PHYTOCHEMICAL ISOLATION AND STANDARDIZATION OF ORTHOSIPHON STAMINEUS BENTH. LEAF EXTRACT FROM DIFFERENT LOCATIONS AND SELECTED ANTIOXIDANT AND TOXICITY STUDIES
NUR FARAH AMALINA BINTI MUGHNI
UNIVERSITI SAINS MALAYSIA 2014
PHYTOCHEMICAL ISOLATION AND STANDARDIZATION OF
ORTHOSIPHON STAMINEUS BENTH. LEAF EXTRACT FROM DIFFERENT
LOCATIONS AND SELECTED ANTIOXIDANT AND TOXICITY STUDIES
by
NUR FARAH AMALINA BINTI MUGHNI
Thesis submitted in fulfillment of the requirements for the degree of Master of Science
February 2014 PENGASINGAN FITOKIMIA DAN PEMIAWAIAN EKSTRAK DAUN
ORTHOSIPHON STAMINEUS BENTH. DARI LOKASI YANG BERBEZA
UNTUK KAJIAN ANTIOKSIDAN DAN TOKSISIITI
oleh
NUR FARAH AMALINA BINTI MUGHNI
Tesis yang diserahkan untuk memenuhi keperluan bagi Ijazah Sarjana Sains
Februari 2014 ACKNOWLEDGEMENTS
First and foremost praise to Allah the almighty who gave me the knowledge, strength and the determination to finish my thesis successfully.
This success and final outcome of this project required a lot of guidance and assistance from many people. I am extremely fortunate to have gotten this along the completion of my project work. I would like to express the deepest appreciation to my supervisors
Prof. Dr. Zhari Ismail for his commitment and invaluable guidance throughout the study.
I offer my sincerest gratitude to my co-supervisor Associate Prof. Dr Nornisah
Mohamed, without her invaluable assistance and support, this study would not have been completed.
I express my special thanks to Majlis Amanah Rakyat (MARA) for providing financial support and scholarship. I am also grateful to School of Pharmaceutical Science , USM for providing a good research environment and all of the staff at this school for their kindness help and contribution in this study. My sincere thanks to Dr Abdul Rahim and
Dr Beh for helping me and give many ideas for completing my research and Dr Lee
Chong Yew for helping me elucidate NMR structure.
My sincere appreciation goes to all of my friends and lab mates especially Fatin Fathiah for their constant encouragement and investing their time and energies in this project.
ii
TABLE OF CONTENTS
ACKNOWLEDGEMENT ii
TABLE OF CONTENTS iii
LIST OF TABLES x
LIST OF FIGURES xii
LIST OF ABBREVIATION xv
ABSTRAK xvii
ABSTRACT xx
CHAPTER 1 – INTRODUCTION
1.1 General Introduction 1
1.2 Justification of the Study 4
1.3 Objectives of the Study 6
CHAPTER 2 – LITERATURE REVIEW
2.1 Orthosiphon stamineus 7
2.1.1 Classification and Description 7
2.1.2 Botanical Description 8
2.1.3 Plant Habitat and Cultivation 10
2.1.4 Chemical Constituents 10
2.1.5 Traditional uses of Orthosiphon stamineus 17
2.1.6 Review of Biological Activity of 17
Orthosiphon stamineus
iii
2.2 Antioxidant 27
2.2.1 Types of Antioxidant 28
2.2.2 Natural Antioxidants 28
2.2.3 Antioxidant Assays for Plant 29
2.2.4 Literature Study of Antioxidant Activity 29
2.2.5 Analysis and Quantification of Phenolic Compounds 30
2.3 Cytotoxicity 30
2.4 Malaysian Herbal Monograph 32
2.5 The Challenges of Standardizing Herbs
2.5.1 The Plant Physiology 32
2.5.2 Variability on Individual Plant 33
2.5.3 Adulteration and Deterioration 35
CHAPTER 3 – EXPERIMENTAL METHODOLOGY
3.1 Materials and Chemicals 37
3.2 Apparatus and Instrumentation 37
3.2.1 High Performance Liquid Chromatography 37
3.2.2 Ultra Violet (UV) Spectroscopy 38
3.2.3 Fourier Transform Infrared (FTIR) Spectroscopy 38
3.2.4 High Performance Thin Layer Chromatography (HPTLC) 38
3.2.5 Extraction 38
3.2.5 Nuclear Magnetic Resonance (NMR) 38
3.3 Procurement of Raw Material
3.3.1 The Source of Orthosiphon stamineus leaf 39
iv
3.4 Phytochemical Screening
3.4.1 Alkaloids 41
3.4.2 Terpenes 41
3.4.3 Flavonoids 42
3.4.4 Anthraquinones 42
3.4.5 Tannins 42
3.4.6 Saponins 43
3.5 Quality Control Analysis
3.5.1 Ash Content 43
3.5.1.1 Total Ash 43
3.5.1.2 Acid Insoluble Ash 44
3.5.2 Extractive Values (Hot extraction)
3.5.2.1 Water 44
3.5.2.2 Ethanol 44
3.5.3 Extractive Value (Cold Maceration)
3.5.3.1 Water 45
3.5.3.2 Ethanol 45
3.5.4 Loss of Drying 45
3.5.5 Nutritive Value 46
3.5.5.1 Protein 46
3.5.5.2 Fat 49
3.5.6 Determination of Heavy Metals 49
3.5.6.1 Preparation of samples 49
v
3.5.7 Microbial Limit Test (MLT) of Orthosiphon stamineus 49
Leaf
3.5.7.1 Procedure for Total Aerobic Microbial, 50
Yeast and Mold Count (via pour plate)
3.5.7.2 Procedures for Specific Microorganism Test 50
3.6 Plant Extraction 51
3.7 Chemical Profiling
3.7.1 High Performance Thin Layer Chromatography (HPTLC) 52
3.7.2 High Performance Liquid Chromatography 53
3.7.2.1 Preparation of Standards and Test Samples 53
3.7.2.2 Chromatographic Condition 53
3.7.3 Ultra violet-Visible (UV-Vis) Spectrophotometry 53
3.7.4 Attenuated Total Reflection-Fourier Transform
Infrared (ATR-FTIR) Spectrophotometry 54
3.8 Chemometric Classification of Orthosiphon stamineus
by Fourier Transform Infrared (FT-IR) 54
3.8.1 Sample Analysis 54
3.8.2 Data Analysis 55
3.9 Isolation of Chemical Components 56
3.9.1 Extraction 56
3.9.2 Separation of Chemical Components from 57
Chloroform Extract
3.9.3 Qualitative Analysis of F1 using Spectroscopic 58
and Chromatographic Techniques
vi
3.9.4 Development of High Performance Liquid
Chromatography (HPLC) Methanol of F1 Compound in
Water Extract of Orthosiphon stamineus Leaf 59
3.9.5 Validation of HPLC Method 60
3.9.6 Quantification of F1 Compound in Methanol Extract of
