KR20090095134A - Optimized extract of schizandrin from omija - Google Patents
Optimized extract of schizandrin from omija Download PDFInfo
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- KR20090095134A KR20090095134A KR1020080020275A KR20080020275A KR20090095134A KR 20090095134 A KR20090095134 A KR 20090095134A KR 1020080020275 A KR1020080020275 A KR 1020080020275A KR 20080020275 A KR20080020275 A KR 20080020275A KR 20090095134 A KR20090095134 A KR 20090095134A
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, 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
- A23L2/00—Non-alcoholic beverages; Dry compositions or concentrates therefor; Their preparation
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, 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
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/105—Plant extracts, their artificial duplicates or their derivatives
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/79—Schisandraceae (Schisandra family)
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/30—Extraction of the material
- A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
Abstract
Description
오미자(Schizandra Chinensis Baillo)는 목련과(Magnoliaceae)에 속하는 낙엽성, 만성 목질등본식물의 성숙한 과실이다. 도란형으로 끝은 뾰족하고 치아 모양의 거치가 있으며 잎 뒷면에는 약간의 털이 있다. 꽃은 홍백색으로 6-7월에 피며 과실은 장과로서 이삭 모양으로 6-8월에 개화하여 9-10월에 열리고 서리가 내린 후 채취하여 이 열매를 약용으로 쓴다. Schizandra Chinensis Baillo) is a mature fruit of deciduous, chronic woody, etc. belonging to the Magnoliaceae family. Obovate with pointed tip and tooth-shaped pedestal with some hairs on the back of leaf. The flower is red-white and blooms in June-July. The fruit is berry, which blooms in June-August, opens in September-October, collects after frost, and uses this fruit for medicinal purposes.
오미자는 독특한 방향성과 단맛, 신맛, 쓴맛, 짠맛, 매운맛 등 다섯 가지의 맛이 있는데, 껍질은 달콤하고 살은 시며 씨는 맵고 쓰고 떫은맛이 나며 잘 익은 열매는 단맛이 있고 독특한 향기가 난다.Omija has five flavors: unique fragrance, sweet, sour, bitter, salty, and spicy. The skin is sweet and sour, the seeds are spicy, bitter and astringent, and the ripe fruit has a sweet and unique fragrance.
문헌상 자료를 살펴보면, 약용식물사전에 열매는 한방에서 자양강장제, 진해거담제 또는 수렴제로 정(精)을 증진시켜 내분비의 호르몬 분비를 촉진시킨다고 한다. 본초서(本草書)에서는 허로(虛勞)와 몸을 보하고 눈을 밝게 하고 신장을 데우며 음을 강하게 하고 남자의 정력을 증진시킬 뿐만 아니라 소갈(消渴)을 그치고 번 열(煩熱)을 없엔다. 또한 주독(酒毒)을 풀고 기질 해소를 다스린다고 기록되어 있다.According to the literature, the medicinal plant dictionary promotes the secretion of hormonal secretions by enhancing jeong (로) in herbal medicine, nourishing tonic, antitussive expectorant, or astringent. In the main book, it looks after the body and body, brightens the eyes, warms the kidneys, strengthens the sound, enhances the man's energy, and stops the heat and burns. To get rid of. It is also said that it relieves the poison and controls the dissolution of temperament.
최근 시대가 변화하고 현대인들의 생활방식과 식생활, 질병형태 다양화 등 여러 사회적인 여건 변화와 건강에 대한 관심이 증대하면서 생약재를 소재로 한 우리 전통차에 대한 관심과 시장 규모가 커지고 있는 추세이다.Recently, as the times change and modern people's lifestyles, dietary lifestyles, and disease forms are diversified, their interest in health and the health of Korean traditional teas based on herbal medicines is increasing.
오미자 과실에 함유된 성분은 쉬잔드린(schizandrin), 쉬잔드란(schizandran), 고미신(gomisin)류 등과 같은 주로 리그난 화합물이며, 이는 오미자의 다양한 생리활성을 나타낸다.The components contained in Schizandra fruit are mainly lignan compounds such as schizandrin, schizandran, gomisin, etc., which exhibit various biological activities of Schizandra chinensis.
오미자의 주요 기능성분인 쉬잔드린(schizandrin)은 특히, 약리 효과가 강하다고 보고되고 있으며 간 보호 효과, 신경안정작용, 진통, 위액분비억제, 담즙분비촉진 등의 효능이 있는 것으로 알려져 있다. Schizandrin, the main functional ingredient of Schizandra chinensis, has been reported to have strong pharmacological effects, and is known to be effective in protecting liver, neurostable function, analgesic, gastric juice secretion, and biliary secretion.
