ANTIOXIDANT ACTIVITY OF N-HEXANE, ETHYL ACETATE, AND ETHANOL EXTRACT FROM LAKOOCHA LEAVES (Artocarpus lacucha Buch-Ham) BY USING DPPH (1,1-DIPHENYL-2-PICRYLHYDRAZYL) ASSAY
UNDERGRADUATE THESIS
Submitted in Partial Fulfillment of the Requirements for the Degree of Sarjana Farmasi in Faculty of Pharmacy University of Sumatera Utara
BY:
MARDIANA NIM 141501233
PHARMACY BACHELOR’S PROGRAMME FACULTY OF PHARMACY UNIVERSITAS SUMATERA UTARA MEDAN 2018
i Universitas Sumatera Utara ANTIOXIDANT ACTIVITY OF N-HEXANE, ETHYL ACETATE, AND ETHANOL EXTRACT FROM LAKOOCHA LEAVES (Artocarpus lacucha Buch-Ham) BY USING DPPH (1,1-DIPHENYL-2-PICRYLHYDRAZYL) ASSAY
UNDERGRADUATE THESIS
Submitted in Partial Fulfillment of the Requirements for the Degree of Sarjana Farmasi in Faculty of Pharmacy University of Sumatera Utara
BY:
MARDIANA NIM 141501233
PHARMACY BACHELOR’S PROGRAMME FACULTY OF PHARMACY UNIVERSITAS SUMATERA UTARA MEDAN 2018
ii Universitas Sumatera Utara
iii Universitas Sumatera Utara ACKNOWLEDGEMENT
I thank all who in one way or another contributed to the completion of this undergraduate thesis. First, I give thanks to God for protection and ability to do work. The praise and gratitude of mine to the Almighty God has given his mercy and grace, so that I can compile and finalize undergraduate thesis entitled
"Antioxidant Activity of n-Hexane, Ethyl Acetate and Ethanol Extract from
Lakoocha Leaves (Artocarpus lacucha Buch.-Ham) by using DPPH (1,1-
Diphenyl-2-Picrylhydrazyl) Assay”. This undergraduate thesis is proposed as one of the requirements to obtain the title of Undergraduate of Pharmacy, Universitas
Sumatera Utara, Medan. This would not be possible without the help, support and guidance from various parties and I would like to take this opportunity to show my sincere gratitude and thank to:
1. Prof. Dr. Masfria, M.S., Apt., as Dean of Faculty of Pharmacy, Universitas
Sumatera Utara.
2. Dr. Sumaiyah, S.Si., M.Si., Apt., as Head of the undergraduate study
program, Universitas Sumatera Utara.
3. Dr. Poppy Anjelisa Zaitun Hasibuan, S.Si., M.Si., Apt., as supervisor who has
guided and directed me in writing this undergraduate thesis.
4. Imam Bagus Sumantri, S.Si., M.Si., Apt., as an academic advisor who has
given me advice and direction in academics every semester.
5. Lecturer of Universitas Sumatera Utara, for all knowledge, education and
guidance that given to me throughout my college days.
iv Universitas Sumatera Utara 6. The administrative staffs of Universitas Sumatera Utara who have provided
administrative ease during my college days.
Nobody has been more important to me in the pursuit of this project than the members of my family. I would like to express my gratitude to my beloved parents Sudarno and Sunirah, and my awesome siblings Rian, Mery and Melly who have always been inspirational, encouraging, provides prayers and support me in whatever I pursue. I am extending my thanks to thanking Denny Satria
S.Farm., Apt., and my friend Satyodjiwo, Lordiana, Darshieny, Muh. Hilmi,
Nurul, kak Riri, Ranti, kak Della and kak Sangkot for the assistance and genuine support throughout this research work. I would like to say thanks to all my friends for their constant encouragement.
Finally, I thank the examiners for the feedbacks that were given to me during the presentation, which really helped me to complete my undergraduate thesis successfully. I really hope this undergraduate thesis can provide benefits to all parties who require. Finally, my thanks go to all the people who have supported me to complete the research work directly or indirectly.
Thank you very much. May the Almighty God richly bless all of you.
