KR20120019574A - A method of extracting antioxidants from pine trees - Google Patents

Abstract

PURPOSE: A method for extracting antioxidant materials from pine trees is provided to widely use the pine extract in various industries relating to medicine, functional food, or cosmetic product. CONSTITUTION: A method for extracting antioxidant material from pine trees comprises: a step of adding water to pine needle powder and extracting by hot water extraction at 80°C for 12 hours; a step of removing extract liquid and drying pine needle powder residue; and a step of adding hexane to the residue and performing solvent extraction at 60°C for 12 hours.

Description

A METHOD OF EXTRACTING ANTIOXIDANTS FROM PINE TREES}

The present invention relates to a method for extracting antioxidants from pine, and more particularly, to a method for extracting proanthocyanidins from pine.

With the development of medicine, the average life expectancy has increased and the elderly population has increased. According to the latest statistics released by the National Statistical Office, the proportion of the elderly aged 65 or older accounted for 9.1% of the total population in 2005. It is estimated that by 2020, it will increase to 13.2% of the total population, which is a serious problem due to aging. In addition, Korea's GNP per capita was $ 14,162 in 2004, and Korea's GNP per capita increased by 10% every year. There is a tendency to decrease intake and increase the intake of fat-rich foods such as meat. Due to changes in social conditions, including dietary changes, the pattern of the disease has also changed, and the number of deaths from infectious diseases, which are the major causes of death in the past, is rapidly decreasing. The number of deaths from cardiovascular diseases such as vascular disease or coronary artery disease is steadily increasing.

It is recognized that the causes of diseases such as aging, cancer, cerebrovascular disease, cardiovascular diseases, and the like are free radicals or reactive oxygen species (ROS). The reactive oxygen species may refer to oxygen having high reactivity of oxygen, which is essential for biological maintenance, due to stress such as biological stress caused by germs invading living organisms or various environmental stresses caused by deterioration of the global environment. High oxygen can cause serious physiological disorders. For example, a superoxide radical, a hydroxyl radical, hydrogen peroxide or singlet oxygen.

Specifically, the free radicals or reactive oxygen species destroy cells, cut off the connective tissue of the dermal layer of the skin, or cause cross-linking, so that not only skin-related diseases such as wrinkles, atopic dermatitis, acne or skin cancer, but also cancer, myocardial infarction ( cardiovascular diseases including myocardial infarction, stroke, and atherosclerosis, degenerative neurological diseases such as amyotrophic laternal sclerosis or Parkin's disease (PD) neurodegenerative disease). In addition, free radicals or reactive oxygen species can produce a harmful substance called lipid peroxide by peroxidating lipids in the human body, and the lipid peroxide acts on blood vessels and causes various adult diseases such as arteriosclerosis or blood serum. It is.

As mentioned above, as life expectancy increases and society ages, interest in aging-related diseases such as aging and degenerative diseases is increasing, and interests in metabolic syndrome and adult diseases due to economic growth and dietary changes are increasing. It is increasing. As interest in these diseases increases, interest in free radicals or reactive oxygen species reported as the cause of the diseases increases, and interest in substances having antioxidant effects is increasing.

It is an antioxidant system that removes free radicals or reactive oxygen species in the body, and macromolecular antioxidant enzymes and vitamins such as superoxide dismutase (SOD), peroxidase (POD) or catalase (CAT) Antioxidants such as C, vitamin E, or glutathione.

However, due to pollution, ultraviolet rays or various causes including the biological stress and aging, the antioxidant system of the human body gradually becomes unable to operate normally, and in this case, the active oxygen species can be removed to protect by ingesting antioxidants. There is a need for an in vivo supply of a substance that is capable of antioxidants. In accordance with the demand for supplying such antioxidants in the body, efforts to prevent or treat the disease or delay aging by taking antioxidants or foods having an antioxidant effect and the like are increasing.

Conventionally, synthetic antioxidants having excellent antioxidant power have been reported, such as butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT) or tertiary butylhydropuinone (TBHQ).

However, the synthetic antioxidants are toxic to some of the absorbent substances in the body, can be teratogenic factors and carcinogens, can cause liver hypertrophy or increase the microsomal enzyme activity in the liver, Safety problems such as causing pathological damage to organ tissues such as lungs and kidneys have been raised.

Therefore, in recent years, many studies have been conducted to develop an antioxidant that is safer to the human body and excellent in antioxidant power. As part of this research, many studies are being conducted on natural antioxidants, that is, antioxidants obtained from natural substances.