본 발명은 오미자의 리그난 성분 중 하나인 쉬잔드린(Schizandin)을 지표물질로 선정하여 이를 최대로 하는 추출방법 및 관련 조건을 확립하고, 이를 이용한 고기능성 오미자음료를 개발하고자 한다.The present invention is to select one of the lignan components of Schizandra chinensis (Schizandin) as an indicator material to establish the extraction method and related conditions to maximize this, and to develop a high-functional Schizandra beverage using the same.
현재 직면한 과제는 최저비용으로 최대의 수율을 얻는 것이다. 이를 위해서는 기능성분을 중심으로 한 최적 추출조건의 확립이 필요하다. The challenge now is to get maximum yield at the lowest cost. For this purpose, it is necessary to establish the optimum extraction condition centering on the functional ingredient.
건강기능식품으로 업그레이드 하는 과정에서 필수적인 요건은 제품의 효능이 다. 제품의 효능을 일차적으로 입증하는 과정에서 필요한 것은 함유된 기능성분의 양으로서, 타 제품과 비교하여 기능성분의 함량이 떨어지면 높은 부가가치는 기대할 수 없다. 따라서 입증된 기능성분의 함량을 최대로 하는 추출조건 확립이 필요하다. An essential requirement for upgrading to dietary supplements is the efficacy of the product. What is needed in the process of primarily demonstrating the efficacy of the product is the amount of functional ingredients contained, and high added value cannot be expected when the content of the functional ingredients is lower than other products. Therefore, it is necessary to establish extraction conditions that maximize the content of proven functional ingredients.
오미자의 리그난 성분 중 하나인 쉬잔드린을 지표물질로 선정하여 이를 최대로 하는 추출방법 및 관련 조건을 확립하고, 고기능성 오미자음료를 개발하고자 한다.One of the lignan components of Schizandra chinensis is selected as an indicator and the extraction method and related conditions are maximized, and high functional Schizandra chinensis is developed.
오미자는 그 고유의 붉은색을 가지고 있다. 오미자의 붉은색은 오미자의 관능적인 요소 중의 중요한 부분이며, 오미자의 붉은 색을 잃지 않고 오미자추출액을 만드는 것이 관건이다. Schisandra has its own red color. The red color of Schizandra chinensis is an important part of Schizandra's sensual element, and it is key to make Schizandra extract without losing the Schizandra red color.
오미자의 기능성분과 오미자의 붉은 색을 잃지 않으면서 최적의 추출점을 도출하기 위하여 추출액의 오미자 기능성분을 분석하고 갈색도(BI)를 측정하여 오미자 관능성이 높은 추출액을 제조하고자 하였다.In order to derive the optimal extraction point without losing the functional components of Schizandra chinensis and the red color of Schizandra chinensis, we analyzed the Schizandra chinensis functional components and measured the brownness (BI) to prepare extracts with high Schizandra functionalities.
일반적으로 오미자의 추출은 열수 또는 설탕물을 이용하여 추출하게 된다. 열수로 추출할 경우 약간의 기능성분이 추출되나 대부분 유기산과 색소가 추출된다. 그러나 갈색도가 높아져서 관능성이 떨어진다. 설탕물을 이용하여 추출할 경우 관능성은 높아지나 기능성분의 추출이 거의 이루어지지 않는다.In general, the extract of Schizandra chinensis extract is extracted using hot or sugar water. When extracted with hot water, some functional ingredients are extracted, but most organic acids and pigments are extracted. However, the brownness is higher, so the sensuality is reduced. When extracted with sugar water, the functionality is increased, but the extraction of functional components is hardly made.
본 발명의 추출방법을 사용할 경우 기능성분이 많이 추출되고 관능성 또한 높은 추출물을 만들 수 있다.When the extraction method of the present invention is used, a lot of functional ingredients can be extracted and the functionality can also be made high.
이하 본 발명을 구체적으로 설명하고자 한다.Hereinafter, the present invention will be described in detail.
가. 일반성분 분석end. General Ingredient Analysis
오미자의 일반성분 분석은 조지방, 수분, 회분, 조단백질, 조섬유를 분석하였으며, 탄수화물의 함량은 앞의 다섯 가지 성분의 합을 뺀 나머지로 계산하였다. 조지방은 산분해법에 의하여 실시하였으며, 수분과 회분 함량은 미국공인분석화학회(AOAC)법에 준하여 105℃ 상압건조법과 직접 회화법을 이용하여 측정하였다. 조단백질은 자동질소분해증류장치를 사용하여 측정하였으며, 조섬유는 식이섬유자동분석기 (Fibertec 2010)를 사용하여 측정하였다.The general component analysis of Schizandra chinensis was analyzed for crude fat, moisture, ash, crude protein and crude fiber. Carbohydrate content was calculated by subtracting the sum of the five components. Crude fat was determined by acid digestion, and moisture and ash content were measured using 105 ° C atmospheric pressure drying and direct ashing in accordance with the American Institute of Analytical Chemistry (AOAC). Crude protein was measured using an automatic nitrogen digestion distillation apparatus and crude fiber was measured using a dietary fiber automatic analyzer (Fibertec 2010).
