Ethnobotanical study of medicinal plants used by Sidama people of Hawassa Zuria district, Sidama Zone, Southern Ethiopia

BANCHIAMLAK NIGUSSIE TEFERA 한림대학교 대학원 2019 국내박사

Ethiopia is one of the species-rich countries in the world and the center of origin with regard to the diversity of many plant species. Ethnobotanical studies are vital to investigate these diverse biological resources for medicinal purposes. Thus, the aim of this study was to document the indigenous knowledge of Sidama people on the use of medicinal plants to treat human and livestock diseases in Hawassa Zuria district of Southern Ethiopia. Ethnobotanical survey was conducted in ten kebeles of Hawassa Zuria districts. A total of 150 informants (118 men and 32 women) were selected to collect ethnobotanical information from ten kebeles by means of a stratified purposive sampling method. Among the informants, 30 key informants were selected purposefully. Ethnobotanical data were collected through semi-structured interviews and group discussions and were analyzed by descriptive statistics, informant consensus factor (ICF), fidelity level (FL) and ranking methods. A total of 105 medicinal plants distributed across 52 families and 96 genera were collected. Fabaceae (11 species) was represented by the highest number of plant species, followed by Lamiaceae (7 species). Herbs (34%) were dominant growth habit of plants,followed by trees (33%). Leaves (56%) were the dominant plant part used in the preparation of remedies,followed by fruit (15%). The most common methods of remedy preparation were grinding (39%), followed by chewing anf boiling (11% each). Oral (74%) was the major routes of administration, followed by dermal (20%). There was a significant knowledge difference (p<0.05) between social groups regarding the use of traditional medicinal plants. Insects and ectoparasites disease category (ICF = 0.95) had higher informant consensus factor value followed by fever disease category (ICF = 0.91). Eucalyptus globulus Labill. (FL =100%), had a higher fidelity level to treat stomach-ache, followed by Ensete ventricosum (Welw.) Cheesman. (FL =87.27%) to treat placenta delay. Ensete ventricosum (Total score = 73) was ranked highest as the most preferable medicinal plant for various purposes by local people, followed by Olea welwitschii (Knobl.) Gilg (Total score = 72). The present study revealed the existence of indigenous knowledge of medicinal plants to treat human and livestock ailments. However, agricultural expansion, firewood collection, environmental degradation, and deforestation are the main threats to medicinal plants. Therefore, there should be mentoring for the local people in the study area to conserve their indigenous knowledge resources and prevent the extinction of medicinal plants 에티오피아는 식물 종의 다양성이 풍부한 곳이며 많은 식물 종의 원산지이다. 의약학 분야에서 다양한 생물 자원을 조사하는 것은 민속식물학 연구의 중요한 분야이다. 이 연구의 목적은 에티오피아 남부 하와사 주리아 지역에서 시다마 사람들이 인간과 가축의 질병을 치료하는 데 사용하는 약용식물의 전통지식을 수집하는 것이다. 민속식물학 연구는 하와사 주리아 지역의 열 개 케벨 (마을)을 대상으로 수행하였다. 총 150명의 정보 제공자(남자 118명, 여자 32명)를 대상으로 반계층적 표본추출 방법을 사용하여 전통지식 정보를 수집하였다. 정보 제공자 중 30명은 특별히 선택되었다. 인터뷰와 그룹 토론을 통해 수집한 정보를 정보 합치도, 충실도 레벨 및 순위 결정 등의 통계적 방법으로 분석하였다. 총 52개 과 96개 속에 포함되는 105 종의 약용식물에 대한 정보를 수집하였다. 이 가운데 콩과 (11종)가 가장 많았고, 꿀풀과 (7종)가 그 뒤를 이었다. 초본 (34%)이 가장 많았고 교목 (33%)이 그 뒤를 이었다. 사용 부위는 잎이 56%로 가장 많았고 열매 (15%)가 그 뒤를 이었다. 이들 약용식물의 가장 일반적인 처리 방법은 연삭이었으며 (39%) 저작과 끓이는 것이 각각 11%로 그 뒤를 이었다. 투여 로는 경구 (74%)가 주를 이뤘고 피부 도포(20%)가 그 뒤를 이었다. 약용식물의 전통지식에 있어서 집단 간에 유의미한 차이(p <0.05)가 존재하였다. 곤충 및 체외 기생충이 일으키는 질병 범주가 정보 합치도(0.95)가 가장 높았고 발열질환 질병 범주의 정보 합치도(0.91)가 그 뒤를 이었다. 충실도의 경우 Eucalyptus globulus가 복통을 치료하는 것과 관련하여 가장 높았고 (100%) Ensete ventricosum이 태반 지연을 치료하는 것과 관련하여 그 뒤를 이었다 (87.27%). Ensete ventricosum은 지역 주민들이 가장 선호하는 약용식물이었고(총점 73), Olea welwitschii가 그 뒤를 이었다(총점 72). 본 연구를 통하여 인간과 가축의 질병을 치료하기 위한 약용식물에 대한 전통지식의 존재가 확인되었다. 한편 농지 확장, 땔감 수집, 환경 파괴 및 삼림 벌채는 약용식물의 주된 위협이다. 따라서 지역 주민들이 전통지식 자원을 보존하고 약용식물의 멸종을 막기 위한 계도 노력이 필요하다. 키워드: 약용식물, 전통지식, 하와사 주리아 지역, 시다마족, 에티오피아

