Chemical and Biochemical Evaluation in Five Varieties of Piper betle L. Leaves from Bangladesh
- Issued Date
- 2021
- Keyword
- Piper betle L
- Abstract
- Piper betle L. leaves have been extensively consumed for mastication, mouth freshening, and medicinal purposes throughout the world. These leaves still lacked data, especially on their quality, safety, and efficacy in Bangladesh. This study aimed at evaluation of chemical (elemental: major, minor, trace, and toxic element; volatile and non-volatile organic compounds) and biochemical (antioxidant, antimicrobial, and cytotoxicity) properties in five varieties of Piper betle L. leaves (FBL) var. Bangla, Sanchi, Misti, Khasia, and BARI Paan 3 from Bangladesh.
Inductively coupled plasma-optical emission spectrometry (ICP-OES) and inductively coupled plasma-mass spectrometry (ICP-MS) analysis of elemental concentrations in FBL were found to be decreasing order of elements (major: K > Ca > Mg > P > Na; minor: Mn > Fe > Sr > Ba > Zn > Cu > Cr > Ni; trace: Ga > Li > V > Cs > Se > U > Be; toxic: Pb > As > Cd > TI). Among the nutritionally important elements, all examined betel leaves were good sources of K (3.99 – 5.86 g/kg), Ca (2.64 – 2.80 g/kg), and Mg (1.26 – 1.33 g/kg). Whereas, potentially all minor and toxic elements were present in safe limit except Mn (21.94 – 25.09 mg/kg) and Pb (129.36 – 965.91 µg/kg) specified by World Health Organization (WHO) / Food and Agriculture Organization (FAO).
Simultaneous distillation extraction and gas chromatography-mass spectrometry (SDE/GC-MS) were applied to analyze the volatile organic compound in FBL. The betel leaf variety Misti showed the highest amount of volatile organic compounds (13958.90 mg/kg) followed by BARI Paan 3 (11684.10 mg/kg), Khasia (11109.70 mg/kg), Sanchi (6958.51 mg/kg), and Bangla (4346.91 mg/kg). A total of 101 compounds were identified in this analysis. Out of those, 42 were common in all varieties with different quantities, and the other 59 compounds were not available in each variety. The present research reported 50 new volatile organic compounds in betel leaves for the first time compared to published literature. Eugenol was found in all varieties as a major compound and other main compounds were β-caryophyllene, γ-muurolene, valencene, eucalyptol, chavicol, and caryophyllene oxide.
The non-volatile organic compound screening tests confirmed the presence of different non-volatile organic compound classes like phenol, flavonoids, terpene, steroids, phytosterols, and saponin in FBL. Finding out the optimum parameters for the ultrasonic extraction from betel leaves using response surface methodology (RSM) with central composite circumscribed (CCC) design to maximize the total phenolic content. The solvent was selected as ethanol: acetic acid: water (70%: 5%: 25%, v/v) and the optimal extraction condition was: time 90 min; extraction temperature, 75 °C; solid/liquid ratio, 1:15.41. Followed by, FBL were extracted by the above optimized ultrasonic extraction conditions and their total phenolic and flavonoid contents showed high variation ranging from 110.51 to 322.8 mg GAE/g DW and 46.79 to 57.09 mg QE/g DW, respectively. The major phenolic compound hydroxychavicol was quantified 14.19 mg/g to 38.19 mg/g in FBL by high-performance liquid chromatography–diode array detector (HPLC-DAD) analysis.
The antioxidant activity of IC50 value of 2,2-Diphenyl-1-pycrilhydrazil (DPPH) and 2,2’-Azino-(bis 3-ethylbenzthiazoline-6-sulphonic acid) (ABTS) assay was ranged from 0.17 to 0.43 mg/mL and 0.04 to 0.11 mg/mL, respectively, in FBL extract. The IC50 values of DPPH and ABTS assay were 10.88 to 14.47 µg/mL and 7.66 to 18.00 µg/mL for hydroxychavicol and eugenol, individually, which are the major phenolic compounds. The same extracts were displayed good inhibitory properties against S. typhimurium, P. aeruginosa, E. coli, A. faecalis, and MRSA bacteria compared to other tested bacteria. Khasia and BARI Paan 3 were showed the highest antioxidant and antimicrobial activity than other varieties (Bangla, Sanchi, and Misti). The cytotoxicity activity in FBL extract in respect of 50% cell viability in human epithelial cell (HeLa), human neuroblastoma clonal cell (SH-SY5Y), and mesenchymal stem cells (MSCs) were 0.19 to 0.32 mg/mL, 0.41 to 0.54 mg/mL, and 0.46 to 0.96 mg/mL, respectively. On the other hand, the cytotoxicity activity of 50% cell viability showed 3.14 to 3.21 µg/mL, 5.81 to 6.06 µg/mL, 28.47 to 32.13 µg/mL in HeLa, SH-SY5Y, and MSCs cells; separately for hydroxychavicol and eugenol. In cancer cell lines, an increased cell death rate was observed with an increase in the betel leaf extract concentration but normal cells were unaffected. These results suggested that Piper betle L. leaves could be a potentially good source of bioactive anticancer agents.
