An ethnobotanical, phytochemical and metabolomics investigstion of plants from the Paulshoek Communal Area, Namaqualand

Includes bibliographical references.The aim of this thesis is to investigate medicinal plants from different perspectives in an attempt to arrive at a new, integrated and streamlined method for the discovery of bioactive secondary metabolites of plant origin. This will be done through a focused study of the traditionally used medicinal plants of the Paulshoek region of Namaqualand and a demographic study of the people who use them. Trends in traditional medicinal plant choice will be investigated and methods of traditional knowledge acquisition and transfer will be examined. Additional assessment of bioactivity and trends in bioactivity will be conducted and a variety of physico-chemical and computational techniques will be used to determine the major metabolites present in selected plant species. These different approaches to medicinal plants will be brought together in a single holistic method put forward as a possible way of conducting future studies into discovering active metabolites for potential drug development

DEVELOPMENT OF FINGERPRINTING APPROACH FOR IDENTIFICATION AND DETECTION OF ADULTERATION IN FOOD

Ph.DDOCTOR OF PHILOSOPH

Phytomedicinal and Pharmacological Studies of Selective Plants of Kolli Hills and Development of Standardisation Methods for its Formulation

The thesis entitled “Phytomedicinal and Pharmacological Studies of Selective Plants of Kolli hills and Development of Standardization Methods for its Formulation” is an attempt to investigate and carry out a scientific study on medicinal plants of Kolli hills. From the initial study, the plants of Kolli hills such as Justicia gendarussa Burm, Rungia pectinata Nees, Strobilanthes ciliatus Nees and Rhinacanthus nasutus Linn belonging to the family Acanthaceae were investigated for its phytochemical, ethnomedicinal and pharmacological properties. Based on the comparative analysis Strobilanthes ciliatus Nees was chosen for further studies from the selected plants. Further, this thesis work embodies pharmacognostic, phytochemical and pharmacological study of whole plant of Strobilanthes ciliatus Nees. In addition formulation development, evaluation of formulation parameters and development of standardization method using an isolated phytochemical marker was studied. A detailed pharmacognostical evaluation of the leaves, stem and root was carried out for Strobilanthes ciliatus Nees. Morphoanatomy has been studied to aid pharmacognostic and taxonomic species identification using physiochemical determinations and standard phytochemical procedures. The physiochemical, morphological and histological parameters presented may be proposed as parameters to establish the authenticity of Strobilanthes ciliatus Nees. Non-communicable diseases (NCD’s) are not passed from person to person. They are of long duration and generally slow progression. According to the WHO report, of 57 million deaths that occurred globally in 2008, 36 million – almost two thirds – were due to NCD’s, comprising mainly cardiovascular diseases, cancers, diabetes and chronic lung diseases (23). A total of 57 million deaths occurred in the world during 2008; 36 million (63%) were due to NCD’s, principally cardiovascular diseases, diabetes, cancer and chronic respiratory diseases (89). Nearly 80% of these NCD deaths (29 million) occurred in low- and middle-income countries. The studies indicate the therapeutic potential of whole plant of Strobilanthes cliatus Nees and justify the traditional uses of this plant. The plant may prove to be promising in the management and alleviation of painful inflammatory conditions, hyperglycemia, hepatic diseases, cough, cytotoxic and aging related disorders. All the studies performed provides a strong evidence for the use of the Strobilanthes ciliatus Nees as a potent medicinal plant and novel solid dosage forms containing Strobilanthes ciliatus Nees extract that can be used as an alternative remedy for management and treatment of various Non-communicable diseases