Novel computational methods for natural product (NP) research : development of natural compound molecular fingerprint (NC-MFP) & construction of metabolite and plant networks

서명원 Graduate School, Yonsei University 2020 국내박사

천연물 성분은 살아 있는 유기체에 의해 생성된 천연 화합물을 말한다. 많은 신약 후보 물질들이 천연 화합물로부터 유래되었고 특히 식물은 영양 성분 및 의약적 특성이 풍부하기 때문에, 천연물과 생물학적 활성 사이의 관계에 대해 컴퓨터를 이용한 연구가 진행되어 왔다. 컴퓨터를 이용하여 천연물을 현실적으로 연구하기 위해서는 새로운 계산 방법이 필요하다. 본 학위 논문에서는 천연물 기반 약물 개발 및 잠재적인 약용식물 탐색을 위해 컴퓨터를 이용한 새로운 두 가지의 계산 방법을 제안하였다. 첫 번째는 천연 화합물 구조 분류 및 천연 화합물 구조와 생물학적 활성과의 관계를 설명하기 위한 천연 화합물 분자지문 (NC-MFP)을 개발하였다. 두 번째는 잠재적인 약용식물 탐색을 위해 대사물질 네트워크와 식물 네트워크를 구축한 내용이다. 분자 구조를 표현하기 위한 방법은 약물 개발에 활용되어 왔다. 특히 합성 화합물을 기반으로 하는 분자 구조의 생물학적 특성을 설명하기 위해서 여러가지의 위상 설명자(topological descriptor)들이 개발되었다. 천연 화합물 구조는 합성화합물과 비교하여 독특한 구조적 특성을 가지기 때문에 이전에 개발되었던 위상 설명자들은 천연물의 분자구조를 설명하는데 한계가 있다. 이번 연구에서는 생물학적 활성과 관련된 천연 화합물 구조를 설명하기 위해 천연 화합물 분자 지문 (Natural compound molecular fingerprint, NC-MFP)이라고 하는 새로운 분자 지문을 개발하였다. 천연 화합물 분자 지문은 분자 골격 (Scaffold), 분자 골격-조각 연결 지점 (Scaffold-fragment connection point), 그리고 분자 조각 (Fragment)로 구성된 분자 골격 기반의 분자 지문이다. 천연물의 분자 골격 정보는 Bemis and Murko 가 제안한 방법과 천연물 사전 데이터베이스(Dictionary of Natural Product, DNP)의 천연 화합물 분류 시스템을 기반으로 구성하였다. 새로운 천연 화합물 분자 지문에 대한 평가를 위해 2 가지의 이진 분류 작업을 진행하였다. 첫 번째 분류 작업은 상용화 된 화합물 데이터베이스에 있는 정보들로 훈련하여 천연 화합물 구조를 합성 화합물 또는 천연 화합물 구조로 분류하는 작업이다. 두 번째 분류 작업은 7 개의 표적 단백질에서 억제 활성 정보를 포함하는 천연 화합물 구조에 대해 활성인지 비활성인지 분류하는 작업이다. 두 가지 분류 작업에 대한 결과에서 천연 화합물 분자 지문(NC-MFP)이 다른 분자 지문들보다 우수한 성능을 보였다. 따라서 천연물 화합물 분자 지문(NC-MFP)은 천연 화합물 기반 약물 개발에서 가상 스크리닝에 적용 가능한 강력한 분자 지문이다. 약용식물은 다양한 특성을 가지고 치료 목적으로 사용되어 왔다. 약용식물에는 물질 대사로부터 생성된 다양한 대사 산물이 포함되어 있다. 대사 물질들은 물리화학적 및 다양한 화학구조를 가지는 활성 대사물질이기 때문에, 약용식물은 생물학적 또는 치료적인 특성을 가진다. 두 식물 간에 공통 대사 산물을 공유하는 것은 생물학적 또는 치료적인 특성을 공유할 수 있기 때문에, 생물학적 활성과 관련된 식물 관계는 대사물질 함량 유사성 (Metabolite-content similarity)을 기초로 계산될 수 있다. 대사체학의 발전에 따라 대사 산물–식물 연관 정보와 관련된 데이터베이스가 구축되었다. 그러나 연구된 식물에 따라서 알려진 대사 물질의 숫자가 다르기 때문에, 식물의 대사물질 함량 데이터는 불균형 하다. 불균형한 대사물질 함량 데이터를 극복하기 위해서, 대사 물질 네트워크 (Metabolite networ, MN)를 대사물질의 구조적 유사성, 대사 경로 유사성 및 생물학적 기원 유사성을 기반으로 구축하였다. 그 다음 대사 물질 네트워크로부터 식물의 대사 물질을 추론하고 대사 물질 함량 유사성에 기초한 타니모토 거리 (Tanimoto distance)로 식물 간의 관계를 계산하였다. 전 세계 식물들의 잠재적인 약용식물을 탐색하기 위해서 식물 네트워크 (Plant network, PN)를 구축하였다. 잠재적인 약용식물은 식물 네트워크와 예측 점수 (Prediction coefficient score)를 사용하여 탐색되었다. 결과적으로, 예측 점수를 기준으로 10 개의 잠재적인 약용식물을 탐색하였고, 그 중 상위 5 개의 예측된 생물학적 활성은 문헌을 통해 검증하였다. 따라서 대사 산물과 식물을 기반으로 네트워크 구축 및 분석은 잠재적 약용식물 탐색에서 유용한 방법이다. An ingredient of a natural product (NP) is a natural compound (NC) produced by a living organism. Since many of new drugs candidates are analogous to or derived from the NCs and the plants of NPs usually have rich in the nutritional component or medicinal properties, computer-aided research on the relationship between the NPs and their biological activities have been widely studied. To physically realistically research for NPs using a computer, it is necessary for the novel computational methods. In this thesis, two novel computational methods were developed for exploring new NP-based drugs and the potential medicinal plants. The first study is about the development of Natural Compound Molecular Fingerprint (NCMFP) to classify NC structures and explain between NC structures and their biological activities. The second study is about the construction of metabolite and plant networks to explore potential medicinal plants. Computational approaches for describing molecular structure have been widely using for drug discovery research. Notably, several topological descriptors have been developed to describe the biological properties of molecular structure based on synthetic compounds. Since the structure of NC has distinctive structural characteristics compared to synthetic compound, developed topological descriptors have limitations in expressing the molecular structure of specific NC. In the first study, novel molecular fingerprint, called Natural Compound Molecular Fingerprints (NC-MFP) was developed for explaining NC structures related to biological activities. The NC-MFP is a scaffold-based molecular fingerprint method that consists of scaffolds, scaffold-fragment connection points (SFCP), and fragments. The scaffolds of NC-MFP constructed based on both the Bemis and Murko (BM) method and the classification system of the Dictionary of Natural Product database (DNP). Two kinds of binary classification tasks were introduced for evaluation purposes. In the result of tasks, NC-MFP outperformed compared with other molecular fingerprints. Therefore, NC-MFP is a potent molecular descriptor for the virtual screening of NC for NP-based new drug development. Medicinal plants have various medicinal properties and use for therapeutic purposes. Medicinal plants contain various metabolites produced from the metabolism. Since these metabolites are bioactive metabolites with physicochemical and diverse chemical structures, medicinal plants have biological or therapeutic properties. Therefore, since sharing common metabolites between two plants can share biological or therapeutic properties. With the rapid development of metabolomics, the database related to metabolite-plant relation has been created. However, since the number of known metabolites is different according to the researched plants, the metabolite-content data of plants is unbalanced. In order to overcome the unbalanced metabolite-content data, the metabolite network (MN) was constructed. Then, metabolites of plants were inferred by the MN, and the plant relationship was calculated by Tanimoto distance (Td) based on metabolite content similarity. To explore the potential medicinal plants from the plants in the world, the plant network (PN) was constructed. The potential medicinal plants were explored from the plants by using the prediction coefficient scores in the PN. As a result, ten potential medicinal plants were explored according to the prediction coefficient score. Of that, the biological activities of the top five plants were validated using the literature. Therefore, the construction of networks based on metabolites and plants would be a useful method for exploring potential medicinal plants.

