KR102699337B1 - Immune-boosting composition comprising Angelica extract and Aster glehni extract - Google Patents

Abstract

The present invention relates to an immune-enhancing composition containing an Angelica gigas Nakai extract and an Aster japonica extract, and more particularly, to an immune-enhancing composition containing an Aster japonica extract together with an Angelica gigas Nakai extract as an effective ingredient, thereby greatly enhancing an immune-enhancing effect. The immune-enhancing composition of the present invention contains a mixture of an Angelica gigas Nakai extract and an Aster japonica extract in a weight ratio of 1:1 to 5:1 as an effective ingredient. The Angelica gigas Nakai extract is preferably a water extract of Angelica gigas Nakai or a fermented Angelica extract obtained by extracting Angelica gigas Nakai and then fermenting it. According to the present invention, by mixing an Aster japonica extract with an Angelica gigas Nakai extract as an effective ingredient for immune-enhancing, the immune-enhancing effect can be greatly enhanced compared to the use of Angelica gigas Nakai alone or Aster japonica Nakai alone. The immune-enhancing composition of the present invention uses an 'Angelica gigas Nakai extract' extracted with water or fermented with edible yeast after water extraction as a main ingredient for immune-enhancing, and therefore can be used both in the form of medicines and foods such as health functional foods.

Description

{Immune-boosting composition comprising Angelica extract and Aster glehni extract}

The present invention relates to an immune-enhancing composition containing an extract of Angelica gigas Nakai and an extract of Aster japonica root, and more particularly, to an immune-enhancing composition containing an extract of Aster japonica root together with an extract of Angelica gigas Nakai as effective ingredients, thereby significantly enhancing the immune-enhancing effect.

Angelica is a representative medicinal plant belonging to the Umbelliferae family and the genus Angelica . In the past, it was collected from the mountains, but recently, it is mostly harvested through cultivation. To cultivate Angelica for medicinal purposes, Angelica must be harvested before it has grown flower stalks. In other words, although it is a perennial plant, it must be dug up before the flowers bloom for herbal use. It grows very slowly at first, but after June, when it has grown to a certain extent, it grows vigorously.

Angelica is used to treat cough and upper gastrointestinal symptoms, fever and chills caused by febrile diseases, and uterine bleeding and infertility in women. In modern clinical practice, Angelica is widely used to treat arrhythmia, ischemic stroke, chorea in the elderly, cerebral arteriosclerosis, sickle cell anemia, arrhythmia, thromboembolic vasculitis, vascular headache, hypertension, uterine prolapse, and insomnia.

Aster glehni, a representative indigenous plant of Ulleungdo, is a perennial plant belonging to the Asteraceae family and is similar in shape to wild chives. The stem grows to a height of about 1 m and has short hairs all over its body, giving it a rough texture. The leaves grow alternately and have petioles, and are shaped like willow leaves or broad willow leaves, with a narrow base and sharp tip, coarsely serrated edges, and coarsely hairy. Aster glehni blooms white flowers from August to September, and the lower leaves fall over when the flowers bloom.

Ulleungdo Island's Aster has larger leaves and grows faster than those found on the mainland, so it has been produced in large quantities and has been eaten as a vegetable since long ago. It has also been reported to have the highest protein content among domestic wild vegetables.

The whole plant of Aster japonica is known to have pharmacological effects on cough, diuresis, and asthma. It has also been used as a medicinal plant to reduce fever in colds and as an expectorant for tonsillitis. Patent Publication No. 10-2018-0120604 discloses a composition for improving cognition or memory containing Aster japonica extract as an effective ingredient.

Republic of Korea Patent Registration No. 10-0252194 Republic of Korea Patent Registration No. 10-0998225 Republic of Korea Patent Registration No. 10-1192280 Republic of Korea Patent Registration No. 10-1279598 Republic of Korea Patent Registration No. 10-1303688 Republic of Korea Patent Registration No. 10-1746388

The inventors of this invention focused on the immune-enhancing effect of Angelica gigas. However, in order to use Angelica gigas in the form of food such as an immune-enhancing medicine, health functional food, or health supplement, there were two challenges. First, the immune-enhancing effect must be greatly improved so that a clear effect can be achieved, and it must be sufficiently effective even through water extraction or edible yeast fermentation.

To this end, the inventors of the present invention conducted many experiments and studies to find a combination plant and a combination method that can bring about an excellent immune-boosting synergistic effect when used in combination with an angelica extract, and as a result, they reached the present invention.

The purpose of the present invention is to provide an immune-enhancing composition having a significantly improved immune-enhancing effect compared to the use of the Angelica gigas extract alone or the Aster japonica extract alone by mixing the Aster japonica extract with the Angelica gigas extract.

In addition, the present invention aims to provide a composition that exhibits an excellent immune-enhancing effect while using as a main ingredient an extract of Angelica gigas Nakai extracted only with water without using an unsuitable organic solvent when used as a food.

In order to achieve the above purpose, in the present invention,

Provided is an immune-enhancing composition containing as an effective ingredient a mixture of an Angelica gigas Nakai extract and an Aster japonica extract in a weight ratio of 1:1 to 5:1. Preferably, the Angelica gigas Nakai extract and the Aster japonica extract are mixed in a weight ratio of 2:1 to 5:1.

