KR100394577B1 - Crude extracts from Schizandra chinensis RUPRECHT seed and process for preparation thereof - Google Patents

Crude extracts from Schizandra chinensis RUPRECHT seed and process for preparation thereof Download PDF

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KR100394577B1
KR100394577B1 KR10-2000-0067978A KR20000067978A KR100394577B1 KR 100394577 B1 KR100394577 B1 KR 100394577B1 KR 20000067978 A KR20000067978 A KR 20000067978A KR 100394577 B1 KR100394577 B1 KR 100394577B1
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extract
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schisandra chinensis
methanol
kccm
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KR20020037968A (en
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정기태
주인옥
최정식
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전라북도(농업기술원)
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    • AHUMAN NECESSITIES
    • A23FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23LFOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L3/00Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs
    • A23L3/34Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals
    • A23L3/3454Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals in the form of liquids or solids
    • A23L3/3463Organic compounds; Microorganisms; Enzymes
    • A23L3/3472Compounds of undetermined constitution obtained from animals or plants
    • AHUMAN NECESSITIES
    • A23FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23LFOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L3/00Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs
    • A23L3/40Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by drying or kilning; Subsequent reconstitution
    • A23L3/44Freeze-drying
    • AHUMAN NECESSITIES
    • A23FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23VINDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2200/00Function of food ingredients
    • A23V2200/10Preserving against microbes
    • AHUMAN NECESSITIES
    • A23FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23VINDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2300/00Processes
    • A23V2300/14Extraction

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  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Botany (AREA)
  • Microbiology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
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  • Health & Medical Sciences (AREA)
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  • Food Science & Technology (AREA)
  • Polymers & Plastics (AREA)
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Abstract

본 발명은 오미자 종자 기능성 추출물 및 그 제조방법에 관한 것으로 다양한 유기용매로 추출한 오미자 종자 추출물과 그 분획물들은 돈지와 대두유에 대해 높은 항산화력을 나타내고 DPPH 자유 라디칼 소거 활성이 우수하며 식품 부패 미생물에 대해서는 항균활성을 나타내는 뛰어난 효과가 있고 또 높은 아질산염 소거능을 나타내는 뛰어난 효과가 있다.The present invention relates to Schisandra chinensis seed functional extract and its manufacturing method. Schisandra chinensis seed extract and its fractions extracted with various organic solvents show high antioxidant activity against lard and soybean oil, and have excellent DPPH free radical scavenging activity and antimicrobial against food decay microorganisms. It has an excellent effect of showing activity and an excellent effect of showing high nitrite scavenging ability.

Description

오미자 종자 기능성 추출물 및 그 제조방법{Crude extracts from Schizandra chinensis RUPRECHT seed and process for preparation thereof}Schizandra chinensis RUPRECHT seed and process for preparation according to Schizandra chinensis extract

본 발명은 오미자 종자 기능성 추출물 및 그 제조방법에 관한 것이다. 더욱 상세하게는, 본 발명은 오미자 종자로부터 유기용매를 사용하여 추출한 항산화성, 항균성 및 아질산염 소거능이 있는 오미자 종자 추출물 및 그 제조방법에 관한 것이다.The present invention relates to Schisandra chinensis seed functional extract and a method for producing the same. More specifically, the present invention relates to a schizandra seed extract with antioxidant, antimicrobial and nitrite scavenging ability extracted using an organic solvent from Schizandra seeds and a method for producing the same.

오미자(Schizandra chinensisRUPRECHT)는 목련과(Magnoliaceae) 식물로 6 ∼ 7월에 꽃이 피어 열매는 9 ∼10월에 성숙하여 심홍색을 띈다. 오미자는 오대산, 지리산, 발왕산 지역에서 군락을 이루어 자생하고, 강원도의 화천, 인제, 평창, 경상북도의 봉화, 전라북도 무주, 진안, 장수 및 경상남도의 함양 등지에서 재배되고 있으며 1998년도 재배면적은 260 ha이고 생산량은 141 M/T이다. Schizandra chinensis RUPRECHT is a Magnoliaceae plant that blossoms in June-July and matures in September-October and becomes magenta. Omija grows in colonies in Odaesan, Jirisan, and Balwangsan areas, and is cultivated in Hwacheon, Inje, Pyeongchang, Gyeongsangbuk-do, Bonghwa, Jeollabuk-do, Muju, Jinan, Jangsu, and Gyeongsangnam-do. The yield is 141 M / T.

오미자는 예로부터 한방에서 전신쇠약, 정신 육체적 피로, 기관지염, 기관지천식, 신경쇠약, 저혈압, 심장기능저하, 영양실조궤양과 상처의 치료 및 시력을 증진시키는데 이용되며, 차 또는 하절기의 화채재료 및 그 색소를 이용한 녹말다식과 오미자주로 가공, 이용되고 있다.Schisandra chinensis has been used in traditional medicine to improve systemic weakness, mental and physical fatigue, bronchitis, bronchial asthma, nervous breakdown, hypotension, cardiac dysfunction, malnutrition ulcers and wounds, and to improve visual acuity in tea or summer. It is processed and used mainly in starch foods and Schisandra chinensis using pigments.