약용식물의 발효 균주 선발을 통한 반추위 발효 개선에 관한 연구

이아름 전북대학교 일반대학원 2014 국내석사

This study was conducted to improvement of rumen fermentation via microbial strain selection for fermenting medicinal plants. There were three experiments in this study, and their biological activity was determined with antibacterial activity and antioxidant activity assay. In first experiment, the β-glucosidase - producing lactic acid bacteria was firstly isolated from salted fish and Korean traditional fermented cabbage called Kim-chi. The isolation was based on MRS - enriched medium and twenty-six colony showing β-glucosidase activity. For the identification of isolated β-glucosidase producing bacteria. The 16S rRNA gene sequence was analysed. Finally those bacteria separated by high β-glucosidase activity were identified as six isolates such as Weissella confusa, Weissella cibaria, Lactobacillus curvatus, Lactobacillus brevis, Lactobacillus plantarum, and Lactobacillus sakei, respectively. In second experiment, ten medicinal plants such as Eucommia ulmoides Oliv., Allium hookeri, Dioscorea quinqueloba Thunb., Asarum sieboldii, Hovenia dulcis Thunb., Morus alba L., Acanthopanax senticosus, Leonurus japonicus Houtt., Glycyrrhiza uralensis and Platycodon grandiflorum Nakai were used, and they were fermented with the six lactic acid bacteria as we used in the first experiment. The medicinal plants were extracted by ethanol, and antibacterial activity was investigated. Antibacterial activity was improved in L. curvatus NJ40 and L. plantarum NJ45 compared with control which fermented without lactic acid bacteria. Thus, L. curvatus NJ40 and L. plantarum NJ45 could be recommendable starter culture. In third experiment treatments, there were six treatments; 1) control, 2) garlic oil, 3) ethanol, 4) medicinal plants extract only, 5) medicinal plants + L. curvatus NJ40 extract, and 6) medicinal plants + L. plantarum NJ45 extract, respectively. The incubation time was 24 h only at 39℃. The in vitro ruminal pH was similar across the treatments. It appears that volatile fatty acid production was relatively higher in fermented medicinal plants with lactic acid bacteria treatments compared with control. Based on the present result, fermenting medicinal plants with L. curvatus and L. plantarum more desirable methane inhibitor compared with others.