Orthosiphon stamineus Leaf by HPLC Method 61
3.10 Antioxidant activity 62
3.10.1 Chemicals and Apparatus 62
3.10.2 Plant Samples 62
3.10.3 Assay for Total Phenolic 62
3.10.4 Assay for Total Flavonoids 63
3.10.5 Scavenging Effect on 1,1-diphenyl-2-picrylhydrazyl 64
(DPPH)
3.10.6 Antioxidant Assay Using a β-carotene-linoleate 64
Model System
3.11 Toxicity Assay 65
3.12 Statistical Analysis 66
CHAPTER 4 – RESULTS AND DISCUSSION
4.1 Phytochemical Screening 67
4.2 Quality Control Analysis 68
4.2.1 Ash Content 68
4.2.2 Loss of Drying 70
vii
4.2.3 Extractive Values 71
4.2.4 Nutritive Value 75
4.2.5 Determination of Heavy Metals 76
4.2.6 Microbial Limit Test (MLT) of Orthosiphon stamineus 77
Leaf
4.3 Chemical Profiling 80
4.3.1 High Performance Thin Layer Chromatography (HPTLC) 80
4.3.2 High Performance Liquid Chromatography 81
4.3.3 Ultra violet-Visible (UV-VIS) Spectrophotometry 86
4.3.4 Attenuated Total Reflectance-Fourier Transform
Infrared (ATR-FTIR) spectroscopy 87
4.4 Chemometric Classification of Orthosiphon stamineus by
Fourier Transform Infrared FT-IR 92
4.5 Environmental Effects 101
4.6 Isolation using Aqueous Extract of Orthosiphon stamineus Leaf 115
4.7 Antioxidant Activity 131
4.7.1 Total Phenolic 131
4.7.2 Total Flavonoids 132
4.7.3 Scavenging Effect on 1,1-diphenyl-2-picrylhydrazyl 136
(DPPH)
4.7.4 Antioxidant Assay Using a β-carotene-linoleate 140
Model System
4.7.5 Statistical Analysis 145
4.8 Toxicity using Brine Shrimp Lethality Assay 146 viii
CHAPTER 5 – CONCLUSION
5.1 Conclusion 150
5.2 Suggestion for Further Studies 152
REFERENCES 153
APPENDICES 168
LIST OF PUBLICATION 197
ix
LIST OF TABLES
Pages
Table 1.1 Products Orthosiphon stamineus registered under National Pharmaceutical Control Bureau (NPCB) 4
Table 1.2 Patents published based on the World International Property Organization (WIPO) 5
Table 2.1 Chemical constituents of Orthosiphon stamineus 12
Table 2.2 Summary of scientific studies on the Orthosiphon stamineus from the reviewed literature 22
Table 3.1 Orthosiphon stamineus from various localities used in this study 39
Table 3.2 Microwave digestion setting 49
Table 4.1 Preliminary phytochemical test of leaves of dried powder Orthosiphon stamineus 68
Table 4.2 Summary of physicochemical analysis of leaf of Orthosiphon stamineus 74
Table 4.3 Nutritive value (energy, carbohydrate, protein and fat) of dried powder leaf of Orthosiphon stamineus Benth. 75
Table 4.4 Heavy metal content of Orthosiphon stamineus leaf 77
Table 4.5 Contamination of microbial in the leaf of Orthosiphon stamineus 79
Table 4.6 Percentage of marker content in of Orthosiphon stamineus leaf 85
Table 4.7 Major characteristic IR absorbtion bands of Orthosiphon stamineus 91
Table 4.8 Type of soil for 10 different locations of Orthosiphon stamineus plant 102
x
Table 4.9 Temperature of location for the samples of Orthosiphon stamineus collected 104
Table 4.10 Percentage of humidity of the samples from ten different locations 105
Table 4.11 Altitude of the samples collected from ten different locations 107
13 Table 4.12 Comparison C NMR (CDCl3) spectral data of F1 compound 119
Table 4.13 Precision of the HPLC methods for the determination of F1 compound in Orthosiphon stamineus leaf water extract 126
Table 4.14 Percentage F1 compound in the water extract of Orthosiphon stamineus in different locations 127
Table 4.15 IC50 values of different extracts of Orthosiphon stamineus in DPPH scavenging assay 138
Table 4.16 Correlation berween total phenolic, total flavonoid, DPPH scavenging assay and β-carotene linoleic acid 145
Table 4.17 Toxicity activity using brine shrimp assay 148
xi
LIST OF FIGURES
Pages
Figure 2.1 Picture of Orthosiphon stamineus leaf 8
Figure 2.2 Flower of Orthosiphon stamineus 9
Figure 2.3 Chemical structure of Orthosiphon stamineus (diterpenes) 13
Figure 2.4 Chemical structure of Orthosiphon stamineus (triterpenes) 14
Figure 2.5 Chemical structure of Orthosiphon stamineus (flavones) 15
Figure 2.6 Chemical structure of Orthosiphon stamineus (phenolic acid) 16
Figure 2.7 Chemical structure of Orthosiphon stamineus (benzochromene) 16
Figure 3.1 Orthosiphon stamineus from various localities use in this study 40
Figure 3.2 Scheme of extraction of Orthosiphon stamineus dried leaves 56
Figure 3.3 Scheme of isolation F1 compound from Orthosiphon stamineus 58
Figure 4.1 Total ash, water soluble ash and acid insoluble ash of dried powder leaf Orthosiphon stamineus from 10 different locations 70
Figure 4.2 Comparable of percentage of loss of drying between 10 different locations 71
Figure 4.3 Percentage of cold maceration and hot maceration on raw material of Orthosiphon stamineus 72
Figure 4.4 Percentage of cold maceration and hot maceration (ethanol) on raw material of Orthosiphon stamineus 73
Figure 4.5 TLC profile of methanolic extract of Orthosiphon stamineus leaf from 10 different locations using sinensetin as standard (365nm) 80
Figure 4.8 HPLC chromatogram of standard; a) romirinic acid, b) sinensetin and c) eupatorin 82
xii
Figure 4.9 HPLC chromatogram of methanolic extract of Orthosiphon stamineus leaf from 10 different locations 83
Figure 4.10 UV spectra of leaf of Orthosiphon stamineus from 10 locations 87
Figure 4.11 FTIR spectra of methanolic extract of Orthosiphon stamineus leaf 89
