Isolation And Nmr Characterization Of Ursane-Type Triterpenoid And The Bioprotective Investigations Of Secondary Metabolites From The Leaves Of Peperomia pellucida

ABSTRACT

Peperomia pellucida (L.) Kunth is a plant of immense medicinal value. It is consumed as vege and also used in herbal medicine for the treatment of different ailments such as abdominal pain, abscesse, acne, boils and colic rhematic joint pain. This study investigates bioactive constituents of the leaves of P. pellucida. An ursane-type triterpenoid was isolated from the leaves of P. pellucida and was characterized using ¹H, C-13, COSY, DEPT and HSQC NMR Spectroscopic techniques. The quantitative phytochemical screening of the extract revealed the presence of alkaloids (2.49 ± 0.02 %), phenols (0.05 ± 0.01 %), flavonoids (0.59 ± 0.01 %), saponins (0.64 ± 0.02 %) and tannins (0.08 ± 0.01 %). The results showed that the alkaloid content was the highest. Vitamins detected included thiamine (0.21 ± 0.03 mg/100g), niacin (0.57 ± 0.01 mg/100g), riboflavin (0.34 ± 0.01 mg/100g), ascorbic acid (8.74 ± 0.12 mg/100g) and β-carotene (2.33 ± 0.15 mg/100g), with ascorbic acid and thiamin having the highest and lowest concentrations respectively. Minerals detected included calcium (2.82 ± 0 mg/100g), magnesium (1.19 ± 0.06 mg/100g), potassium (0.63 ± 0.01 mg/100g), phosphorus (0.49 ± 0.01 mg/100g), sodium (0.03 ± 0.05mg/100g) and nitrogen (3.14 ± 0.03 mg/100g). Nitrogen had the highest concentration, while sodium was the lowest. The antioxidant activity of P. pellucida as determined by scavenging effect on 2,2-diphenyl-1-picryhydrazyl radical (DPPH) revealed that the fractions possessed antioxidant activity when compared with antioxidant standard ascorbic acid. Antimicrobial activity was investigated at varying concentration of 100mg/ml, 50mg/ml/, 40mg/ml, 20mg/ml and 10mg/ml against Escherichia coli, Bacillus cereus, Staphylococcus aereus and Salmonella typhi using the Agar diffusion method. The result showed that P. pellucida extract inhibited the growth of the test organisms, with the highest growth of inhibition Staphylococcus aureus and the lowest growth of inhibition against Bacillus cereus. The antimicrobial properties indicate the potential usefulness of this plant in the treatment of various pathogenic diseases which in future can be developed as a potential antimicrobial agent used in the treatment of infectious diseases. The results of this study revealed the presence of various pharmacologically active principles of medicinal importance including alkaloids, tannins, flavonoids, phenols and saponins. P. pellucida leave extracts are also good source of vitamins and minerals.  

 TABLE OF CONTENTS

Title Page                                                                                                                    i

Declaration                                                                                                                  ii

Certification                                                                                                                iii

Dedication                                                                                                                  iv

Acknowledgements                                                                                                    v

Table of Contents                                                                                                       vi

List of Tables                                                                                                              x

List of Figures                                                                                                             xi

List of Plate                                                                                                                xii

Abstract                                                                                                                      xii

                                     

CHAPTER 1 INTRODUCTION                                                                              

1.1       Background of Study                                                                                     1

1.2       Statement of Problem                                                                                     3

1.3       Aim and Objectives                                                                                        3

1.4       Justification of Study                                                                                     4

1.5       Scope of Study                                                                                               4

1.6       Significance of Study                                                                                     4

 

CHAPTER 2  LITERATURE REVIEW                                                                          5

2.1       Origin and Geographical Distribution of Peperomia pellucida                      5

2.2       Description of the Plant                                                                                  6

2.3       Piperaceae Family                                                                                           7

2.4       Ethnomedicinal Uses of Peperomia pellucida                                                8

2.5       Pharmacological Activitiy of Peperomia pellucida                                        9

2.5.1    Antioxidant activity                                                                                        9

2.5.2     Anti-microbial activity                                                                                   10

2.5.3     Pharmacological activity                                                                                10

2.6        Economic Uses                                                                                              11

2.7        Phytochemicals                                                                                              11

2.7.1     Phenolic                                                                                                         12

2.7.2     Flavonoids                                                                                                     13

2.7.3    Tannins                                                                                                            15

2.7.4    Alkaloids                                                                                                         16

2.7.5    Terpenoids                                                                                                      17

2.7.6    Saponins                                                                                                          18

2.8        Spectroscopy                                                                                                  18

2.8.1     Nuclear magnetic resonance (NMR) spectroscopy                                        19

2.8.2     One-dimensional nuclear magnetic resonance (1d NMR) spectroscopy       20

2.8.3    Two-dimensional nuclear magnetic resonance (2d NMR) spectroscopy        20

2.9        Chemical Constituents of Peperomia pellucida                                            20

2.10      Phytochemical Screening on Peperomia pellucida                                        23

 