Medan, 09 August 2018 Author
Mardiana 141501233
v Universitas Sumatera Utara PLAGIARISM DECLARATION
With my signature, I confirmed that, Name : Mardiana Student’s ID number : 141501233 Study programme : S-1 Pharmacy Title of undergraduate thesis : Antioxidant Activity of n-Hexane, Ethyl Acetate and Ethanol Extract from Lakoocha Leaves (Artocarpus lacucha Buch.-Ham) by using DPPH (1,1-Diphenyl-2-Picrylhydrazyl) Assay Hereby, I declare that this undergraduate thesis is written based on the data and the results of my own work, which never been submitted by others to obtain a degree in other colleges. It is also not a plagiarism because the quotation is written after the source is mentioned in the bibliography. I also confirmed that, I have documented all the methods, data and processes truthfully and not manipulated any data. If in the future, there is a complaint from another party because of this undergraduate thesis found plagiarism due to its own mistake, then I am willing to accept any sanction by the Faculty of Pharmacy of Universitas Sumatera Utara and not be the responsibility of my supervisor. This declaration I do truthfully to be used if necessary as necessary.
Medan, 09 August 2018 Declaration by,
Mardiana ID 141501233
vi Universitas Sumatera Utara ANTIOXIDANT ACTIVITY OF N-HEXANE, ETHYL ACETATE AND ETHANOL EXTRACT FROM LAKOOCHA LEAVES (Artocarpus lacucha Buch.-Ham) BY USING DPPH (1,1-DIPHENYL-2-PICRYLHYDRAZYL) ASSAY
ABSTRACT
Antioxidants are compounds that can contribute one or more electrons to free radicals to inhibit or prevent harmfull effect of free radicals. One of the plants known to have antioxidant is lakoocha (Artocarpus lacucha Buch.-Ham). The aim of this study was to investigate phytochemical screening and antioxidant activity of n-hexane, ethyl acetate and ethanol extract of lakoocha leaves. The powdered simplicia was standardized and screened phytochemically. The extract was obtained by maceration with n-hexane, ethyl acetate and ethanol 96% successively then concentrated using rotary evaporator to obtain concentrated of n-hexane extract, ethyl acetate extract and ethanol extract. Phytochemical screening and antioxidant activity was performed against these extracts. Antioxidant activity was determined by DPPH (1,1-diphenyl-2- picrylhydrazyl) radical scavenging method using ultravilet-visible spectrophotometer at wavelength of 516 nm after incubated for 60 minutes in dark place. Quercetin was used as positive control. Statistical analysis was done by One way ANOVA. The result of standardization obtained water content was 8%, water-soluble extractive was 26.58%, ethanol-soluble extractive was 10.38%, total ash was 8% and acid-insoluble ash was 4.29%. Simplicia contained secondary metabolite compounds including flavonoid, saponin, tannin, glycoside and steroid. The n- hexane extract contained steroid. The ethyl acetate extract contained steroid, tannin, glycoside, flavonoid and saponin. The ethanol extract contained tannin, glycoside, flavonoid and saponin. The IC50 values of n-hexane extract, ethyl acetate extract and ethanol extract was 1062.03±1.42 µg/ml, 323.18±0.02 µg/ml and 99.23±0.07 µg/ml respectively, whereas for quercetin was 2.32±0.01 µg/ml, showing there was a significant difference which was 0.00 (p<0.05). If could be concluded of this study showed that ethanol extract of lakoocha leaves has antioxidant activity with strong category whereas n-hexane extract and ethyl acetate extract have inactive antioxidant activity with very weak categories.