Figure 4.12 Overlay spectra of Orthosiphon stamineus methanol extract analyzed at different time 93
Figure 4.13 Principle Component Analysis (PCA) of leaf of Orthosiphon stamineus from 10 locations 94
Figure 4.13a FTIR spectra for KBPP, CJPM and DSJM (Group I) 95
Figure 4.13b FTIR spectra for PPKM, HLSM and SKTM (Group III) 96
Figure 4.13c FTIR spectra of SNSM, PNSM and SUMM (Group IV) 97
Figure 4.13d FTIR spectra for TPPM (Group II) 98
Figure 4.14 Comparison FTIR spectra for Group I and Group IV 100 Figure 4.15 Percentage of marker concentration (sinensetin and eupatorin) content with the increasing of altitude 107
Figure 4.16 Comparison of heavy metal content (lead and cadmium) from different locations 109
Figure 4.17 Percentage of marker compound (sinensetin and eupatorin) for each locations 110
Figure 4.18 Mercury content for ten locations 111
Figure 4.18a Principle Component Analysis (PCA) of leaf of Orthosiphon stamineus from 10 locations based on nutritive profile 113
Figure 4.17 Chemical structure of F1 compound 115 (5-hydroxy- 6,7,3’,4’tetrametoxyflavone)
Figure 4.18 UV spectra of F1 compound 116
Figure 4.19 IR spectra of F1 compound 117
Figure 4.20 13C NMR data of F1 compound 120
xiii
Figure 4.21 1H NMR data of F1 compound 121
Figure 4.22 DEPT45 NMR data of F1 compound 122 Figure 4.23 DEPT90 NMR data of F1 compound 122
Figure 4.24 DEPT135 NMR data of F1 compound 123 Figure 4.25 HPLC chromatogram of methanol extract of Orthosiphon stamineus leaf for different locations 128
Figure 4.26 HPLC chromatogram of F1 compound 130
Figure 4.27 Gallic acid calibration curve 131
Figure 4.28 Total phenolic content in Orthosiphon stamineus leaf 132
Figure 4.329 Quercetin calibration curve 133
Figure 4.30 Total flavanoid content in Orthosiphon stamineus leaf 134
Figure 4.31 Percentage of inhibition of different extracts of 137 Orthosiphon stamineus leaves in DPPH scavenging assay
Figure 4.32 Comparative DPPH radical scavenging activity of Orthosiphon stamineus leaves extracts and quercetin 138
Figure 4.33 -carotene test of Orthosiphon stamineus leaf 141 Figure 4.34 Brine shrimp picture (A) before treatment and (B) after treatment For PNSM sample at concentration 125 μg/ml 146
xiv
LIST OF ABBREVIATIONS
AAS Atomic Absorption Spectroscopy
AlCl3 Aluminium chloride
As Arsenic
ATR-FTIR Attenuated Total Reflection-Fourier Transform Infrared
BHA Butylated hydroxylanisole
BHP British Herbal Pharmacopoeia
BSA Bovin serum albumin
BSLT Brine shrimp lethality test
BuOH Butanol
CAT Catalase
Cd Cadmium
CHCl3 Chloroform
CJPK Changkat Jering Perak
DMSO Dimethyl sulfoxide
DSJM Desaru Johor
DPPH 2,2-Diphenyl-1-Picrylhydrazil
EUP Eupatorin
FCR Folin-Ciocalteu’s reagent
FTIR Fourier transform infrared
GC Gas chromatography
GC-MS Gas chromatography-mass spectrometry
xv
Hg Mercury
HLSM Hulu Langat Selangor
HMPs Herbal Medicinal Products
HPLC High performance liquid chromatography
HPTLC High performance thin layer chromatography
KBPP Kepala Batas Pulau Pinang
MLT Microbial limit test
UV Ultra-violet
NPCB National Pharmaceutical Control Bureau
PPKM Pasir Puteh Kelantan
PNSM Pantai Negeri Sembilan
RA Rosmarinic acid
ROS Reactive oxygen species
SIN Sinensetin
SKTM Sungai Kok Terengganu
SNSM Sendayan Negeri Sembilan
SUMM Sungai Udang Melaka
TLC Thin Layer Chromatography
TNC Total nonstructural carbohydrates
TPPM Taiping Perak
WHO World Health Organization
WIPO World International Property Organization
xvi
PENGASINGAN FITOKIMIA DAN PEMIAWAIAN EKSTRAK DAUN ORTHOSIPHON STAMINEUS BENTH. DARI LOKASI YANG BERBEZA UNTUK KAJIAN ANTIOKSIDAN DAN TOKSISITI
ABSTRAK
Kajian ini bertujuan untuk pemiawaian Orthosiphon stamineus daripada sepuluh lokasi berbeza di seluruh Semenanjung Malaysia termasuk ujian farmakognosi, profil kimia dan aktiviti biologi (antioksidan dan toksisiti).
Analisis gravimetrik had logam berat dan ujian had mikrob telah dilakukan ke atas serbuk daun kering untuk memeriksa kualiti dan keselamatan bahan mentah sebelum meneruskan analisis ke peringkat yang lain. Tenaga, karbohidrat, protein dan lemak dalam semua sampel telah menunjukan keputusan (per 1g bahan mentah) dalam lingkungan masing-masing 305-339 kkal, 53.1-59.9 g, 12.4-17.8 g dan 3.3-5.2 g.
Pemeriksaan awal fitokimia pada bahan mentah mendedahkan kehadiran terpenoid, flavonoid, tanin dan saponin.
Profil kimia yang telah ditentukan secara kualitatif dengan menggunakan teknik spektroskopi (UV-Vis dan IR) dan kromatografi (HPLC). Keputusan menunjukkan bahawa spektrum yang sama telah diperolehi dalam Orthosiphon stamineus daun ekstrak metanol dari 10 lokasi yang berbeza. Asid rosmarinik, sinensetin dan eupatorin telah dipilih sebagai bahan penanda untuk dianalisis oleh HPLC. Kandungan mereka adalah dalam julat antara 2.12 + 0.94 % - 14.61 + 2.23 % (asid rosmarinik), 0.25 + 2.29 %- 1.04
+ 0.78 % (sinensetin) dan 0.23 + 2.05 % - 1.00 + 1.12 % (eupatorin). Analisis komponen utama (PCA) telah digunakan untuk mendapatkan maklumat dengan menggunakan data
xvii yang diperolehi daripada ujian inframerah. Perbezaan yang ditunjukkan mungkin disebabkan oleh kepelbagaian dalam faktor-faktor alam sekitar. Perbezaan suhu antara lokasi yang berbeza adalah kecil (Purata 270C - 290C) manakala perbezaan dalam lembapan adalah lebih jelas (70% - 89%). Perubahan dalam ketinggian juga boleh dilihat pada setiap lokasi yang dikaji iaitu dalam lingkungan 3.84 m-112.92 m.