오미자 성분에 관한 연구는 김 등이 오미자의 일반성분, 유기산 및 안토시아닌(anthocyanin) 색소 등에 대하여 보고하였으며[Kim, K.I., Nam, J.H. and Kwon, T.W. On the proximate composition, organic acid and anthocyanins of Omija,Schizandra chinensisBaillon. Korean J. Food Sci. Technol. 5: 178-182 (1973)], 양 등은 안토시아닌 색소의 안정성에 대하여 보고하였다[Yang, H.C., Lee, J.M. and Song, K.B. Anthocyanins in cultured Omija(Schizandra chinensisBaillon) and its stabililty. Korean Soc. Agri. Chem. Biotechnol. 25: 35-43 (1982)]. 또한 이 등은 오미자의 부위별 유리당, 지질 및 비휘발성 유기산 조성에 대한 일련의 연구를 수행하였다[Lee, J.S. and Lee, S.W. The studies of composition of free sugar, fatty acid and nonvolatilitic organic acid in part of Omija(Schizandra chinensisBaillon). Korean J. Dietary culture 4: 177-181 (1989)]. 오미자의 약리학적 작용에 관한 연구로서 Hikino 등이 간장보호 작용에 대하여 보고하였으며[Hikino, H., Kios, Y., Takuchi, H. and Ikeya, Y. Validity of the oriental medicines 60. Liver-protective drugs. II Antihepatotoxicaction of lignoids fromS. chinensisfruits. Planta Med. 50: 213-216 (1984)] 이 등은 알콜 해독작용[Lee, J.S. and Lee, S.W. Effect of water extract in fruits of Omija(Schizandra chinensisBaillon) on alcohol metabolism. Korean J. Dietary culture 5: 259-262 (1990)]에 대하여 서 등은 항 당뇨 작용에 대하여 보고하였다[Sheo, H.J., Lee, M.Y. and Hwang, J.S. Effect ofSchizandrae fructusextract on blood constituents of alloxan induced diabetic rabbits. J. Korean Soc. Food Sci. Nutri. 16: 262-268 (1987)]. 오미자 가공 연구는 반응표면 방법에 의한 오미자 음료 제조에 대한 강 등의 연구[Kang, K.C., Park, J. H., Beak, S. B., Jhin, H.S. and Rhee, K.S. Optimization of beverage preparation formSchizandra chinensisBaillon by response surface methodlogy. Korean J. Food Sci. Technol. 24: 74-81 (1992)]와 건조 오미자 추출 과실즙을Saccharomyces cerevisiae ellipsoideusSaccharomyces coreanus으로 발효시켜 품질 특성을 검토한 장의 연구[Chang, E.J. Studies on the production of Omija Wine. M.S. Thesis, Korea Univ., Korea (1985)]와 정 등의 오미자 건조와 저장에 관한 연구가 보고되었다[Jung, G. T., Ju, I.O. and choi, J.S. Studies on drying and preservation of Omija(Schizandra chinensis). Korean Postharvest Sci. Technol. 5: 217-223 (1998)]. 오미자의 항균성에 대하여는 이 등의 병원성미생물[Lee, S.H. and Lim, Y.S. Antimicrobial effects ofSchizandra chinesisextract on pathogenic microorganism. J. Korean Soc. Food Sci. Nutri. 27: 239-243 (1998)], 김치에서 분리한 유산균[Lee, S.H. and Lim, Y.S. Effect ofOmija(Schizandra chinesis) extract on the growth of lactic acid bacteria isolated from Kimchi. Korean J. applied Microbiol. Biotechnol. 25: 224-228 (1997)] 그리고Listeria monocytogenes에 대한 항균 효과가 보고[Lee, S.H. and Lim, Y.S. Antimicrobial effects ofSchizandra chinesisextract againstListeria monocytogenes. Korean J. applied Microbiol. Biotechnol. 25: 442-447 (1997)]되었을 뿐 오미자 종자에 대한 항산화, 항균 및 아질산염 소거 등의 생리활성에 관한 연구는 아직 미비한 실정이다.In the study of Schizandra chinensis, Kim et al. Reported on Schizandra chinensis, organic acid and anthocyanin pigment [Kim, K.I., Nam, J.H. and Kwon, T.W. On the proximate composition, organic acid and anthocyanins of Omija,Schizandra chinensisBaillon. Korean J. Food Sci. Technol. 5: 178-182 (1973)], Yang et al. Reported the stability of anthocyanin pigments [Yang, H.C., Lee, J.M. and Song, K.B. Anthocyanins in cultured OmijaSchizandra chinensisBaillon) and its stabililty. Korean Soc. Agri. Chem. Biotechnol. 25: 35-43 (1982). In addition, Lee et al. Conducted a series of studies on the composition of free sugars, lipids and nonvolatile organic acids by sites of Schizandra chinensis [Lee, J.S. and Lee, S.W. The studies of composition of free sugar, fatty acid and nonvolatilitic organic acid in part of Omija (Schizandra chinensisBaillon). Korean J. Dietary culture 4: 177-181 (1989). As a study on the pharmacological action of Schizandra chinensis, Hikino et al reported on hepatoprotective action [Hikino, H., Kios, Y., Takuchi, H. and Ikeya, Y. Validity of the oriental medicines 60. Liver-protective drugs . II Antihepatotoxicaction of lignoids fromS. chinensisfruits. Planta Med. 50: 213-216 (1984)] and the like have been described in alcohol detoxification [Lee, J.S. and Lee, S.W. Effect of water extract in fruits of Omija (Schizandra chinensisBaillon) on alcohol metabolism. Korean J. Dietary culture 5: 259-262 (1990)] reported on antidiabetic activity [Sheo, H.J., Lee, M.Y. and Hwang, J.S. Effect ofSchizandrae fructusextract on blood constituents of alloxan induced diabetic rabbits. J. Korean Soc. Food Sci. Nutri. 16: 262-268 (1987). Schisandra chinensis research has been conducted by Kang et al. [Kang, K.C., Park, J. H., Beak, S. B., Jhin, H.S. and Rhee, K.S. Optimization of beverage preparation formSchizandra chinensisBaillon by response surface methodlogy. Korean J. Food Sci. Technol. 24: 74-81 (1992)] and dried Schisandra chinensis fruit juiceSaccharomyces cerevisiae ellipsoideusWowSaccharomyces coreanusA study on the quality of fermented rice by fermentation [Chang, E.J. Studies on the production of Omija Wine. M.S. Thesis, Korea Univ., Korea (1985)] and studies on the drying and storage of Schizandra chinensis [Jung, G. T., Ju, I.O. and choi, J.S. Studies on drying and preservation of OmijaSchizandra chinensis). Korean Postharvest Sci. Technol. 5: 217-223 (1998). The antimicrobial activity of Schizandra chinensis against pathogenic microorganisms such as Lee, S.H. and Lim, Y. S. Antimicrobial effects ofSchizandra chinesisextract on pathogenic microorganism. J. Korean Soc. Food Sci. Nutri. 27: 239-243 (1998)], lactic acid bacteria isolated from kimchi [Lee, S.H. and Lim, Y. S. Effect of Omija (Schizandra chinesis) extract on the growth of lactic acid bacteria isolated from Kimchi. Korean J. applied Microbiol. Biotechnol. 25: 224-228 (1997); andListeria monocytogenesThe antimicrobial effect of this has been reported [Lee, S.H. and Lim, Y. S. Antimicrobial effects ofSchizandra chinesisextract againstListeria monocytogenes. Korean J. applied Microbiol. Biotechnol. 25: 442-447 (1997)], but studies on the physiological activities such as antioxidant, antibacterial and nitrite scavenging of Schizandra chinensis seeds are still insufficient.

본 발명자들은 상기와 같은 점에 착안하여 오미자 종자를 각종 유기용매로 추출하고 분획한 후 각 추출물의 POV(peroxide value), TBA(thiobarbituric acid), 전자공여 측정 방법에 의한 항산화성, 식품부패 미생물에 대한 항균성, 발암유도물질인 아질산염 소거능을 조사하여 오미자 종자 추출물이 항산화성과 항균성이 우수하고 아질산염 소거능이 있음을 확인함으로서 본 발명을 완성하였다.In view of the above, the present inventors extracted and fractionated Schisandra chinensis seeds with various organic solvents, and then, each of the extracts was subjected to antioxidant, food decay microorganisms by POV (peroxide value), TBA (thiobarbituric acid), and electron donor measurement methods. The present invention was completed by investigating nitrite scavenging ability, which is an antimicrobial and carcinogenic substance, against Schisandra chinensis seed extract having excellent antioxidant and antibacterial properties and nitrite scavenging ability.

따라서, 본 발명의 목적은 오미자 종자로부터 추출한 항산화성, 항균성, 아질산염 소거능이 있는 추출물을 제공함에 있다.Accordingly, an object of the present invention is to provide an extract with antioxidant, antimicrobial and nitrite scavenging ability extracted from Schisandra chinensis seed.

본 발명의 다른 목적은 상기 오미자 종자 추출물의 제조방법을 제공함에 있다.It is another object of the present invention to provide a method for preparing the Schisandra chinensis extract.

본 발명의 또 다른 목적은 오미자 종자 추출물을 용매별로 분획한 분획물을 제공함에 있다.Still another object of the present invention is to provide a fraction obtained by dividing Schisandra chinensis seed extract for each solvent.

본 발명의 상기 목적은 오미자 종자를 분쇄하여 물, 메탄올, 에틸아세테이,트, 클로로포름을 각각 가하여 추출한 추출물을 다시 클로로포름, 에틸아세테이트, 부탄올 등으로 분획하고, 상기 오미자 종자 추출물의 POV, TBA를 각각 측정하고 DPPH법에 의해 DPPH 자유 라디칼 소거활성을 측정하여 항산화력을 조사하였다. 이어서, 추출용매별 오미자 종자 추출물이 각종 미생물의 생장을 억제하는 항균활성을 클리어 존의 직경을 측정하는 방법으로 조사하여 아질산염 소거능이 있음을 확인함으로서 달성하였다.The object of the present invention is to crush the Schisandra chinensis seed, adding water, methanol, ethyl acetate, tet, chloroform and extract the extract extracted into chloroform, ethyl acetate, butanol and the like again, POV, TBA of the schisandra seed extract The antioxidant activity was investigated by measuring DPPH free radical scavenging activity by DPPH method. Subsequently, the extract of Schizandra chinensis seed per extractant was achieved by investigating the antimicrobial activity of inhibiting the growth of various microorganisms by measuring the diameter of the clear zone to confirm that it had nitrite scavenging ability.

이어서, 본 발명의 구성을 설명한다.Next, the structure of this invention is demonstrated.

도 1은 본 발명 오미자 종자 메탄올 추출물의 부탄올 분획물(50㎍/mL)에 의해 처리된 미생물(오른쪽)과 처리되지 않은 미생물(왼쪽)을 주사전자 현미경으로 관찰한 결과 사진도이다.1 is a photograph showing the results of observing microorganisms (right) and untreated microorganisms (right) treated with butanol fraction (50 μg / mL) of the Schizandra chinensis seed methanol extract of the present invention under a scanning electron microscope.

