Antibacterial Activity and Chemical Composition Analysis of Ruta graveolens L. Leaves and Stems Extract Restricted; Files Only

Fang, Xin (Spring 2023)

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The promising bioactivity of the secondary metabolites plants produced, such as alkaloids and coumarins, serve as inspiration for developing new antibiotics. Ruta graveolens L. (Rutaceae) is a traditional herbal medicine originated from the Balkan Peninsula and has been used to treat infections and skin inflammations since the Middle Ages. Past studies have shown that the crude extract of Ruta graveolens L. enriched with secondary metabolites and has antibacterial properties against multiple bacteria strains (Al-Majmaie et al., 2021; Das & Deka, 2017). However, there is limited research to determine which compounds are responsible for these antibacterial properties. Hence, this project aims to isolate and characterize the structures of secondary metabolites from Ruta graveolens and test their antibacterial properties against bacteria strains with established antibiotic resistance.

The air-dried leaves and stems of Ruta graveolens L. were first extracted in 80% aqueous ethanol (v/v) and partitioned into four layers: ethyl acetate, dichloromethane, n-butanol, and aqueous layers. The major alkaloids in Ruta graveolens L. were present in the dichloromethane and n-butanol layers confirmed by silica TLC spraying with Dragendorff’s reagent. Fractions containing alkaloids of interest were separated via column chromatography and preparative high-performance liquid chromatography. Compound 1, with similar structure of isorutarin and leptophylloside, have been isolated from the n-butanol layer and its structure was identified via LC-MS, 1H-NMR, 13C-NMR, 1H-13C HMBC, 1H-13C Edited HSQC, and NOESY. However, this compound did not exhibit significant inhibitory activity against Staphylococcus aureus strain (AH0845 and UAMS1) nor Escherichia coli strain (CDC015).

A fraction with IC50 6.50 μg/mL against Staphylococcus aureus strain (AH0845) was obtained from dichloromethane layer. The major chemical composition of this fraction is hypothesized to be composed of two reported alkaloids skimmianine and kokusaginine in an estimated ratio of 1:2 based on LC-MS, 1H-NMR, 13C-NMR, 1H-1H COSY, 1H-13C HSQC, 1H-13C HMBC, and 1H-15N HMBC. The growth inhibitory effects against Staphylococcus aureus strain (AH0845 and UAMS1) and Escherichia coli strain (CDC015) were also evaluated on all remaining fractions. Multiple subfractions from the dichloromethane layer have inhibitory effects against Staphylococcus aureus strain (AH0845 or UAMS1), but all n-butanol subfractions lacked significant inhibition activity against either bacterium.

Table of Contents

Chapter 1: Introduction 1

1.1 Antibiotic Resistance 1

1.2 Natural Products and Their Roles in Drug Discovery 2

1.2.1 Class of Compounds with Diverse Bioactivities: Alkaloids 3

1.2.2 The Discovery Process of Bioactive Natural Products and Persisting Challenges 3

1.3 Plant of Interest: Ruta graveolens L. (common rue), Rutaceae 4

1.4 Project Aims 9

Chapter 2 Materials and Methods 11

2.1 Plant Collection and Processing 11

2.2 Plant Extraction and Compounds Isolation 11

2.2.1 Plant Extraction: Maceration 11

2.2.2 Acid-Base Partition 11

2.2.3 Flash Chromatography 12

2.2.4 Sephadex LH-20 Column Chromatography 13

2.2.5 Preparative High Performance Liquid Chromatography (HPLC) 13

2.2.6 Normal Phase Column Chromatography 15

2.3 Chemical Composition Visualization and Structure Analysis 15

2.3.1 Normal Phase Thin Layer Chromatography (TLC) 15

2.3.2 Analytical High Performance Liquid Chromatography (HPLC) 16

2.3.3 Liquid Chromatography Mass Spectrometry (LC-MS) 17

2.3.4 Nuclear Magnetic Resonance (NMR): 1H, 13C, 1H-13C HMBC, 1H-13C HSQC 19

2.4 Biological Assays 19

2.4.1 Single-dose Growth Inhibition Assay 19

2.4.2 Dose Response Growth Inhibition Assay - Minimum Inhibitory Concentration (MIC) 20

Chapter 3 Results and Discussion 22

3.1 Extraction and Isolation 22

3.2 Structure Elucidation and Chemical Composition Analysis 23

3.2.1 Structure Elucidation of Compound 1 24

3.2.2 Chemical Composition Analysis of 2716-2-PF15 33

3.2.3 Isolation of potential unreported alkaloids 42

3.3 Growth Inhibitory Activities of Compound 1 and Subfractions 44

3.3.1 2716-2-PF15 exhibited promising inhibitory activities against Staphylococcus aureus (AH0845 strain) 44

3.3.2 Screening of Growth Inhibitory Activities on Fractions Derived from Dichloromethane Layer 46

3.3.3 Screening of Growth Inhibitory Activities on Compound 1 and Fractions Derived from n-butanol Layer 47

Chapter 4: Conclusion 48

References 49

Supplementary Materials 56

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