The above-mentioned Angelica extract is, in one preferred embodiment, an aqueous extract of Angelica gigas, and more preferably, a hot water extract of Angelica gigas. In another preferred embodiment, the Angelica gigas extract is a fermented Angelica extract obtained by extracting Angelica gigas and then fermenting it.

In a preferred embodiment,

The above angelica extract,

Step of washing and crushing the angelica root;

A step of adding 8 to 12 times the weight of water to the crushed Angelica gigas and extracting at 110 to 130°C for 50 to 70 minutes;

A step of filtering the extract after extraction; and

It can be obtained by a method including a step of spray drying the filtered extract.

The above-mentioned Angelica extract fermentation product is preferably obtained by fermenting Angelica gigas by extracting it with water, and more preferably by fermenting Angelica gigas by extracting it with hot water. The above-mentioned Angelica extract fermentation product is preferably obtained by fermenting it with yeast.

In a preferred embodiment,

The above fermented extract of Angelica gigas Nakai is

In Article 10,

The above fermented extract of Angelica gigas Nakai is

Step of washing and crushing the angelica root;

A step of adding 8 to 12 times the weight of water to the crushed Angelica gigas and extracting at 110 to 130°C for 50 to 70 minutes;

Step of cooling the extract to room temperature after extraction;

A step of adding active dry yeast to the cooled extract at 0.08 to 0.12 wt% of the extract weight and culturing at 20 to 35°C for 15 to 30 hours;

Step of sterilizing the culture medium after culturing;

A step of filtering the sterilized culture solution; and

It can be obtained by a method including a step of spray drying the filtered culture solution.

The above-mentioned Artemisia capillaris extract is preferably an aqueous or ethanol aqueous solution extract of Artemisia capillaris.

In a preferred embodiment, the extract of the wormwood is

The step of harvesting, washing, and then cutting or crushing the mugwort;

A step of mixing an extraction solvent in an amount 13 to 17 times the weight of the cut or crushed mugwort;

A step of mixing the extractant and the extracting solvent and extracting at 70-80℃ for 5-7 hours;

A step of filtering the extract after extraction;

Step of concentrating the filtrate to 14-16 brix;

A step of adding excipients in an amount of 0.8 to 1.2 times the weight of the solid content to the concentrated filtrate;

A step of sterilizing after adding excipients; and

It can be obtained by a method including a step of spray drying after sterilization.

In the present invention, the term 'a mixture of an extract of Angelica gigas Nakai and an extract of Aster japonica' is defined to include both 'a mixture of extracts of Angelica gigas Nakai and Aster japonica' and 'a mixture obtained by mixing Angelica gigas Nakai and Aster japonica and then extracting them together.'

In the present invention, the term 'Angelica gigas extract' is defined to include both an extract obtained by extracting Angelica gigas with a solvent and a fermented product of an Angelica gigas extract obtained by extracting Angelica gigas with a solvent and then fermenting it.

According to the present invention, by mixing and using an Artemisia capillaris extract with an Angelica gigas Nakai extract as an effective ingredient for enhancing immunity, the immune-enhancing effect can be greatly improved compared to using Angelica gigas Nakai alone or Asteria capillaris Nakai alone.

The composition for enhancing immunity of the present invention containing extracts of Angelica gigas Nakai and Artemisia capillaris as effective ingredients has an excellent effect of enhancing immunity, as confirmed by the synergistic effect of increasing NO concentration and the synergistic effect of increasing iNOS and COX-2 expression. And this effect is significantly increased compared to extracts of Angelica gigas Nakai alone or extracts of Artemisia capillaris alone.

In addition, since the present invention uses an extract of angelica tree extracted with water or fermented with edible yeast after extraction with water as a main ingredient for enhancing immunity, it can be used in both food forms such as medicines and health functional foods.