관동화 유래 세스퀴테르펜 화합물의 성분프로파일 및 유방암 세포주에서의 oplopane 세스퀴테르펜의 표적 단백질에 관한 연구

학위논문(박사)--서울대학교 대학원 :약학대학 약학과,2019. 8. 김영식.관동(Tussilago farfara L.)은 국화과의 다년생 약초로서 관동의 말린 꽃봉오리(관동화, Farfarae Flos)는 전통 의학에서 기침, 기관지염 및 천식과 같은 호흡기 질환을 치료하기 위해 사용되었다. 주요 생리활성성분으로는 플라보노이드, 테르페노이드, 퀸산유도체 등이 보고되었으며, 특히 세스퀴테르펜 화합물군이 항염증, 세포증식억제, 뇌신경보호 등에서 높은 효능을 나타내었다. 따라서 본 연구에서는 관동화 유래 세스퀴테르펜 화합물에 대한 1) 대량 분리법, 2) LC-MS 기반의 성분프로파일링, 3) 유방암 세포주에서의 표적단백질 규명을 수행하였다. 항류크로마토그래피를 이용하여 관동화 유래 세스퀴테르펜 화합물의 대량분획법(직접연속주입법, Direct and Continuous Injection mode)을 고안하였다. 추출액 자체를 이동상으로 사용함으로써 유기용매의 사용량을 크게 줄였으며, 관동화 1 kg의 추출물 315.9 g으로부터 6.8 g의 세스퀴테르펜 강화분획을 한 번에 획득하였다. 기존의 항류크로마토그래피 분리방법은 1~5 g의 추출물을 주입하기 때문에 직접연속주입법을 통해 분리 시간과 비용을 절감할 수 있었다. 또한, 주요 세스퀴테르펜 단일화합물의 정량분석을 수행하였고 용매분획법이나 컬럼크로마토그래피를 이용해 얻어진 분획물에 비해 높은 함량을 확인하였다. UPLC-MS/MS 기법을 이용하여 관동화의 oplopane 및 bisabolane 계열의 세스퀴테르펜에 대한 성분프로파일을 제시하였다. 해당 화합물군은 질량분석기의 ESI 이온화과정에서 쉽게 In-source fragmentation (IS-CID) 되는 화합물로서 모분자 질량값을 얻기 위해 QqQ-MS의 Precursor ion scan을 적용하였다. 구조적 특성을 기반으로 네 가지의 특이적 이온(diagnostic ions, [M+H]+ 215, 217, 229, 231)을 선정하여 총 74종의 화합물에 대한 모분자 질량값을 확인하였고, Q-TOF MS의 Product ion scan을 이용하여 각 모분자 이온의 특징적인 쪼개짐 양상(fragmentation pattern)을 고분해능 수준에서 확인하였다. 또한 11종의 화합물을 분리 및 구조동정하여 고안된 동시분석법을 검증하였고, MRMHR 분석법을 이용하여 관동화 추출물에 함유된 주요 세스퀴테르펜 화합물의 함량을 확인하였다. 유방암 세포주 MDA-MB-231과 MCF-7에 대한 ECN (7β-(3´-ethyl cis-crotonoyloxy)-1α-(2´-methyl butyryloxy)-3,14-dehydro-Z-notonipetranone)의 높은 세포증식억제능을 확인하였고, Chemical Proteomics 기법을 이용하여 표적단백질을 제시하였다. In vitro 스크리닝 결과, 관동화 유래 세스퀴테르펜 화합물군은 항염증효능 뿐만 아니라 세포증식억제능을 보였으며 그 중 ECN이 가장 높은 효능을 나타냈다. ECN 기반의 clickable probe를 합성하여 세포 내에서의 click 반응으로 표적 단백질을 분획하였고, 가수분해된 펩타이드 혼합물의 TMT isobaric label 및 Orbitrap MS/MS 분석을 통하여 음성대조군 대비 3배 이상의 선택성을 갖는 17종의 표적단백질을 규명하였다. 또한, 높은 선택성을 갖는 두 종의 표적단백질 14-3-3 protein zeta, peroxiredoxin-1에 대한 ECN의 작용 위치(binding site)와 결합친화도(ITC)를 확인하였다. 본 연구 결과들을 종합하여 볼 때, 관동화 유래 세스퀴테르펜 화합물군에 대한 LC-MS/MS 성분프로파일은 관동화를 포함하는 생약제제의 품질관리에 적용될 수 있으며, oplopane 골격의 세스퀴테르펜에 대한 표적단백질을 규명함으로써 세포증식억제능에 한하여 유효성분과 약효의 상관관계를 확인하는데 기초가 되는 연구로 사료된다.Farfarae Flos is the dried buds of Tussilago farfara L., a perennial plant of the family Asteraceae, and has been used to treat coughs, bronchitis, and asthmatic conditions in traditional herbal medicine. Among its bioactive compounds, sesquiterpenoids exhibit various biological activities such as anti-inflammative, anti-proliferative, and neuroprotective effects. In the present study, preparative separation, chemical profiling, and activity-based proteome profiling of sesquiterpenoids from Farfarae Flos were performed. Firstly, a novel fractionation and purification method of counter-current chromatography (CCC), called direct and continuous injection (DCI) mode, was developed to fractionate and preparatively separate sesquiterpenoids from Farfarae Flos. Since the extraction solution was used as a mobile phase in this method, solvent consumption was greatly reduced. 6.8 g of sesquiterpenoid-enriched (STE) fraction was obtained from the crude extract (315.9 g) of Farfarae Flos (1 kg) in a single CCC run with a separation time of 8.5 hrs. The sample injection capacity of CCC-DCI was greater than 300 grams which could not be handled in conventional CCC methods. Moreover, quantification study indicated that the fractionation efficiency of CCC-DCI was higher than those of conventional fractionations: solvent partitioning and open column chromatography. The developed method demonstrates that CCC is a useful technique for enriching target components from natural products. Secondly, a liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS)-based dereplicative method was developed to identify and quantify oplopane- and bisabolane-type sesquiterpenoids of Farfarae Flos. The MS-based nontargeted metabolomic approach for these chemical analogues, sesquiterpene esters, is challenging because of their in-source fragmentation and structural diversity. In order to profile these sesquiterpenoids, four diagnostic ions (m/z 215.143, 217.158, 229.123, and 231.138) were suggested in the positive ion mode and the developed method utilized two sequential MS/MS scan modes to characterize common skeletons and investigate the fragmentation