약용식물에서 UPLC-ESI-Q-TOF/MS를 이용한 주요 알칼로이드 분석법 개발 및 응용

정원태 충북대학교 2020 국내박사

Alkaloids are organic compounds containing nitrogen, which are mainly present as secondary metabolites in plants and show distinct physiological activity in the human body. Their qualitative and quantitative analyses are difficult owing to their diverse chemical structures. Some alkaloids are analyzed using liquid chromatography, but the number is limited depending on the system used. Recently, ultra-performance liquid chromatography-electrospray ionization quadrupole time of flight mass spectroscopy (UPLC-ESI-Q-TOF/MS) has attracted attention as an excellent technique for the rapid and accurate identification and quantification of natural source, including alkaloids. In this study, UPLC-ESI-Q-TOF/MS has been proposed as a quick and accurate method to analyze the major alkaloids belonging to six chemical groups (isoquinoline, indole, pyrrolizidine, quinoline, pyridine, and tropane), and has been applied to medicinal plants. Furthermore, a chemical profiling of alkaloids obtained from the extracts of medicinal plants has been proposed. Alkaloids containing isoquinoline, pyrrolizidine, and quinoline groups show excellent peak resolution upon elution with a 0.1% formic acid solution. Indole, nicotine, and tropane were rapidly separated into peaks with high-resolution resolution when the LC column was eluted with buffered aqueous solution containing ammonium formate. The fragmentation ions generated by ESI-Q-TOF/MS undergoing collision-induced dissociation (CID) were observed and the molecular structure fragmentation pathway was identified. The mass spectra were used as a library to identify alkaloids and as an important source for non-target alkaloids. The UPLC-ESI-Q-TOF/MS method for each alkaloid group was validated. Representative plants containing alkaloids of each series were selected and their chromatograms and mass spectra of them were obtained. Their constituent alkaloids were identified and their contents calculated by matching the retention time, ionization form, and fragment ions of the standard samples. In addition, eight isoquinoline alkaloids found in Chelidonium majus L., six terpenoid indole alkaloids in Catharanthus roseus, three pyrrolidine alkaloids in four plants, and one tobacco alkaloid found in tobacco products were identified. In particular, pyrrolizidine alkaloids provided a table capable of qualitatively identifying 73 alkaloids by their fragmentation ions. Interpretation of the data obtained using UPLC-ESI-Q-TOF/MS minimized the ambiguity of the quality of the compounds and reduced the time used to identify the target and non-target compounds. It has been described in detail from sample processing to the determination of the compounds. This approach has been found to provide excellent chromatogram and mass information for the structural elucidation of compounds in complex extracts and is expected to be useful for the identification of alkaloids in medicinal plants.

EFFECTS OF FERMENTED MEDICINAL PLANTS (GYNURA PROCUMBENS, REHMANNIA GLUTINOSA, SCUTELLARIA BAICALENSIS) ON GROWTH PERFORMANCE IN NONRUMINANT ANIMALS

정미경 단국대학교 2016 국내석사

EXPERIMENT 1: Effect of fermented medicinal plants on growth performance, nutrient digestibility, fecal noxious gas emissions, and diarrhea score in weanling pigs Antibiotics used as growth promoters in livestock has been banned in European Union since 2006. Alternatives of antibiotics have focused on phytogenic plants, such as herb and medicinal plant. No studies have evaluated use of fermented medicinal plants (FMP) made up of Gynura procumbens, Rehmannia glutinosa, and Scutellaria baicalensis in weanling pigs. Therefore, an experiment was conducted to determine the effects of FMP on growth performance, nutrient digestibility, fecal noxious gas emissions, and diarrhea score in weanling pigs. FMP supplementation increased (P < 0.05) average daily gain, average daily feed intake, gain:feed, apparent total tract digestibility of dry matter, nitrogen, and gross energy compared with NC treatment, while a linear effect (P < 0.05) was observed on those criteria. Ammonia, total mercaptans, and hydrogen sulfide concentrations were decreased (P < 0.05) by the supplementation of FMP compared with NC. Additionally, diarrhea score was lower (P < 0.05) by FMP addition compared with NC during d 0 to 7 and d 8 to 14. These results suggested that FMP could be used as alternative of antibiotics by enhancing growth performance, nutrient digestibility, and decreasing fecal noxious gas emission and early diarrhea score of weanling pigs. EXPERIMENT 3: Egg production, Egg quality, and Cecal Microbial Populations of Layers fed Diets Supplemented with Fermented Medicinal Plant A study was undertaken to examine the effect of fermented medicinal plant (FMP) containing G. procumbens and R. glutinosa on egg production, cecal microflora population and egg quality in layers. In total, 288 Hy-line brown layers (35 wks of age) were randomly divided into 4 dietary treatment groups with 6 replicate pens (12 birds/replicate) per treatment group for 5 wks. The dietary treatments were a control basal diet without FMP or 0.5, 1, 2 % of FMP. Egg production and egg weight during experiment during 35-39 wks improved linearly (P <0.05) by the supplementation of FMP. The results of the cecal microflora population of Lactobacillus showed a linear increase, whereas cecal microflora population of E. coli showed a linear decrease related to the level of FMP addition in the diet (P <0.05). Eggshell strength, albumen height and Haugh unit were significantly higher in birds fed FMP compared with the control groups (P <0.05). These results indicate that adding FMP to the diet of layer can improve their laying performance, egg weight, eggshell strength, albumen height, Haugh unit and cecal microflora; therefore, diets containing FMP may enhance the physiologic condition of layers during laying period. EXPERIMENT 2: Effect of fermented medicinal plants (Gynura procumbens, Rehmannia glutinosa, Scutellaria baicalensis) as alternative performance enhancers in broilers In the present study, the effect of dietary supplementation of mixed fermented medicinal plants (FMP) on growth performance in broilers and whether FMP have potential to serve as antibiotic growth promoter (AGP) alternatives was investigated. A total of 765 1-d-old male broiler chicks (ROSS strain) were used in a 5 wk feeding experiment and randomly divided into 5 groups with 9 replicates of 17 birds each. The treatments were NC (negative control, basal diet), PC (positive control, basal diet with 5 ppm enramycin), FMP 0.05 (basal diet with FMP 0.05%), FMP 0.1 (basal diet with FMP 0.1%), and FMP 0.2 (basal diet with FMP 0.2%). Both body weight gain and feed conversion ratios were improved, as compared with NC or in response to FMP supplementation, respectively. Additionally, dry matter and nitrogen retention, metabolizable energy, and excreta noxious gas emission of ammonia, hydrogen sulfide, and total mercaptans were improved as compared with NC or in response to FMP supplementation, demonstrating a positive correlation. The amount of intestinal microflora was improved (increased Lactobacillus spp counts and reduced E.coli counts) as compared with NC, PC, and a dose response increase of FMP supplementation, in both the small and large intestines. Taken all together, our results suggest and support the viable possibility that FMPs supplementation can be used as an effective alternative to AGPs for improving the performance of broilers.