Figure 1 shows the results of confirming the NO concentration when 'Angelica gigas extract (ANE)' and 'Artemisia aster extract (EAG)' were treated alone and when they were mixed and treated at a weight ratio of 1:5.
Figure 2 shows the results of confirming the NO concentration when 'Angelica gigas extract (ANE)' and 'Artemisia aster extract (EAG)' were treated alone and when they were mixed and treated at a weight ratio of 1:2.
Figure 3 shows the results of confirming the NO concentration when 'Angelica gigas extract (ANE)' and 'Artemisia aster extract (EAG)' were treated alone and when they were mixed and treated at a weight ratio of 1:1.
Figure 4 shows the results of confirming the NO concentration when 'Angelica gigas extract (ANE)' and 'Artemisia aster extract (EAG)' were treated alone and when they were mixed and treated at a weight ratio of 2:1.
Figure 5 shows the results of confirming the NO concentration when 'Angelica gigas extract (ANE)' and 'Artemisia aster extract (EAG)' were treated alone and when they were mixed and treated at a weight ratio of 3:1.
Figure 6 shows the results of confirming the NO concentration when 'Angelica gigas extract (ANE)' and 'Artemisia aster extract (EAG)' were treated alone and when they were mixed and treated at a weight ratio of 4:1.
Figure 7 shows the results of confirming the NO concentration when 'Angelica gigas extract (ANE)' and 'Artemisia aster extract (EAG)' were treated alone and when they were mixed and treated at a weight ratio of 5:1.
Figure 8 shows the results of confirming the NO concentration when 'Fermented Angelica Extract (FAN)' and 'Artemisia princeps extract (EAG)' were treated alone and when they were mixed and treated at a weight ratio of 1:5.
Figure 9 shows the results of confirming the NO concentration when 'Fermented Angelica Extract (FAN)' and 'Artemisia princeps extract (EAG)' were treated alone and when they were mixed and treated at a weight ratio of 1:2.
Figure 10 shows the results of confirming the NO concentration when 'Fermented Angelica Extract (FAN)' and 'Artemisia princeps extract (EAG)' were treated alone and when they were mixed and treated at a weight ratio of 1:1.
Figure 11 shows the results of confirming the NO concentration when 'Fermented Angelica Extract (FAN)' and 'Artemisia princeps extract (EAG)' were treated alone and when they were mixed and treated at a weight ratio of 2:1.
Figure 12 shows the results of confirming the NO concentration when 'Fermented Angelica Extract (FAN)' and 'Artemisia princeps extract (EAG)' were treated alone and when they were mixed and treated at a weight ratio of 3:1.
Figure 13 shows the results of confirming the NO concentration when 'Fermented Angelica Extract (FAN)' and 'Artemisia princeps extract (EAG)' were treated alone and when they were mixed and treated at a weight ratio of 4:1.
Figure 14 shows the results of confirming the NO concentration when 'Fermented Angelica Extract (FAN)' and 'Artemisia princeps extract (EAG)' were treated alone and when they were mixed and treated at a weight ratio of 5:1.
Figures 15 and 16 show the results of confirming the synergistic effects of iNOS and COX-2 when Angelica gigas extract (ANE) and Artemisia capillaris extract (EAG) were treated alone and when they were treated together.
Figures 17 and 18 show the results of confirming the synergistic effects of iNOS and COX-2 when ‘Fermented Angelica Extract (FAN)’ and ‘Artemisia princeps extract (EAG)’ were treated alone and when they were treated together.

The composition for enhancing immunity of the present invention contains a mixture of an extract of Angelica gigas Nakai and an extract of Artemisia capillaris as an effective ingredient. The present invention is described in detail below.

The above 'mixture of Angelica gigas extract and Aster japonica extract' can be obtained by mixing the respective extracts of Angelica gigas and Aster japonica, or by first mixing Angelica gigas and Aster japonica and then extracting them together.

The above 'mixture of Angelica gigas extract and Aster japonica extract' is preferably a mixture of Angelica gigas extract and Aster japonica extract in a weight ratio of 1:1 to 5:1. More preferably, it is a mixture of Angelica gigas extract and Aster japonica extract in a weight ratio of 2:1 to 5:1. In this case, the mixture can be obtained by mixing Angelica gigas extract and Aster japonica extract in the above ratio, or by first mixing Angelica gigas and Aster japonica in the above ratio and then extracting them together.

In a preferred embodiment of the present invention, the Angelica gigas extract is an aqueous extract of Angelica gigas, more preferably, an aqueous extract of Angelica gigas. In another preferred embodiment of the present invention, the Angelica gigas extract is an Angelica gigas extract fermented by extracting Angelica gigas and then fermenting it. The Angelica gigas extract can be obtained by adding water to Angelica gigas and then extracting it at 110 to 130°C. A preferred embodiment of producing the Angelica gigas extract is described below. The Angelica gigas extract fermented can be obtained by preferably fermenting Angelica gigas by extracting it with aqueous water and then fermenting it, more preferably, by fermenting it with yeast. A preferred embodiment of producing the Angelica gigas extract fermented is described below.

Preferred embodiment: Preparation of Angelica extract

After washing the angelica root, it is crushed and used as the raw material for angelica extract.

Add water to the crushed Angelica and extract the water. All commonly used extraction methods, such as cold extraction, hot water extraction, ultrasonic extraction, reflux extraction, and cooling extraction, can be used. However, the hot water extraction method is preferable.

Preferably, water is mixed in an amount 8 to 12 times the weight of the Angelica gigas, and extracted at 110 to 130°C for 50 to 70 minutes. At this time, the extraction temperature is more preferably 115 to 125°C. In one embodiment of the present invention, extraction was performed at a temperature of 121°C for 1 hour.

After extraction, the extract is filtered through a 60 mesh filter.

The filtered extract is spray-dried to obtain Angelica extract.

Preferred embodiment: Preparation of fermented extract of Angelica gigas Nakai

Wash the angelica root and crush it coarsely.

Add water to the crushed Angelica and extract the water. All commonly used extraction methods, such as cold extraction, hot water extraction, ultrasonic extraction, reflux extraction, and cooling extraction, can be used. However, the hot water extraction method is preferable.

Preferably, water is added in an amount of 8 to 12 times the weight of the Angelica gigas to the crushed Angelica gigas, and extracted at 110 to 130°C for 50 to 70 minutes. At this time, the extraction temperature is more preferably 115 to 125°C. In one embodiment of the present invention, extraction was performed at a temperature of 121°C for 1 hour.