Pengasingan oleh kromatografi dengan menggunakan ekstrak air daripada daun
Orthosiphon stamineus menghasilkan pengasingan satu sebatian. Ujian analisis kimia dan analisis spektrum telah dilakukan dan memberi satu struktur yang telah dijelaskan sebagai 5-hidroksi-6,7,3’,4’-tetrametoksiflavon.
Jumlah kandungan fenolik sampel termasuk dalam julat 71,63 mg asid galik/g berat kering (Kepala Batas) kepada 171 mg asid galik/g berat kering (Hulu Langat).
Kandungan flavonoid jumlah adalah dalam lingkungan 105.63+0.09 mg kuersetin/g berat kering (Sungai Udang) to 245.96+0.02 mg kuersetin/g berat kering (Kepala Batas).
Antara ekstrak dikaji, aktiviti antioksidan tertinggi menggunakan ujian DPPH adalah ekstrak dari Desaru dengan nilai IC50 2.778 μg/ml. Dalam ujian β-karotena, aktiviti antioksidan yang tertinggi telah diperolehi dalam ekstrak daripada Hulu Langat dengan peratusan 96.46 % pada kepekatan 1000 μg/ml. Kajian ini juga membuktikan bahawa tidak wujud hubungan antara jumlah fenolik atau jumlah flavonoid dengan ujian DPPH dan β-karotena.
Kajian toksisiti daripada ekstrak dikaji dengan menggunakan ujian anak udang.
Kematian anak udang dengan 50% kepekatan (LC50) adalah dalam lingkungan 106.09-
234.7 μg/ml.
xviii
Kesimpulannya, kajian yang telah dilakukan menunjukkan bukti kualiti dan keselamatan, aktiviti antioksidan dan toksisiti ekstrak Orthosiphon stamineus adalah seragam dari 10 lokasi yang berbeza.
xix
PHYTOCHEMICAL ISOLATION AND STANDARDIZATION OF ORTHOSIPHON STAMINEUS BENTH. LEAF EXTRACT FROM DIFFERENT LOCATIONS AND SELECTED ANTIOXIDANT AND TOXICITY STUDIES
ABSTRACT
This study aimed to standardize Orthosiphon stamineus extract from ten different locations throughout Peninsular Malaysia that includes pharmacognosical tests, chemical profile and biological activity (antioxidant and toxicity using a brine shrimp bioassay).
Gravimetric analysis, heavy metal limit and microbial limit test were done on the dried leaf powder to determine the quality and safety of the raw material. Energy, carbohydrate, protein and fat content were estimated in all samples and the results (per
1g of raw material) were in the range of 305-339 kcal, 53.1-59.9 g, 12.4-17.8 g and 3.3-
5.2 g respectively. Preliminary phytochemical screening revealed the presence of terpenoids, flavonoids, tannins and saponins.
The chemical profile was determined qualitatively using spectroscopic (UV-Vis and IR) and chromatographic techniques (HPLC). The results showed that similar spectra were obtained in the methanolic leaf extracts from all locations. Rosmarinic acid
(RA), sinensetin (SIN) and eupatorin (EUP) marker compounds were analyzed by
HPLC. Their content was in the range of 2.12+0.94 % - 14.61+2.23 % (RA),
0.25+2.29% - 1.04+0.78 % (SEN) and 0.23+2.05 % - 1.00+1.12 % (EUP). The similarity and difference between spectra (which has been done using FT-IR) were analyzed using Principle Component Analysis (PCA) as tools for extracting relevant chemical information from obtaining infrared data. These differences may be explained
xx due to the variations in the environmental factors. The difference in temperature between the different locations was marginal (average 270C – 290C) whereas the difference in relative humidity was more obvious (70 % - 89 %). Changes in altitude can also be seen amongst the studied locations, the altitude values were in the range of 3.84m-112.92m.
Separation by column chromatography from the Orthosiphon stamineus leaf water extract resulted in the isolation of one compound. On the basis of chemical and spectral data, the structures was elucidated as 5-hydroxy-6,7,3’,4’-tetramethoxyflavone.
The total phenolic content of the samples from ten locations are within the range of 71.63+0.02 mg Gallic acid/g dry weight (Kepala Batas) to 171+0.02 mg Gallic acid/ g dry weight (Hulu Langat). The total flavonoid content was in the range of 105.63+0.09 mg quercetin/g dry weight (Sungai Udang) to 245.96+0.02 mg quercetin/g dry weight
(Kepala Batas). Methanolic leaf extracts of the Orthosiphon stamineus were then screened for antioxidant activity. Amongst the extracts studied, the highest antioxidant activity in DPPH scavenging assays was obtained in the extract prepared from Desaru with IC50 values 277.8+0.10 µg/ml. In the β-carotene assay, the highest antioxidant activity was obtained in the extract prepared from Hulu Langat, with the percentage inhibition of 96.46+0.02 % at 1000 µg/ml. Statistical analysis was done and resulted no correlation exist between total phenolic or total flavonoid with DPPH assay and β- carotene assay.
Toxicity of the extracts was studied using the brine shrimp lethality assay. The median lethal concentration (LC50) was in the range of 106.09+1.45 – 323.47+4.43
µg/ml.
xxi
In conclusion, the current study provides evidence on quality and safety, antioxidant activity and toxicity assay using a brine shrimp assay of the standardized
Orthosiphon stamineus extract from 10 different locations.
xxii
CHAPTER 1
INTRODUCTION
1.1 General introduction
Ever since decades ago, herbs have been proven to have potentials to cure and heal diseases and illnesses. Besides decorative plants and flavor enhancing, herbs are widely used as healing sources for diseases and illness prior to the discovery of modern medicine. Throughout the centuries, herbs are found to be able to treat various illnesses involving digestive problems, toothaches, open wounds, etc. Extensive researches have then been carried out to further develop and explore the potentials of these herbs.
Based on the statistics carried out by the World Health Organization (WHO), 80% of the world population prefers to choose herbs as medicine to treat ailments. WHO states that about 119 medicinal substances are extracted from various plants and 74% of them are still used without any modification. Up to date, the extracted substances from herbs have become the basis of manufactured medication for diseases involving cardiovascular problems, asthmas and hypertensions (WHO, 1998).
Originally, the word herb is derived from the word “Herba”, a Latin word referred to grasses, green crops and other leafy plants. Nowadays, herbs extend to plants, trees or shrubs possessing culinary, medicinal and aesthetic properties. Generally, a plant can be used as a whole, by part or even with combination with other plants with the purpose of the treatments. The World Health Organization (WHO) stated that herbal
1 medicines (plants or parts of plants) contain an incipient as an active ingredient, whether it is in the crude or processed state (WHO, 1998).