Characterization of Aroma Extracts Isolated from Medicinal Plants : 약용식물체에서 분리한 향기 성분의 분석과 항산화 능에 관한 연구

가미현 東國大學校 大學院 2003 국내석사

건조된 감국, 고본 (稿本), 독활, 박하 (薄荷), 석창포, 송엽 (松葉), 우슬, 정향 (丁香), 창이자 (蒼耳子), 총백 (蔥白), 형개 (荊芥), 홍화 (紅花), 흑두 (黑豆)는 독특한 향을 지닌 한국의 전통적인 약용 식물체이다. 그들이 지닌 향기 성분의 항산화 능에 대하여 연구하였다. 약용 식물체의 향기 성분은 감압하의 증기 증류와 dichloromethane을 용매로한 액체-액체 연속 추출을 이용하여 추출하였다. 추출된 향기 성분은 gas chromatography/mass spectrometry (GC/MS)와 gas chromatography-flame ionization detector (GC-FID)를 이용하여 정성 및 정량 분석하였다. 향기 추출물의 항산화 능은 aldehyde/carboxylic acid assay를 사용하여 평가되었다. 200 ㎍/mL 수준에서, 30일 동안 고본, 독활, 박하, 석창포, 형개, 홍화의 향기 추출물의 항산화 능은 홍화 > 석창포 > 고본 > 박하 > 독활 > 형개 순이었다. 향기 추출물의 항산화 능은 잘 알려져 있는 항산화제인 α-tocopherol (Vitamin E)와 butylated hydroxytoluene (BHT)와 비교하였다. Dried Chrysanthemi Flos (Chrysanthemum morifolium Ramatuelle var. sinense Makino), Angelicae tenuissimae Radix (Angelica tenuissima Nakai), Araliae Radix (Aralia continentalis Kitagawa), Menthae Herba (Mentha arevensis L.), Acori Rhizoma (Acorus gramineus Soland), pine needles (Pinus sylvestris L.), Achyranthis Radix (Achyranthes japonica (Miquel) Nakai), clove buds (Syzygium aromaticum (L.) Merr. et Perry), Xanthii Fructus (Xanthium strumarium L.), Allium (Allium fistulosum L.), Nepetae Herba (Schzonepeta tenuifolia Briq.), Carthami Flos (Carthamus tinctorius L.), and black beans (Phaseolus vulgaris L.) are traditional medicinal plants in Korea and have their own characteristic aromas. Aroma compounds isolated from the medicinal plants were obtained using liquid-liquid continuous extraction following steam distillation under reduced pressure (55℃ and 95 mmHg). Aroma compounds were identified in the extracts using gas chromatography (GC) and gas chromatography/mass spectrometry (GC/MS). Antioxidant properties of the aroma extracts were investigated by aldehyde/carboxylic acid assay. At a concentration of 200 ㎍/mL, the antioxidative activities in the aroma extracts of Angelicae tenuissimae Radix, Araliae Radix, Menthae Herba, Acori Rhizoma, Nepetae Herba and Carthami Flos were in the following descending order: Carthami Flos > Acori Rhizoma > Angelicae tenuissimae Radix > Menthae Herba > Araliae Radix > Nepetae Herba, for 30 days. The antioxidative activities in aroma extracts were comparable to that of the known antioxidants, α-tocopherol (Vitamin E), and butylated hydroxytoluene (BHT).