To date, natural antioxidants, such as tocopherols, flavone derivatives, isoflavones, flavonoids and sesamol (sesamol), gossypol, phenolic acids, phytic acid, saponin, trypsin inhibitor, and the like.

In particular, a component known to be excellent in functionality among the flavonoids is proanthocyanidin. The proanthocyanidins are polyphenolic substances of anthocyanidins, and as shown in FIG. 1, generally, condensation of flavon-3-ols and flavan-3,4-diols into structural units or It has a structure of a multimer by polymerization.

The proanthocyanidins correspond to the secondary metabolites of plants synthesized through the flavonoid biosynthetic pathway, and are present in many kinds of plants and are polymer materials (molecular weights of about 500 to 3,000 Da) that precipitate alkaloids or proteins. The proanthocyanidins have been reported as antioxidants that are recognized for their excellent antioxidant activity, and in addition, antibacterial, antiviral, anticancer, anti-inflammatory, anti-allergic, anti-aging effects or vasodilation have been reported. Activity and anticancer activity are attracting attention. Specifically, in vivo experiments ( in In vivo test, proanthocyanidins have been found to be more active than the vitamin C, vitamin E or beta-carotene free radical scavenging ability, it is reported that the ability to inhibit oxidative tissue damage is also significantly higher. In addition, since it can improve the normal cell toxicity (chemotherapeutic agent) acting on cancer cells in relation to chemotherapy, it has been studied as a candidate for chemotherapeutic agents.

Pine, Pinus densiflora ) is an evergreen coniferous tree that grows widely in forests in Korea, China, Japan and all Asian regions. In East Asian countries including Korea, various parts of pine, such as pine leaves, fruits, cones, cortices, or pollen, have been widely used as food or various health functional ingredients. It has been used as a therapeutic agent in alternative medicine.

Specifically, pine has been reported anti-inflammatory effect, antimicrobial effect, antiviral effect, antidepressant (antidepressant) and triglyceride reducing effect. In addition, pine trees, especially pine bark, are known to contain a large amount of natural antioxidants, particularly flavonoids, which strongly remove free radicals or active oxygen species, and have an excellent anti-aging effect. Is reported to be present. In addition, pine leaves, that is, pine needles, are known to have an excellent anti-oxidant effect, not only to inhibit DNA damage and apoptosis caused by reactive oxygen species, but also to have an anti-hypertension effect. .

The pine needles include essential oils such as flavonoids such as quercetin or kaempferol, α-oinene, β-pinene, and camphene as main active ingredients. It is known that tannins and resins, sugars, carotenes, or vitamin C are contained in large amounts.

Therefore, research is being conducted to develop functional foods having excellent physiological activities such as antioxidant activity from pine needles having an advantage of being easily obtained at low cost.

Proanthocyanidins contained in plants are mainly obtained by extraction. Conventionally, the solvent used for the extraction of proanthocyanidins is mainly an organic solvent. When extracting proanthocyanidins using an organic solvent such as methanol, acetone or ethyl acetate, or mixtures thereof, the use of the extract as a food, medicine or raw material is subject to various restrictions and recovery of the organic solvent. And there was a problem in that a lot of costs such as purification. On the other hand, when extracting the proanthocyanidin from the plant using water, there is no restriction on the use range of the extract, carbohydrates are extracted together, there was a problem that the purity and yield of the proanthocyanidin is lowered.

In order to solve the problems of the prior art as described above, an object of the present invention is to provide a method for extracting high purity antioxidants by a relatively simple process with high efficiency.

In order to achieve the above object, the present invention provides a method for extracting an antioxidant from pine. More specifically, adding water to the pine leaf powder and performing a hydrothermal extraction method; Removing the extract prepared by the hydrothermal extraction, obtaining and drying pine leaf powder residue; And a secondary extraction step of adding hexane to the dried pine leaf powder residue and performing a solvent extraction method.

Hereinafter, the present invention will be described in more detail.

Based on the fact that the extraction yield and purity of a specific substance are different depending on the type of solvent, the present inventors need additional processes such as recovery and purification of an organic solvent, or the purity of proanthocyanidins is too low to purify them with high purity. As a result of studies to solve the problems of the conventional extraction method, which requires additional cost and time due to the additional process, the pine leaf powder residue by the pine hot water extraction process, which is a by-product of the hot water extraction method using water as a solvent, When hexane was added and solvent extraction was performed, it was confirmed that high purity proanthocyanidins can be extracted, thereby completing the present invention.

