study acts effectively for the proliferation and differentiation of immune-related cells, especially T cells and B cells.

Third, NO content of all samples was increased in the nitrogen oxide induction evaluation, especially for RCD-3, at the highest concentration of 1000 μg/mL. NO is an important signal transmitter that acts for defense against foreign substances in the immune system and is an inorganic educt produced from L-arginine by the action of nitric oxide synthase (NOS). It is involved in many biological processes, such as immune reactions and cytotoxicity, and is known as a defense substance in the immune system [24]. However, since it has been reported that the excessive secretion of nitric oxide induces inflammatory diseases to cause damages to cells and tissues, it is very important to maintain an appropriate nitric oxide concentration [29]. Therefore, it can be judged that an increase in nitric oxide production at a concentration without cytotoxicity activates the immune system in the body [30]. In the above-written results, it was observed that the nitric oxide- inducing ability increased in a concentration-dependent manner at concentrations of 50, 250, 500, and 1000 μg/mL and did not affect cytotoxicity. Therefore, the extracts developed in this study are thought to enhance the immuno-stimulating activity of macrophagocytes.

Fourth, for RCD 3, IL-2 and TNF-γ were the highest measured at 45.2 mg/mL and 101 and 6 mg/mL, respectively, in the measurement of the cytokine content in the mouse serum. IL-2 not only induces the differentiation, proliferation, and activity of T cells to the extent that it is known as a T cell growth factor, but also increases the formation of NK cells and the production of antibodies by B cells. Therefore, it is a representative cytokine for immune activities known to be effective for the suppression of tumor metastasis [31,32]. Currently, most of the immunosuppressive agents used clinically in tissue transplantation, etc., have been developed as mechanisms of actions to inhibit the secretion and action of IL-2 and are being distributed worldwide [31]. According to the results of this experiment, the level of sub-critical extract (RCD-3) was the highest, indicating that the administration of the extracts developed in this study is acting effectively for the production of IL-2, which is related to the proliferation and differentiation of immunity-related cells, especially T cells.

Fifth, in NK activation experiments, the positive control group (PC) of the samples was all found to have higher activity than the negative control group (NC), with more than 60% cell activity in RCD-3. Natural killer cells (NK cells), one of the immune cells responsible for innate immunity, play an important role not only in the immune response to infections by viruses or bacteria, but also in the anticancer immune response through the interaction between receptors and ligands without prior sensitization [30]. According to the results of this study, the cellular activity of RCD-3 was found to be the highest at over 60%, indicating that the extracts developed in this study enhance anticancer immunity.

This study has shown that Rubia hexaphylla leaf hydrothermal extract or Rubia hexaphylla leaf ethanol extract did not simultaneously exhibit nitrogen oxide-inducing effects, cytokine secretion, and NK cell activity-inducing effects. In addition, if the weight ratio of Rubia hexaphylla leaves, Cymbopogon citratus and Dioscorea japonica did not meet the range of 1:0.5~1.5:0.5~1.5, there was no simultaneous effects, and also the effects were decreased.

However, if a solid phase construct using Rubia hexaphylla leaves, Cymbopogon citratus and Dioscore japonica contains the sub-critical extract as an active ingredient, the obtained composition provides antioxidant and immune-activating effects by simultaneously improving nitrogen oxide, cytokine secretion, and NK cell activity. Rubia hexaphylla, Cymbopogon citratus, and Dioscorea japonica are widely known for their medicinal utility, but little is known about their effects on the immune system. However, through this study, it is judged that linalool oxide and epoxy-linalool oxide, which are the main components of Cymbopogon citratus, induced IL-6 production [33]. In addition, it seems that saponin, a major component of Dioscorea japonica, increased cytokine and nitric oxide production [29]. Due to these results, the mixed extract of this study was found to be effective in enhancing immunity. In addition, the three extracts were shown to increase IL-6 and TNF-α secretion, which means that the three extracts are effectively working on the immunity, especially the proliferation and differentiation of T cells and B cells. Such proliferation and differentiation of T cells and B cells is expected to increase the production of TNF-α and IL-6 to enhance immunity in the body. Meanwhile, the three extracts were found to activate the action of nitric oxide synthase (NOS). This NOS activation promotes the production of L-arginine and is considered to induce nitric oxide production.

Although this study did not derive the optimal composition for pharmacological purposes, food purpose, and cosmetic purpose, subsequent studies need to present the optimal composition and characteristics for each use. Furthermore, this study for each application would contribute significantly to the commercialization of this optimized composition if the optimized composition for the activation of nitrogen oxide, cytokine secretion, or NK cell activation were determined.

Given that inflammation is the busiest of the three stages of the healing reaction, efforts are made to protect damaged areas and return to normal without damage if possible, and sub-critical extracts are expected to help maximize the net function of these inflammations, further promoting nitride oxide induction, cytokine secretion, and maximizing NK cell activity. The mixture of plant extracts developed from this study could be used as one of the basic materials for the development of new drugs to promote immune and antioxidant functions in the body, which are essential for a sustainable and healthy life in humans. In addition, this experiment is meaningful in that it proved its effectiveness from a sustainable perspective rather than a short-term perspective by using subcritical extracts extracted from eco-friendly Rubia hexaphylla leaves, Cymbopogon citratus, and Dioscorea japonica. It is hoped that it will be a reference study for the development of mankind’s sustainable immune system in times such as these.