나. 추출방법I. Extraction Method
시료는 분쇄한 오미자와 분쇄하지 않은 오미자를 중량비로 10배 (w/v) 용량의 용매를 혼합하여 추출하였다. 분쇄 오미자는 건조된 오미자를 전량 분쇄하였다. 추출용매는 물, 헥산, 에탄올 (25, 50, 75, 100%)을 사용하였으며, 추출시간은 3시간으로 하였다. 온도는 용매 및 시간을 달리하여 40~80℃에서 추출하였다. 추출물 은 와트만(Whatman (No.3))여과지를 이용하여 감압여과한 후 부피를 보정하였다.The sample was extracted by mixing a crushed Schizandra chinensis and uncrushed Schizandra chinensis in a weight ratio of 10 times (w / v). The ground schizandra crushed all of the dried schizandra. Water, hexane, ethanol (25, 50, 75, 100%) was used as the extraction solvent, the extraction time was 3 hours. The temperature was extracted at 40 ~ 80 ℃ by varying the solvent and time. The extract was filtered under reduced pressure using Whatman (No. 3) filter paper and the volume was corrected.
다. 추출물의 쉬잔드린(schizandrin) 분석All. Schizandrin Analysis of Extracts
오미자 추출물의 쉬잔드린(schizandrin)은 고성능 액체크로마토그래피 (HPLC, High Perfomance Liquid Chromatography, 515 pump, Waters, USA)를 이용하여 분석하였다. 컬럼은 워터스(Waters Sunfire (C18, 5 ㎛, 4.6×150 mm))를 이용하였으며, 검출기는 유브이 디텍터(UV Detector)를 사용하였다. 이 때 크로마토그램은 252 nm의 파장에서 얻었다. 이동상의 조성은 아세토니트릴 55%와 물 45%를 조성 변화 없이 등용매 (isocratic)로 전개하였으며, 흐름속도는 1.0 ㎖/min로 유지하였다. 추출물은 시린지 필터 (syringe filter, Minisart RC15, 0.45 ㎛, Sartorius, Germany)를 사용하여 여과한 후 10 ㎕를 HPLC에 주입하여 20분간 전개시켰다. 쉬잔드린(schizandrin)의 표준시료는 20 mg을 70% 메탄올에 녹인 후, 1, 10, 100, 1000 ppm으로 제조하여 전개시켰다. 얻어진 크로마토그램 피크의 적분 값을 토대로 한 검량곡선 (calibration curve)을 이용하여 정량분석 하였다.Schizandrin of Schisandra chinensis extract (schizandrin) was analyzed using high performance liquid chromatography (HPLC, High Perfomance Liquid Chromatography, 515 pump, Waters, USA). The column used Waters (Waters Sunfire (C18, 5 μm, 4.6 × 150 mm)) and a detector using a UV detector. At this time, the chromatogram was obtained at a wavelength of 252 nm. The composition of the mobile phase was 55% acetonitrile and 45% water was developed in isocratic without changing the composition, the flow rate was maintained at 1.0 mL / min. The extract was filtered using a syringe filter (syringe filter, Minisart RC15, 0.45 μm, Sartorius, Germany), and then 10 μl was injected into HPLC and developed for 20 minutes. The standard sample of schizandrin was prepared by dissolving 20 mg in 70% methanol and then preparing 1, 10, 100, 1000 ppm. Quantitative analysis was performed using a calibration curve based on the integral of the obtained chromatogram peaks.
라. 갈색도에 의한 안토시아닌(Anthocyanin) 색소 비교la. Comparison of Anthocyanin Pigments by Brownness
오미자 안토시아닌(anthocyanin) 색소의 추출조건별 변색에 대한 안정성 지표로 갈색도(BI:Browning Index)를 사용하였다. 분광광도계를 이용하여 오미자 안 토시아닌 색소와 갈색 색소가 각각 최대 흡광도를 나타내는 525 nm와 420 nm에서의 흡광도 비로부터 BI를 계산하여 오미자 안토시아닌 색소를 비교하였다.The brownness index (BI: Browning Index) was used as a stability index for discoloration of extracts of Schisandra chinensis anthocyanin. Using spectrophotometer, BI was calculated from the absorbance ratios at 525 nm and 420 nm, respectively, where Schisandra chinensis anthocyanin pigment and brown pigment exhibited maximum absorbance.