In the present invention, pine ( Pinus) densiflora ) is an evergreen coniferous tree that grows widely in forests in Korea, China, Japan and other Asian regions, and has the advantage of easily obtaining pine needles at low cost. The pine is an example of pine ( Pinus densiflora Sieb & Zucc.), Pinus thumbergii Parlatore, Pinus densiflora Sieb & Zucc. For. erecta Uyeki), Scots pine (Pinus sylvestris L.), Sol pine (Pinus pinea Cupressus Sempervirens), and preferably red pine.

The present invention relates to a method for extracting antioxidants from pine. More specifically, the present invention relates to a method of extracting proanthocyanidins from pine with high purity.

More specifically, the present invention comprises the steps of adding water to the pine leaf powder and performing a hot water extraction method; Removing the extract prepared by the hydrothermal extraction, obtaining and drying pine leaf powder residue; And a secondary extraction step of adding hexane to the dried pine leaf powder residue and performing a solvent extraction method.

The antioxidant may be a substance having an antioxidant activity, specifically, a polyphenolic compound, preferably proanthocyanidin.

Adding water to the pine leaf powder and performing the hot water extraction method is to increase the extraction yield, while minimizing the destruction of the active ingredient, 60 ℃ to 100 ℃, preferably 70 ℃ to 90 ℃, more preferably It may be carried out at 75 ℃ to 85 ℃, most preferably at 80 ℃.

In extracting antioxidants from pine leaves, pine leaf powder may be used to increase the extraction area by maximally increasing the contact area between pine leaves and the extraction solvent.

The method for preparing the pine leaf powder includes the steps of washing the pine leaf; It may be carried out by a method comprising the step of drying the washed pine leaves and the process of powdering the dried pine leaves.

The process of pulverizing the pine leaves is to increase the extraction yield for the antioxidant material, while maintaining the antioxidant activity of the extract obtained in the final step to maximize the extraction yield, the pine leaves are 50 to 70 ℃, preferably Preferably it may be carried out by hot air drying at 60 ℃ and a method of crushing. More specifically, the process of drying the pine leaves may be carried out by a hot air drying method at 60 ℃ conditions, the powdering process may be performed using a mixer or a pulverizer, the powdering process is a mixer or pulverizer After grinding the dried pine leaves using may be carried out by the method of homogenization using a sieve.

In this aspect, the method may further comprise the step of powdering pine leaves.

More specifically, the invention Pinus ( Pinus) densiflora ) preparing leaf powder; Adding water to the pine leaf powder and performing hot water extraction; Removing the extract prepared by the hydrothermal extraction, obtaining and drying pine leaf powder residue; And it may be a method of extracting the antioxidant from the pine including a secondary extraction step of adding hexane to the dried pine leaf powder residue and performing a solvent extraction method.

Performing the hot water extraction method is more specifically, hot water by adding 750 ml to 1,850 ml, preferably 1,000 ml to 1,600 ml, and more preferably 1,200 ml to 1,400 ml of hydrothermal water based on 1 kg of the pine leaf powder. This can be done by performing extraction. The temperature condition of the hydrothermal extraction method may be 60 ℃ to 100 ℃, preferably 70 ℃ to 90 ℃, more preferably 75 ℃ to 85 ℃, most preferably 80 ℃, the duration of the hydrothermal extraction process is 4 It may be from hours to 24 hours, preferably 6 hours to 20 hours, more preferably 8 hours to 16 hours, most preferably 12 hours.

The step of obtaining and drying the pine leaf powder residue is performed after the hydrothermal extraction method, removing the liquid including the pine hydrothermal extract, obtaining the remaining pine leaf powder residue and drying the obtained residue. It can be carried out in a method including a process to.

The process of removing the liquid including the pine hot water extract may be performed by filtering the resultant of the hot water extraction method to remove the liquid and obtaining a solid, ie, pine leaf powder residue. The filtration process may be performed by, for example, a filtration method using filter paper.

The pine leaf powder residue obtained by the above process is generally performed after the extraction process to improve the purity or product quality in the pine hot water extract manufacturing process, and then further performing a filtration process, and by-products removed in the process Although accepted as the same, in the present invention, since the pine leaf powder residue obtained in the hydrothermal extraction process further produces a material having an antimicrobial activity and an antioxidant activity, that is, an extract, in terms of resource saving and environmental protection Its economic and environmental effects will be greater.

Drying the obtained residue may be carried out through a conventional powder drying method, for example, a drying method using a dryer.