Traditional medicine is commonly practiced as it is a natural and safe medical approach since centuries ago. Nowadays, herbal products play important roles due to their compatibility with the human body, besides being able to prevent diseases from occurring rather than to cure a post-occurred disease. The chemical constituents present in herbs are part of the physiological function (Kamboj, 2000).
Although traditional medicines involving herbal remedies are trusted and oftenly safe, negative side effects might arise if these remedies are consumed together with prescriptive drugs (Benzie and Watchel-Galor, 2011). Several case reports of the inverted effect after consuming herbal products such as allergic reactions and direct toxification. Fairbairn (1980) suggests that standardization of herbal remedies should be prioritized in producing herbal products to prevent unnecessary side effects.
Standardization is defined as the establishment of pharmaceutical quality that is repeated through comparison with reference materials and to determine the amount of established qualitative or quantitative for one or more compounds or groups of compounds. In phytomedicine field, standardization is used in extraction (Gurib-Fakim et al., 2005). Mosihuzzaman and Choudhary (2008) defined standardization as the control on quality of herbal medicinal products by means of modifying the herbal medicine planning for describing content constituent or group of substances with known therapeutic activity. Standardization is the most important step which allows the identification of known active constituents. However, there are still unknown active
2 constituents in the herbs. For these cases, herbal products need to be standardized using certain marker compounds. Based on Ong (2004), chemical standardization usually contains chemical identifications (using spectroscopic or chromatographic fingerprint) and chemical assays (active constituents or marker compounds).
It is very important to maintain the quality of herbal products to ensure the safety and effectiveness of the products (Busse, 2000). There are some variables that give effect to the safety, efficiency and quality of the herbal medicines such as the freshness of the plants, temperature, water availability, light exposure, nutrients and the time of collection (Calixto, 2000).
In traditional mediciation, old folks believe that Orthosiphon stamineus possesses diuretic properties, antiallergic, antiinflammatory, antihypertensive and antitumor. It is also used to treat diabetes, rheumatism and gout (Burkhill, 1966).
In this study, the leaves of Orthosiphon stamineus Benth plant from different locations were studied. This plant is locally known as “Misai kucing” or literally cat’s whiskers due to its unique flower which resembles a cat’s whisker. The entire plant possesses therapeutic properties; such as the leaves of the plant are believed to contain antioxidant property (Akowuah et al.,2005 (b) ; Khamsah et al.,2006; Yam et al.,2007), antiinflammatory property (Masuda et al.,1992; Yam et al.,2008) and diuretic property
(Arafat et al., 2008).
3
1.2 Justification of study
Orthosiphon stamineus Benth is the source of many secondary metabolites compounds. Secondary metabolites perform a significant part in the adoption of plants to the surroundings besides being able to overcome stress conditions. The concentration of secondary metabolites in Orthosiphon stamineus plants are regulated by physiological, genetic, environmental and chemical factors such as light, rainfall, temperature, chemicals, soil and plant growth regulators (Ibrahim and Jaafar, 2013).
Various products derived from Orthosiphon stamineus were registered under
National Pharmaceutical Control Bureau (NPCB) (Table 1.1). According to the World
International Property Organization (WIPO), there are several patents available for
Orthosiphon stamineus (Table 1.2).
Table 1.1: Products Orthosiphon stamineus registered under National Pharmaceutical Control Bureau (NPCB)
Product Name MAL No Company Name
Misai kucing MAL20012878T Herbal Science Sdn. Bhd.
Misai kucing plus MAL20040150T Herbal Science Sdn. Bhd.
Golden leaf cat's whisker tea plus MAL05101755T Herbal Land Manufacturing Sdn. Bhd.
Yang yen bao capsule MAL07101275TC Kangyew Healthcare
Forest'secret precious misai kucing MAL09111786TC Forest Secret Sdn. Bhd. herbal extract Orthosiphon stamineus plus MAL09021103T Duha Sdn. Bhd. Misai kuching herbal tea 3 gm MAL07061365T Natural Wellness Industries Sdn. Bhd. Naturax misai kucing 2g tea MAL09071080T Biotropics Malaysia Berhad http://www.portal .bpfk.gov.my/product (27/3/2013)
4
Table 1.2: Patents published base of the World International Property Organization (WIPO)
Title Applicant WO/2011/078652 Orthosiphon stamineus extracts with beneficial use Biotropics Malaysia Berhad as cognition enhancer Wo/2011/129680 A use of an effective amount of a composition Universiti Sains Malaysia Usm comprising o. Stamineus leaf extract 2010143862 Sunstar Inc. Composition containing plant extract 2005139136 National Institute Of Advanced Insulin secretagogue Industrial & Technology 02180232 Gorshkov Anatolij Nikolaevich Agent eliciting antitumor activity 2001224330 Food comprising lactobacillus symbiotic culture Uechi Hideko product and medicinal plant and method for producing the same http://www.patentscope.wipo.int/search (3/4/2013)
This study aimed to:-
1. Provide a comparative study of Orthosiphon stamineus leaf selected from Peninsular
Malaysia based on a humidity, temperature, altitude, collection season and soil
condition.
2. Provide information regarding free radical scavenging and brine shrimp lethality
bioassay of Orthosiphon stamineus leaf extract.
3. Prove the potential relationship between the complex chemical constituent and
geographical origin of the samples.
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1.3 Objectives of study
1. To analyse the quality (microscopic, macroscopic, colour and gravimetric), safety
(microbial and heavy metal) and nutritive value of Orthosiphon stamineus from 10
different locations based on the Malaysian Herbal Monograph Volume 2.
2. To profile and standardize methanol extracts of Othosiphon stamineus Benth. leaf by
using spectroscopic and chromatographic methods of selected marker compounds
from all locations.
3. To isolate and identify selected compound from the water extract of Orthosiphon
stamineus leaf.
4. To evaluate the selected antioxidant and toxicity (brine shrimp lethality assay)
properties of the methanolic extracts.
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CHAPTER 2
LITERATURE REVIEW
2.1 Orthosiphon stamineus
Orthosiphon stamineus Benth, also known as “Misai Kucing” is a genus in the family of
Lamiaceae. “Misai Kucing” plant is an herbal species originated from the South-East
Asia region. In Malaysia, the plant’s leaves are used as a treatment to diuretic, diabetes and hypertension. In Vietnam, the arterial part of the plant is used to cure urinary lithiasis, eruptive fever, oedema, influenza, rheumatism, hepatitis, jaundice and biliary lithiasis. Since 1930s, phytochemical and pharmacological studies of this plant have been studied (Tezuka et al., 2000).