Keywords: Antioxidant activity, DPPH, Lakoocha Leaf (Artocarpus lacucha Buch.-Ham)
vii Universitas Sumatera Utara AKTIVITAS ANTIOKSIDAN DARI N-HEKSAN, ETIL ASETAT DAN ETANOL EKSTRAK DARI DAUN MOBE (Artocarpus lacucha Buch.-Ham) DENGAN MENGGUNAKAN UJI DPPH (1,1-DIPHENYL-2- PICRYLHYDRAZYL)
ABSTRAK
Antioksidan adalah senyawa yang dapat memberikan satu atau lebih elektron ke radikal bebas yang mana dapat menghambat dan mencegah efek buruk dari radikal bebas. Salah satu tanaman diketahui mempunyai antioksidan adalah mobe (Artocarpus lacucha Buch.-Ham). Tujuan dari penelitian ini adalah untuk meneliti skrining fitokimia dan aktivitas antioksidan dari ekstrak n-heksan, etil asetat dan ethanol dari daun mobe. Serbuk simplisia distandardisasi dan diskrining secara fitokimia. Ekstrak diperoleh dengan maserasi menggunakan pelarut n-heksan, etil asetat dan etanol 96% berturut-turutkemudian dipekatkan menggunakan rotary evaporator untuk mendapatkan ekstrak n-heksan, etil asetat dan etanol yang pekat. Skrining fitokimia dan aktivitas antioksidan dilakukan terhadap ekstrak-ekstrak ini. Aktivitas antioksidan ditentukan dengan metode pemerangkapan radikal DPPH (1,1-diphenyl-2-picrylhydrazyl) menggunakan spektrofotometer ultraviolet-visible pada panjang gelombang 516 nm setelah diinkubasi selama 60 menit di tempat gelap. Kuersetin digunakan sebagai kontrol positif. Analisis statistik dilakukan dengan One-way ANOVA. Hasil standardisasi didapat kadar air 8%, kadar sari larut air 26,58%, kadar sari larut etanol 10,38%, total abu 8% dan abu tidak larut asam 4,29%. Simplisia mengandung senyawa-senyawa metabolit sekunder diantaranya flavonoid, saponin, tannin, glikosida, dan steroid. Ekstrak n-heksan mengandung steroid. Ekstrak etil asetat mengandung steroid, tannin, glikosida, flavonoid dan saponin. Ekstrak etanol mengandung tannin, glikosida, flavonoid dan saponin. Nilai IC50 ekstrak n-heksan, etil asetat dan etanol masing-masing sebesar 1062,03±1,42 µg/ml, 323,18±0,02 µg/ml dan 99,23±0,07 µg/ml, sedangkan untuk kuersetin sebesar 2.32±0.01 µg/ml. Ini menunjukkan terdapat perbedaan yang signifikan dengan nilai sebesar 0,00 (p<0,05). Jika dapat disimpulkan dari penelitian ini menunjukkan bahwa ekstrak etanol daun mobe memiliki aktivitas antioksidan dengan kategori kuat sedangkan ekstrak n-heksan dan etil asetat memiliki antioksidan yang tidak aktif dengan kategori sangat lemah.
Kata kunci: Aktivitas antioksidan, daun mobe (Artocarpus lacucha Buch.-Ham), DPPH
viii Universitas Sumatera Utara CONTENTS
Pages
TITLE ...... i
VERIFICATION SHEET ...... ii
ACKNOWLEDMENT ...... iii
PLAGIARISM DECLARATION ...... v
ABSTRACT ...... vi
ABSTRAK ...... vii
CONTENTS ...... viii
LIST OF TABLES ...... xiii
LIST OF FIGURES ...... xiv
LIST OF APPENDICES ...... xv
CHAPTER I INTRODUCTION ...... 1
1.1 Background ...... 1
1.2 Problem Statement ...... 3
1.3 Hypothesis ...... 3
1.4 Objectives ...... 4
1.5 Benefits ...... 4
1.6 Research Framework ...... 5
CHAPTER II THEORY ...... 6
2.1 Description of Plant ...... 6
2.1.1 Toxonomy ...... 6
2.1.2 Local name...... 6
2.1.3 Native name ...... 7
ix Universitas Sumatera Utara 2.1.4 Plant morphology ...... 7
2.1.5 Plant benefit ...... 8
2.1.6 Chemical constituents ...... 9
2.2 Extraction ...... 9
2.2.1 Cold method...... 9
2.2.2 Hot method ...... 10
2.3 Free radical ...... 10
2.4 Antioxidant ...... 12
2.4.1 Flavonoid ...... 15
2.4.2 Quercetin...... 16
2.5 UV-Visible Spectrophotometer ...... 17
2.6 Method of Antioxidant Capacity Assessment ...... 17
2.6.1 DPPH free radical scavenging ...... 18
2.6.2 Maximum absorption wavelength ...... 19
2.6.3 Reaction time ...... 19
CHAPTER III METHODOLOGY ...... 20
3.1 Apparatus and Materials ...... 20
3.1.1 Apparatus ...... 20
3.1.2 Materials ...... 20
3.2 Sample Preparation ...... 21