The secondary extraction step may be performed by adding hexane to the dried pine leaf powder residue and solvent extraction.

In detail, the solvent extraction method is 40 ℃ to 80 ℃, preferably 50 ℃ to 70 ℃, more preferably in order to minimize the destruction of the active ingredient while increasing the extraction yield in consideration of the characteristics of the solvent and the stability of the active ingredient. Preferably from 55 ° C to 65 ° C, most preferably 60 ° C. More specifically, the secondary extraction step is based on 1 kg of pine leaf powder used before the hot water extraction, 750 ml to 1,850 ml of hexane, preferably 1,000 ml to 1,600 ml, more preferably 1,200 ml to 1,400 ml It can be carried out by the method of performing a solvent extraction method by adding. The temperature condition of the solvent extraction method using the hexane is 40 ℃ to 80 ℃, preferably 50 ℃ to 70 ℃, more preferably 55 ℃ to 65 ℃, most preferably 60 ℃, the duration of the hydrothermal extraction process is 4 It may be from hours to 24 hours, preferably 6 hours to 20 hours, more preferably 8 hours to 16 hours, most preferably 12 hours.

The antioxidant extracted by the secondary extraction process may be additionally subjected to a filtration process using filter paper, a concentration process using a reduced pressure concentration method, or a drying process using a freeze-drying method, etc., according to the use condition and method. Can be.

As described above, the extraction method of the present invention by performing only the solvent extraction process, the manufacturing method is simple, the cost required for the manufacturing process is low, and the treatment such as disposal as a by-product in the conventional pine hydrothermal extract manufacturing process By reusing the residue, not only the environmental and economic aspects are excellent, but also the extract prepared by the extraction method of the present invention is a substance having antioxidant activity prepared by the conventional solvent extraction method, specifically proanthocyanidins The purity of the remarkably high, the antioxidant activity is also very good.

Therefore, since the substance having antioxidant activity is very excellent in antioxidant activity, it is a cause of skin aging such as cell destruction and wrinkle formation by cutting of connective tissue of dermal dermal layer, causing cardiovascular disease, degenerative neurological disease or various adult diseases. Free radicals and free radical species can be removed, and since they are derived from natural products, they are not only safe, but also can suppress immunomodulatory activity, in particular, excessive generation of NO due to side reactions, so that antioxidants can be used. It is expected that the present invention can be applied in various ways as additives for functional foods, functional cosmetics and medicines, and foods, cosmetics or medicines requiring long-term preservation.

Compared to the conventional method, the present invention is a method capable of obtaining a material having excellent antioxidant ability, in particular, proanthocyanidins with a significantly improved high purity, and is a natural antioxidant proanthocyanidin that is safe for human body and has excellent antioxidant power. Can be obtained from low-cost pine trees distributed throughout Korea with excellent efficiency, and the process is simple and simple by using residues that have been treated as by-products in the process of extract preparation using the existing hot water extraction method. Since it is an extraction method, it is not only economically effective but also environmentally friendly, and it is an antioxidant that can inhibit various diseases or aging associated with free radicals or free radicals, specifically health functional foods and medicines related to the use of proanthocyanidins. Or foods requiring active ingredient and long-term preservation of cosmetics, It can be widely applied in various industries such as additives of cosmetics or pharmaceuticals.

1 is a flow chart showing a process for preparing an extract of the present invention.
Figure 2 is a graph showing the DPPH radical scavenging ability of the extract according to an embodiment of the present invention. In Figure 2, BHT means butylated hydroxytoluene, Proanthocyanidin means 96% grape seed proanthocyanidin.
Figure 3 is a graph showing the results of performing a FRAP assay (Ferric-reducing antioxidant power) of the extract according to an embodiment of the present invention. In Figure 3, BHT means butylated hydroxytoluene, Proanthocyanidin means 96% grape seed proanthocyanidin.
Figure 4 is a graph showing the measurement of the ferrous ion chelating activity (ferrous ion chelating activity) of the extract according to an embodiment of the present invention.
5 is a comparison of the immune control activity (immunomodulatory effect) through the inhibitory effect of LPS-induced NO production (100%) of the extract according to an embodiment of the present invention (100%) A graph showing the results.

Hereinafter, the preparation examples and examples are presented in order to explain the present invention in more detail. However, the following Preparation Examples and Examples are merely illustrated to aid the understanding of the present invention, and may be modified in various other forms, and the scope of the present invention is not limited to the following Examples.