2.1.1 Classification and description
Taxonomically, this plant is categorized into the following scheme:
Family : Lamiaceae
Genus : Orthosiphon
Species : Stamineus
Scientific name : Orthosiphon stamineus (Benth)
Local name : Misai Kucing
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According to Anon (2001), Orthosiphon aristatus, Orthosiphon grandiflorum and
Orthosiphon spicatus are synonyms to the scientific term, Orthosiphon stamineus. Every country has its own vernacular name for this plant such as Thé de Java (France), kumis kucing (Indonesia), kumis kucing (Sudan), kumis kucing or misai kucing (Malaysia), balbas-pusa and kabling-gubat (The Phillippines), kapen prey (Cambodia), hnwàd méew
(Laos), yaa nuat maeo (Thailand) and r[aa]u m[ef]o (Vietnam).
2.1.2 Botanical description
Orthosiphon stamineus is a quadrangular perennial herb, with a height of 25-200 cm, inadequately ramified and ascending stems. The plant is an herbaceous shrub, grows to a stature of 1.5 m and can be found in tropical and subtropical regions. The stem is intensely arranged in a quadrangular manner, reddish in colour, erect and branches profusely. As shown in Figure 2.1, the leaves are organized in inverse sets, straightforward, sleek, hued green, glabrous with a lanceolate leaf cutting edge and serrate edge. The leaf apices are sharpened with an intense leaf base. The petiole is moderately short, measuring around 0.3 cm in the ballpark and ruddy purple in the shade.
Figure 2.1: Picture of Orthosiphon stamineus leaf
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In Figure 2.2, it is observed that the flowers are borne on the verticals and length of about 16 cm. The terminal inflorescence is borne on a maroon pubescent. The flowers are campanulas, white or blue in colour, with long filament over mid-green foliage, making the flowers resembles cat’s whiskers. The calyx is gamosepalous with two lips
(bilabiate), which is greenish red in colour and measuring about 6 mm in length. One of the calyx lips has a toothed margin while the other lip is enclosed with minute white hairs. The corolla is also partially gamapetalous with two lips (bilabiate) covered with minute hairs. The corolla is pale violet in colour with lips shorter than the corolla tube.
The labellum is pale violet in colour, hairy and pinkish on the under surface. Four stamens are inserted near the base of the corolla tube. The stamens are not the same in length, measuring from 4.7 to 5.2 cm. There is a single, central terete style with a clavate stigma (Muhamad and Mustafa, 1994).
Figure 2.2: Flower of Orthosiphon stamineus
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2.1.3 Plant habitat and cultivation
Jaganath and Ng (2000) explained that Orthosiphon stamineus has been cultured for quite a while. Wild Orthosiphon stamineus grow along roadsides, forest sides and on wasteland. This plant is easily propagated through three or four nodded stem cuttings.
These stem cuttings are obtained from the mother plant of more than five months age.
The middle portion of the stem is then chosen to obtain a higher rate of success in propagation. Before transferring to the fields, the stem cuttings are propagated for about a month. The leaves and branches can be harvested after 3 to 4 months after the field transfer. Orthosiphon stamineus thrives in well-drained soils in full sunlight. These plant branches more profusely and generally do better with regular applications of organic fertilizers such as chicken dung. Orthosiphon stamineus is not significantly susceptible to disease, but it's prone to insect attack (Jaganath and Ng, 2000).
2.1.4 Chemical constituent
Orthosiphon stamineus plants have been reported to contain several chemical compounds. Orthosiphon stamineus has been investigated phytochemically and pharmacologically since 1989, when oil namely methylripariochromene A was isolated from this plant (Guerin et al., 1989). Phytochemically investigation of the dry leaves of
Orthosiphon stamineus has led to the isolation of highly-oxygenated isopimarane-type diterpenes, orthosiphol A-E (Masuda et al., 1992; Sumaryono et al.,1991; Takeda et al.,
1993), and also reported on monoterpenes, triterpenes, saponins, flavonoids, hexoses, organic acids, rosmarinic acid, chromene and myo-inositol. Other bioactive compounds
10 have also been isolated such as sterols organic acid and caffeic acid derivatives (Olah et al., 2003). Olah et al. (2003) stated that the most important compound present in the
Orthosiphon stamineus leaves is the polyphenols which are polymethoxylated flavonoids: sinensetin, eupatorin, etc. and caffeic acid derivatives: rosmarinic acid, cichoric acid, caffeic acid, etc. The detail of chemical constituents identified in
Orthosiphon stamineus is listed in Table 2.1 while the structures of some compounds are shown in Fig. 2.3 to Fig. 2.7.
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Table 2.1: Chemical constituents of Orthosiphon stamineus
Class of Chemical constituents Reference compounds Diterpenes Orthosiphols A Staminols A [9] Tezuka et al (2000) [1] Staminols B [10] Awale et al. (2001) Orthosiphols B Staminolactones A Masuda et al. (1992) [2] [11] Orthosiphols C-J Staminolactones B [3-7] [12] Orthosiphols K-N Norstaminol A [24] Orthosiphols R-T Orthosiphonone A Orthosiphols S [25] [8] Orthosiphonone B [26] Secoorthosiphol A-C Triterpenes Oleanolic acid Betulinic acid [29] Tezuka et al. (2000) [27] Β-sitosterol [13] Hossain and Ismail Ursolic acid [28] Maslinic acid (2003) Orthosiphonic acid Flavones Eupatorin [15] Tezuka et al. (2000) Sinensetin [16] Sumaryono et al. Rutin (1991) Salvigenin [18] Hossain and Ismail Ladanein (2003) Tetramethylscutallarein [19] 3’-hydroxy-5,6,7,4’-tetramethoxyflavone [17] 7,3’, 4’-tri-O-methylluteolin [14] 6-hydroxy-5,7,4’-trimethoxyflavone [20] Kaempferol-3-O-β-glycoside [21] Quercetin-3-O-β-glucoside [22] Phenolic Acid Caffeoyl tartrate [23] Sumaryono et al. Rosmarinic acid [30] (1991) Vomifoliol [32] Aurantiamide acetate [31] 2,3-dicaffeoyl tartrate Benzochromene Methylripariochromene A [35] Shibuya et al. (1999) Acetovanillochromene [36] Orthochromene A [34]
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Orthosiphol A [1] Orthosiphol B [2]
R1 = COCH3, R2=H, R3=COPh, Orthosiphol F [3] R=H , Orthosiphol I [6] R1 = COCH3, R2= R3=H , Orthosiphol G [4] R=COCH3, Orthosiphol J [7] R1 = COCH3, R3= COPh , Orthosiphol H [5]
R= COCH3, Staminol A [9] Orthosiphol S [8] R= H , Staminol B [10]
Staminolactone A [11] Staminolactone B [12] Figure 2.3: Chemical structures of Orthosiphon stamineus (diterpenes) 13