본 발명은 오미자 종자를 마쇄하여 탈지한 후 3 ∼ 8배량의 유기용매를 가하고 70 ∼ 95℃ 온도로 1 ∼ 3회 추출 후 여과하여 회전진공 증발기(rotary vacuum evaporator)로 농축하고 동결건조하여 제조되고, DPPH 자유 라디칼을 소거하며, 락토바실러스 플란타룸[Lactobacillus plantarum(KCCM 11322)], 바실러스 서브틸리스 [Bacillus subtilis(KCCM 11314)], 스타필로코쿠스 아우레우스[Staphylococcus aureus(KCCM 12103]), 살모넬라 타이피무리움[Salmonella typhimurium(KCCM 11806) ], 에스케리키아 콜리[Escherichia coli(ATCC 10536)], 페니실리움 시트리눔 [Penicillium citrinum(KCCM 11663)] 균주의 생장을 억제하고, 아질산염을 소거하는 오미자 종자 기능성 추출물이다.본 발명은 오미자 종자를 분쇄한후 물, 메탄올, 에탄올, 에틸아세테이트, 클로로포름을 각각 가하여 추출하고 추출물을 클로로포름, 에틸아세테이트, 부탄올, 물로 순차적으로 분획하는 단계; 돈지와 대두유 각각에 상기 용매별로 추출한 오미자 종자 추출물을 첨가하고 과산화물가를 측정하여 오미자 종자 추출물의 항산화력을 조사하는 단계; 리놀레산에 상기 용매별로 추출한 오미자 종자 추출물을 첨가하고 TBA 값을 측정하여 오미자 종자 추출물의 항산화력을 조사하는 단계; 상기 용매별로 추출한 오미자 종자 추출물을 용해한 메탄올을 DPPH 용액에 혼합하고 흡광도를 측정하는 DPPH 법에 의해 오미자 종자 추출물의 항산화력을 조사하는 단계; 시험균주 각각을 배양한 배양액을 플레이트에 떨어뜨려 얇게 깔은 후 용매별로 추출한 오미자 종자 추출물을 흡수시킨 필터페이터 디스크를 올려놓고 배양한 다음 클리어 존의 직경을 측정하여 오미자 종자 추출물의 항균력을 조사하는 단계; 식품부패균을 48시간 배양한 배지에 오미자 메탄올 추출물의 부탄올 분획물을 처리하고 주사전자 현미경으로 균체의 형태변화를 관찰하는 단계 및; 용매별로 추출한 오미자 종자 추출물에 NaNO2용액을 첨가하고 pH를 1.2, 3.0, 6.0으로 조정하여 반응시킨 반응용액에 초산용액과 Griess 시약을 가하고 혼합한 후 흡광도를 측정하여 아질산염 소거능을 조사하는 단계로 구성된다.The present invention is prepared by grinding the Schisandra chinensis seed, degreasing it, adding 3 to 8 times the amount of an organic solvent, extracting 1 to 3 times at a temperature of 70 to 95 ° C., filtration, concentrating with a rotary vacuum evaporator, and lyophilizing. , Scavenging DPPH free radicals, Lactobacillus plantarum (KCCM 11322), Bacillus subtilis (KCCM 11314), Staphylococcus aureus (KCCM 12103) , Salmonella typhimurium (KCCM 11806), Escherichia coli (ATCC 10536), Penicillium citrinum (KCCM 11663)] inhibit the growth and inhibit nitrite The present invention extracts Schisandra chinensis seed. The present invention is pulverized Schisandra chinensis seed, and extracted with water, methanol, ethanol, ethyl acetate and chloroform, respectively. The method comprising in sequence fractions Tate, butanol, and water; Adding the Schisandra chinensis seed extract extracted for each solvent to lard and soybean oil and measuring the peroxide value to investigate the antioxidant power of the Schisandra chinensis extract; Adding the Schisandra chinensis seed extract extracted for each of the solvents to linoleic acid and measuring the TBA value to investigate the antioxidant activity of the Schisandra chinensis extract; Investigating the antioxidant power of the Schisandra chinensis seed extract by mixing the methanol dissolving Schisandra chinensis seed extract extracted for each solvent into a DPPH solution and measuring the absorbance; Drop the culture medium of each of the test strains on a plate and lay it thinly, and then put on a filter paper disk which absorbed the Schisandra chinensis extract extracted for each solvent, incubate it, and measure the diameter of the clear zone to investigate the antimicrobial activity of the Schisandra chinensis extract. ; Treating the butanol fraction of Schizandra chinensis extract in a medium cultured with food rot bacteria for 48 hours and observing the morphological changes of the cells under a scanning electron microscope; NaNO 2 solution was added to the extract of Schizandra chinensis seed extracted by solvent, and acetic acid solution and Griess reagent were added to the reaction solution by adjusting the pH to 1.2, 3.0, and 6.0. do.

본 발명에서 마쇄한 오미자 종자에 유기용매를 3 ∼ 8배 가량 첨가하고 바람직하게는 5배량 첨가한다.In the present invention, about 3 to 8 times the amount of the organic solvent is added to the schizandra seed, which is ground.

본 발명에서 오미자 종자 추출은 70 ∼ 95℃ 조건에서 1 ∼ 3회 실시하며 바람직하게는 85℃에서 2회 실시한다.In the present invention, Schisandra chinensis seed extraction is carried out 1-3 times at 70-95 ° C., preferably twice at 85 ° C.

본 발명에서 사용한 오미자 종자는 전북 무주산 건조오미자(Schizandra chinensisRUPRECHT)를 하룻밤 동안 물에 불려 과육을 제거하고 60℃에서 열풍 건조한 종자를 사용하였다. Schizandra chinensis RUPRECHT in Schizandra chinensis RUPRECHT was soaked in water overnight to remove pulp and used hot-dried seeds at 60 ° C.

본 발명에서 사용한 유지는 어떤 항산화제도 첨가되지 않은 대두유와 돈지를 이용하였다.The fats and oils used in the present invention used soybean oil and lard without any antioxidants.

본 발명에서 사용한 오미자 종자 추출 및 분획용 시약은 1급, 나머지 시약은 특급을 사용하였다.The reagent for extracting and fractionating Schisandra chinensis seed used in the present invention was used as the first grade, and the rest of the reagent was used as an express.

본 발명에서 항균력 조사시에 사용한 균주는 락토바실러스 플란타룸[Lactobacillus plantarum(KCCM 11322)], 바실러스 서브틸리스[Bacillus subtilis(KCCM 11314)], 스타필로코쿠스 아우레우스[Staphylococcus aureus(KCCM 12103]), 살모넬라 타이피무리움[Salmonella typhimurium(KCCM 11806)], 에스케리키아 콜리[Escherichia coli(ATCC 10536)], 페니실리움 시트리눔[Penicillium citrinum(KCCM 11663)]을 사용하였다.In the present invention, the strains used for the investigation of antimicrobial activity were Lactobacillus plantarum (KCCM 11322), Bacillus subtilis (KCCM 11314), Staphylococcus aureus (KCCM 12103). ]), Salmonella typhimurium (KCCM 11806), Escherichia coli (ATCC 10536) and Penicillium citrinum (KCCM 11663) were used.

이하, 본 발명의 구체적인 방법을 실시예를 들어 상세히 설명하고자 하지만 본 발명의 권리범위는 이들 실시예에만 한정되는 것은 아니다.Hereinafter, the specific method of the present invention will be described in detail with reference to Examples, but the scope of the present invention is not limited only to these Examples.

실시예 1: 오미자 종자 추출물 및 분획물 제조Example 1 Preparation of Schisandra chinensis Seed Extract and Fraction

오미자 종자를 분쇄기(CEMOTEC 1090)로 마쇄한 시료를 헥산(hexane)으로 탈지하고 시료 5배 량의 추출용매로 물(water), 메탄올(methanol), 에탄올(ethanol), 에틸아세테이트(ethyl acetate), 클로로포름(chloroform)을 가하여 환류냉각관을 부착시킨 플라스크에 넣고 85℃ 워터배스(water bath)에서 3시간씩 2회 추출 후 여과하여 회전 진공 증발기(rotary vacuum evaporator)로 농축하고 동결건조하여 추출물을 얻었다. 여러 추출물 중 가장 활성이 높은 추출물을 클로로포름, 에틸아세테이트, 부탄올, 물 등의 용매로 순차 분획 후 농축하고 동결건조하여 분획물을 얻었다.A sample of Schisandra chinensis seed pulverized (CEMOTEC 1090) was degreased with hexane, and 5 times the amount of the sample was extracted using water, methanol, ethanol, ethyl acetate, ethyl acetate, Chloroform was added to the flask to which the reflux condenser was attached, followed by extracting twice in an 85 ° C. water bath for 3 hours, filtered, concentrated in a rotary vacuum evaporator, and lyophilized to obtain an extract. . Among the various extracts, the most active extract was sequentially fractionated with a solvent such as chloroform, ethyl acetate, butanol, and water, concentrated and lyophilized to obtain a fraction.