Example  1: Preparation of Extract

The pine leaves used in this experiment were red pine leaves collected from Jirisan in July 2007. The red pine leaves were washed with water, hot-air dried at 60 ° C., and then powdered with a mixer.

Hot water hexane extract (Hot water-hexane, HWH) and Comparative Examples 1 to 4 of the present invention were prepared by the method described in FIG.

More specifically, the red pine leaf hydrothermal extract of Comparative Example 1 was prepared by adding 4,000 ml of water to 3 kg of the red pine leaf powder and standing by extracting at 80 ° C. for 12 hours. The hexane extract of Comparative Example 2 was prepared by adding 4,000 ml of hexane to 3 kg of the red pine leaf powder and standing by extracting at 60 ° C. for 12 hours. The ethanol extract of Comparative Example 3 was prepared by adding 4,000 ml of ethanol to 3 kg of the red pine leaf powder and standing at 80 ° C. for 12 hours for extraction. Hot water ethanol extract (Comparative Example 4) (HWE) is added to 4,000 ml of water to 3 kg of the red pine leaf powder, extracted for 12 hours at 80 ℃ and filtered, to remove the liquid containing the extract and remaining After collecting water and drying, it was further prepared by adding 4,000 ml of ethanol and performing solvent extraction for 12 hours at 80 ℃.

In the hydrothermal hexane extract of the experimental example of the present invention, 4,000 ml of water was added to 3 kg of the red pine leaf powder, and extracted at 80 ° C. for 12 hours, followed by filtration, to remove the liquid including the extract solution and to collect and dry the remaining residue. Further, 4,000 ml of hexane was added and prepared by performing solvent extraction at 60 ° C. for 12 hours.

Each of the extracts was filtered using a filter paper (Whatman No 3, England), and then concentrated under reduced pressure at 60 ℃ using a rotary evaporator (freeze-dried). Each of the lyophilized extracts was stored at −20 ° C. until the experiment was performed, including the measurement of antioxidant activity.

Example  2: determination of the concentration of antioxidants

The total polyphenolic compounds (TPC) concentrations of the extracts of Examples and Comparative Examples were colorimetrically determined by the Folin-Ciocalteu method. After diluting each extract with 99% methanol solution, 300 μl of diluted sample and 1.5 ml of 0.25 N Folin Ciocalteau's reagent (10 x dilutions, Sigma, USA) and 1.2 ml of sodium carbonate (7.5% w / v, Fisher Scientific, UK) Were mixed and reacted at room temperature for 30 minutes. After the reaction, the absorbance was measured at 765 nm. Total polyphenolic component was calculated as the concentration of total polyphenolic component (mg) per 100 g of sample using gallic acid (MP biomedicals LLC, USA) as a standard.

In addition, the concentration of proanthocyanidins was measured by the method of Brand-Williams. More specifically, 0.1 ml of each extract was diluted with 0.9 ml of 99% methanol solution, followed by 1 ml of diluted sample and 6 ml of 5% (v / v) HCl (in butanol) solution and 2% (w / v) FeNH ₄ 0.2 ml of (SO ₄ ) ₂ H ₂ O (in 2M HCl) solution was mixed, reacted at 95 ° C. for 40 minutes, and cooled to room temperature.

After the reaction, the mixture was centrifuged at 14,000 xg for 5 minutes, the supernatant was separated to remove insoluble components, diluted 10 times with 5% (v / v) HCl (in butanol) solution, and then 550. Absorbance was measured immediately at nm. Determination of total proanthocyanidin content was calculated using a concentration of 96% grape seed proanthocyanidin (Naturex, France) as standard.

The measured total polyphenolic component and total proanthocyanidin content measurement results and the calculated results are shown in Table 1 below.

Total Polyphenol Concentration (mg / g extract) Proanthocyanidin Content (mg / g extract) Purity of Proanthocyanidins (%) relative to the total polyphenols Experimental Example 199.5 ± 0.4 186.3 ± 0.3 93.8 ± 0.3 Comparative Example 1 124.4 ± 0.3 4.5 ± 0.06 3.6 ± 0.03 Comparative Example 2 143.6 ± 0.3 128.6 ± 0.4 93.2 ± 0.2 Comparative Example 3 810.0 ± 0.6 360.0 ± 0.5 44.4 ± 0.1 Comparative Example 4 88.3 ± 0.1 54.2 ± 0.1 61.4 ± 0.2

As shown in Table 1 above, in relation to the purity of proanthocyanidins relative to the total polyphenol components, the hydrothermal hexane extract as an experimental example was the best, whereas in the case of Comparative Example 1 in which the hydrothermal extraction method was performed, proanthocyanidins Purity was found to be the worst.