Norstaminol A [24] Orthosiphonone A [25]
Orthosiphonone B [26]
Figure 2.3 (Continued): Chemical structures of Orthosiphon stamineus (diterpenes)
B- sitosterol [13] Oleanolic acid [27]
Ursolic acid [28] Betulinic acid [29] Figure 2.4: Chemical structures of Orthosiphon stamineus (triterpenes)
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R1=R2=H, R4=OCH3, 7,3’,4’ –Tri-O-methylluteolin [14] R1=R3=OH, R2=OCH3, R4=H , Eupatorin [15] R1=R2=R3=OCH3, R4= H, Sinensetin [16] R1=OH, R2=R3=OCH3, R4=H, 3-hydroxy-5,6,7,4-tetramethoxyflavone [17] R1=R4=H, R2= OCH3 , Salvigenin [18] R1=CH3, R2 =OCH3, R4= H, Tetramethylscuteliarein [19] R1=CH3, R2 =OH, R4= H, 6-Hydroxy-5,7,4-trimethoxyflavone [20]
Kaempferol-3-O-β-glucoside [21] Quercetin-3-O-β-glucoside [22] Figure 2.5: Chemical structures of Orthosiphon stamineus (flavones)
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Caffoeul tartrate [23] Rosmarinic acid [30]
Aurantiamide acetate [31] Vomifoliol [32] Figure 2.6: Chemical structures of Orthosiphon stamineus (phenolic acid)
Methylripariochromene A Orthochromene A [34] Acetovanillochromene [36] [35]
Figure 2.7: Chemical structures of Orthosiphon stamineus (benzochromene)
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2.1.5 Traditional uses of Orthosiphon stamineus
Orthosiphon stamienus is a useful traditional medicine herbs as it can be used to treat many illnesses which involve diuretic, antiinflammatory, anticholestatic, urolithiatic, antirheumatic, and antidiabetic activities (Wiart, 2002). Orthosiphon stamineus have been used differently in different countries. Decoction of Orthosiphon stamineus leaf in certain places especially in Malaysia and Indonesia are used to treat arteriosclerosis, alleviate bladder and kidney discomfort, gout and rheumatism (Awale et al., 2001; Tezuka et al., 2000; Wiart, 2002). The Vietnamese uses this plant to treat urination, reduce eruptive fever urination, rheumatism, influenza, hepatitis, biliary lithiasis and jaundice. In India, Orthosiphon stamineus issued to treat diabetes and this plant is mixed with Andrographis paniculata (Wiart, 2002).
2.1.6 Review of biological activity of Orthosiphon stamineus
Aqueous extract from the leaves of Orthosiphon stamineus in Germany exhibited diuretic effect (Englert and Harnischfeger, 1992). In Vietnam, urine output or on the sodium excretion shows no influence after 24 hours of treatment under consistent conditions (Doan et al., 1992). After oral administration of aqueous extracts of the plant in different doses (400 mL/day of 3.75% extract, 400 ml/day of 15% of extract, and 500 mL/day of 3.3% extract) in volunteers, increased the diuresis (Schuman, 1927). In another study, 14 patients were treated (dose 9500 ml/day of 12% infusion of leaves for
10 days) and increase in diuresis and excretion of chloride and urea was observed. In more recent research conducted on 40 volunteers for diuretic effects showed no influence on urine output or sodium excretion recorded in a placebo-controlled double
17 blinded crossover study at a dose of 0.6 liter/day of an infusion equivalent to 10 g of the dried leaves (Doan et al., 1992). Increased acidity in the urine has been reported in a study which was carried out on 6 healthy male volunteers who consumed 250 ml of
Orthosiphon stamineus tea every 6-hour for one day (Nirdnoy and Muangman, 1991).
In Thailand, research conducted has shown that the leaves of Orthosiphon stamineus have both favorable and non favorable effects on stone prevention. The study showed that uric acid containing stones may be prevented after drinking the tea but higher risk of stone formation may occur due to the excretion of oxalate in the urine.
However, since the research was done on a healthy patient, where all the parameters were normal and may not be applicable to kidney stone patients (Nirdnoy and
Muangman, 1991). In another research conducted in Russia, it was shown that the administration of dried extract in experimental and therapeutic doses to white rats having post-ischemic acute renal insufficiency was observed to significantly decrease the concentration of lipid peroxidation in the kidneys. The preparation of the extract inhibits hemolysis induced by the Fenton reagent and photoactive chlorpromazine. The inhibition may be due to scavenging of free radicals by phenolic compounds of the extract contained in the preparations (Shantova, 1998).
Orthosiphon stamineus Benth is believed to also contain antihypertensive properties (Hossain et al., 2008; Sriplang et al., 2007; Yam et al., 2009). Shibuya et al.
(1999) studied the water decoction of the air-dried Orthosiphon Arristatus leaf in the mixture of water and chloroform and resulted in an inhibitory effect which is caused by the major constituent of the leaf (methylripariochromene A) on the contractive responses in the rat thoracic aorta smooth muscle stimulated with KCI beforehand (Shibuya et al.,
18
1999). Azizan et al. (2012) studied the systolic blood pressure of spontaneously hypertensive rats (SHR) using Orthosiphon stamineus leaf. This study showed result in a significant mean reduction of systolic blood pressure with SHR and its efficacy is comparable to a modern recent antihypertensive agent (Azizan et al., 2012).
HPLC method has been designed for separation of three methoxylated flavones
(eupatorin, sinensetin and 3’-hydroxy-5,6,7,4’-tetramethoxyflavone) and rosmirinic acid derivatives using a methanol extract of Orthosiphon stamineus from locations in
Malaysia. It was observed that there was a difference in antioxidant activities (ranging from 55.5% to 84.2%) and variation of total phenolics (ranging from 6.7 to 10.1 mg caffeic acid/g dry weight) (Akowuah et al., 2004). In further study, Orthosiphon stamineus methanol extract was used to determine the antioxidant activity by measuring the scavenging effect on 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical and on superoxide anion which resulted in variation of free-radical (ranging from 62.82% to
92.34%) and superoxide-anion scavenging activities (ranging from 53.29% to 75.88%).
This study concluded that methanol extract was active. Its antioxidative efficiency was comparable to that of pure synthetic antioxidant butylated hydroxylanisole (BHA) and quercetin (Akowuah et al., 2005). Khamsah et al. (2006) stated that the antioxidant activity of Orthosiphon stamineus extract was not only due to the phenolic compounds but also staminane-types diterpenes, triterpenes and some other components present in the plant’s leaf.