CHAPTER 3: MATERIALS AND METHODS                                                   25

3.1       Materials for Extraction                                                                                  25

3.2       Sample Collection and Preparation                                                                 26

3.3       Solvent Extraction                                                                                          26

3.4       Concentration of Peperomia Pellucida  Using Digital Heidolph

            Rotary Evaporator.                                                                                         26

3.5       Packing the Column                                                                                        27

3.6       Thin Layer Chromatography                                                                           28

3.6.1    Spotting, development and visualization                                                        28

3.7       Quantitative Phytochemical Screening                                                           29

3.7.1    Determination of alkaloids                                                                             29

3.7.2    Determination of flavonoids                                                                           30

3.7.3    Determination of tannins                                                                                30

3.7.4    Determination of saponins                                                                              31

3.7.5    Determination of phenols                                                                               32

3.8       Determination of Vitamin Content                                                                 32

3.8.1    Determination of ascorbic acid                                                                       32

3.8.2    Determination of niacin                                                                                  33

3.8.3    Determination of riboflavin                                                                            34

3.8.4    Determination of thiamin                                                                                35

3.8.5    Determination of b-carotene                                                                           35

3.9       Determination of Mineral Elements                                                                36

3.9.1    Determination of potassium and sodium                                                        37

3.9.2    Determination of magnesium and calcium                                                     37

3.9.3    Determination of phosphorus                                                                         38

3.9.4    Determination of copper and zinc                                                                  39

3.10      Proximate Analysis of Peperomia pellucida                                                  40

3.10.1  Determination of protein content                                                                    40

3.10.2  Determination of crude fibre content                                                             40

3.10.3  Determination of lipid content                                                                        41

3.10.4  Determination of ash content                                                                          42

3.10.5  Determination of moisture content                                                                 42

3.10.6  Determination of carbohydrate content                                                          43

3.11     Determination of Antioxidant Activity                                                          43

3.12     Antibacteriall Assay of P. pellucida Leaf Methanol Extract                          44

3.12.1   Obtaining and confirming the test organisms                                                44

3.12.2   Antibacterial susceptibility test                                                                      44

3.12.3   Determination of minimum inhibitory concentration (MIC)                         45

 

 CHAPTER 4: RESULTS AND DISCUSSIONS                                                             46

4.1       Phytochemical Screening Result                                                                     46

4.2       Mineral Content Results                                                                                 48

4.3       Vitamin Content Results                                                                                50

4.4       Proximate Composition Of P. pellucida Leaves                                             52

4.5       Column Chromatography Results                                                                   55

4.6       Thin Layer Chromatograph (TLC) Results                                                     56

4.7       Spectral Analysis Result                                                                                 57

4.7.1   ¹H-NMR spectra result                                                                                    57

4.7.2    2-Dimensional NMR spectra interpretation                                                    59

4.8       Antioxidant Activity of P .pellucida Leaves Extract                                                 63

4.9       Antimicrobial Properties of P. pellucida Leave Extract                                 64       

CHAPTER 5: CONCLUSION AND RECOMMENDATIONS                                     67

5.1       Conclusion                                                                                                      67

5.2       Recommendations                                                                                          67

            References                                                                                             

 

 

 

 

 

 

 

 

 

LIST OF TABLES

4.1:      Phytochemical compostition of the leaf of P. pellucida expressed

            in percentage (%)                                                                                            46

 4.2:     Mineral element composition of  P. pellucida leaves                          48

 4.3.     Quantitative determination of vitamin contents of P. pellucida

            leaves                                                                                                              50

 4.4      Proximate composition of P. pellucida leaves                                                52

 4.5:     Fractionation of chloroform extract of P .pellucida leaves by

            column chromatograph                                                                                   55

 4.6      ¹H-NMR chemical shift for fraction C20                                                       58

 4.7:     ¹³C (DEPT) chemical shift of fraction C20                                                    61

 4.8:     Antioxidant determination of P. pellucida leaves                                          63

 4.9:     Zones of growth inhibition at varying concentrations of

            methanolic  extract of P. pellucida leaves against the test organisms            65

4.10:    Minimum inhibitory concentration of methanol leaves extract of

             P. pellucida                                                                                                    65

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

LIST OF FIGURES

4.1:      ¹H-NMR for fraction C20                                                                              57

4.2:      ¹³C (DEPT) NMR spectrum for fraction C20                                                 59

4.3:      ¹H-¹H-COSY spectrum for fraction C20                                                        60

4.4:      ¹H-¹³C HSQC spectrum for fraction C20                                                       60

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

                                

LIST OF PLATE

2.1:      Peperomia pellucida plant                                                                             5