patterns of their parent molecules. Under the optimized UHPLC-MS/MS method, 74 sesquiterpenoids were identified from the Farfarae Flos and 11 compounds were isolated for the method validation. Furthermore, the diagnostic ions and the MS/MS fragment behaviors were applied to accurate quantification of the 8 isolated sesquiterpenoids. Consequently, the developed LC-MS/MS-based dereplicative method highlighted the chemical composition of the Farfarae Flos and could be applied to quality control of the herbal medicine. Finally, target proteins of ECN (7β-(3´-ethyl cis-crotonoyloxy)-1α-(2´-methyl butyryloxy)-3,14-dehydro-Z-notonipetranone) in human breast cancer cells were identified by chemical proteomics methodology. ECN showed potent anti-proliferation activity in MDA-MB-231 and MCF-7 human breast cancer cells based on its α,β-unsaturated carbonyl moiety. Therefore, the potential cellular target proteins of ECN were identified using ECN-based clickable probe and quantitative MS/MS analysis. Among more than 200 identified proteins, 17 proteins showed more than 3 enrichment ratio in both cell lines. Furthermore, recombinant 14-3-3 protein zeta and peroxiredoxin-1 were further verified by isothermic calorimetry and their alkylation sites. Taking the interaction between α,β-unsaturated carbonyl moiety of ECN and cysteine residues of proteins into account, peptides containing Cys25, Cys94 of 14-3-3 protein zeta and Cys83 of peroxiredoxin-1 were significantly reduced by ECN. Although these results could not confirm the role of ECN in the breast cancer cells, this suggestion of multiple target proteins contributed to understand the ECN-mediated anti-proliferative and anti-inflammatory effects, leading to further studies.CONTENTS ABSTRACT ...................................................................................................... I CONTENTS ................................................................................................... IV LIST OF FIGURES ................................................................................... IX LIST OF TABLES ..................................................................................... XII I. INTRODUCTION .................................................................................... 1 1. Farfarae Flos .................................................................................................. 2 1.1. Constituents and bioactivities ................................................................... 2 1.2. Oplopane and bisabolane sesquiterpenoids ............................................. 4 2. Counter-current chromatography (CCC) .............................................. 6 2.1. Background ................................................................................................. 6 2.2. Solvent selection and application .............................................................. 9 3. Chemical profiling ...................................................................................... 12 3.1. Liquid chromatography and mass spectrometry (LC-MS) .................. 12 3.2. Scan modes of tandem mass spectrometry (MS/MS) ............................ 14 3.3. LC-MS/MS-based dereplication methodology ...................................... 17 4. Activity-based proteome profiling .......................................................... 20 4.1. Background ............................................................................................... 20 4.2. Click chemistry ......................................................................................... 22 4.3. Quantitative proteome profiling based on mass spectrometry ............ 24 II. STATE OF THE PROBLEM .......................................................... 26 III. MATERIALS AND METHODS ................................................. 29 1. Materials ....................................................................................................... 30 1.1. Farfarae Flos ............................................................................................. 30 1.2. Chemicals and reagents ........................................................................... 