실험결과, 표 1에 나타낸 바와 같이 용매별 추출 수율을 보면 에틸 아세테이트에 의한 추출 수율은 12.0%로 가장 높았고 클로로포름 추출 수율은 11.7%, 물 추출 수율은 8.2%, 메탄올 추출 수율은 6.4%이었고 에탄올 추출 수율은 5.0%로써 가장 낮았다.As a result, as shown in Table 1, the extraction yield by solvent showed the highest extraction yield with ethyl acetate (12.0%), the chloroform extraction yield was 11.7%, the water extraction yield was 8.2%, the methanol extraction yield was 6.4%, and the ethanol extraction. The yield was the lowest as 5.0%.

다양한 추출용매로 추출한 오미자 종자 추출물의 수율Yield of Schisandra chinensis Extract Extracted with Various Extraction Solvents 용매menstruum water 메탄올Methanol 에탄올ethanol 에틸아세테이트Ethyl acetate 클로로포름chloroform 추출수율(%)Extraction yield (%) 8.28.2 6.46.4 5.35.3 12.012.0 11.711.7

실험예 1: POV 측정Experimental Example 1: POV Measurement

본 실험예에서는 돈지와 대두유 100 g에 DMSO(dimethyl sulfoxide) 용액에 녹인 상기 실시예 1에서 얻은 추출 용매별 오미자 종자 추출물을 500 ppm 되게 가하여 60℃ 항온기에 저장하면서 경시적으로 1 g씩 공전삼각플라스크에 평취하여 분석하였다. 시료에 클로로포름 10 mL, 아세트산 15 mL 및 KI 포화용액 1 mL를 가하여 1분간 진탕시켜 5분간 암소에 방치시킨 후, 증류수를 75 mL 첨가하여 진탕시킨 다음 0.01 N Na2S2O3용액으로 적정하여 POV(peroxide value)로 하였다.In this Experimental Example, 100 g of lard and soybean oil were added to 500 ppm of Schisandra chinensis seed extract according to the extraction solvent obtained in Example 1 dissolved in DMSO (dimethyl sulfoxide) solution, and stored at 60 ° C. in a constant temperature flask for 1 g each. Was analyzed by leveling up. The sample in chloroform 10 mL, was added to 15 mL and KI saturated solution of 1 mL of acetic acid was shaken for 1 minutes then allowed to stand for 5 minutes cow, was shaken by the addition of distilled water to 75 mL and then titrated with 0.01 N Na 2 S 2 O 3 solution It was set as POV (peroxide value).

실험결과, 표 2에 나타낸 바와 같이 시험전 돈지의 과산화물가는 1.23 meq/kg이었고 무첨가시 16일 후에 22.6 meq/kg까지 과산화물가가 증가되었다. 돈지에 대한 오미자 종자 추출물의 항산화력은 메탄올 추출물 > 에탄올 추출물 > 물 추출물 > 클로로포름 추출물 > 에틸아세테이트 추출물 순이었으며 천연항산화제인 δ-토코페롤(δ-tocopherol) 보다 모든 용매 추출물들이 과산화물가가 낮아 항산화 효과가 높게 나타났다. 또 표 3에는 대두유의 과산화물가를 나타낸 것으로 과산화물가가 1.11 meq/kg인 돈지 보다 대두유가 빠르게 산패가 일어나 추출물의항산화력이 비교적 효과가 낮았다. 천연항산화제인 δ-토코페롤은 효과가 거의 없었으나 오미자 종자 추출물에서는 효과가 있었는데 그중 에탄올 추출물이 44.58 meq/kg으로 가장 효과적이었고 그 다음으로 메탄올 추출물(46.71 meq/kg)이었다.As a result, as shown in Table 2, the peroxide value of the piglet before the test was 1.23 meq / kg and the peroxide value increased to 22.6 meq / kg after 16 days without addition. Antioxidant activity of Schisandra chinensis seed extract was in order of methanol extract> ethanol extract> water extract> chloroform extract> ethyl acetate extract, and all solvent extracts had lower antioxidant effect than δ-tocopherol, a natural antioxidant. appear. In addition, Table 3 shows the peroxide value of soybean oil, and the antioxidative power of the extract was relatively low because soybean oil rancidated faster than that of lard with peroxide value of 1.11 meq / kg. The natural antioxidant δ-tocopherol had little effect, but it was effective in Schisandra chinensis extract, of which ethanol extract was the most effective at 44.58 meq / kg, followed by methanol extract (46.71 meq / kg).

오미자 종자 추출물이 함유된 돈지를 60℃에서 저장하면서 시간경과에 따라 측정한 과산화물가 변화(단위: meq/kg)Changes in the Peroxide Values Measured over Time with Storage of Pork Containing Schisandra chinensis Seed at 60 ° C (Unit: meq / kg) 용매menstruum 저장시간(일)Save time (days) 00 88 1616 water 1.231) 1.23 1) 10.9110.91 13.7613.76 메탄올Methanol 1.231.23 8.778.77 13.4313.43 에탄올ethanol 1.231.23 9.829.82 13.8313.83 에틸아세테이트Ethyl acetate 1.231.23 14.9414.94 17.3217.32 클로로포름chloroform 1.231.23 11.2211.22 14.0514.05 δ-토코페롤δ-tocopherol 1.231.23 13.8413.84 18.8018.80 대조군Control 1.231.23 12.4612.46 22.6022.60 [주] 1)은 3번 반복한 평균값이다.단위: meq/kgNOTE 1) is the average value repeated three times.Unit: meq / kg

오미자 종자 추출물이 함유된 대두유를 60℃에서 저장하면서 시간경과에 따라 측정한 과산화물가 변화Changes of Peroxides Measured over Time with Soybean Oil Containing Schisandra chinensis Seed Extract at 60 ° C 용매menstruum 저장시간(일)Save time (days) 00 88 1616 water 1.111) 1.11 1) 24.8824.88 53.4653.46 메탄올Methanol 1.111.11 23.7923.79 46.7146.71 에탄올ethanol 1.111.11 15.2715.27 44.5844.58 에틸아세테이트Ethyl acetate 1.111.11 28.9728.97 56.8956.89 클로로포름chloroform 1.111.11 28.1528.15 58.4058.40 δ-토코페롤δ-tocopherol 1.111.11 32.6432.64 60.1260.12 대조군Control 1.111.11 30.8730.87 62.9862.98 [주] 1)은 3번 반복한 평균값이다.단위: meq/kgNOTE 1) is the average value repeated three times.Unit: meq / kg

실험예 2: TBA 측정Experimental Example 2: TBA Measurement

본 실험예에서 기질 용액은 0.1 M 인산버퍼(phosphate buffer;pH 7.0)와 에탄올을 4:1로 혼합한 용매에 리놀레산(linoleic acid)를 0.03 M이 되도록 첨가하였다. 이 기질 용액 20 mL에 0.1 M 인산버퍼(phosphate buffer;pH 7.0) 19.2 mL, 실시예 1에서 얻은 추출용매별 오미자 종자 추출물을 각각 500 ppm되게 0.8 mL 첨가한 후 40℃ 진탕배양기에 저장하면서 경시적으로 시료액 2.0 mL를 취하여 분석하였다.In this experiment example, the substrate solution was added so that linoleic acid became 0.03 M in a solvent in which 0.1 M phosphate buffer (pH 7.0) and ethanol were mixed 4: 1. To 20 mL of this substrate solution, 19.2 mL of 0.1 M phosphate buffer (pH 7.0) and 0.8 mL of Schizandra seed extract for each extraction solvent obtained in Example 1 were added to 500 ppm, and then stored at 40 ° C in a shaker. 2.0 mL sample solution was taken and analyzed.