In a nutshell, betel leaves were found to be good sources of essential elements, rich sources of volatile and non-volatile organic compounds, with several biochemical activities. Further research studies are needed on the betel leaf to isolate and characterize the identified major volatile and non-volatile organic compounds. The present study will be important for its more valuable and target-specific applications in controlling several diseases related to humans and other animals.|Piper betle L. 잎은 전 세계적으로 저작, 구강청결 및 약용 목적으로 널리 사용된다. 이러한 Piper betel L. 잎은 방글라데시에서 품질, 안전성 및 효능에 대한 데이터가 부족한 실정이다. 본 연구는 5 종류의 Piper betle L. 잎 (FBL) 인 Bangla, Sanchi, Misti, Khasia 및 BARI Pann3 의 품종별 화학적 (무기 원소 : 다량, 미량, 초미량, 독성원소, 휘발성 및 비휘발성 유기화합물) 및 생화학적 (항산화, 항균 및 세포 독성) 특성 평가를 목표로 하였다.
유도결합플라즈마 분광분석기 (ICP-OES) 및 유도결합플라즈마 질량분석기 (ICP-MS) 을 이용한 5종류의 Piper betle L. 의 무기원소 분석결과 (다량 무기원소 : K> Ca> Mg> P> Na; 미량 무기원소 : Mn> Fe> Sr> Ba> Zn> Cu> Cr> Ni; 초미량 무기원소 : Ga> Li> V> Cs> Se> U> Ba; 독성원소 : Pb> As> Cd> Tl) 으로 확인하였다. 영양학적으로 중요한 무기원소 중 분석한 모든 betel 잎에서 K (3.99-5.86 g/kg), Ca (2.64-2.80 g/kg) 및 Mg (1.26-1.33 g/kg) 으로 확인되었다. 반면, 세계 보건기구 (WHO) / 식량 농업기구 (FAO) 에서 지정한 한계 기준 Mn (21.94-25.09 mg/kg) 및 Pb (129.36-965.91 µg/kg) 를 제외한 모든 무기원소에서 안전한 수치로 확인되었다.
동시증류추출 및 가스크로마토그래피 질량분석법 (SDE/GC-MS) 을 사용하여 FBL의 휘발성 유기화합물 분석을 하였다. Betel 잎 중 Misti 종에서 가장 많은양의 휘발성 유기화합물 (13958.90 mg/kg) 이 확인되었으며, BARI Paan3 (11684.10 mg/kg), Khasia (11109.70 mg/kg), Sanchi (6958.51 mg/kg) 및 Bangla (4346.91 mg/kg) 순으로 확인되었다. 휘발성 유기성분 분석을 통하여 총 101종의 화합물을 확인하였다. 이 중 42종의 화합물은 공통으로 발견되었으며, 현재까지 연구된 타 문헌과 비교 시 본 연구를 통하여 betel 잎에서 새로운 50종의 휘발성 화합물을 확인하였다. Eugenol 은 모든 betel 잎에서 주요 화합물로 확인되었으며, 기타 주요 화합물은 β-caryophyllene, γ-muurolene, valencene, eucalyptol, chavicol 및 caryophyllene oxide 이었다.