BI=A525/A420 BI = A 525 / A 420
A525 = 흡광도(Absorbance at 525 nm)A 525 = Absorbance at 525 nm
A420 = 흡광도(Absorbance at 420 nm)A 420 = Absorbance at 420 nm
마. 최적추출조건 설정을 위한 반응표면 분석법hemp. Response Surface Methodology for Optimal Extraction Conditions
본 실험에서는 오미자의 추출조건 최적화를 위하여 반응표면분석법 (Response Surface Methodology)을 이용하였다. 추출용매, 추출온도를 독립변수로 설정하였으며, 이 독립변수에 영향을 받는 종속변수로서 쉬잔드린의 농도와 갈색도를 측정하여 표 1과 같이 최적점 도출에 사용하였다.In this experiment, response surface methodology was used to optimize the extraction conditions of Schizandra chinensis. The extraction solvent and extraction temperature were set as independent variables. As the dependent variables affected by the independent variables, the concentration of xanthrin and the brownness were measured and used to derive the optimum point as shown in Table 1.
건조 오미자의 일반성분을 분석한 결과 수분 12.23%, 회분 3.25%, 조단백질 7.17%, 조지방 23.0%, 조섬유 14.54% 및 탄수화물이 39.78%로 각각 나타났다. As a result of analyzing common ingredients of dried Schizandra chinensis, water 12.23%, ash 3.25%, crude protein 7.17%, crude fat 23.0%, crude fiber 14.54% and carbohydrate 39.78%.
분쇄한 오미자의 쉬잔드린은 온도의 영향은 크지 않았으나 에탄올의 농도가 증가할수록 쉬잔드린 함량도 증가하였다. 쉬잔드린에 대한 추출조건을 최적화하고자 반응표면분석(RSM) 프로그램을 이용 하여 분석한 결과, 도 1과 같이 추출온도 40℃, 추출용매 75% 에탄올에서 7.0 mg/g으로 최적 점을 보였다. 이 최적점을 바탕으로 추출한 추출물의 쉬잔드린 함량은 도 2에서 보는바와 같이 높은 추출효율을 보였다.In the shredded Schizandra chinensis, the effect of temperature was not significant, but as the concentration of ethanol increased, the xanthrin content increased. As a result of analysis using the reaction surface analysis (RSM) program to optimize the extraction conditions for xanthrin, the optimum temperature was 7.0 mg / g in the extraction temperature of 40 ℃, 75% ethanol extraction solvent. The xanthrin content of the extract extracted on the basis of this optimum point showed a high extraction efficiency as shown in FIG.
BI(Browning Index)는 오미자 추출물의 최대흡수파장인 525nm 부근에서의 흡광도와 갈색 색소의 최대흡수파장인 420 nm에서의 흡광도의 비로 나타낸 것이다. 안토시아닌(anthocyanin)이 파괴되면 붉은 색이 퇴색되어 500~535nm 부근에서 흡광도가 감소하고, 독립적인 반응에 의해 갈색 색소가 생성되어 420 nm 부근에서 흡광도가 증가한다. 따라서 이번 연구에서는 이를 이용하여 오미자 안토시아닌 색소를 비교하였다. 갈색도 측정 결과, 도 3과 같이 추출용매의 에탄올 함량과 추출 온도가 낮을수록 높게 나타났으며, 40℃ 물 추출에서 2.6으로 최적점을 보였다. 이는 가열온도 및 에탄올 함량이 증가할수록 BI가 감소하여 안토시아닌 색소가 파괴되고 갈색색소가 생성되었음을 시사하고 있다. 헥산추출의 경우 쉬잔드린 함량은 7.1 mg/g으로 높게 나타났으나, 갈색도 값이 현저하게 낮아 오미자 추출물의 관능성을 고려할 때 극성용매를 사용하여 추출하는 것이 음료화 과정에서 유리하다고 사료된다.The Browning Index (BI) is expressed as the ratio of absorbance near 525 nm, the maximum absorption wavelength of Schizandra chinensis extract, and absorbance at 420 nm, the maximum absorption wavelength of brown pigment. When anthocyanin is destroyed, the red color fades and the absorbance decreases in the vicinity of 500 to 535 nm, and a brown pigment is generated by an independent reaction to increase the absorbance in the vicinity of 420 nm. Therefore, this study used this to compare Schisandra chinensis anthocyanin pigment. As a result of the brownness measurement, as shown in Figure 3, the lower the ethanol content and the extraction temperature of the extraction solvent was higher, and showed an optimum point of 2.6 at 40 ℃ water extraction. This suggests that BI decreased with increasing heating temperature and ethanol content, resulting in the destruction of anthocyanin pigment and the formation of brown pigment. In the case of hexane extraction, the xanthrin content was high as 7.1 mg / g, but the brownness value was very low, and it is considered that it is advantageous to extract using polar solvent in consideration of the functionality of Schizandra chinensis extract.