In addition, in the case of Comparative Example 4 using the residue subjected to the hydrothermal extract method, that is, in the case of the hydrothermal ethanol extract, the purity of proanthocyanidin was 61.4%, which was about 30% lower than that of the experimental example. In addition, in the case of Comparative Example 3 using ethanol as a solvent, the purity was 44.4%, which was about 50% of the experimental example.

Therefore, the experimental example was found to be most suitable in that proanthocyanidin can be extracted with high purity.

Example  3: antioxidant activity of extract Measurement 1 ( DPPH Electronic by law Donation ability  Research)

In relation to the antioxidant activity of the extract prepared in Example 1, free radical scavenging was measured by the DPPH method applying the Brand-Williams method.

More specifically, each extract was diluted in 99% methanol at various concentrations (final concentration: 10 μg / ml, 50 μg / ml, 100 μg / ml, 250 μg / ml and 500 μg / ml). .

The electron donating ability of red pine leaf extract was measured using 1,1-diphenyl1-2-picrylhydrayl (DPPH, Sigma). 1 ml of each extract prepared in Example 1 and 2 ml of DPPH solution (5.9 mg / 100 ml in methanol) were mixed, followed by incubation for 30 minutes at room temperature and light shielding conditions. After the reaction, absorbance was measured at 517 nm. The DPPH free radical scavenging ability was calculated by the following Equation 1. In Formula 1, A ₀ means absorbance of a control before reacting with the extract, and A ₁ means absorbance measured after reacting with the extract. The measured absorbance is shown in Figure 2 and Table 2 below.

[Calculation 1]

DPPH free radical scavenging capacity (%) = [(A ₀ -A ₁ ) / A ₀ ] X 100

Extract Type Antioxidant activity (IC ₅₀ , μg / ml) Experimental Example (HWH) 53 ± 0.49 Comparative Example 1 (Hot water) 102 ± 0.96 Comparative Example 2 (Hexane) 82 ± 0.73 Comparative Example 3 (Ethanol) 120 ± 1.37 Comparative Example 4 (HWE) 140 ± 0.62

As shown in FIG. 2, the hydrothermal hexane extract of Experimental Example was found to have the best antioxidant activity. More specifically, it was confirmed that the antioxidant activity was increased in a concentration-dependent range in the concentration range of 10 μg / ml to 250 μg / ml, as shown in Table 2, it was confirmed that the best at 53 μg / ml even in IC ₅₀ . On the other hand, in Comparative Example 4 (HWE), which was found to be excellent antibacterial activity among the comparative examples, the antioxidant activity was confirmed to be the worst. In addition, the hydrothermal extract (Comparative Example 1) and ethanol extract (Comparative Example 3) were found to be over 100 μg / ml even in IC ₅₀ , and the hydrothermal hexane extract of the experimental example of the present invention was applied to extracts prepared by other extraction methods. In comparison, the antioxidant activity was remarkably excellent.

Example  4: antioxidant activity of extract Measurement 2 ( FRAP assay Investigation by

In order to measure the antioxidant activity of the extract, FRAP assay was performed by applying the method of Benzie and Strain. The FRAP assay measures the degree of reduction of ferric oxide (Fe ^{3 +} ) to ferric oxide (Fe ^{2 +} ) by the antioxidant, to determine the antioxidant capacity of the antioxidant.

More specifically, after diluting each extract to various concentrations (final concentration: 10 μg / ml, 50 μg / ml, 100 μg / ml, 250 μg / ml and 500 μg / ml) in 99% methanol, diluted sample 1 2.5 ml of 0.2 M potassium phosphate buffer (pH 6.6) and 2.5 ml of 1% (w / v) potassium ferricyanide (MP biomedicals LLC, USA) were added and mixed. The mixed solution was reacted at 50 ° C. for 20 minutes and then cooled to room temperature. Thereafter, 2.5 ml of 10% trichloroacetic acid (Junsei Kagaku, Japan) was added, 2.5 ml of the sample was separated, and mixed with 2.5 ml of secondary distilled water (ddH ₂ O). 0.5 ml of 0.1% (w / v) FeCl ₃ was added to the mixed sample of distilled water, and reacted for 30 minutes. After the reaction, the absorbance was measured at 700 nm. Vitamin C (L-(+)-ascorbic acid, Sigma, USA), gallic acid, proanthocyanidin (PA), and BHT (butylated hydroxytoluene, Sigma, USA) were identified as comparative groups. It was. The measurement results are shown in FIG. 3.