30 1.3. Apparatus .................................................................................................. 31 1.4. Cell lines .................................................................................................... 32 2. Methods ......................................................................................................... 33 2.1. An efficient fractionation method for the preparative separation of sesquiterpenoids from Farfarae Flos by CCC ........................................ 33 2.1.1. Measurement of the partition coefficient (KD) ................................. 33 2.1.2. Preparation of the extract solution and solvent system ................... 33 2.1.3. CCC-DCI fractionation ...................................................................... 34 2.1.4. Solvent partitioning and open column chromatography ................ 35 2.1.5. Isolation of three major sesquiterpenoids ........................................ 36 2.1.6. Preparation of sample solution .......................................................... 36 2.1.7. HPLC analysis and calibration curve ............................................... 37 2.2. Chemical profiling of sesquiterpenoids from Farfarae Flos based on LC-MS/MS analysis .................................................................................. 38 2.2.1. Sample preparation from Farfarae Flos ........................................... 38 2.2.2. UHPLC separation ............................................................................. 38 2.2.3. MS/MS analysis ................................................................................... 39 2.2.4. Separation of sesquiterpenoids and structural determination ....... 40 2.2.5. Validation parameters for quantification ......................................... 41 2.3. Activity based proteome profiling: Identification of target proteins of an oplopane sesquiterpenoid in breast cancer cells ..................................... 43 2.3.1. Fractionation of Farfarae Flos extract ............................................. 43 2.3.2. Cell viability assay .............................................................................. 43 2.3.3. Synthesis of ECN-based clickable probe .......................................... 44 2.3.4. Gel-based proteome profiling ............................................................ 45 2.3.5. Preparation of probe-labeled proteome for MS-based analysis ..... 46 2.3.6. LC-MS/MS analysis and data processing ......................................... 48 2.3.7. Modification sites of identified proteins by ECN ............................. 49 2.3.8. Isothermal titration calorimeter ........................................................ 50 IV. RESULTS AND DISCUSSION ..................................................... 52 1. An efficient fractionation method for the preparative separation of sesquiterpenoids from Farfarae Flos by CCC ..................................... 53 1.1. Principle of CCC-DCI fractionation ...................................................... 53 1.2. Selection of the extraction and elution solvents based on KD values ... 55 1.3. CCC-DCI fractionation ........................................................................... 59 1.3.1. Four stages of CCC-DCI .................................................................... 59 1.3.2. Preparative separation of three major sesquiterpenoids ................ 62 1.4. Quantification study ................................................................................ 67 1.4.1. Validation parameters ........................................................................ 68 1.4.2. Comparison of CCC-DCI with conventional methods .................... 70 1.5. Discussion .................................................................................................. 73 2. Chemical profiling of sesquiterpenoids from Farfarae Flos based on LC-MS/MS analysis .................................................................................... 75 2.1. Characterization of diagnostic ions ......................................................... 