3.2.1 Sample collection...... 21
3.2.2 Sample identification ...... 21
3.2.3 Simplicia preparation ...... 21
3.2.4 Extract preparation...... 21
x Universitas Sumatera Utara 3.3 Simplicia Standardization ...... 22
3.3.1 Macroscopic observation ...... 22
3.3.2 Microscopic observation ...... 22
3.3.3 Water content ...... 22
3.3.4 Water-soluble extractive ...... 23
3.3.5 Ethanol-soluble extractive ...... 23
3.3.6 Total ash...... 24
3.3.7 Acid-insoluble ash ...... 24
3.4 Phytochemical Screening ...... 24
3.4.1 Phytochemical screening using simplicia powder ...... 25
3.4.1.1 Alkaloid ...... 25
3.4.1.2 Glycoside ...... 25
3.4.1.3 Flavonoid ...... 26
3.4.1.4 Saponin ...... 26
3.4.1.5 Tannin...... 26
3.4.1.6 Steroid/triterpenoid...... 26
3.4.2 Phytochemical screening using extract ...... 27
3.4.2.1 Alkaloid ...... 27
3.4.2.2 Glycoside ...... 27
3.4.2.3 Flavonoid ...... 27
3.4.2.4 Saponin ...... 28
3.4.2.5 Tannin...... 28
3.4.2.6 Steroid/triterpenoid...... 28
3.5 Reagent Preparation ...... 28
xi Universitas Sumatera Utara 3.5.1 Dragendorff reagent ...... 28
3.5.2 Liebermann-Burchardreagent ...... 29
3.5.3 Meyer reagent ...... 29
3.5.4 Bouchardate reagent ...... 29
3.5.5 Molisch reagent ...... 29
3.5.6 Hydrochloric acid 2N reagent ...... 29
3.5.7 Iron (III) chloride 10% reagent ...... 29
3.5.8 Aluminium (III) chloride 5% reagent ...... 29
3.5.9 Sulphuric acid 2N reagent ...... 30
3.5.10 Vanillin-sulphuric acid reagent...... 30
3.5.11Lead (II) acetate 0.4M reagent...... 30
3.5.12 Chloral hydrate solution ...... 30
3.5.13 Nitrate acid 0.5N reagent ...... 30
3.6 Preparation of Stock Solution ...... 30
3.6.1 Extract stock solution ...... 30
3.6.2 Quercetin stock solution ...... 30
3.6.3 DPPH 0.5 mM stock solution ...... 31
3.7 Antioxidant Activity Assay by using Uv-vis Spectrophotometer ...... 31
3.7.1 The principle of DPPH radical scavenging assay ...... 31
3.7.2 DPPH blank solution ...... 31
3.7.3 Determination of maximum absorption wavelength ...... 31
3.8 Preparationof Test Samples ...... 31
3.8.1 Test sample of extract ...... 31
xii Universitas Sumatera Utara 3.8.2 Test sample of quercetin ...... 32
3.8.3 The percentage of radical scavenging assay ...... 32
3.8.4 IC50 values ...... 33
CHAPTER IV RESULT AND DISCUSSION ...... 34
4.1 Plant Identification ...... 34
4.2 Simplicia Standardization ...... 34
4.3 Pyhtochemical Screening ...... 36
4.4 Antioxidant Activity of Lakoocha Leaves Extract using DPPH Method ...... 37
4.4.1 Determination of maximum absorption wavelength ...... 37
4.4.2 Antioxidant activity of test samples ...... 37
4.4.3 The IC50 values ...... 40
CHAPTER V CONCLUSION AND SUGGESTION ...... 42
5.1 Conclusion ...... 42
5.2 Suggestion ...... 42
REFERENCES ...... 43
xiii Universitas Sumatera Utara LIST OF TABLES
Table Pages
4.1 The Result of Standardization of Lakoocha Leaves Simplicia ...... 35
4.2 The Result of Phytochemical Screening of Simplicia and Extract ...... 36
4.3 The Percentage of Scavenging from n-Hexane Extract ...... 38
4.4 The Percentage of Scavenging from Ethyl Acetate Extract ...... 38
4.5 The Percentage of Scavenging from Ethanol Extract ...... 38
4.6 The Percentage of Scavenging from Quercetin ...... 38
4.7 The Result of Linear Regression and Mean±SD of Lakoocha Leaves Extract and Quercetin...... 40
xiv Universitas Sumatera Utara LIST OF FIGURES
Figure Pages
1.1 Research Framework ...... 5
2.1 Lakoocha Plant ...... 8
2.2 Vitamin C Structure ...... 14
2.3 Flavonoid Structure ...... 15
2.4 Quercetin Structure ...... 15
2.5 DPPH and Antioxidant Reaction ...... 19
4.1 The Curve of Maximum Absorption of DPPH 40 ppm Solution using Uv-visible Spectrophotometer ...... 37
4.2 The Percentage of Scavenging of n-Hexane Extract ...... 39
4.3 The Percentage of Scavenging of Ethyl Acetate Extract ...... 39