비휘발성 유기화합물 스크리닝 테스트를 통해 FBL 의 phenol, flavonoids, terpene, steroids, phytosterols 및 saponin을 확인하였다. 또한, CCC 설계(central composite circumscribed)와 RSM (response surface methodology) 을 사용하여 betel 잎에서 초음파 추출을 통한 최적의 총 페놀 함량을 추출하기 위한 방법을 마련하였다. 최종 사용된 용매로 ethanol : acetic acid : water (70 : 5: 25 v/v) 를 선정하였으며, 최적의 추출 조건은 추출 시간 90분, 추출 온도 75℃; 고체/액체 비율 1:15.41로 지정하였다. 확립한 최적의 초음파 추출 조건에 의해 FBL를 추출하였으며, 총 페놀 및 플라보노이드 함량은 각각 110.51-322.8 mg GAE/g DW 및 46.79-57.09 mg QE/g DW로 확인되었다. 이후 주요 페놀 화합물인 hydroxychavicol 를 액체크로마토그래피-다이오드 검출기 (HPLC-DAD) 를 이용하여 FBL의 14.19 mg/g – 38.19 mg/g 으로 정량하였다.
2,2-Diphenyl-1-pycrilhydrazil (DPPH) 및 2,2'-Azino- (bis 3-ethylbenzthiazoline-6-sulphonic acid) (ABTS) 분석 시 FBL 추출물에서 IC50 값의 각각 황산화 활성 0.17-0.43 mg/mL, 0.04-0.11 mg/mL 으로 확인되었다. DPPH 및 ABTS 의 주요 페놀 화합물 IC50 값은 hydroxychavicol 및 eugenol 에 대해 각각 10.88-14.47 µg/mL 및 7.66-18.00 µg/mL이었다. 또한, Betle 잎 추출물은 S. typhimurium, P. aeruginosa, E. coli, A. faecalis, and MRSA 박테리아에 대하여 우수한 억제 특성을 나타냈다. Khasis 와 BARI Paan 3 은 다른 품종 (Bangla, Sanchi, 및 Misti) 보다 높은 항산화 및 항균 활성을 보였다. 인간 상피 세포 (HeLa), 인간 신경 모세포종 클론세포 (SH-SY5Y) 및 중간엽줄기세포 (MSCs) 의 IC50 값에 따른 FBL 추출물의 세포독성 활성은 각각 0.19-0.32 mg/mL, 0.41-0.54 mg/mL, 0.46-0.96 mg/mL 으로 확인되었다. 반면, hydroxychavicol 및 eugenol 의 세포독성활성 (IC50) 은 HeLa, SH-SY5Y 및 MSCs 세포에서 각각 3.14-3.21 µg/mL, 5.81-6.06 µg/mL, 28.47-32.13 µg/mL 을 나타냈다. 암세포중에서 betle 잎의 추출물 농도가 증가하면 암세포의 사멸률이 증가하였으며, 정상세포는 영향을 받지 않았다. 본 연구를 통하여 Piper betle L. 잎이 잠재적으로 생체 활성 항암제 공급원으로 사용될 수 있음을 기대볼 수 있다.
Betle 잎은 여러 가지 생화학적 효능과 함께 필수영양소의 좋은 공급원, 휘발성 및 비휘발성 유기화합물의 공급원으로 확인되었다. Betle 잎에 대한 추가적 연구로 확인된 주요 휘발성 및 비휘발성 유기화합물을 분리 및 특성화하고자한다. 또한, 현재의 연구는 건강 및 기타 동물과 관련된 여러 질병을 제어하는데 있어 가치 있고 특정 분야에 중요할 것으로 판단된다
- Alternative Title
- 방글라데시산 Piper betle L. 잎의 품종별 화학적 평가
- Alternative Author(s)
- 이슬람 아티쿨
- Affiliation
- 조선대학교 일반대학원
- Department
- 일반대학원 식품영양학과
- Advisor
- 김경수
- Awarded Date
- 2021-08
- Table Of Contents
- CONTENTS
List of tables VII
List of figures X
List of abbreviations XII
Abstract XIV
CHAPTER I Analysis of major, minor, trace, and toxic element in five varieties of Piper betle L. leaves (FBL) from Bangladesh 1
1.1. Introduction 1
1.1.1. Importance of mineral elemental analysis in Piper betle L. leaves 3
1.1.1.1. Major mineral elements 4
1.1.1.2. Minor mineral elements 5
1.1.1.3. Trace mineral elements 5
1.1.1.4. Toxic mineral elements 6
1.1.2. Analysis of mineral elements 6
1.1.2.1. Microwave digestion 7
1.1.2.2. Inductively coupled plasma-optical emission spectrometry 7
1.1.2.3. Inductively coupled plasma-mass spectrometry 8
1.1.3. Justification of this study 8
1.2. Materials and methods 9
1.2.1. Sample collection 9
1.2.2. Reagent and chemicals 11
1.2.3. Analytical apparatus of mineral elements 12
1.2.4. Samples preparation and digestion for mineral elements 12
1.2.5. Calibration procedure 13
1.2.6. Quality assurance 14
1.2.7. Statistical analysis 15
1.3. Results and discussion 15
1.3.1. Validation of analytical methods 15
1.3.2. Major mineral elements in FBL 17
1.3.3. Minor mineral elements in FBL 18
1.3.4. Trace mineral elements in FBL 19
1.3.5. Toxic mineral elements in FBL 20
1.3.6. Multivariate statistical analysis based on mineral element results obtained in FBL 21
1.4. Conclusion 23
CHAPTER II Analysis of volatile organic compounds in five varieties of Piper betle L. leaves (FBL) from Bangladesh 24