75 2.1.1. Diagnostic filtering .............................................................................. 78 2.1.2. Fragmentation patterns of the diagnostic ions ................................. 80 2.2. Precursor ion scan for the diagnostic ions .............................................. 82 2.3. Method validation ..................................................................................... 86 2.3.1. Separation of 11 sesquiterpenoids ...................................................... 86 2.3.2. Structural elucidation ......................................................................... 88 2.4. CID-fragmentation behavior of sesquiterpenoids ............................... 105 2.5. Quantification of sesquiterpenoids by MRMHR ................................... 109 2.6. Discussion ................................................................................................ 114 3. Activity-based proteome profiling: Identification of target proteins of an oplopane sesquiterpenoid in breast cancer cells ..................... 116 3.1. Anti-proliferation activities of Farfarae Flos ....................................... 116 3.2. Synthesis of ECN-based clickable probe .............................................. 119 3.3. Gel-based proteome profiling of clickable probe ................................. 125 3.4. MS-based profiling of target proteins of ECN ..................................... 127 3.5. Thermodynamics and binding sites of ECN for target proteins ........ 129 3.6. Discussion ................................................................................................ 133 V. CONCLUSION ..................................................................................... 135 REFERENCES ........................................................................................... 138 ABSTRACT IN KOREAN ................................................................... 157 LIST OF FIGURES Fig. 1. Tussilago farfara L. and Farfarae Flos .............................................. 3 Fig. 2. Chemical structures of reported oplopane and bisabolane type sesquiterpenoids from Farfarae Flos ................................................. 5 Fig. 3. A schematic diagram of CCC system ................................................. 8 Fig. 4. A schematic diagram of CCC separation based on KD value ..... 10 Fig. 5. Polarity correlation between HEMWat systems and isolates ..... 11 Fig. 6. A schematic diagram of LC-MS system .......................................... 13 Fig. 7. A schematic diagram of MS/MS scan modes ................................. 16 Fig. 8. A schematic diagram of LC-MS/MS based dereplication ........... 18 Fig. 9. A schematic diagram of dereplication using MS/MS database ... 19 Fig. 10. A schematic diagram of activity-based proteome profiling ...... 21 Fig. 11. Click chemistry reaction ................................................................... 23 Fig. 12. A schematic diagram of LC-MS based quantitative proteome profiling ................................................................................................ 25 Fig. 13. A schematic diagram of CCC-DCI mode ...................................... 54 Fig. 14. CCC-DCI chromatogram of Farfarae Flos extract .................... 61 Fig. 15. Preparative separation of three major sesquiterpenoids .......... 63 Fig. 16. 1H and 13C spectrum of TG .............................................................. 64 Fig. 17. 1H and 13C spectrum of AECN ........................................................ 65 Fig. 18. 1H and 13C spectrum of ECN ........................................................... 66 Fig. 19. HPLC-UV chromatograms of extract and fraction ................... 71 Fig. 20. In-source fragmentation of sesquiterpenoids ............................... 76 Fig. 21. Proposed diagnostic ions under LC-ESI-MS analysis ............... 77 Fig. 22. Ion chromatogram of STE fraction and diagnostic filtering ..... 79 Fig. 23. Fragmentation patterns of diagnostic ions ................................... 81 Fig. 24. Total ion chromatograms of precursor ion scan ......................... 83 Fig. 25. Preparation of 11 sesquiterpenoids from STE fraction ............. 