The extract obtained by extraction is inoculated with a fermentation strain and cultured and fermented to obtain a fermented extract of Angelica gigas Nakai. Preferably, the extract is cooled to room temperature, and then the fermentation strain is inoculated and cultured and fermented. More preferably, it is cooled to 27 to 33°C, and then active dry yeast is added and cultured at 20 to 35°C for 15 to 30 hours. The active dry yeast is added in an amount of 0.08 to 0.12 wt% relative to the weight of the extract. The culture is more preferably performed at 27 to 33°C for 18 to 24 hours.

After culturing, the culture solution is sterilized. A conventional sterilization method can be applied for sterilization. In the embodiment of the present invention, sterilization was performed at 95 to 100°C for 30 minutes to 1 hour.

The sterilized culture medium is filtered through a 60 mesh filter.

The filtered culture solution is spray-dried to obtain a fermented extract of Angelica gigas Nakai.

The above-mentioned Asteraceae extract is preferably an Asteraceae water or ethanol aqueous solution extract. A preferred embodiment of preparing the Asteraceae extract is as follows.

Preferred embodiment: Preparation of extract of Aster japonica

The Asteraceae plant uses one or more parts selected from the whole plant, leaves, stems, flowers, and roots. It is preferable to extract the aerial parts of the Asteraceae plant, such as leaves and stems.

The mugwort is harvested, washed, and then cut or crushed before use.

Mix the cut or crushed mugwort with an extraction solvent in an amount of 13 to 17 times the weight of the mugwort, more preferably 14 to 16 times the weight of the solvent.

Water or ethanol aqueous solution is used as the extraction solvent for extracting Artemisia capillaris. It is preferable to use 50 to 80 wt% ethanol aqueous solution.

After mixing the extractant and the wormwood extract, extract at 70-80℃ for 5-7 hours.

As the extraction method, commonly used extraction methods such as cold extraction, hot water extraction, ultrasonic extraction, reflux extraction, and cooling extraction can be used. In the embodiment of the present invention, extraction was performed by reflux extraction. The extraction can be performed once or repeatedly twice or more.

After extraction, the extract is filtered and the filtrate is concentrated to 14-16 brix.

Add excipients in an amount of 0.8 to 1.2 times the weight of the solid content of the filtrate to the concentrated filtrate. The reason for adding excipients is to prevent the extract from absorbing the surrounding moisture and hardening or turning into a taffy-like substance, and to maintain a powder form. Preferably, add excipients in an amount equal to the weight of the solid content of the filtrate. It is preferable to use dextrin as the excipient.

After adding the excipients, sterilization is performed. A conventional sterilization method can be applied for sterilization. In the embodiment of the present invention, sterilization was performed at 90 to 100°C for 50 to 70 minutes.

After sterilization, spray drying is performed to obtain the extract of Artemisia capillaris.

The composition for enhancing immunity of the present invention is obtained by mixing the extract of Angelica gigas Nakai and the extract of Artemisia capillaris obtained as described above at a certain ratio. A preferred embodiment of obtaining the composition for enhancing immunity of the present invention is as follows.

Preferred embodiment: Preparation of a composition for enhancing immunity

The composition for enhancing immunity of the present invention is obtained by mixing the Angelica gigas extract and the Aster japonica extract in a weight ratio of 1:1 to 5:1. More preferably, the Angelica gigas extract and the Aster japonica extract are mixed in a weight ratio of 2:1 to 5:1.

In the case of the hot water extract of Angelica obtained above, it is most preferable to mix it with the extract of Artemisia capillaris at a weight ratio of 4:1 to 5:1. In particular, the immune-enhancing effect was the best when mixed at a weight ratio of 5:1.

In the case of the fermented extract of Angelica gigas Nakai obtained above, when mixed with the extract of Aster japonica in the range of 2:1 to 5:1, the NO enhancement significantly increased by more than 2-fold and the expression of iNOS and COX-2 significantly increased by more than 100%. The immune enhancement effect was more excellent in the mixing range of 3:1 to 5:1, and was the best in the mixing range of 4:1 to 5:1.

The present invention will be described in more detail below through specific examples. These examples are intended to illustrate the present invention and are not intended to limit the scope of the present invention.

<Example 1>

Preparation of Angelica Extract

The Angelica root was washed and crushed. 50 kg of the crushed Angelica root was added to 500 ℓ of tap water and extracted at 121℃ for 1 hour. The extract was filtered through a 60 mesh filter. The filtrate was spray-dried to obtain the Angelica extract.

<Example 2>

Production of fermented extract of Angelica gigas Nakai

Angelica root was washed and crushed. 50 kg of the crushed Angelica root was added to 500 ℓ of tap water and extracted at 121℃ for 1 hour. Active dry yeast was added to the extract at 0.1 (w/w)% of the extract amount and cultured at 30℃ for 20 hours. After culture, it was sterilized at 95℃ for 1 hour. After sterilization, it was filtered through a 60 mesh filter. The filtrate was spray dried to obtain a fermented Angelica extract.

<Example 3>

Preparation of Artemisia capillaris extract

The aerial parts of Aster japonica were collected and crushed. 2,000 ℓ of 70% ethanol solution was added to 185 kg of the crushed aerial parts of Aster japonica and extracted at 75℃ for 6 hours. A filter aid was added to the extract and filtered using a filter press. The filtrate was concentrated to 15 brix, and an equal amount of dextrin was added based on the solid content. After adding dextrin, it was sterilized at 95℃ for 1 hour. After sterilization, it was spray-dried to obtain an Aster japonica extract.