2.1. Introduction 24
2.1.1. Importance of volatile organic compounds in Piper betle L. leaves 24
2.1.2. Analysis of volatile organic compounds 25
2.1.2.1. Simultaneous distillation extraction 25
2.1.2.2. Gas chromatography-mass spectrometry 28
2.1.3. Justification of this study 28
2.2. Materials and methods 29
2.2.1. Sample collection 29
2.2.2. Reagent and chemicals 29
2.2.3. Analytical apparatus of volatile organic compounds 30
2.2.4. Extraction of volatile organic compounds in FBL 30
2.2.5. Establishment of retention index 32
2.2.6. Analysis of volatile organic compounds by GC-MS 32
2.2.7. Statistical analysis 33
2.3. Results and discussion 34
2.3.1. Establishment of retention index of n-alkane 34
2.3.2. Volatile organic compound in FBL from Bangladesh 35
2.3.2.1. Volatile organic compound in Piper betle L. var. Bangla from Bangladesh 35
2.3.2.2. Volatile organic compound in Piper betle L. var. Sanchi from Bangladesh 39
2.3.2.3 Volatile organic compounds in Piper betle L. var. Misti from Bangladesh 43
2.3.2.4. Volatile organic compounds in Piper betle L. var. Khasia from Bangladesh 47
2.3.2.5. Volatile organic compounds in Piper betle L. var. BARI Paan 3 from Bangladesh 51
2.3.2.6. Comparison of volatile organic compounds in FBL from Bangladesh 54
2.3.2.7. Multivariate statistical analysis in FBL from Bangladesh 66
2.4. Conclusion 69
CHAPTER III Non-volatile organic compounds screening, optimization of ultrasonic method for the extraction of total phenol, and HPLC-DAD analysis from five varieties of Piper betle L. leaves (FBL) from Bangladesh 70
3.1 Introduction 70
3.1.1. Importance of non-volatile organic compound in Piper betle L. leaves 70
3.1.1.1. Phenolic compound 71
3.1.1.2. Flavonoid compound 73
3.1.1.3. Terpene compound 73
3.1.1.4. Phytosterol compound 74
3.1.1.5 Saponin compound 74
3.1.2. Method of extraction 75
3.1.2.1. Heat reflux extraction (HRE) 76
3.1.2.2. Soxhlet extraction 76
3.1.2.3. Ultrasound-assisted extraction (UAE) 77
3.1.2.4. Supercritical fluid extraction (SFE) 77
3.1.2.5. Ultrasonic-assisted extraction (SFE) 78
3.1.3. Analytical methods for non-volatile organic compounds 79
3.1.4. Optimization of extraction of total phenol 80
3.1.4. Justification of this study 80
3.2. Materials and methods 81
3.2.1. Sample collection 81
3.2.2. Reagent and chemicals 81
3.2.3. Analytical apparatus of non-volatile organic compound 82
3.2.4. Extraction procedure for non-volatile organic compound screening 83
3.2.5. Qualitative screening of non-volatile organic compound in FBL from Bangladesh 83
3.2.5.1. Detection of phenolic compound 84
3.2.5.2. Detection of flavonoids compound 84
3.2.5.3. Detection of terpene compound 84
3.2.5.4. Detection of phytosterol compound 84
3.2.5.5. Detection of saponin compound 84
3.2.6. Optimization of ultrasonic methods for the extraction of total phenol from Piper betle L. leaves from Bangladesh 85
3.2.6.1. Ultrasonic bath and centrifuge condition 85
3.2.6.2. Extraction solvent selection 85
3.2.6.3. Extraction time selection 85
3.2.6.4. Extraction solid/liquid ratio selection 86
3.2.6.5. Extraction temperature selection 86
3.2.6.6. Extraction of central composite circumscribed (CCC) design 86
3.2.7. Determination of total phenol 86
3.2.8. Determination of total flavonoid 87