상기 시료액 2.0 mL에 35% 트리클로로아세트산(trichloroacetic acid) 1.0 mL과 0.75% TBA시약 2.0 mL를 가한 다음 30초 동안 진탕시킨 후 95℃ 수욕 상에서 40분 동안 반응시켰다. 이 반응액을 실온까지 냉각시켜 아세트산 1.0 mL, 클로로포름 2.0 mL를 가하여 진탕시킨 후, 3,000 rpm에서 5분 동안 원심분리하여 상징액의흡광도를 532 nm에서 측정하여 이를 TBA(thiobarbituric acid) 값으로 하였다.1.0 mL of 35% trichloroacetic acid and 2.0 mL of 0.75% TBA reagent were added to 2.0 mL of the sample solution, followed by shaking for 30 seconds, followed by reaction for 40 minutes on a 95 ° C. water bath. The reaction solution was cooled to room temperature, shaken by adding 1.0 mL of acetic acid and 2.0 mL of chloroform, followed by centrifugation at 3,000 rpm for 5 minutes to measure the absorbance of the supernatant at 532 nm, which was determined as a TBA (thiobarbituric acid) value.

실험결과, 표 4에 나타낸 바와 같이 대조구는 6일 후 1.557이었고 메탄올 추출물이 0.065로 TBA값이 매우 낮아 항산화력이 가장 우수하였는데 δ-토코페롤 (0.229) 보다 높았으며 BHA(0.014)와는 비슷하였다. 따라서, 리놀레산(Linoleic acid)에 대한 추출물의 항산화 효과는 메탄올 추출물 > 클로로포름 추출물 > 에틸아세테이트 추출물 > 에탄올 추출물 > 물 추출물 순이었다.As a result, as shown in Table 4, the control was 1.557 after 6 days and methanol extract was 0.065, and the TBA value was very low, which was the best antioxidant activity, which was higher than δ-tocopherol (0.229) and was similar to BHA (0.014). Therefore, the antioxidant effect of the extract on linoleic acid was methanol extract> chloroform extract> ethyl acetate extract> ethanol extract> water extract.

오미자 종자 추출물이 함유된 리놀레산을 40℃에서 보관하면서 측정한 TBA 값의 변화Changes in TBA Values of Linoleic Acid Containing Schisandra chinensis Seed Extracts at 40 ° C Storage 용매menstruum 저장시간(일)Save time (days) 1One 22 44 66 water 0.1191) 0.119 1) 0.3530.353 0.5370.537 1.2621.262 메탄올Methanol 0.0220.022 0.0850.085 0.1080.108 0.0650.065 에탄올ethanol 0.0230.023 0.1460.146 0.2230.223 0.3690.369 에틸 아세테이트Ethyl acetate 0.0360.036 0.1810.181 0.2060.206 0.2980.298 클로로포름chloroform 0.0040.004 0.1330.133 0.1920.192 0.1570.157 BHABHA 0.0020.002 0.0650.065 0.0590.059 0.0140.014 δ-토코페롤δ-tocopherol 0.1320.132 0.2130.213 0.2450.245 0.2290.229 대조군Control 0.2940.294 0.5690.569 1.4741.474 1.5571.557 [주] 1)은 3번 반복한 평균값이다.NOTE 1) is the average value repeated three times.

실험예 3: DPPH법에 의한 항산화력 조사Experimental Example 3: Investigation of Antioxidant Capacity by DPPH Method

본 실험예는 DPPH(1,1-diphenyl-2-picrylhydrazyl)를 사용한 항산화 활성 검정법으로 실시예 1에서 추출한 여러 농도의 오미자 종자 추출물 시료를 4 mL의 메탄올에 녹여 1.5×10-4M DPPH 메탄올 용액 1 mL를 첨가한 후, 30분간 실온에 방치하여 517 nm에서 흡광도를 측정하였다. 상기 시료를 첨가하지 않은 대조군의 흡광도를 1/2로 감소시키는데 필요한 시료의 양(㎍)을 RC50으로 나타냈으며, 기존의 항산화제인 δ-토코페롤 및 BHA와 비교하였다.This Experimental Example is an antioxidant activity assay using DPPH (1,1-diphenyl-2-picrylhydrazyl), and the sample of Schisandra chinensis seed extract extracted in Example 1 was dissolved in 4 mL of methanol and 1.5 × 10 -4 M DPPH methanol solution. After adding 1 mL, the solution was left at room temperature for 30 minutes and the absorbance was measured at 517 nm. The amount (μg) of the sample required to reduce the absorbance of the control group without adding the sample by 1/2 was expressed as RC 50 , and compared with the existing antioxidants δ-tocopherol and BHA.

실험결과, 표 5에 나타낸 바와 같이 DPPH를 50% 환원시키는데 필요한 추출물의 첨가 농도(RC50)를 보면 상업용 BHA와 δ-토코페롤이 각각 19.8 ㎍/mL와 18.7 ㎍/mL이었는데 비해 오미자 종자 추출물이 약간 많았다. 이중 메탄올 추출물이 33.2 ㎍/mL으로 가장 활성이 높았고 에틸 아세테이트와 클로로포름 추출물은 465 ㎍/mL로 거의 활성이 없었다.As a result, as shown in Table 5, the concentration of extract (RC 50 ) required for 50% reduction of DPPH was 19.8 ㎍ / mL and 18.7 ㎍ / mL for commercial BHA and δ-tocopherol, respectively. Many. Among the methanol extracts, the highest activity was 33.2 ㎍ / mL, and the ethyl acetate and chloroform extracts were 465 ㎍ / mL.

상기 실험예 1 ∼ 3의 결과를 종합해 보면 오미자 종자의 항산화물질 추출을 위하여 메탄올과 에탄올이 효과적이었으며, 특히 메탄올 추출물이 여러 항산화 측정 방법에서 높은 활성을 나타냈다. 추출물중 가장 활성이 높은 메탄올 추출물을 클로로포름, 에틸 아세테이트, 부탄올, 물 용매로 순차 분획하여 추출 수율을 검토한 결과 표 6에 나타낸 바와 같이 클로로포름이 48.2%로 가장 높았고 물이 25.5%, 부탄올 15.8%, 에틸 아세테이트 10.6% 순이었다. 또 분획별 자유 라디칼(free radical) 소거 활성을 비교한 결과는 표 7과 같은데 에틸 아세테이트 분획에서 DPPH를 50% 환원시키는데 필요한 추출물의 첨가 농도(RC50)가 17.9 ㎍/mL으로 BHA(19.8 ㎍/mL)나 δ-토코페롤(18.7 ㎍/mL) 보다 항산화 활성이 컸으며, 부탄올 분획에서도 22.8 ㎍/mL로 상당한 활성을 나타냈으나 물 분획에서는 활성이 아주 미미했다.To summarize the results of Experimental Examples 1 to 3, methanol and ethanol were effective for extracting antioxidants of Schisandra chinensis seeds. In particular, methanol extract showed high activity in various antioxidant measurement methods. Among the extracts, methanol extract with the highest activity was sequentially fractionated with chloroform, ethyl acetate, butanol and water solvent, and the extraction yield was examined. As shown in Table 6, chloroform was the highest as 48.2%, water was 25.5%, butanol 15.8%, Ethyl acetate followed by 10.6%. The results of comparing the free radical scavenging activity of each fraction are shown in Table 7. The concentration of extract (RC 50 ) required to reduce DPPH by 50% in the ethyl acetate fraction was 17.9 ㎍ / mL and BHA (19.8 ㎍ / mL). mL) and δ-tocopherol (18.7 ㎍ / mL) was more antioxidant activity, but the butanol fraction showed a significant activity of 22.8 ㎍ / mL, but very little in the water fraction.