<Experimental Example 1>

Analysis of NO concentration by mixed treatment of Angelica gigas extract and Artemisia capillaris extract

To evaluate the effects of mixed treatment of Angelica gigas extract and Artemisia capillaris extract on NO concentration and its mechanism, the following experiment was conducted.

As test materials, ‘Angelica gigas Nakai extract (ANE)’ obtained in Example 1 and ‘Artemisia princeps extract (EAG)’ obtained in Example 3 were used.

The mixing ratio of ‘Angelica gigas extract (ANE)’ and ‘Artemisia princeps extract (EAG)’ for each sample is shown in Table 1 below.

Sample name Mixing ratio
(Angelica: Aster) Mixture concentration
( ㎍/㎖ ) Concentration of Angelica extract ( ㎍/㎖ ) Extract of Artemisia capillaris
Concentration ( ㎍/㎖ ) Sample 1
1:5 31.3 5.2 26.1 Sample 2 62.5 10.4 52.1 Sample 3 125 20.8 104.2 Sample 4 250 41.7 208.3 Sample 5
1:2 31.3 10.4 20.9 Sample 6 62.5 20.8 41.7 Sample 7 125 41.7 83.3 Sample 8 250 83.3 166.7 Sample 9 1:1 31.3 15.6 15.6 Sample 10 62.5 31.3 31.3 Sample 11 125 62.5 62.5 Sample 12 250 125.0 125.0 Sample 13
2:1 31.3 20.9 10.4 Sample 14 62.5 41.7 20.8 Sample 15 125 83.3 41.7 Sample 16 250 166.7 83.3 Sample 17
3:1 31.3 23.5 7.8 Sample 18 62.5 46.9 15.6 Sample 19 125 93.8 31.3 Sample 20 250 187.5 62.5 Sample 21
4:1 31.3 25.0 6.3 Sample 22 62.5 50.0 12.5 Sample 23 125 100.0 25.0 Sample 24 250 200.0 50.0 Sample 25
5:1 31.3 26.1 5.2 Sample 26 62.5 52.1 10.4 Sample 27 125 104.2 20.8 Sample 28 250 208.3 41.7

The experimental method is as follows.

RAW264.7 cells were seeded in a 96-well plate at a density of 1 × 10 ⁵ cells per well. After 24 hours of seeding the cells, each sample was treated singly or mixed at different concentrations.

After 24 hours of sample processing, each medium was transferred to a 1.7㎖ microtube and centrifuged for 5 minutes at 3,000 rpm and 4℃. After centrifugation, the supernatant was collected and 100㎕ was dispensed into a new 96-well plate. Before starting the experiment, 100㎕ of pre-prepared Griess reagent (1g/25㎖) was added to each 96-well plate containing the supernatant.

The 96-well plate was incubated at room temperature for 15 minutes after blocking light. The absorbance was measured at 540 nm, and the NO concentration was calculated using a standard graph.

The NO concentration increasing effect was compared when the Angelica gigas extract and Aster japonica extract were mixed and treated, compared to when the Angelica gigas extract was treated alone or when the Aster japonica extract was treated alone. The NO increasing effect was expressed as a % compared to the Angelica gigas extract alone or the Aster japonica extract alone. The results are shown in Table 2 and Figures 1 to 7 below.

At this time, the significance was shown in comparison with the group treated with the Angelica gigas Nakai extract alone ( ^# p < 0.05, ^## p < 0.01, ^### p < 0.001) and the group treated with the Aster japonica root extract alone ( ^* p < 0.05, ^** p < 0.01, ^*** p < 0.001).

Sample name Mixing ratio
(Angelica: Aster) Mixture concentration
( ㎍/㎖ ) Angelica extract
Single comparison (%) Artemisia capillaris extract
Single comparison (%) Sample 1 1:5 31.3 18.2 -3.2 Sample 2 62.5 38.9 11.8 Sample 3 125 52.2 29.4 Sample 4 250 24.0 7.7 Sample 5 1:2 31.3 54.8 26.7 Sample 6 62.5 59.4 28.2 Sample 7 125 85.6 57.8 Sample 8 250 56.5 36.0 Sample 9 1:1 31.3 118.7 79.1 Sample 10 62.5 108.5 67.8 Sample 11 125 110.7 79.1 Sample 12 250 80.3 56.7 Sample 13 2:1 31.3 253.9 189.8 Sample 14 62.5 176.3 122.3 Sample 15 125 138.9 103.1 Sample 16 250 93.8 68.4 Sample 17 3:1 31.3 411.5 318.8 Sample 18 62.5 243.0 175.9 Sample 19 125 182.8 140.5 Sample 20 250 113.0 85.1 Sample 21 4:1 31.3 604.0 476.4 Sample 22 62.5 325.3 242.2 Sample 23 125 188.8 145.6 Sample 24 250 146.6 114.3 Sample 25 5:1 31.3 657.5 520.2 Sample 26 62.5 345.9 258.7 Sample 27 125 300.6 240.5 Sample 28 250 189.3 151.4

As can be seen in Figures 1 to 7, when Angelica gigas Nakai extract (ANE) was treated alone at concentrations of 31.3 μg/mL, 62.5 μg/mL, 125 μg/mL, and 250 μg/mL, the NO concentration increased in a concentration-dependent manner compared to the normal group, and when Aster japonica extract (EAG) was treated alone at concentrations of 31.3 μg/mL, 62.5 μg/mL, 125 μg/mL, and 250 μg/mL, the NO concentration also increased in a concentration-dependent manner compared to the normal group.