87 Fig. 26. Precursor ion scans for isolated sesquiterpenoids ...................... 90 Fig. 27. HSQC spectrum of compound No. 7 ............................................. 91 Fig. 28. HSQC spectrum of compound No. 11 ............................................ 92 Fig. 29. HSQC spectrum of compound No. 12 ........................................... 93 Fig. 30. HSQC spectrum of compound No. 14 ........................................... 94 Fig. 31. HSQC spectrum of compound No. 23 ........................................... 95 Fig. 32. HSQC spectrum of compound No. 36 ........................................... 96 Fig. 33. HSQC spectrum of compound No. 39 ........................................... 97 Fig. 34. HSQC spectrum of compound No. 45 ........................................... 98 Fig. 35. HSQC spectrum of compound No. 60 ........................................... 99 Fig. 36. HSQC spectrum of compound No. 68 ......................................... 100 Fig. 37. HSQC spectrum of compound No. 72 ......................................... 101 Fig. 38. NOESY spectrum of compound No. 7 ......................................... 102 Fig. 39. NOESY spectrum of compound No. 23 ....................................... 103 Fig. 40. NOESY spectrum of compound No. 45 ....................................... 104 Fig. 41. Representative MS/MS fragmentation behaviors .................... 107 Fig. 42. Fragmentation behaviors of mono- and hetero-isotopic ions ... 108 Fig. 43. Herbal materials for quantification study .................................. 110 Fig. 44. Dereplication of 8 sesquiterpenoids by UHPLC-MRMHR ...... 111 Fig. 45. Anti-proliferation activities of fractions from Farfarae Flos .. 117 Fig. 46. Anti-proliferation activities of compounds from Farfarae Flos 118 Fig. 47. Synthesis of ECN-based clickable probe and anti-proliferation activity ................................................................................................. 120 Fig. 48. HSQC spectrum of ECN ................................................................. 121 Fig. 49. HSQC spectrum of ECN-E ............................................................ 122 Fig. 50. HSQC spectrum of ECN-N3 ........................................................... 123 Fig. 51. 15N-HMBC spectrum of ECN-N3 .................................................. 124 Fig. 52. Gel-based profiling of ECN-N3 labeled proteome in situ ........ 126 Fig. 53. Thermograms and parameters for interaction of ECN with identified target proteins ................................................................ 131 Fig. 54. Alkylation of cysteine residues in 14-3-3 protein zeta by ECN .. 132 Fig. 55. Alkylation of cysteine residues in peroxiredoxin-1 by ECN ..... 133   LIST OF TABLES Table 1. The partition coefficients (KD) of three major sesquiterpenoids in different solvent composition ..................................................... 57 Table 2. Comparison of the extraction efficiency of 45% acetonitrile and methanol .............................................................................................. 58 Table 3. The linear range, linearity, LOD, and LOQ of three major sesquiterpenoids by UV detection ................................................. 69 Table 4. The comparison of the fractionation efficiency of CCC-DCI, solvent partitioning, and open column chromatography ........ 72 Table 5. Identified sesquiterpenoids of Farfarae Flos by precursor ion scan of UHPLC-QqQ-MS/MS ........................................................ 84 Table 6. Quantitative parameters for sesquiterpenoids by UHPLC- MRMHR .............................................................................................. 112 Table 7. Intra-day and inter-day precision of UHPLC-MRMHR ......... 112 Table 8. Extraction yield of herbal materials ........................................... 113 Table 9. Contents of 8 sesquiterpenoids in Tussilago farfara by UHPLC- MRMHR .............................................................................................. 113 Table 10. Identified target proteins of ECN in breast cancer cells ...... 129Docto