As shown in FIG. 3, the hydrothermal hexane extract of Experimental Example was found to have the highest antioxidant activity with the highest reducing power. More specifically, the hydrothermal hexane extract of the experimental example was confirmed to be superior to proanthocyanidins and BHT among the comparative group known to have excellent antioxidant activity. On the other hand, compared to 500 μg / ml of iron oxide reduction capacity of Comparative Example 3, the ethanol extract was found to have the highest content of proanthocyanidins in the comparative example, it was confirmed that the iron oxide reduction capacity of 250 μg / ml of the experimental example is better It became. From the above results, it was confirmed that the antioxidant capacity of the proanthocyanidins produced by the hydrothermal hexane extraction method of the present invention is superior to the antioxidant capacity produced by other extraction methods.

Example  5: Antioxidant Activity of Extracts Measurement 3 ( FIC assay Investigation by

In order to measure the antioxidant activity of the extract, Dinis' method was applied to confirm the metal chelating activity (Metal chelating activity). Chelating agents are known to be effective as secondary antioxidants in terms of reducing reducing power and stabilizing the oxidized form of metal ions. Ferric oxide (Fe ^{2 +)} is Pantone reaction (Fenton reaction) the through hydroxyl radicals (hydroxyl radical, OH ^-) can be maintained and the generation, spread hydroxyl radical (peroxyl radical) and know the lipid (lipid hydroperoxide) peroxide By decomposing into an alkoxyl radical, it is possible to increase the lipid peroxidation by initiating a chain reaction of lipid peroxidation. Thus, by the iron chelating ability to view and compare ferric oxide (Fe ^{+ 2)} Ferrozine capable of forming a complex with (complexe), it is possible to measure the antioxidant activity as the secondary antioxidant.

The experiment was specifically carried out in the following manner. First, FeSO ₄ (2 mM, Sigma, USA) and Ferrozine (5 mM) were diluted 20-fold, and each extract was diluted in 99% methanol at various concentrations (final concentration: 10 μg / ml, 50 μg / ml, 100 μg /). ml, 250 μg / ml and 500 μg / ml), and then mixed with 1 ml of diluted sample, 1 ml of FeSO _{4 and} 1 ml of diluted Ferrozine. The mixed solution was reacted at room temperature (15 ° C. to 20 ° C.) for 10 minutes. After the reaction, the absorbance was measured at 562 nm. Chelating of iron ions was calculated by the following formula (2). In Formula 2, A ₀ means absorbance of the control before reacting with the extract, and A ₁ means absorbance measured after reacting with the extract. As a comparison group, vitamin C (ascorbic acid), gallic acid (gallic acid), proanthocyanidin (PA), and BHT, which were identified as antioxidant, were used. The measured and calculated results are shown in FIG. 4.

[Equation 2]

Chelating effect (%) = [1- (A ₁ / A ₀ )] X100

As shown in FIG. 4, the hydrothermal hexane extract of Experimental Example was found to have remarkably superior chelating ability (FIC activity) compared to Comparative Example. More specifically, as confirmed in FIG. 4, the chelating ability is more excellent in the range of 50 μg / ml and 100 μg / ml, and the hydrothermal hexane extract of Experimental Example has further antioxidant activity as a secondary antioxidant in other extracts. It was confirmed to be excellent. In addition, the hydrothermal hexane extract of the experimental example was confirmed to be superior to BHT among the comparative group known to have excellent antioxidant activity, and confirmed the antioxidant activity similar to vitamin C (ascorbic acid) and gallic acid (gallic acid). On the other hand, the hydrothermal ethanol extract of Example 4 undergoing an extraction process similar to the example of the comparative example was found to have the lowest chelating ability, the lowest antioxidant capacity.

From the above results, it was confirmed that the antioxidant material extracted by the hydrothermal hexane extraction method of the present invention is significantly superior to the antioxidant material produced by other conventional extraction methods.

Example  6: immunomodulatory activity of extracts Immunomodulatory activity ) Measure

In order to confirm the immunomodulatory activity of the extract, it was confirmed that the NO production control ability. When stimulated in the macrophage (macrophage) and the immune response proceeds, NO is produced. By applying this, the immunomodulatory activity of the extract was confirmed.