In addition, when Angelica gigas Nakai extract (ANE) and Aster japonica extract (EAG) were mixed and treated, when mixed in weight ratios of 1:1, 2:1, 3:1, 4:1, and 5:1, the NO concentration increased compared to when each substance was treated alone, and a statistically significant synergistic effect occurred at all concentrations. When Angelica gigas Nakai extract (ANE) and Aster japonica extract (EAG) were mixed in weight ratios of 4:1 and 5:1, the NO concentration increased by more than 2-fold (100%) compared to the single treatment group at all concentrations, and the synergistic effect was the best when mixed in a weight ratio of 5:1.

<Experimental Example 2>

Analysis of NO concentration by mixed treatment of Angelica gigas extract fermentation and Artemisia capillaris extract

In order to evaluate the effect of mixed treatment of Angelica gigas Nakai extract fermentation and Artemisia capillaris extract on NO concentration and its mechanism, the following experiment was conducted.

As test materials, ‘Fermented Angelica Extract (FAN)’ obtained in Example 2 and ‘Artemisia princeps extract (EAG)’ obtained in Example 3 were used.

The mixing ratio of the fermented extract of Angelica gigas Nakai and the extract of Artemisia capillaris for each sample is shown in Table 3 below.

Sample name Mixing ratio
(Angelica: Aster) of the mixture
Concentration ( ㎍/㎖ ) Fermented extract of Angelica gigas Nakai
Concentration ( ㎍/㎖ ) Extract of Artemisia capillaris
Concentration ( ㎍/㎖ ) Sample 29 1:5 31.3 5.2 26.1 Sample 30 62.5 10.4 52.1 Sample 31 125 20.8 104.2 Sample 32 250 41.7 208.3 Sample 33 1:2 31.3 10.4 20.9 Sample 34 62.5 20.8 41.7 Sample 35 125 41.7 83.3 Sample 36 250 83.3 166.7 Sample 37 1:1 31.3 15.6 15.6 Sample 38 62.5 31.3 31.3 Sample 39 125 62.5 62.5 Sample 40 250 125.0 125.0 Sample 41 2:1 31.3 20.9 10.4 Sample 42 62.5 41.7 20.8 Sample 43 125 83.3 41.7 Sample 44 250 166.7 83.3 Sample 45 3:1 31.3 23.5 7.8 Sample 46 62.5 46.9 15.6 Sample 47 125 93.8 31.3 Sample 48 250 187.5 62.5 Sample 49 4:1 31.3 25.0 6.3 Sample 50 62.5 50.0 12.5 Sample 51 125 100.0 25.0 Sample 52 250 200.0 50.0 Sample 53 5:1 31.3 26.1 5.2 Sample 54 62.5 52.1 10.4 Sample 55 125 104.2 20.8 Sample 56 250 208.3 41.7

The experimental method is the same as Experimental Example 1, and the NO concentration increase effect was compared when the Angelica gigas Nakai extract fermentation was treated alone or when the Artemisia princeps flower extract was treated alone, and when the Angelica gigas Nakai extract fermentation and Artemisia princeps flower extract were mixed and treated. The NO increase effect compared to the Angelica gigas Nakai extract fermentation alone or the Artemisia princeps flower extract alone was expressed as a %. The results are shown in Table 4 and Figures 8 to 14 below.

At this time, the significance was shown in comparison with the group treated only with the Angelica gigas Nakai extract fermentation product ( ^# p < 0.05, ^## p < 0.01, ^### p < 0.001) and the group treated only with the Aster japonica root extract ( ^* p < 0.05, ^** p < 0.01, ^*** p < 0.001).

Sample name Mixing ratio
(Angelica: Aster) mixture
Concentration ( ㎍/㎖ ) Fermented Angelica Extract
Single comparison (%) Artemisia capillaris extract
Single comparison (%) Sample 29 1:5 31.3 81.8 26.7 Sample 30 62.5 14.2 -4.7 Sample 31 125 23.8 9.9 Sample 32 250 32.6 8.8 Sample 33 1:2 31.3 210.5 116.5 Sample 34 62.5 101.0 67.8 Sample 35 125 75.8 56.0 Sample 36 250 77.7 45.8 Sample 37 1:1 31.3 231.9 131.4 Sample 38 62.5 140.4 100.7 Sample 39 125 125.8 100.4 Sample 40 250 93.6 58.9 Sample 41 2:1 31.3 401.8 249.8 Sample 42 62.5 230.5 175.8 Sample 43 125 171.3 140.8 Sample 44 250 156.9 110.9 Sample 45 3:1 31.3 594.8 384.4 Sample 46 62.5 364.5 287.7 Sample 47 125 243.3 204.7 Sample 48 250 198.0 144.6 Sample 49 4:1 31.3 916.4 608.6 Sample 50 62.5 482.8 386.5 Sample 51 125 295.2 250.8 Sample 52 250 217.9 160.9 Sample 53 5:1 31.3 982.7 654.8 Sample 54 62.5 471.8 377.3 Sample 55 125 315.7 268.9 Sample 56 250 250.1 187.4