용매별 오미자 종자 추출물을 각각의 DPPH 자유 라디칼 환원활성DPPH Free Radical Reduction Activities of Solvent Seed Extracts by Solvents 용매menstruum 추출물 농도(㎍/mL)Extract concentration (µg / mL) RC50 1)(㎍/mL)RC 50 1) (μg / mL) 00 2525 5050 100100 water 0.2792) 0.279 2) 0.2590.259 0.2450.245 0.2020.202 174.7174.7 메탄올Methanol 0.2790.279 0.1740.174 0.0880.088 0.0440.044 33.233.2 에탄올ethanol 0.2790.279 0.2470.247 0.2240.224 0.1240.124 93.093.0 에틸아세테이트Ethyl acetate 0.2790.279 0.2360.236 0.2570.257 0.2400.240 465.0465.0 클로로포름chloroform 0.2790.279 0.2170.217 0.2070.207 0.2330.233 465.0465.0 BHABHA 0.2790.279 0.0930.093 0.0880.088 0.0900.090 19.819.8 δ-토코페롤δ-tocopherol 0.2790.279 0.0830.083 0.0820.082 0.0810.081 18.718.7 [주] 1) 30분 후 DPPH의 환원 50%에 대해 요구되는 양2) 3번 반복한 평균값NOTE 1) Amount required for 50% reduction of DPPH after 30 minutes 2) Average value repeated three times

오미자 종자 메탄올 추출물의 분획 수율Fraction Yield of Schisandra chinensis Seed Methanol Extract 용매menstruum 클로로포름chloroform 에틸아세테이트Ethyl acetate 부탄올Butanol water 분획수율(%)Fraction yield (%) 48.248.2 10.610.6 15.815.8 25.525.5

다양한 용매로 추출한 오미자 종자 추출물로부터 얻은 여러 용매 분획의 DPPH 자유 라디칼 환원 활성DPPH Free Radical Reduction Activity of Various Solvent Fractions from Schizandra Seed Extracts Extracted with Various Solvents 용매menstruum 추출물 농도(㎍/mL)Extract concentration (µg / mL) RC50 1)(㎍/mL)RC 50 1) (μg / mL) 00 2525 5050 100100 클로로포름chloroform 0.2542) 0.254 2) 0.2200.220 0.1860.186 0.1240.124 37.737.7 에틸아세테이트Ethyl acetate 0.2540.254 0.0770.077 0.0670.067 0.0560.056 17.917.9 부탄올Butanol 0.2540.254 0.1110.111 0.0900.090 0.0650.065 22.822.8 water 0.2540.254 0.2390.239 0.2290.229 0.2010.201 254.0254.0 BHABHA 0.2540.254 0.0930.093 0.0880.088 0.0910.091 19.819.8 δ-토코페롤δ-tocopherol 0.2540.254 0.0830.083 0.0820.082 0.0810.081 18.718.7 [주] 1) 30분 후 DPPH의 50% 환원을 위해 요구되는 양2) 3번 반복하여 측정한 평균NOTE 1) Amount required for 50% reduction of DPPH after 30 minutes 2) Average measured over 3 times

실험예 4: 항균성 측정Experimental Example 4: Antibacterial Measurement

사면배양된 시험 균주를 락토바실러스 플란타룸(Lactobacillus plantarum)은 MRS broth, 바실러스 서브틸리스(Bacillus subtilis)와 에스케리키아 콜리 (Escherichia coli)(ATCC 10536)는 영양배지(nutrient broth), 스타필로코쿠스 아우레우스(Staphylococcus aureus)와 살모넬라 타이피무리움(Salmonella typhi murium)(KCCM 11806)은 트립틱 소이 브로스(tryptic soy broth), 페니실리움 시트리눔(Penicillium citrinum)(KCCM 11663)은 PD broth에 접종하여 30℃에서 24시간 동안 배양하여 활성화시키고 이 배양액 0.3 mL를 각각의 MRS, nutrient, 트립틱 소이(tryptic soy) 및 PD 아가 플레이트(PD agar plate)에 떨어뜨린 후 스프래더(spreader)로 균일하게 도포하였다. 각 시험균이 접종된 플레이트(plate) 위에 오미자 종자 추출물을 50 ㎍ 흡수시킨 ø6 mm 필터 페이퍼 디스크(ø6 mm filter paper disc;Whatman No. 2)를 놓고 30℃에서 48시간 동안 배양 후 디스크(disc) 주위에 나타난 클리어 존(clear zone)의 직경을 측정하여 항균력을 비교하였다.The tested strains were Lactobacillus plantarum , MRS broth, Bacillus subtilis and Escherichia coli (ATCC 10536), nutrient broth, Staphylo Staphylococcus aureus and Salmonella typhi murium (KCCM 11806) are tryptic soy broth, Penicillium citrinum (KCCM 11663) Inoculate broth and incubate at 30 ° C for 24 hours to activate and drop 0.3 mL of this culture onto each MRS, nutrient, tryptic soy and PD agar plate and spreader The coating was applied uniformly. Place a ø6 mm filter paper disc (Whatman No. 2) containing 50 μg of Schizandra seed extract on a plate inoculated with each test strain and incubate for 48 hours at 30 ° C. The antimicrobial activity was compared by measuring the diameter of the clear zone appearing around.

실험결과, 표 8에 나타낸 바와 같이, 오미자 종자 추출물의 항균 활성은 합성항균제인 소르브산(sorbic acid) 보다 락토바실러스 플란타룸,바실러스 서브틸리스, 살모넬라 타이피무리움 및 에스케리키아 콜리 등에 대하여 항균력이 같거나 높았으며 스타필로코쿠스 아우레우스와 페니실리눔 시트리눔에 대하여는 낮았다. 모든 추출물이 실험에 사용된 식품 부패 균주에 대해 항균 효과를 나타냈는데 락토바실러스 플란타룸, 바실러스 서브틸리스, 에스케리키아 콜리 및 페니실리눔 시트리눔은 메탄올 추출물의 클리어 존이 각각 12.7 mm, 14.0 mm, 11.3 mm, 10.6 mm로 가장 항균활성이 컸으며, 스타필로코쿠스 아우레우스와 살모넬라 타이피무리움은 에틸아세테이트 추출물의 클리어 존(clear zone)이 각각 11.6 mm와 12.0 mm로 용매별 추출물 중에 가장 항균 효과가 우수하였다. 오미자 종자 추출물이 모든 시험 균주에 대하여 항균 활성을 나타냈는데 특히 바실러스 서브틸리스에 대하여 가장 강한 항균 활성을 나타냈고 락토바실러스 플란타룸과 살모넬라 타이피무리움에 대해서도 다른 시험 균주보다 높은 경향이었다. 상기 결과에 따라 모든 시험균에 전반적으로 강한 항균 활성을 나타내는 메탄올 추출물을 이용하여 클로로포름, 에틸아세테이트, 부탄올, 물층으로 분획하여 항균력을 검토하였으며 이 결과를 표 9에 나타냈다. 물 분획에서 대체적으로 항균 활성이 낮았으며 스타필로코쿠스 아우레우스와 살모넬라 타이피무리움은 전혀 항균 효과가 나타나지 않았다. 부탄올 분획의 경우 락토바실러스 플란타룸,바실러스 서브틸리스, 스타필로코쿠스 아우레우스, 살모넬라 타이피무리움 및 에스케리키아 콜리에서 클리어 존의 직경이 각각 13.1 mm, 14.2 mm, 12.4 mm, 12.4 mm 및 13.0 mm로 소르브산 보다 강한 항균 활성을 나타내었고 페니실리움 시트리눔에 대한 항균력은 물 분획이 11.9 mm의 클리어 존을 형성하여 가장 우수하였으나 합성항균제인 소르브산(13.8 mm)보다 떨어지는 경향이었다.Experimental results, as shown in Table 8, the antimicrobial activity of Schisandra chinensis seed extract was higher than that of sorbic acid, a synthetic antimicrobial agent, Lactobacillus plantarum.,Bacillus subtilis, Salmonella typhimurium and Escherichia coli The antimicrobial activity was the same or higher, and lower for Staphylococcus aureus and penicillin citrinum. All extracts showed antimicrobial effects against the food decay strains used in the experiments: Lactobacillus plantarum, Bacillus subtilis, Escherichia coli And penicillin citrinum had the highest antibacterial activity of 12.7 mm, 14.0 mm, 11.3 mm, and 10.6 mm, respectively, and the clear zones of methanol extracts were ethyl acetate extracts from Staphylococcus aureus and Salmonella typhimurium. The clear zone was 11.6 mm and 12.0 mm, respectively. Schisandra chinensis extract showed antimicrobial activity against all test strains, especially against Bacillus subtilis, and showed higher tendency than other test strains for Lactobacillus plantarum and Salmonella typhimurium. According to the above results, the antimicrobial activity was examined by fractionation into chloroform, ethyl acetate, butanol, and water layer using methanol extract showing strong antibacterial activity on all test bacteria. The results are shown in Table 9. Antibacterial activity was generally low in the water fraction, and Staphylococcus aureus and Salmonella typhimurium showed no antimicrobial effect. Lactobacillus plantarum for butanol fraction,Antibacterial stronger than sorbic acid in Bacillus subtilis, Staphylococcus aureus, Salmonella typhimurium and Escherichia coli, with clear zone diameters of 13.1 mm, 14.2 mm, 12.4 mm, 12.4 mm and 13.0 mm, respectively The antimicrobial activity against penicillium citrineum was the best as the water fraction formed a clear zone of 11.9 mm, but it was lower than the synthetic antibacterial sorbic acid (13.8 mm).