3.2.9. HPLC instrument and conditions 87
3.2.10. HPLC method validation 88
3.2.10.1. Linearity and sensitivity 88
3.2.10.2. Specificity and precision 89
3.2.10.3. Accuracy and spike recovery 89
3.2.11. Statistical analysis 90
3.3. Results and discussion 91
3.3.1 Qualitative screening of non-volatile organic compounds by chemical test in FBL 91
3.3.2 Qualitative screening of non-volatile organic compounds by HPLC-DAD analysis in FBL 98
3.3.3. Single-factor experimental analysis 102
3.3.3.1. Influence of solvent ratio to extract total phenol 102
3.3.3.2. Influence of extraction time to extract total phenol 102
3.3.3.3. Influence of extraction solid/solvent ratio to extract total phenol 103
3.3.3.4. Influence of extraction temperature to extract total phenol 103
3.3.4. Optimization of the variable by central composite circumscribed design 105
3.3.4.1. Model fitting with statistical analysis 105
3.3.4.2. Optimization of ultrasonic extraction 106
3.3.4.3. Verification of the predicted mode 111
3.3.5. Determination of total phenol and flavonoid in FBL 111
3.3.6. Validation of the HPLC-DAD analytical method 113
3.3.6.1. HPLC-DAD analytical method validation 113
3.3.6.2. Linearity and sensitivity 113
3.3.6.3. Specificity and precision 115
3.3.6.4. Accuracy and spike recovery 116
3.3.7. Determination of hydroxychavicol in FBL by HPLC-DAD 117
3.4. Conclusion 118
CHAPTER IV Analysis of biochemical activities in five varieties of Piper betle L. leaves (FBL) from Bangladesh 119
4.1 Introduction 119
4.1.1. Importance of biological activity in Piper betle L. leaves 119
4.1.1.1. In vitro antioxidant activity 120
4.1.1.1.1. DPPH radical scavenging assay 121
4.1.1.1.2. ABTS radical scavenging assay 122
4.1.2. Antibacterial activity 122
4.1.3. Cytotoxicity activity 123
4.1.4. Justification of study 123
4.2 Material and Methods 124
4.2.1. Sample Collection 124
4.2.2. Reagent and chemicals 124
4.2.3. Analytical apparatus of biochemical properties test 125
4.2.4. Extraction of sample 125
4.2.4.1. Antioxidant and cytotoxicity activity 125
4.2.4.2. Antimicrobial activity 125
4.2.5. Determination of antioxidant activity 126
4.2.5.1. DPPH assay procedure 126
4.2.5.2. ABTS assay procedure 127
4.2.6. Determination antibacterial activities 127
4.2.6.1 Microorganism for antimicrobial activity 127
4.2.6.2. Preparation of bacterial culture 127
4.2.6.3. Determination of zone of inhibition 127
4.2.7. Determination of cytotoxicity activity 128
4.2.7.1. Cytotoxicity screening 128
4.2.7.2. Cell count 129
4.2.7.3. MTT assay 129
4.2.8. Statistical analysis 130
4.3. Results and discussion 130
4.3.1. Antioxidant activity in FBL 130
4.3.2 Correlation between total phenolic content and antioxidant activity 133
4.3.3. Antibacterial activity 135
4.3.4. Cytotoxicity activity 138
4.4. Conclusion 142
Summary 143
References 145
Appendices 167
Curriculum vita 170
Acknowledgments 176
- Degree
- Doctor
- Publisher
- 조선대학교 대학원
- Citation
- Md. Atikul Islam. (2021). Chemical and Biochemical Evaluation in Five Varieties of Piper betle L. Leaves from Bangladesh.
- Type
- Dissertation
- URI
- https://oak.chosun.ac.kr/handle/2020.oak/16999
http://chosun.dcollection.net/common/orgView/200000503225
- Appears in Collections:
- General Graduate School > 4. Theses(Ph.D)
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