Characterization of antioxidant properties of aroma constituents isolated from medicinal plants

이광근 Office of Graduate Studies of University of Califo 2000 해외박사

Aroma extracts isolated from medicinal plants have been widely used in aromatherapy and their aroma components are believed to possess medicinal properties, as well as antioxidant activity. Aroma extracts isolated from medicinal plants such as clove buds, eucalyptus leaves, and various beans, were prepared and examined for antioxidant activity by three different assays. The antioxidative activity of aroma extracts of mung beans, soybeans, and cloves as well as major aroma chemicals like eugenol, eugenyl acetate, and thymol were comparable to that of the natural antioxidant, α-tocopherol (vitamin E). The aroma extracts isolated from soybeans, mung beans, kidney beans, and azuki beans inhibited the oxidation of hexanal for nearly one month at a level of 250 μL/mL. Mung bean and soybean extracts inhibited malonaldehyde (MA) formation from cod liver oil by 86% and 88%, respectively, at 250 μL/mL level. Aroma compounds contained in the extracts of soybean and mung bean were identified by gas chromatography (GC) and gas chromatography/mass spectrometry (GC/MS). Among identified chemicals, eugenol, maltol, benzyl alcohol and 1-octen-3-ol inhibited the oxidation of hexanal by 100%, 93%, 84%, and 32%, respectively, for a period of 40 days at concentrations of 500 μg/mL. Eugenol, maltol, benzyl alcohol, and 1-octen-3-ol inhibited MA formation from cod liver oil by 91%, 78%, 78%, and 78%, respectively, at the 160 μg/mL level.

Molecular pharmacological mechanisms of ethnopharmacologically valuable medicinal plants from bangladesh

Muhammad Jahangir, Hossen Sungkyunkwan university 2016 국내박사

Despite the lacking of scientific verification of the effectiveness and the safety of natural products but due to cost-effectiveness and lacking of side effects, the using trend of ethnomedicine is becoming more popular as a complementary and alternative medicine. Besides the documenting of ethnomedicinal value of medicinal plants and compound, molecular evidence based scientific validation of traditional medicinal plant has been important path of modern research. Therefore, the immunopharmacological activities of the methanolic extract of the aerial parts of Phyllanthus acidus L (Pa-ME), Persicaria chinensis L (Pc-ME), and Xanthium strumarium L (Xs-ME), and ethanolic extract of panax ginseng root (BGF1K) were evaluated. Codonopsis lanceolata (Campanulaceae) is dicotyledonous herbaceous perennial plant has been widely used in traditional medicine and is considered to have medicinal properties to treat diseases and symptoms such as bronchitis, coughs, spasm, psychoneurosis, cancer, obesity, hyperlipidemia, edema, hepatitis, colitis, and lung injury. Therapeutic potentials of C. lanceolata and its previously reported molecular mechanisms are described here. Pa-ME and Pc-ME inhibited LPS-induced nitric oxide (NO), prostaglandin E2 (PGE2), interleukin-1 (IL-1), interleukin-6 (IL-6), inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX)-2 and tumor necrosis factor-(TNF-) release by RAW264.7 macrophages and strongly ameliorated HCl/EtOH-induced gastric ulcers, and LPS/D-GalN-intoxicated acute liver injury in mice. Moreover, these extracts suppressed activator protein-1 (AP-1) activation and Mitogen activated protein kinase (MAPK) phosphorylation in LPS-stimulated RAW264.7 macrophages cells and a U937 monocyte-like human cell line. BGF1K down regulated the NF-B pathway by suppressing Inhibitor of kappa B alpha kinase (IKK)and Interferon regulatory factor 3 (IRF3) pathway by suppressing TANK-binding kinase 1 (TBK1) according to gene reporter assay, immunoblot analysis and AKT overexpression experiment. Overall, data suggested that the suppression of IKK and TBK1, which mediates transcriptional activation of NF-B and IRF3 respectively, contributes to BGF1K broad spectrum inhibitory activity, and this extract can be further developed as a lead anti-inflammatory and antioxidative therapy. The production of NO and PGE2 were suppressed by Xs-ME. Moreover, orally administered Xs-ME renovated HCl/EtOH-induced gastric lesion and LPS/D-GalN-induced acute liver injury. Furthermore, this extracts downregulated the expression of iNOS, COX-2, IL-1, IL-6 and TNF-production and reduced the nuclear levels of NF-B and AP-1. Here, firstly identified resveratrol as one of polyphones of this extract which exhibited one of active anti-inflammatory components in Xs-ME and likely targeted as PDK1of NF-B and AP-1. By blocking PDK1 and MAPK kinase activity and their downstream transcription factor of NF-B and AP-1, Xs-ME yields anti-inflammatory activity in vitro and in vivo.

Development of analytical method for esculetin in medicinal plants using LC-MS/MS and esculetin-induced growth inhibition effect in vascular smooth muscle cells