4.4 The Percentage of Scavenging of Ethanol Extract ...... 39
4.5 The Percentage of Scavenging of Quercetin ...... 40
xv Universitas Sumatera Utara LIST OF APPENDICES
Appendix Pages
1 The Result of Plant Identification ...... 48
2 The Picture of Artocarpus lacucha Buch,Ham ...... 49
3 The Result of Microscopic Observation of Lakoocha Leaf ...... 50
4 The Flowchart of Simplicia Production...... 51
5 The Flowchart of the Production of Lakoocha Leaves Extract with a Successive Maceration Method ...... 52
6 The Flowchart of Antioxidant Activity Assay ...... 54
7 The Calculation of Lakoocha LeavesStandardization ...... 55
8 The Result of Phytochemical Screening of Simplicia Powder of Lakoocha leaves ...... 58
9 The Result of Phytochemical Screening of Lakoocha Leaves Extract ...... 59
10 The Calculation of Scavenging Percent and IC50 Values ...... 60
11 The Result of Statistical Analysis ...... 64
xvi Universitas Sumatera Utara CHAPTER I
INTRODUCTION
1.1 Background
Since long time ago, medicinal plants have been used to improve health, recovery health, prevention and healing of disease by the people in Indonesia
(Angelina et al., 2015). According to WHO (World Health Organization) in 2008, registered that 68% of the world of population still relied on traditional treatment systems and mostly, plants involve to cure diseases and over 80% of the world of population using herbal remedies to support their health. Various medicinal plants and thousands of plants being potential drug in Indonesia contain a variety of natural chemical compounds (Saifudin et al., 2011). Entering the 21st century as the globalization era, in Indonesia, the development of technology and the utilization of medicinal plants in health services have known and used extraction as a concept (Depkes RI, 2000).
Nowadays, the world of medicine and healthy discuss about free radicals and antioxidants. This is happened because mostly disease is begun by an excessive oxidation reaction in the body (Winarsi, 2007). A free radical is a molecule or compound having a single unpaired electron in an outer orbit from the result of normal cell metabolism or external sources (pollution, cigarette smoke, radiation). Reactive Oxygen Species (ROS) is a collective term used for oxygen-derived free radical included superoxide anion (O2•-), peroxyl (ROO•), hydroxyl (OH•), nitric oxide (NO•) and non-radical include hydrogen peroxide
1 (H2O2), singlet oxygen ( O2) and hypochlorous acid (HOCl) (Pietta, 2000).
Almost all organisms have many antioxidants to protect them from a free radical
1 Universitas Sumatera Utara attacking that can reduce the rate of free radical formation and as chain-breaking antioxidants that scavenge and stabilize free radicals. If the amount of antioxidant in the body is less than the production of free radicals, it leads to an oxidative stress condition. Oxidative stress may risk for chronic disease such as cancer, aging, diabetes, atherosclerosis, ischaemic heart disease, immunosuppression, neurodegenerative diseases and others (Hasan et al., 2009).
Antioxidants are compounds that can inhibit or prevent oxidation of compounds which result free radicals. Antioxidants can reduce the chance or percent of the chronic diseases, including cancer and heart disease (Shekhar and
Anju, 2014). There are two kinds of antioxidants: Natural antioxidant commonly found in plants such as fruits, grains and vegetables. Some natural antioxidant compounds are derivatives of phenol, coumarin, tocopherol, hydroxycinnamic, flavonoids, diphenol, dihydroflavon, catechines and ascorbic acid. Synthetic antioxidants include butyl hydroxylanisole (BHA), butyl hydroxytoluene (BHT), propyl gallate, ethoxyquin and others (Mustarichie et al., 2017).