쉬잔드린 함량과 오미자 색의 지표인 갈색도값을 동시에 충족시켜주는 최적 추출조건을 찾기 위해 분쇄 오미자의 두 결과 그래프를 겹쳐 도 4와 같이 등고선 그래프로 나타내었다.In order to find the optimum extraction condition that satisfies the brownish value, which is an indicator of the Schizandrarin content and Schizandra color, two graphs of the shredded Schizandra chinensis were overlaid as shown in Fig. 4.
쉬잔드린은 추출용매로서 에탄올의 함량이 높을 때 많이 추출되었으며, 갈색도 값은 반대로 에탄올의 함량이 적을 때 증가하였다. 오미자의 기능성분인 쉬잔드린함량을 높게 해야 하지만, 쉬잔드린의 함량이 높을 조건은 갈색도값이 낮아져 제품의 관능성을 떨어뜨린다. 따라서 오미자 고유의 색깔을 충분히 유지하면서 쉬잔드린의 함량을 높일 수 있는 최적점을 도출하였다. 그 결과, 온도는 46℃, 에탄올의 농도는 24%에서 쉬잔드린의 함량은 최고점의 76%인 5.4 mg/g을 나타내었으며, 갈색도값은 최고점의 76%인 1.824를 나타내었다. Xjandrin was extracted much when the ethanol content was high as an extractant, and the brownness value was increased when the ethanol content was low. It is necessary to increase the content of Xuzanrin, a functional ingredient of Schizandra chinensis, but high Xuzanrin content lowers the sensory value of the product due to the lower brown value. Therefore, the optimum point to increase the content of xyzandrin while maintaining sufficient color of Schizandra chinensis was derived. As a result, the temperature was 46 ° C, the concentration of ethanol was 24%, and the content of xanthrin was 5.4 mg / g, which was 76% of the highest point, and the brownness value was 1.824, which is 76% of the highest point.
도 1은 쉬잔드린에 대한 분쇄오미자의 추출조건 최적화 그래프이다.1 is a graph illustrating the optimization of extraction conditions of ground Schizandra chinensis against Xanthrin.
도 2는 쉬잔드린 표준물질과 분쇄오미자 40℃, 75% 에탄올 추출 시 쉬잔드린 함량을 액체크로마토그램으로 나타내어 비교한 것이다.Figure 2 is a comparison of the Xuzanrin standard and the crushed
도 3은 갈색도(BI:browning index)에 대한 분쇄오미자의 추출조건 최적화 그래프이다.3 is a graph illustrating optimization of extraction conditions of ground Schizandra against browning index (BI).
도 4는 오미자의 추출조건 최적화 그래프이다.Figure 4 is a graph of the optimization condition of Schizandra chinensis.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR101054903B1 (en) | 2009-09-10 | 2011-08-05 | 부산대학교 산학협력단 | Extraction method of lignans from Schisandra chinensis seeds |
| CN102731271A (en) * | 2011-12-25 | 2012-10-17 | 大兴安岭林格贝有机食品有限责任公司 | New method for purifying schisandrin |
| KR20170009470A (en) | 2015-07-17 | 2017-01-25 | 종근당건강 주식회사 | Composition for protectng liver cells comprising Schisandra chinensis Concentrate containing elevated amounts of effective ingredients and method for manufacturing the same |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR101054903B1 (en) | 2009-09-10 | 2011-08-05 | 부산대학교 산학협력단 | Extraction method of lignans from Schisandra chinensis seeds |
| CN102731271A (en) * | 2011-12-25 | 2012-10-17 | 大兴安岭林格贝有机食品有限责任公司 | New method for purifying schisandrin |
| CN102731271B (en) * | 2011-12-25 | 2014-11-19 | 大兴安岭林格贝有机食品有限责任公司 | New method for purifying schisandrin |
| KR20170009470A (en) | 2015-07-17 | 2017-01-25 | 종근당건강 주식회사 | Composition for protectng liver cells comprising Schisandra chinensis Concentrate containing elevated amounts of effective ingredients and method for manufacturing the same |
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