윤은선 건국대학교 대학원 2012 국내박사

A liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis method was developed to determine the contents of esculetin in medicinal plants. The analysis was performed using multiple reaction monitoring (MRM) in negative mode, and an XBridgeTM C18 column (2.1×100 mm, 3.5 m) was used. Methanol and 0.1% formic acid were used for gradient analysis. The calibration curve showed good linearity (r2>0.9993). The limit of detection (LOD) and limit of quantitation (LOQ) were 0.02 and 0.07 ng/ml respectively. The intra- and inter-day precisions were 1.5-6.8% and 2.0-5.3%, respectively, and the accuracy was 102.0-110.2%. The contents of esculetin in 35 different plants were determined, and Fraxini cortex showed the highest content of esculetin (761.5-5,475.0 mg/kg). In Mori folium and Artemisiae capillaris herba, 5.2-21.5 mg/kg and 7.0-17.6 mg/kg of esculetin were found, respectively. In other medicinal plants, no esculetin was detected, or it was present at a concentration less than 10 mg/kg. The analysis method appears to be simple, sensitive, and reproducible. The pharmacological effects of esculetin isolated from medicinal plants were investigated as part of the new medicines development. Esculetin is known to inhibit the proliferation of vascular smooth muscle cells (VSMC). However, the signaling pathway by which esculetin mediates its molecular effects in VSMC remains to be identified. The present results suggest an unexpected role of the p38 MAPK signaling pathway in esculetin-induced inhibition of VSMC growth. Treatment of VSMC with esculetin resulted in significant growth inhibition and G1-phase cell cycle arrest, which was followed by down-regulation of cyclins and cyclin-dependent kinase (CDK) expression. This G1-phase cell-cycle arrest was due to up-regulation of p21WAF1 expression. In addition, esculetin treatment activated p38 MAPK and ERK1/2. Pretreatment with SB203580, which is a p38 MAPK specific inhibitor, or expression of the dominant negative p38 MAPK (DN p38 MAPK) gene blocked esculetin-induced p38 MAPK activation and p21WAF1 expression. Finally, both the growth inhibition and the down-regulation of CDKs induced by esculetin were suppressed by either SB203580 or the DN p38 MAPK mutant gene. In conclusion, these results demonstrate that activation of p38 MAPK contributes to esculetin-induced p21WAF1 expression in VSMC by decreasing both the cyclin D1/CDK4 and cyclin E/CDK2 complexes.

A Study for the Fermentation of Medicinal Plant to Develop Natural Skin Cosmetics