다양한 용매로 추출한 오미자 종자 추출물에 의한 항균력Antimicrobial Activity of Schizandra koji Extracts Extracted with Various Solvents 균주Strain 플레이트상의 클리어 존(mm)Clear zone on plate (mm) water 메탄올Methanol 에탄올ethanol 아세테이트acetate 소르브산Sorbic acid L.plantarumL.plantarum 11.51) 11.5 1) 12.712.7 12.012.0 12.512.5 12.712.7 B.subtilisB.subtilis 11.911.9 14.014.0 12.612.6 11.711.7 13.813.8 S.aureusS.aureus 10.410.4 10.610.6 10.010.0 11.611.6 12.312.3 S.typhimuriumS.typhimurium 10.510.5 11.111.1 10.510.5 12.012.0 11.311.3 E.coliE.coli 9.19.1 11.311.3 9.79.7 9.79.7 11.311.3 P.citrinumP.citrinum 9.09.0 10.610.6 9.99.9 9.09.0 13.813.8 [주] 1) 3번 반복 측정한 평균값1) Average value measured 3 times

오미자 종자 메탄올 추출물로부터 얻은 다양한 용매 분획의 항균력Antimicrobial Activity of Various Solvent Fractions from Schizandra chinensis Seed Extracts 균주Strain 플레이트 상의 클리어 존(mm)Clear Zone on Plate (mm) 클로로포름chloroform 에틸아세테이트Ethyl acetate 부탄올Butanol water 소르브산Sorbic acid L.plantarumL.plantarum 12.71) 12.7 1) 12.512.5 13.113.1 8.48.4 12.712.7 B.subtilisB.subtilis 12.812.8 13.113.1 14.214.2 10.210.2 13.813.8 S.aureusS.aureus 12.012.0 12.512.5 12.412.4 00 12.312.3 S.typhimuriumS.typhimurium 11.511.5 11.711.7 12.412.4 00 11.311.3 E.coliE.coli 12.312.3 12.312.3 13.013.0 9.89.8 11.211.2 P.citrinumP.citrinum 9.29.2 9.89.8 10.810.8 11.911.9 13.813.8 [주] 1) 3번 반복 측정한 평균값1) Average value measured 3 times

실험예 5: 미생물 형태변화Experimental Example 5: Change of Microorganism Morphology

실시예 1에서 얻은 오미자 종자 메탄올 추출물의 부탄올 분획물을 48시간 배양된 식품 부패균에 첨가하여 3시간 방치한 다음 이를 원심분리로 균체를 분리하여, 2% 글루타르알데하이드 용액에 침지하고 4℃, 2시간 동안 전고정(prefixing) 시킨 후, 0.1 M 인산 버퍼로 3회 세척하고 1% 오스뮴사산화물(osmium tetroxide) 용액으로 4℃에서 2시간 동안 후고정(post-fixing)시켜 0.1 M 인산버퍼로 3회 세척하였다. 고정시킨 시료를 에탄올 50%, 70%, 80%, 90%, 95%, 100%를 사용하여 순차적으로 20분씩 탈수시킨 다음, 이소아밀아세테이트(isoamylacetate)에 하룻밤 침지시켰다. 그 후 액체 질소를 사용하여 크리티칼 포인트 건조기(critical point dryer)로 건조 후 이온 코팅기(ion coater)를 사용하여 금으로 코팅(coating)시켜 주사전자 현미경(Scanning Electron Microscope;JSM-5410LV, JEOL, Japan)으로 15 ∼ 20 KV에서 1000과 3500배로 관찰하였다.Butanol fraction of the Schizandra chinensis extract obtained in Example 1 was added to the food decay culture incubated for 48 hours, and left for 3 hours. The cells were separated by centrifugation, immersed in 2% glutaraldehyde solution, and then heated at 4 ° C. for 2 hours. After pre-fixing for 3 hours, washed three times with 0.1 M phosphate buffer and post-fixed with 1% osmium tetroxide solution at 4 ° C. for 2 hours three times with 0.1 M phosphate buffer. Washed. The fixed sample was dehydrated sequentially for 20 minutes using 50%, 70%, 80%, 90%, 95%, and 100% of ethanol, and then immersed in isoamylacetate overnight. Thereafter, the liquid was dried in a critical point dryer using liquid nitrogen and coated with gold using an ion coater to scan a scanning electron microscope (JSM-5410LV, JEOL, Japan). ) And 1000 and 3500 times at 15-20 KV.

실험결과, 도 1에 나타낸 바와 같으며 대조구에 비하여 오미자 분획물을 처리하였을 때 균체가 팽윤되고, 세포벽이 붕괴되어 세포가 파괴되는 형태적 변화를 나타내었다.As a result, as shown in Figure 1, when the Schisandra chinensis fraction treatment compared to the control cells showed a morphological change swelling, cell wall collapsed, cells were destroyed.

실험예 6: 아질산염 소거능 측정Experimental Example 6: Measurement of nitrite scavenging ability

실시예 1에서 각종 추출용매로 추출한 오미자 종자 추출물 1 mL(1 ㎎)에 1 mM NaNO2용액 2 mL를 첨가하고 0.2 N 구연산 완충액으로 반응 용액의 pH를 1.2, 3.0, 6.0으로 각각 조정하여 반응 용액의 부피를 10 mL로 한 후, 37℃에서 1시간 동안 반응시켜 얻은 반응 용액을 각각 1 mL씩 취하고 여기에 2% 초산 용액을 5 mL를 첨가한 다음, Griess 시약 0.4 mL를 가하여 잘 혼합시켜 15분간 방치시킨 후 520 nm에서 흡광도를 측정하여 잔존하는 아질산량을 구하였다.2 mL of 1 mM NaNO 2 solution was added to 1 mL (1 mg) of Schisandra chinensis extract extracted with various extracting solvents in Example 1, and the pH of the reaction solution was adjusted to 1.2, 3.0, and 6.0 with 0.2 N citric acid buffer, respectively. After the volume was adjusted to 10 mL, take 1 mL of each reaction solution obtained by reacting at 37 ° C. for 1 hour, add 5 mL of 2% acetic acid solution, add 0.4 mL of Griess reagent, and mix well. After leaving for one minute, absorbance was measured at 520 nm to determine the amount of residual nitrite.