Orthosiphon stamineus plays the role in bacteriostatic activity and antibiotic activity due to the presence of the saponins and ceffeic acid derivatives, respectively
(Chen, 1989). The oral administration of Orthosiphon stamineus extract has been
19 performed on patients and it was reported to increase choleresis and cholekinesis, together with an antibacterial action in cholecystitis (Chen, 1989). 3-hydroxy-5,6,7,4 tetramethoxyflavone and flavones sinensetin did not confirm these findings, which administered intravenously at a dose of 10 mg/kg body weight (Schut and Zwavig,
1993).
The antiinflammatory activity was verified using Orthosiphon stamineus leaf chloroform extract by using carrageenan-induced hind paw edema method. HPLC method was done to identify the active compounds contributing to its antiinflammatory activity and the result showed that the flavonoids rich chloroform extract fraction (CF2) which contained eupatorin, sinensetin and 3’-hydroxy-5,6,7,4’-tetramethoxyflavone may affect this activity because of the presence of flavonoid compounds fit for influencing the nitric oxide pathway (Yam et al., 2010).
Sripalang et al. (2007) suggested that Orthosiphon stamineus (aqueous extract) is advantageous for enhancing the profile in diabetic rats. The study carried out showed the effects of Orthosiphon stamineus aqueous extract on plasma glucose concentration, lipid profile in normal and stretozotocin-induced diabetic rats. In the oral glucose tolerance tests which were dose-dependent in both normal and diabetic rats, the extract (0.2-1.0 g/kg) significantly decreased plasma glucose concentration. The most effective decrease in plasma glucose concentration occurred when the extract at 1 g/kg and it was compared with the result of glibenclamide (5 mg/kg). The extract had significantly reduced plasma glucose concentration. The plasma glucose concentration was found to be lower in the extract-treated diabetic rats (at day 7 and 14) that after rehashed every day oral administration of the extract (0.5 g/kg) for 14 days. Towards the conclusion of
20 the study, the plasma triglyceride concentration was lower in the extract- treated diabetic rats and plasma HDL-cholesterol concentration was significantly increased in diabetic rats which were treated with extract (Sriplang et al., 2007).
Isolation of bioassay-guided on Orthosiphon stamineus aqueous extract was used to carry out antiangiogenic studies and showed that hexane fraction gave three componds which were oleanolic acid, betulinic acid and ursolic acid. The extract exhibited 20% antiangiogenic activity while the hexane fraction exhibited 80% antiangiogenic activity. The three isolated compounds gave 100% antiangiogenic activity. It can be concluded that the isolated compounds and the hexane fraction had promising antiangiogenic activity (Hussain et al., 2012).
The methanol extract of the plant (dried aerial parts) was studied for cytotoxicity.
The fractions of the extract, hexane, chloroform, etanoic acid, butanol and water showed that the cytotoxicity at IC50 was less than 100 µg/mL. Five new isopimarane-type diterpenes were given in chloroform and etanoic acid fractions using chromatography. It showed mild to weak antiproliferative activities toward highly liver metastatic colon 26-
L5 carcinoma and human HT-1080 fibrosarcoma cell lines (Tezuka et al., 2000).
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Table 2.2: Summary of scientific studies on the Orthosiphon stamineus from the reviewed literature
Biological activity Methodology Compound present (positive results) References Antioxidant In vitro assay : Sinensetin (SEN) Akowuah et al., 2004 Eupatorin (EUP) Β-carotene linoleic acid system 3’-hydroxy-5,6,7,4’- tetramethoxyflavone (TMF) Rosmirinic Acid (RA) In Vitro assay: Sinensetin (SEN) Akowuah et al., 2005 Free radical scavenging using DPPH Eupatorin (EUP) 3’-hydroxy-5,6,7,4’- tetramethoxyflavone (TMF) Rosmirinic Acid (RA) In vitro assay: Diterpenes Khamsah et al., 2006 Free radical scavenging using DPPH, β- Triterpenes carotene linoleic acid system In vitro assay: Sinensetin (SEN) Yam et al., 2007 Free radical scavenging using DPPH, Eupatorin (EUP) 3+ Fe -induced lipid peroxidation 3’-hydroxy-5,6,7,4’- inhibiting activities, Trolox equivalent tetramethoxyflavone antioxidant capacity (TEAC) (TMF)
In vitro assay: Polyphenols Zakaria et al., 2008 Free radical scavenging using DPPH
Antiinflammatory In vivo assay: Orthosiphol A Masuda et al., 1992 The inflammation induced by tumor Orthosiphol B promoters, 12-O- tetradecanoylphorbol-13-acetae, on mouse ears
22
In vitro assays: Oleanolic acid Hsu et al.,2010 Lipopolysaccharide (LPS) -stimulates Ursolic acid nitric oxide (NO), prostaglandin E2 (PGE2) and intracellular reactive oxygen species (ROS) production RAW 264.7 cells. In vivo assay: Flavonoids Yam et al., 2008 Carrageenan-induced hind paw edema Phenolic compounds method In vivo assay: Sinensetin Yam et al., 2010 Carrageenan-induced hind paw edema Eupatorin (EUP) method 3’-hydroxy-5,6,7,4’- In vitro assay: tetramethoxyflavone Nitric oxide (NO) inhibition (TMF) Diuretic In vivo assay: Flavonoids Doan et al., 1992 Placebo controlled double-blind crossover model In vivo assay: Flavonoids Englert and Harnischfeger, Enhances ion excretion in rat to a level Triterpenes 1992 comparable to that obtained with furosemide In vivo assay: Flavonoids Arafat et al., 2008 Diuretic induced by Triterpenes hydrochlorothiazide and hypouricemic Caffeic acid derivatives endorsed by allopurinol In vivo assay: Diterpene Beaux et al., 1999 Induced by hydrochlorothiazide Triterpene Glycosides Phenolic compounds (flavonoids, tannins and coumarins)
23
Antibacterial In vitro assay : Rosmarinic acid Ho et al., 2010 Antibacterial activity against food- borne bacteria such as: B. subtilis, B. cereus, S. Areas, Listeria monocytogenes, Klebsiella pneumonia, E. coli, V. parahaemolyticus, Slamonella enteritidis and Salmonella typhimurium, compared to inhibition of 5% lactic acid ( the natural food preservative) Antifungal In vitro assay: β-caryophyllene Hossain et al., 2008 Disc diffusion and minimum inhibitory caryophyllene oxide concentration (MIC) determination α-humulene method β-pinene Limonene Β-elemene 1-octan-3-ol Antipyretic In vivo assay: Rosmarinic acid Yam et al., 2009 The yeast-induced pyrexia model and compared with paracetamol (acetaminophen in U.S) AntiAngiogenic Ex vivo assay: Rosmarinic acid Siddiqui et al., 2009 Rat aorta assay Sinensetin Other flavonoids Diterpenes Triterpenes
Aisha et al., 2009 Phenolic compounds
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