함영주 건국대학교 대학원 2014 국내박사

인간의 삶에서 즐거움을 주는 요인들 중 하나로 아름다움을 들 수 있다. 피부 관리용 화장품은 이러한 인간의 즐거움에 대한 갈망을 충족시킬 수 있는 것들 중 하나로 여겨져 왔다. 그러나 최근에는 피부 관리용 화장품이 단순한 아름다움에 대한 추구에 그치지 않고 건강한 삶의 유지에까지 그 정의가 확대되고 있다. 다양한 물질들이 피부 관리용 화장품 원료로 적용되어져 왔으며, 최근에는 식물에서 유래된 천연원료가 각광받고 있다. 따라서 현대의 피부 관리용 화장품 원료개발에 있어 식물유래 천연물질의 이해는 매우 중요하다. 본 연구는 주로 한약재 원료로 사용되는 약용식물들의 생리활성에 관한 것으로 다양한 약용식물들이 가지고 있는 생리활성을 분석하고 그 활성향상을 위한 방법으로 발효과정을 적용하는 방법에 관한 것이다. 약용식물에는 다양한 생리활성들이 포함되어 있으나, 대표적인 특징으로 페놀계 화합물에서 기인하는 항산화활성을 들 수 있다. 항산화활성이란 몸 안에 생성된 활성산소와 같은 자유기를 제거하는 능력을 말한다. 활성산소란 체내에서 영양소의 산화를 통한 에너지 생성과정에서 필수적으로 발생하는 것이나, 과도한 생성이나 연령이 증가하여 몸에 축적되게 되면 다양한 질병을 초래할 수 있다. 이에 적절한 활성산소의 제거는 건강유지와 질병예방에 필수적이라고 할 수 있다. 이에 본 연구에서는 주로 약용식물들이 가지고 있는 항산화활성을 조사하였다. 또한 이러한 항산화활성을 향상시키는 방법으로 발효과정을 적용하였다. 첫 번째 실험(Chapter2)은 베타글루코시다아제 활성이 우수한 미생물을 한국 전통막걸리 찌개미로부터 분리하였다. 베타글루코시다아제는 발효과정 중 식물에 포함된 폴리페놀 물질들의 생물전환에 관여하는 효소로 알려져 있어, 본 실험의 균주분리기준으로 사용하였다. 약 1,000여개의 콜로니들을 확보하여 효소활성을 정성적으로 평가한 결과 총 463개의 후보균주를 확보하였고, 다시 정량적인 효소활성평가를 통하여 활성이 우수한 상위 21개 균주를 최종 선발하였다. 이후 현미경관찰을 통하여 효모와 세균으로 분류하였고, 유전자염기서열을 이용하여 분리균주들을 동정한 결과, 분리균주들이 Saccharomyces cerevisiae와 Lactobacillus plantarum인 것으로 나타났다. 두 번째 실험(Chapter3)에서는 애기똥풀(Chelidoniummajusvarasiaticum), 엄나무(Kalopanaxseptemlobus), 칡잎줄기(Puerarialobataroot), 황금(ScutellariabaicalensisGeorgi), 및 대황(RheumrhabarbarumL.)등 총 5가지 약용식물들을 첫 번째 실험에서 얻어진 균주와 이미 확보하고 있는 균주들을 이용하여 발효시킨 후, 발효물의 에탄올 추출물을 제조하고 항산화활성 및 항균활성을 평가하였다. 발효진행 여부는 접종된 종균의 성장을 분석하여 평가하였다. 또한 발효 과정 중 식물에 포함된 항산화 관련물질들의 변화를 조사하기 위하여 thin layer chromatography와 DPPH시약을 이용하여 물질변화를 탐색하였다. 그 결과 대부분의 식물들에서 선발된 균주L. plantarum EJ 43균주가 가장 우수하게 성장하는 것을 알 수 있었으며, 항산화활성 또한 발효 후 향상되는 것을 알 수 있었다. 또한 항균활성도 발효과정 후에 향상됨을 알 수 있었다. 일련의 실험결과 식물발효를 위 종균으로 L. plantarum EJ 43균주가 가장 우수한 것을 알 수 있었으며, 항산화활성 및 항균활성을 종합한 결과, 대황이 가장 우수한 식물임을 알 수 있었다. 이에 선발된 균주와 대황을 이용하여 발효 최적화 실험을 진행하였다. 세 번째 실험(Chapter4)에서는 선발된 균주가 대황을 발효할 때에 가장 우수한 배지성분을 탐색하기 위하여 진행되었다. 총 9가지의 배지성분들을 이용하여 부분요인 실험설계법으로 각 배지성분들의 효과를 분석하였다. 발효효율은 균체성장, 항산화활성 그리고 항균활성을 기준으로 하였다. 그 결과 포도당, 마늘즙, 양파즙, 효모추출물 그리고 magnesium sulfate가 가장 우수한 배지성분으로 나타났다. 일련의 실험결과, 다양한 약용식물들의 항산화활성 및 항균활성을 알 수 있었으며, 특히 발효과정을 통하여 효능이 향상될 수 있음을 알 수 있었다. 특히 향후 피부 관리용 화장품 개발에 있어 효과적인 원료선발에 대한 기초적인 자료로 본 연구결과를 활용할 수 있을 것이다. Beauty is regarded as an element that gives pleasure to human being and on his life. The cosmetics including skin care products have been used for satisfying basic desires of human being toward the pleasure related to beauty. Recently, however, the application of those skin care cosmetics is not only on fulfillment for beauty but also maintenance of health life. Various compounds have been developed and applied to skin care cosmetics and natural products from plants were mostly interested compounds, recently. Therefore the understanding of natural product originated from plant is very important for the development of modern skin care cosmetics. The present study is about biological activity of medicinal plants that are used for oriental medicine and about the analysis of biological activity and about fermentation process to improve their activity. Plant has various compounds and phenolic compounds and their antioxidant activity can be a representative substance and feature that are relevant biological activity of plant. Antioxidant activity can be defined as an activity that can scavenge reactive oxygen and free radicals in our body. Reactive oxygen is produced essentially during oxidative catabolic metabolism to acquire energy, however, unexpectedly over production and/or accumulation along aging can trigger various metabolic disorders. So the present study has an objective for the investigation of antioxidant activity in medicinal plant. Moreover, this study applied the fermentation process to improve their antioxidant activity. In first experiment (Chapter 2), microorganisms showing β-glucosidase activity were screened from the residues produced during the manufacturing of Korean traditional rice wine. The enzyme, β-glucosidase, is known to be involved into the bioconversion for polyphenolic compounds in plant during fermentation process and so this enzyme was used as a criterion for isolation in this study. Almost one thousand colony showing enzyme activity were firstly isolated on qualitative assay and the total 463 were isolated from following quantitative assay procedure. Then bacteria and yeast were separated by microscopic observation, and finally those isolates were identified as Saccharomyces cerevisiae and Lactobacillus plantarum based on genetic information. In second experiment (Chapter 3), total five medicinal plants, Chelidonium majus var asiaticum,Pueraria lobata root,Kalopanax septemlobus, Scutellaria baicalensis Georgi and Rheum rhabarbarum L., were used as materials and they were fermented with microorganism that were previously isolated in Chapter 2. And then preparation of ethanol extracts of them and analysis of biological activities (antibacterial and antioxidant activities) were followed. Whether the fermentation process was proceed or not was determined by analyzing viable cell count after fermentation. And the possible bioconversion of substance in plant during fermentation was monitored by thin layer chromatography combined with DPPH reagent that was used for detection of antioxidant activity. In result, L. plantarum EJ43 showed good growth in most used plants and improved antioxidant activities were found at most plant extract fermented by L. plantarum EJ43. Finally, it could be concluded that L. plantarum EJ43 was the most superior starter culture for the fermentation of plants that were used in this experiment and R. rhabarbarum was determined as predominance plant in the aspects of antibacterial and antioxidant activities. So, R. rhababarum and L. plantarum EJ43 were selected as materials for the following optimization experiment. In third experiment (Chapter 4), effective medium components for the fermentation of R. rhababarum using L. plantarum EJ43 as starter culture were investigated using statistical method. Total nine medium components were used and fractional factorial experimental design was employed for the investigation of the effectiveness of used medium components on fermentation efficiency. After fermentation with different medium composition, ethanol extract was prepared with same procedure that was used in previous experiment (Chapter 3) and then its antibacterial activity and antioxidant activity were assayed. As a result, glucose, garlic juice, onion juice, yeast extract and magnesium sulfate were selected as effective medium components based on statistical significances. Through a series of experiment, it was found that most plants used in this study showed antibacterial and antioxidant activities and their activities could be improved by fermentation process. In particular, the result from the present study can provide useful primary information for the development of natural product as skin care cosmetics.