Artocarpus lacucha Buch.-Ham belongs to the family of Moraceae, popularly regarded as a medicinal plant and commonly called as monkey jack.
This plant is widely distributed in the tropical regions of south and south-east
Asia, mainly Nepal, Srilanka, India, Myanmar, Indonesia, Vietnam and Thailand
(Hari et al., 2014). It has many pharmacological activities such as anti- inflammatory, antiviral, anticancer, antibacterial and anti-HIV (Gautam and Patel,
2014). In Thailand, the dried aqueous extract of its heartwood has been used as a traditional anthelmintic agent (Hari et al., 2014). The fruit is generally sweet-sour pulp eaten fresh but mostly made into arch fish curries by Samosir people and also used as a liver tonic. The leaves are used in treating dropsy (Hossain et al., 2016).
2 Universitas Sumatera Utara The previous study showed hydromethanol extract of lakoocha leaves has antioxidant activity with the IC50 value was 54.74 ppm (Hasan et al., 2009).
However, the antioxidant activity of n-hexane extract of lakoocha leaves (NELL), ethyl acetate extract of lakoocha leaves (EAELL) and ethanol extract of lakoocha leaves (EELL) have not been reported yet. Therefore, the aim of this study is to investigate phytochemical screening and antioxidant activity of NELL, EAELL and EELL by radical scavenging method using DPPH (1,1-diphenyl-2- picrylhydrazyl) radical.
1.2 Problem statement
a. What are secondary metabolites that are presented in lakoocha
(Artocarpus lacucha Buch.-Ham) leaves after phytochemical screening?
b. Do n-hexane extract, ethyl acetate extract and ethanol extract of lakoocha
(Artocarpus lacucha Buch.-Ham) leaves have antioxidant activity with
DPPH (1,1-diphenyl-2-picrylhydrazyl) method?
c. How many are the IC50 values of n-hexane extract, ethyl acetate extract
and ethanol extract of lakoocha (Artocarpus lacucha Buch.-Ham) leaves?
1.3 Hypothesis
a. Secondary metabolites that are presented in lakoocha (Artocarpus lacucha
Buch.-Ham) leaves after phytochemical screening are alkaloid, flavonoid,
tannin, saponin, steroid/triterpenoid and glycoside.
b. Ethyl acetate and ethanol extract have antioxidant activity compare to n-
hexane extract from lakoocha (Artocarpus lacucha Buch.-Ham) leaves
with DPPH (1,1-diphenyl-2-picrylhydrazyl) method.
3 Universitas Sumatera Utara c. The IC50 value of n-hexane extract is more than 200 ppm whereas the IC50
values of ethyl acetate extract and ethanol extract of lakoocha (Artocarpus
lacucha Buch.-Ham) leaves are less than 200 ppm.
1.4 Objectives
a. To identify the secondary metabolites that are presented in lakoocha
(Artocarpus lacucha Buch.-Ham) leaves after phytochemical screening.
b. To know whether the antioxidant activity of n-hexane extract, ethyl acetate
extract and ethanol extract of lakoocha (Artocarpus lacucha Buch.-Ham)
leaves have or not.
c. To know the IC50 values of n-hexane extract, ethyl acetate extract and
ethanol extract of lakoocha (Artocarpus lacucha Buch.-Ham) leaves.
1.5 Benefits
a. Add knowledge to the next researcher about antioxidant in lakoocha
(Artocarpus lacucha Buch.-Ham) leaves.
b. As information sources to the next researcher who will continue
experiment with lakoocha plant (Artocarpus lacucha Buch.-Ham).
c. As supported data to the next researcher who interest in testing antioxidant
in radix, bark, heartwood and fruit of lakoocha with a different method.