Propagation and quality assessment for the introduction of Greyia Radlkoferi into commercialization

Greyia radlkoferi is a South African indigenous tree, which has recently been discovered to be a source of extracts that have a potential in the development of cosmeceutical herbal products having the ability to treat hyperpigmentation disorders. For product development however, G. radlkoferi would need to be available in a commercial scale. Greyia radlkoferi grows naturally in the wild and is often available for cultivation as an ornamental plant. In order to establish this plant into cultivation, suitable propagation techniques must be established for rapid multiplication of trees and thus a sustainable leaf production. For consistent and improved leaf supply to the market, agronomic practices that will enhance leaf production were investigated in the current study. Furthermore, in order to meet market demand in terms of good quality extracts with guaranteed therapeutic efficiency, pre-harvest and post-harvest factors that affect the chemical composition of the extracts were investigated. Recently developed biotechnology techniques such as metabolomics using 1H-NMR and multivariate data analysis offered a platform to study the chemical variation of extracts. Therefore, the current study was aimed at understanding the requirements for propagation and optimum leaf production as well as conditions that favour optimum production of secondary metabolite of G. radlkoferi plant material (at pre and post-harvest) and thus assess its commercial viability. To understand the effects of temperature on seed germination of G. radlkoferi, seeds were exposed to five temperatures (10°C, 15°C, 20°C, 25°C and 30°C) in the incubators in the laboratory. Germination of G. radlkoferi by seeds was discovered to be temperature dependent. The optimum germination temperature of 81% was obtained at 25°C. In the case of vegetative propagation by stem cuttings, the effect of cutting position (basal or apical), exogenous rooting hormone (Seradix1, Seradix 2, 0.1% IBA, 0.3% IBA and 0.8% IBA) and cutting position were investigated in the glasshouse. The cutting position had a significant effect on rooting of G. radlkoferi cuttings with basal cuttings exhibiting 35% rooting as compared to 6% rooting attained for the apical cuttings. A clear trend in rooting response to application of rooting hormones was observed, with 0.1% Indole butyric acid (IBA) showing the highest rooting percentage of 63%. Considering the outcomes of the propagation studies as well as the limited material for vegetative propagation, seed propagation appears to be the most suitable technique for large-scale multiplication of G. radlkoferi. The effect of different pruning techniques as well as harvesting frequencies on fresh and dry weights of G. radlkoferi leaves were evaluated. Factors considered were four pruning treatments (‘pruned but not tipped’, ‘tipped but not pruned’, ‘not pruned nor tipped’ as well as ‘pruned and tipped’) and three harvesting periods (monthly, bimonthly and once–off). Bimonthly harvests highly increased leaf production compared to trees that were harvested monthly and once-off with higher leaf fresh weight yield of 238 g per tree or 2.38 tons/per hectare as well as dry weight yield of 83 g per tree or 0.830 tons/hectare. This outcomes of this study further suggested that a suitable pruning practice for G. radlkoferi would be to either ‘prune only’ or ‘cut back the main stem’ rather than a combination of the two treatments. The influence of seasons (summer, autumn, winter and spring) on the anti-tyrosinase activity and metabolomics profile of G. radlkoferi leaf extracts were investigated. Seasons significantly influenced the chemical composition and the efficacy of the plant extracts. Tyrosinase enzyme inhibition was investigated against monophenolase (tyrosine) with kojic acid as positive control. The highest tyrosinase inhibition concentration with IC50 (50% tyrosinase inhibition concentration) value of 30.3±1.8 μg/ml were obtained in winter harvested leaves compared to the other seasons. The lowest IC50 values were obtained in spring. Metabolomics analysis using orthogonal partial least square discriminant analysis (OPLS-DA) provided a clear class separation according to the harvest season. Extracts from winter harvested leaves contained sucrose, acetamide, alanine and a compound of the catechin group (gallocatechin-(4 alpha->8)-epigallocatechin) as revealed by 1H-NMR metabolomics with assistance of LC-MS. Since compounds of the catechin group are well-known tyrosinase inhibitors, the high tyrosinase activity exhibited in extracts of winter harvested G. radlkoferi leaves could be ascribed to the presence of gallocatechin-(4 alpha->8)-epigallocatechin. Based on the outcomes of the seasonal study, we suggest that in order to obtain extracts with high bioactivity, the best suitable time for harvesting leaf samples is in late autumn-early winter. Processing leaf material using three different drying methods (sun, oven and air drying) significantly influenced chemical composition and the efficacy of the plant extracts. Extracts prepared from air-dried leaf material showed the highest tyrosinase inhibition with IC50 value of 17.80 μg/ml compared to extracts of the other drying methods. Extracts of leaves processed with air drying preserved most metabolites during processing while extracts of sun-dried and oven-dried leaves clearly depleted some metabolites especially amino acids and some aromatic compounds. 1H-NMR metabolomics approach with the assistance of LC-MS data successfully determined a positive association of alanine, acetamide, sucrose and gallocatechin-(4 alpha->8)-epigallocatechin as the chemical constituents contributing to the variation in the air-dried leaves compared to the oven-dried leaves. A positive association of valine, alanine, leucine, isoleucine, gallocatechin-(4 alpha->8)-epigallocatechin and glucose contributed to the variation in air-dried group, compared to the sun-dried group. The highest tyrosinase inhibitory activity exhibited in air-dried samples compared to the other drying methods was associated with the presence of gallocatechin-(4 alpha->8)-epigallocatechin. Because air drying preserved most leaf metabolites compared to sun and oven drying, it was regarded as the most suitable method for processing G. radlkoferi leaf material. This study is the first scientific account that provides guidelines and recommendations to (1) establish G. radlkoferi as a cultivated plant for commercialization, (2) optimize leaf production for sustainable supply to the commercial markets and (3) optimize medicinal content of G. radlkoferi related to harvesting time and post-harvest processing (drying), for enhanced quality of extracts and its productsAgriculture, Animal Health and Human EcologyPh. D. (Agriculture