More specifically, the immunomodulatory activity was measured by checking the degree of NO production as follows. First, RAW 264.7 cells sold from Korea Cell Line Bank were prepared with RPMI-1640 medium (Bio Whittaker, USA) supplemented with 10% FBS (fetal bovine serum, Bio Whittaker, USA) and 100 U / ml penicillin-streptomycin (Bio Whittaker, USA). USA) was incubated in wet air at 37 ° C. and 5% CO ₂ . Dispense the cultured cells into plates (96-well plates) (2 X 10 ⁵ cells per well), 10% FBS (fetal bovine serum, Bio Whittaker, USA) and 100 U / ml penicillin-streptomycin (Bio). Whittaker, USA) was incubated for 18 hours using RPMI-1640 medium added. After the incubation, the extract was diluted with 1 μg / ml LPS (lipopolysaccharide, Sigma, USA) and 99% methanol at various concentrations (final concentration: 10 μg / ml, 50 μg / ml, 100 μg / ml, 250 μg / ml). And 500 μg / ml) were added. After incubation for an additional 24 hours, NO secretion was measured.

The NO secretion was measured by measuring the concentration of nitrite accumulated in the culture. Specifically, 150 μl of the culture solution and 50 μl of Griess reagent (1% sulfanilamide, 0.1% naphthylethylene diamine in 2.5% phosphoric acid solution) were mixed and reacted at room temperature in a plate (96-well microplate) for 10 minutes. After the reaction, the absorbance was measured at 540 nm using an ELISA microplate reader. NaNO ₂ was used as a standard. To determine the background level for nitrite, the absorbance on the cell-free medium (cell-free RPMI-1640) was measured. The measured absorbance is shown in FIG. 5.

As shown in FIG. 5, it was confirmed that the hydrothermal hexane extract of Experimental Example was able to most effectively inhibit NO production induced by LPS as compared to other extracts. More specifically, in the case of the hydrothermal hexane extract of the experimental example, the addition of 250 μg / ml can reduce NO production by 38%, and the addition of 500 μg / ml can reduce NO production by 52%. On the other hand, in the case of other extracts, even when added to 500 μg / ml was confirmed that can suppress about 30% of NO production. In addition, in the case of the hydrothermal hexane extract of the experimental example, the NO production inhibitory ability was significantly increased with increasing concentration, while in other extracts, it was confirmed that the NO production inhibitory ability was gradually increased with increasing concentration. In addition, compared with the hydrothermal hexane extract of the experimental example, the total polyphenol content is about 72%, the proanthocyanidin content is about 69%, and the hexane extract of Comparative Example 2, which is similar to the experimental example, is related to the NO production inhibitory ability. By adding 500 μg / ml of the hexane extract of Comparative Example 2, 100 μg / ml of the hydrothermal hexane extract of Experimental Example was found to be more excellent in NO generation ability, and the extract produced by the hydrothermal hexane extraction method of the present invention. The immunomodulatory activity of was confirmed to be superior to the immunomodulatory activity of extracts produced by other conventional extraction methods.

As described above, in the hot water hexane extraction method of the present invention, as a result of performing the existing hot water extraction method, it is economical and environmentally friendly because it does not use a special machine or process while using a residue which has been treated as a by-product. Proanthocyanidins can be produced with significantly higher purity than other extraction methods, and the proanthocyanidins obtained by the hydrothermal hexane extraction method of the present invention are the DPPH method than the extract extracted by other extraction methods or proanthocyanidins. In addition, the antioxidant activity measured by the FRAP assay and the FIC assay was excellent as well as the immunomodulatory activity measured by the NO assay. Therefore, the method of the present invention is a method that can efficiently obtain a material excellent in antioxidant activity and immunomodulatory activity using pine leaves obtained from pines distributed throughout the country, the development of functional food using pine needles It is expected to be used in various industrial fields, such as the development of food materials and cosmetic materials.

Claims (3)

Pine tree ( Pinus densiflora ) adding water to the leaf powder and performing a hydrothermal extraction method;
Removing the extract prepared by the hydrothermal extraction, obtaining and drying pine leaf powder residue; And
Second extraction step of adding hexane to the dried pine leaf powder residue and performing a solvent extraction method
Extracting antioxidants from the pine tree comprising a. The method of claim 1,
Wherein said antioxidant is proanthocyanidins. The method of claim 1,
The performing of the hot water extraction method is characterized in that for performing the hot water extraction method for 12 hours under the conditions of 80 ℃,
The second extraction step is characterized in that hexane is added to the extract dried pine leaf powder residue and the solvent extraction method is carried out for 12 hours at 60 ℃ condition
How to extract antioxidants.

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