4 Universitas Sumatera Utara 1.6 Research Framework
The research framework can be seen on below:
Independent Variable Dependent Variable Parameter
Extract
NELL Concentration: - 50 ppm Phytoconstituents -Alkaloid - 100 ppm -Tannin - 200 ppm -Saponin - 400 ppm -Flvonoid -Glycoside EAELL -Steroid/Triterpenoid Concentration: - 50 ppm - 100 ppm - 200 ppm - 400 ppm Antioxidant IC50 value EELL activity Concentration: - 25 ppm - 50 ppm - 100 ppm - 200 ppm
Figure 1.1 Research Framework
5 Universitas Sumatera Utara CHAPTER II
THEORY
2.1 Description of Plant
Artocarpus lakoocha is widely distributed in the tropical regions of south and south-east Asia, mainly Nepal, Srilanka, India, Myanmar, Indonesia, Vietnam and Thailand (Hari et al., 2014). As well as being the most wide-ranging species on the Asian continent, lakoocha is also the most tolerant of cool temperatures and dry conditions. It is frequently cultivated for its fruit throughout its range and south as far as Bombay (lat. 19°6 N), so it would seem well-suited to both tropical and subtropical, but frost-free areas (Anonymb, 1988).
2.1.1 Toxonomy
Toxonomy of lakoocha plant is:
Kingdom : Plantae
Division : Tracheophyta
Class : Magnoliophyta
Order : Rosales
Family : Moraceae
Genus : Artocarpus
Species : Lakoocha (Gautam and Patel, 2014)
Synonym : Artocarpus lacucha Buch.-Ham, Artocarpus lacucha Roxb
2.1.2 Local name
Local name of lakoocha plant is Mobe (Batak), Dadak (Pradityo et al.,
2016), Keledang Beruk (Purniawati, 2014)
6 Universitas Sumatera Utara 2.1.3 Native name
Monkey Jack (English), Anjarubi, Asam, Beruni, Beto, Burinik, Dadah,
Darak, Durak, Tampan, Tampang, Tampang wangi (Borneo) (Anonyma, 2018).
Keledang tampang bulu, Cempedak air, Tampang bulu, Tampang manis, Darak,
Tampang dadak, Tampang telor (Malaysia) (Anonym, 2011). Boho't, Dewa
(Assamese), Lakuch, Kshudra panas, Granthiphala, Pitanaasha (Ayurveda), Ye bo luo mi (Chinese), Dahua, Barhal, Beng, Dahu, Lakoocha, Lakuch, Lakuchi, Sans
(Hindi), Lakucha, Otehuli (Kannada), Ma-haad, Puag had, Lokhat (Thailand),
Anubing (Philipine) (Anonym, 2016). Badahar (Nepal) (Tomar et al., 2015).
2.1.4 Plant morphology
A large deciduous tree reaching 15-18m in height with a spreading head bark roughy, grayn, young shoot thin densely clothed with a soft grey, tawny and rusty tomentum (Gautam and Patel, 2014). The leaves stalk is 1.25-2.5 cm long and hairy while the leaves blade is oblong, ovate or elliptic , leathery, 10-30 x 5–
14.5 cm, pointed apex, base is often unequal sided, rounded, wedge-shaped or heart-shaped, entire margin, apex obtuse or acute, alternate. The upper surface labrous except for the sparse short hairs on the midric and lateral veins, lower surface densely pubescent with yellowish to reddish brown soft hairs (Anonym,
2010). Flowers are unisexual, where male and female flowers in separate spherical heads, but on the same tree. Male flowers are yellow-orange while the female are reddish. Fruit is a syncarp (the entire female inflorescence forms a fruit), irregularly rounded, green when young, turning yellow at the time of maturity, later brown. The size differs but the diameter is typically 5-10 cm while fruit weights 200-350 g. The seeds contain sticky white latex, irregular, vary in size like the fruits, 1 cm long, more or less flattened and pointed (Gautam and Patel,
7 Universitas Sumatera Utara 2014). Bark is grey-brown, distantly cracked. Twigs are about 2.5-5 mm thick, densely pubescent with short yellowish to reddish brown hairs (Anonymb, 2018).
2.1.5 Plant benefit
The leaves are used in treating dropsy. The unripe fruit is hot, sour, sweet, causes tridosa impotency, loss of appetite, blood complaint. The fruits are generally sweet sour pulp eaten fresh but mostly made into curries and considered as a liver tonic. Bark contains 8.5 percent tannin that is used to treat cracked skin, heals boils, cure any wound, draw out purulent matter from any abscess and as astringent. The seed and bark of the plant are useful for stomach and liver disease.