As can be seen in Figures 8 to 14, when Angelica gigas Nakai extract fermentation product (FAN) was treated alone at concentrations of 31.3 μg/mL, 62.5 μg/mL, 125 μg/mL, and 250 μg/mL, the NO concentration increased in a concentration-dependent manner compared to the normal group, and when Aster japonica extract (EAG) was treated alone at concentrations of 31.3 μg/mL, 62.5 μg/mL, 125 μg/mL, and 250 μg/mL, the NO concentration also increased in a concentration-dependent manner compared to the normal group.

When Angelica gigas Nakai extract fermentation product (FAN) and Aster japonica extract (EAG) were mixed and treated, the NO concentration increased in all mixed treatment groups except the treatment group mixed at a weight ratio of 1:5 compared to when each substance was treated alone, and a statistically significant synergistic effect occurred at all concentrations. When Angelica gigas Nakai extract fermentation product (FAN) and Aster japonica extract (EAG) were mixed at weight ratios of 2:1, 3:1, 4:1, and 5:1, the NO concentration increased by more than 2-fold (100%) compared to the single treatment group at all concentrations, and the effect was the best when mixed at a weight ratio of 5:1.

<Experimental Example 3>

Expression analysis of iNOS and COX-2 by mixed treatment of Angelica gigas extract and Artemisia capillaris extract

To evaluate the synergistic effect of mixed treatment of Angelica gigas Nakai extract and Aster japonica extract on the expression of iNOS and COX-2, the following experiment was conducted.

Cells treated with 'Angelica gigas Nakai extract (ANE)' obtained in Example 1 and 'Artemisia princeps extract (EAG)' obtained in Example 3, alone or in combination, were used.

After disrupting cells using lysis buffer, proteins were quantified using BCA assay. After loading the quantified proteins, the expression of iNOS and COX-2 was measured by Western blotting.

In Experimental Example 1, the concentration of the mixture with the best NO-elevating effect was 31.3 μg/mL (mixed in weight ratios of 1:5, 1:2, 1:1, 2:1, 3:1, 4:1, and 5:1), and when the Angelica gigas Nakai extract and Aster japonica root extract were treated alone at 31.3 μg/mL, the expression of iNOS and COX-2 was confirmed.

The synergistic effects of iNOS and COX-2 expression when Angelica gigas Nakai extract (ANE) and Aster japonica extract (EAG) were mixed and treated compared to when Angelica gigas Nakai extract (ANE) was treated alone or when Aster japonica extract (EAG) was treated alone. The synergistic effects of iNOS and COX-2 expression compared to Angelica gigas Nakai extract alone or Aster japonica extract alone were expressed as a %. The results are shown in Table 5 and Figures 15 and 16 below.

Mixing ratio
(Angelica: Aster) Angelica extract
Single comparison (%) Artemisia capillaris extract
Single comparison (%) iNOS 1:5 74.9 26.0 1:2 104.4 47.2 1:1 103.7 46.7 2:1 105.7 48.2 3:1 134.2 68.7 4:1 216.7 128.1 5:1 290.2 181.0 COX-2 1:5 41.3 -7.6 1:2 72.8 13.0 1:1 86.4 21.9 2:1 148.2 62.3 3:1 304.3 164.4 4:1 403.5 229.2 5:1 472.1 274.1

When Angelica gigas Nakai extract (ANE) and Aster japonica extract (EAG) were mixed and treated, the expression rates of iNOS and COX-2 increased compared to when each substance was treated alone, resulting in a synergistic effect. When Angelica gigas Nakai extract (ANE) and Aster japonica extract (EAG) were mixed in weight ratios of 4:1 and 5:1, the expression levels increased by more than 2-fold (100%) compared to the single treatment group at all concentrations, and the effect was the best when mixed in a weight ratio of 5:1.

<Experimental Example 4>

Expression analysis of iNOS and COX-2 by mixed treatment of Angelica gigas Nakai extract fermentation and Artemisia capillaris extract

To evaluate the synergistic effect of mixed treatment of Angelica gigas Nakai extract fermentation and Artemisia capillaris extract on the expression of iNOS and COX-2, the following experiment was conducted.

In Experimental Example 1, cells treated with the fermented extract of Angelica gigas Nakai (FAN) of Example 2 and the extract of Aster japonica root (EAG) of Example 3, either alone or in combination, were used.

After disrupting cells using lysis buffer, proteins were quantified using BCA assay. After loading the quantified proteins, the expression of iNOS and COX-2 was measured by Western blotting.

In Experimental Example 2, the concentration of the mixture with the best NO-elevating effect was 31.3 μg/㎖ (mixed in weight ratios of 1:5, 1:2, 1:1, 2:1, 3:1, 4:1, and 5:1), and the expression of iNOS and COX-2 was confirmed when the fermented extract of Angelica gigas Nakai and the extract of Aster japonica root were treated alone at 31.3 μg/㎖.