Terpenoids for medicine

This thesis is concerns research on monoterpenoids, sesquiterpenoids, and diterpenoids with medicinal properties. Terpenoids from commond herbs as well as Cannabis sativa, Inula britannica, Tanacetum parthenium, and Salvia officinalis were investigatedUBL - phd migration 201

Theme Issue Honoring Professor Robert Verpoorte's 75th Birthday: Past, Current and Future of Natural Products Research

This theme issue is to celebrate Professor Robert Verpoorte’s 75th birthday. Prof. Verpoorte has been working in Leiden University over 40 years. There is no need to dwell upon the contributions of Dr. Verpoorte to plant-derived natural products research during his whole life. Dr. Verpoorte was a highly productive scientist throughout his academic career, with over 800 scientific publications in the form of research papers, books, and book chapters. His research interests are very diverse, cover- ing numerous topics related to plant-based natural products such as plant cell biotech- nology, biosynthesis, metabolomics, genetic engineering, and green technology, as well as the isolation of new biologically active compounds. He has left indelible footprints in all these fields, and he is widely recognised as a pioneer in the work of the biosynthesis of indole alkaloids, NMR-based metabolomics, and green technology in natural products production. As close friends and colleagues who have been in nearly daily contact with him over the last 20 years viewing all of these remarkable scientific contributions, we felt compelled to recognize this by the publication of a Special Issue of this journal dedicated to him.Thus, this Special Issue has now finally been released with the help of many of his colleagues and former students as a token of our gratitude to his impressive work.The Special Issue covers five main natural products topics: (1) chemical profiling and metabolomics, (2) separation/isolation and identification of plant specialized metabolites, (3) pharmacognosy of natural products to identify bioactive molecules from natural prod- ucts, (4) novel formulation of natural products, and (5) overview of natural products as a source of bioactive molecules

Investigation of anti-cancer potential of Pleiocarpa pycnantha leaves

Philosophiae Doctor - PhDThe Apocynaceae family is well known for its potential anticancer activity. Pleiocarpamine isolated from the Apocynaceae family and a constituent of Pleiocarpa pycnantha has been reported for anti-cancer activity. Prompted by a general growing interest in the pharmacology of Apocynaceae species, most importantly their anticancer potential together with the fact that there is scanty literature on the pharmacology of P. pycnantha, we explored the anticancer potential of the ethanolic extract of P. pycnantha leaves and constituents. Three known triterpenoids, ursolic acid C1, 27-E and 27-Z p-coumaric esters of ursolic acid C2, C3 together with a new triterpene 2,3-seco-taraxer-14-en-2,3-lactone (pycanocarpine C5) were isolated from an ethanolic extract of P. pycnantha leaves. The structure of C5 was unambiguously assigned using NMR, HREIMS and X-ray crystallography. The cytotoxic activities of the compounds were evaluated against HeLa, MCF-7, KMST-6 and HT-29 cells using the WST-1 assay. Ursolic acid C1 displayed potent cytotoxic activity against HeLa, HT-29 and MCF-7 cells with IC50 values of 5.06, 5.12 and 9.51 μg/ml respectively. The new compound C5 and its hydrolysed open-chain derivative C6 were selectively cytotoxic to the breast cancer cell line, MCF-7 with IC50 values 10.99 and 5.46 μg/ml respectively. We further investigated the mechanism of action of the isolated compounds using specific markers of apoptosis. Exposure of C1-C6 (12.5 μg/ml) to HeLa cells showed a significant increase in reactive oxygen species (ROS) production with the exception of C5. On HT-29, C1, C4, C5 and C6 at 25 μg/ml increased ROS production while on MCF-7 using the same dose, only C5 and C6 caused a significant increase in ROS production compared with a control at P< 0.05. The result on caspase 3/7 activation showed that C1 and C2 (50 μg/ml) caused a marked increase in caspase 3/7 activity between 6-24 h on HeLa cells while only C1 (50 μg/ml) showed a significant increased caspase 3/7 activity on both HT-29 and MCF-7 cell lines when compared with the control, P< 0.05. Some selected compounds were further investigated for their dose-response on caspase 3/7 activity on HeLa and MCF-7 cells. Compounds C2 and C3 activated caspase 3/7 at 12.5 and 25 μg/ml respectively, while on MCF-7only C6 significantly increased caspase 3/7 activity within 24 h of treatment when compared with an untreated control. The result of time -dependent caspase 9 activity showed that C1, C2 and C3 caused an increased activity on HeLa cells between 6-12 h, while only C1 activated caspase 9 on HT-29 cells (3-24 h) and MCF-7 (6-24 h). The dose-response caspase 9 activity showed a significant increase in activation for C6 (12 and 25 μg/ml) on HeLa and C5 (25 μg/ml) on HT-29 cells. All isolated compounds inhibited Topoisomerase I when compared with Camptothecin. Compounds C1-C6 could induce apoptosis on cancer cell lines through an intrinsic pathway and topoisomerase 1 inhibition. This is the first report on the isolation of a 2,3-seco-taraxerene derivative from Apocynaceae family and the anticancer activity of Pleiocarpa pycnantha constituents