실험결과, 표 10에 나타낸 바와 같이 반응 용액이 pH 1.2일 때는 모든 추출물이 아질산염을 56% 이상 분해시킬 수 있었고 메탄올과 에탄올 추출물이 각각 98.6%와 91.9%의 가장 높은 분해능을 나타내었다. pH 3.0일 경우 메탄올 추출물이94.1%로 가장 높았고 그 다음으로 물 추출물(51.7%) > 에탄올 추출물(46.0%) > 클로로포름 추출물(5.9%) > 에틸아세테이트 추출물(3.9%) 순이었다. pH 6.0에서는 메탄올 추출물과 물 추출물에서만 각각 21.3%와 3.8%의 아질산염 분해능이 나타났다. 메탄올 추출물이 다른 추출물보다 모든 pH에서 아질산염 소거작용이 높았으며 pH 변화에 따른 각 용매 추출물의 아질산염 소거작용은 거의 비슷한 경향으로 위내의 pH와 유사한 pH 1.2와 pH 3.0에서 아질산염 소거작용이 높았으나 pH 6.0에서는 거의 없거나 매우 낮게 나타났다. 아질산염과 아민류가 반응하여 결합된 발암성 니트로사민(nitrosamine)은 강산성 조건 특히 인체나 동물 위내의 pH 조건에서 용이하게 생성되므로 본 실험에서와 같이 오미자종자의 메탄올 추출물이 강산성 조건하에서 아질산염 분해능이 크다는 사실은 이 물질이 위내에서 니트로사민의 생성 억제된다.As a result, as shown in Table 10, all the extracts were able to decompose more than 56% of nitrite when the reaction solution was pH 1.2, and methanol and ethanol extracts showed the highest resolution of 98.6% and 91.9%, respectively. At pH 3.0, methanol extract was the highest at 94.1%, followed by water extract (51.7%)> ethanol extract (46.0%)> chloroform extract (5.9%)> ethyl acetate extract (3.9%). At pH 6.0, nitrite resolutions of 21.3% and 3.8% were observed in methanol and water extracts, respectively. The nitrite scavenging effect of methanol extract was higher than that of other extracts at all pHs. The nitrite scavenging effect of each solvent extract was similar to that of the other extracts. Showed little or very low. Since carcinogenic nitrosamines, which are combined with nitrites and amines, are easily produced under strong acid conditions, especially in pH conditions in the human or animal stomach, the fact that methanol extracts of Schisandra chinensis seeds have a high nitrite resolution under strong acid conditions as shown in this experiment. This substance inhibits the production of nitrosamines in the stomach.

또 아질산염 분해능이 가장 우수한 메탄올 추출물을 클로로포름, 에틸아세테이트, 부탄올, 물을 이용하여 분획한 분획물의 아질산염 소거능은 표 11에 나타내었다. 반응 pH가 1.2 일때의 아질산염 분해능은 부탄올 추출물(98.9%) > 에틸아세테이트 추출물(98.4%) > 클로로포름 추출물(84.1%) > 물 추출물(57.4%) 순으로 양호하였고 pH 3.0에서도 같은 경향이었는데 pH 6.0에서는 클로로포름 분획물은 전혀 아질산염을 분해할 수 없었으나 부탄올, 에틸아세테이트, 물 분획물은 각각 61.8%, 48.4%, 18.6% 분해시킬 수 있었다. 부탄올 분획물이 모든 pH 범위 즉 pH 1.2, pH 3.0 및 pH 6.0에서 98.9%, 94.7%, 61.8%로 아질산염 분해능이 가장 높은 값을 나타냈다.In addition, the nitrite scavenging ability of the fraction obtained by distilling methanol extract having the best nitrite resolution using chloroform, ethyl acetate, butanol and water is shown in Table 11. Nitrite resolution at reaction pH of 1.2 was good in the order of butanol extract (98.9%)> ethyl acetate extract (98.4%)> chloroform extract (84.1%)> water extract (57.4%). The chloroform fraction could not degrade nitrite at all, but the butanol, ethyl acetate, and water fractions could degrade 61.8%, 48.4%, and 18.6%, respectively. Butanol fraction showed the highest nitrite resolution of 98.9%, 94.7%, 61.8% in all pH ranges, pH 1.2, pH 3.0 and pH 6.0.

실험예 1 ∼ 6의 결과에 의하면 항산화성 및 항균성이 높은 유기용매 추출물 및 분획물이 같은 경향으로 아질산염 소거작용이 높게 나타났다. 따라서 오미자 종자의 유기용매 용해물질은 항산화성, 항균성 그리고 아질산염 소거작용에 크게 관여함을 알 수 있었다.According to the results of Experimental Examples 1 to 6, the organic solvent extract and the fraction having high antioxidant and antimicrobial activity showed the same tendency and the nitrite scavenging effect was high. Therefore, the organic solvent solubles of Schisandra chinensis seeds were found to be involved in antioxidant, antimicrobial and nitrite scavenging activity.

다양한 용매로 추출한 오미자 종자 추출물에 의한 아질산염 소거능Nitrite Scavenging Ability of Schizandrae Fructus Extracts Extracted with Various Solvents pHpH 아질산염 소거능(%)Nitrite scavenging ability (%) water 메탄올Methanol 에탄올ethanol 에틸아세테이트Ethyl acetate 클로로포름chloroform 1.21.2 60.21) 60.2 1) 98.698.6 91.991.9 55.955.9 56.556.5 3.03.0 51.751.7 94.194.1 46.046.0 3.93.9 5.95.9 6.06.0 3.83.8 21.321.3 00 00 00 [주] 3번 측정한 평균값NOTE 3 average value measured 3 times

오미자 종자 메탄올 추출물로부터 얻은 다양한 용매분획의 아질산염 소거능Nitrite Scavenging Ability of Various Solvent Fractions from Schizandra chinensis Seed Extracts pHpH 아질산염 소거능(%)Nitrite scavenging ability (%) 클로로포름chloroform 에틸아세테이트Ethyl acetate 부탄올Butanol water 1.21.2 84.11) 84.1 1) 98.498.4 98.998.9 57.457.4 3.03.0 35.135.1 83.583.5 94.794.7 40.540.5 6.06.0 00 48.448.4 61.861.8 18.618.6 [주] 1) 3번 측정한 평균값1) Average value measured 3 times

이상, 상기 실시예와 실험예를 통하여 설명한 바와 같이, 다양한 유기용매로 추출한 오미자 종자 추출물과 그 분획물들은 돈지와 대두유에 대해 높은 항산화력을 나타내고 DPPH 자유 라디칼 소거 활성이 우수하며 식품 부패 미생물에 대해서는항균활성을 나타내는 뛰어난 효과가 있고 또 높은 아질산염 소거능을 나타내는 뛰어난 효과가 있으므로 건강식품 산업상 매우 유용한 발명인 것이다.As described through the above examples and experimental examples, Schisandra chinensis seed extract and its fractions extracted with various organic solvents exhibit high antioxidant power against lard and soybean oil, have excellent DPPH free radical scavenging activity, and antibacterial against food decay microorganisms. It is a very useful invention in the health food industry because it has an excellent effect of showing activity and an excellent effect of showing a high nitrite scavenging ability.

Claims (4)

오미자 종자를 마쇄하여 탈지한 후 3 ∼ 8배량의 유기용매를 가하고 70 ∼ 95℃ 온도로 1 ∼ 3회 추출 후 여과하여 회전진공 증발기(rotary vacuum evaporator)로 농축하고 동결건조하여 제조되고, DPPH 자유 라디칼을 소거하며, 락토바실러스 플란타룸[Lactobacillus plantarum(KCCM 11322)], 바실러스 서브틸리스 [Bacillus subtilis(KCCM 11314)], 스타필로코쿠스 아우레우스[Staphylococcus aureus(KCCM 12103]), 살모넬라 타이피무리움[Salmonella typhimurium(KCCM 11806) ], 에스케리키아 콜리[Escherichia coli(ATCC 10536)], 페니실리움 시트리눔 [Penicillium citrinum(KCCM 11663)] 균주의 생장을 억제하고, 아질산염을 소거하는 것을 특징으로 하는 오미자 종자 기능성 추출물.After grinding the Schisandra chinensis seed, degreasing it, adding 3 ~ 8 times of organic solvent, extracting 1 ~ 3 times at 70 ~ 95 ℃ temperature, filtering, concentrating with rotary vacuum evaporator and freeze drying, it is prepared by DPPH free Radical scavenging, Lactobacillus plantarum (KCCM 11322), Bacillus subtilis (KCCM 11314), Staphylococcus aureus (KCCM 12103), Salmonella Ty Inhibition of growth of Salmonella typhimurium (KCCM 11806), Escherichia coli (ATCC 10536), Penicillium citrinum [KCCM 11663], and scavenging of nitrite Schizandra seed functional extract, characterized in. 제 1 항에 있어서, 상기 유기용매는 물(water), 메탄올(methanol), 에탄올(ethanol), 에틸아세테이트(ethyl acetate), 클로로포름(chloroform)중 선택된 어느 하나임을 특징으로 하는 오미자 종자 기능성 추출물.The method of claim 1, wherein the organic solvent Schija seed functional extract, characterized in that any one selected from water (water), methanol (methanol), ethanol (ethanol), ethyl acetate, chloroform (chloroform). 삭제delete 삭제delete
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