The synergistic effects of the expression of iNOS and COX-2 when the fermented Angelica gigas Nakai extract (FAN) was treated alone and when the Aster japonica extract (EAG) was treated alone were compared. The synergistic effects of the expression of iNOS and COX-2 when compared to the fermented Angelica gigas Nakai extract (FAN) alone or the Aster japonica extract (EAG) alone were expressed as a %. The results are shown in Table 6 and Figures 17 and 18 below.

Mixing ratio
(Angelica: Aster) Fermented Angelica Extract
Single comparison (%) Artemisia capillaris extract
Single comparison (%) iNOS 1:5 101.6 59.5 1:2 102.8 60.5 1:1 151.5 99.0 2:1 212.0 146.8 3:1 238.0 167.5 4:1 291.3 209.6 5:1 275.2 196.8 COX-2 1:5 26.4 18.5 1:2 51.1 41.7 1:1 86.5 74.8 2:1 243.5 222.1 3:1 328.8 302.0 4:1 414.0 381.9 5:1 472.6 436.8

When Angelica gigas Nakai extract fermentation product (FAN) and Aster japonica extract (EAG) were mixed and treated, the expression rates of iNOS and COX-2 increased compared to when each substance was treated alone, and a synergistic effect occurred at all concentrations. When Angelica gigas Nakai extract fermentation product (FAN) and Aster japonica extract (EAG) were mixed in weight ratios of 2:1, 3:1, 4:1, and 5:1, the expression increased by more than 2-fold (100%) compared to the single treatment group at all concentrations. The iNOS synergistic effect was the best when Angelica gigas Nakai extract fermentation product (FAN) and Aster japonica extract (EAG) were mixed in a weight ratio of 4:1, and the COX-2 synergistic effect was the best when Angelica gigas Nakai extract fermentation product (FAN) and Aster japonica extract (EAG) were mixed in a weight ratio of 5:1.

The composition for enhancing immunity of the present invention described above and the extract of Angelica gigas Nakai and the extract of Aster japonica root included therein are exemplary, and those skilled in the art will readily understand that various modifications and equivalent other embodiments are possible therefrom. Therefore, it will be readily understood that the present invention is not limited to the forms mentioned in the description of the invention above. The true technical protection scope of the present invention should be determined by the technical idea of the claims, and should be understood to include all modifications, equivalents, and substitutes within the spirit of the present invention defined by the claims of the present invention and its scope. In addition, the terms or words used in this specification and claims should not be interpreted as being limited to their usual or dictionary meanings, and should be interpreted as meanings and concepts that conform to the technical idea of the present invention based on the principle that the inventor can appropriately define the concept of the term in order to explain his own invention in the best way.

Claims (13)

A food composition for enhancing immunity, containing as an effective ingredient a mixture of a fermented extract of Angelica gigas Nakai obtained by fermenting a hot water extract of Angelica gigas Nakai root with yeast and an extract of Aster japonica obtained by extracting the aerial parts including leaves and stems of Aster japonica with ethanol in a weight ratio of 2:1 to 5:1. delete delete In the first paragraph,
The above-mentioned extract of Artemisia capillaris,
The step of collecting the above-ground parts of the Aster japonica, washing them, and then cutting or crushing them;
A step of mixing an extraction solvent in an amount 13 to 17 times the weight of the cut or crushed mugwort;
A step of mixing the extractant and the extracting solvent and extracting at 70-80℃ for 5-7 hours;
A step of filtering the extract after extraction;
Step of concentrating the filtrate to 14-16 brix;
A step of adding excipients in an amount of 0.8 to 1.2 times the weight of the solid content to the concentrated filtrate;
A step of sterilizing after adding excipients; and
A food composition for enhancing immunity, characterized in that it is obtained by a method including a step of spray drying after sterilization. delete delete In the first paragraph,
A food composition for enhancing immunity, characterized in that the hot water extract of the above-mentioned Angelica root is obtained by adding water to the Angelica root and extracting it at 110 to 130°C. In the first paragraph,
The hot water extract of the above angelica root is
Step of washing and crushing the angelica root;
A step of adding 8 to 12 times the weight of water to the crushed Angelica gigas and extracting at 110 to 130°C for 50 to 70 minutes;
A step of filtering the extract after extraction; and
A food composition for enhancing immunity, characterized in that it is obtained by a method including a step of spray drying a filtered extract. In the first paragraph,
The above fermented extract of Angelica gigas is
Step of washing and crushing the angelica root;
A step of adding 8 to 12 times the weight of water to the crushed Angelica gigas and extracting at 110 to 130°C for 50 to 70 minutes;
Step of cooling the extract to room temperature after extraction;
A step of adding active dry yeast to the cooled extract at 0.08 to 0.12 wt% of the extract weight and culturing at 20 to 35°C for 15 to 30 hours;
Step of sterilizing the culture medium after culturing;
A step of filtering the sterilized culture solution; and
A food composition for enhancing immunity, characterized in that it is obtained by a method including a step of spray drying a filtered culture solution. In any one of paragraphs 1, 4, 7 to 9,
The above mixture is a food composition for enhancing immunity, characterized in that it mixes the extract fermentation of Angelica gigas and the extract of Artemisia capillaris in a weight ratio of 4:1 to 5:1.

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