The root is used as refresher (Hossain et al., 2016). The brown powder of heartwood is a product of the aqueous extraction prepared by boiling the wood chips and then evaporating water away. This preparation has been used as a traditional anthelmintic drug for treatment of tapeworm infection in Thailand. The powdered heartwood has been using as an effective and economical skin- whitening agent. The hardwood of lakoocha is comparable to famous teak wood, is used for constructions, furniture, boat making and cabinet work. Seeds are good purgative for childrens and for haemagglutinating. The raw fruits and male flowers spikes (acidic and astringent) are utilized in pickles and chutney. Stem bark contains oxyresveratrol, used for tapeworm (Kumar et al., 2010).
Figure 2.1 Lakoocha Plant
8 Universitas Sumatera Utara 2.1.6 Chemical constituents
The heartwood contains artocarpin, norartocarpin, norcycloartocarpin, cycloartocarpin, resorcinol, β-sitosterol and oxyresveratrol (Gautam and Patel,
2014). The fruit contain vitamin C, β-carotene, β-amyrin acetate, lupeol and some minerals such as zinc, copper, manganese and iron (Hossain et al., 2016).
2.2 Extraction
Extraction is the action of taking out desired component from undesired component in liquid solvent. Knowing that its bioactive component contained in simplicia being easier to select solvent and the right extraction method (Depkes
RI, 2000).
2.2.1 Cold method a. Maceration
Maceration is the extraction method of using solvent with several times shaking or stirring at room temperature. Re-maceration is done repeating the addition of solvent after filtering first macerate and later (Depkes RI, 2000). b. Percolation
Percolation is the extraction method of always using the new solvent until complete extraction of simplicia (exhaustive extraction) which is generally done at room temperature. This process includes several steps such as simplicia preparation, maceration intermediate, actual percolation (dropping/gathering extract) continuously until extract (percolate) obtained which the amount is about
1-5 times of simplicia weight (Depkes RI, 2000).
9 Universitas Sumatera Utara 2.2.2 Hot method a. Reflux
Reflux is the extraction method of using solvent at its boiling point, for a certain time and the amount of solvent are relatively constant in the presence of condenser (Depkes RI, 2000). b. Soxhletation
Soxhletation is the extraction method of always using new solvent, which is generally done with special equipment, so continuously extraction will happen, with the amount of solvent is relatively constant in the presence of condenser
(Depkes RI, 2000). c. Digestion
Digestion is the extraction method of continuously stirring at higher temperature than room temperature which is generally done at 40-50⁰C temperature (Depkes RI, 2000). d. Infusion
Infusion is the extraction method of using water as solvent at 90-98⁰C temperature within 15-20 minutes (Depkes RI, 2000). e. Decoction
Decoction is similar extraction to infusion but the time is longer (30 minutes) and temperature is almost until boiling point of water (Depkes RI, 2000).
2.3 Free radical
A free radical may be defined as an atom, molecule or compound containing one or more unpaired electrons in its outmost atomic or molecular orbital. When a free radical formed can be highly reactive and can start a chain reaction by
10 Universitas Sumatera Utara stealing an electron from another molecule to be a stable form. The sources of free radical can be endogenous and exogenous in nature. Endogenous sources of free radical are intracellularly generated from auto-oxidation or inactivation of small molecules. Exogenous sources of free radical are tobacco smoke, certain pollutants, organic solvents, anesthetics and pesticides (Rao et al., 2011).
Reactive Oxygen Species (ROS) is a collective term used for oxygen- derived free radical include superoxide (O2•-), peroxyl (ROO•), hydroxyl (OH•), nitric oxide (NO•) and non-radical include hydrogen peroxide (H2O2), singlet
1 oxygen ( O2) and hypochlorous acid (HOCl) (Pietta, 2000). Almost all organisms have many antioxidants to protect them from a free radical attacking that can reduce the rate of free radical formation and as chain-breaking antioxidants that scavenge and stabilize free radicals. If the amount of antioxidant in the body is less than the production of free radicals, it leads to an oxidative stress condition
(Hasan et al., 2009). Oxidative stress plays a major part in the development of chronic and degenerative diseases such as cancer, arthritis, aging, autoimmune disorders, cardiovascular and neurodegenerative diseases (Kabel, 2014).
Generally, the steps of free radical formation are: a. Initiation
RH + initiator R• b. Propagation
• • R + O2 ROO
ROO• + RH ROOH + R• c. Termination
R• + R• RR
ROO• + R• ROOR
11 Universitas Sumatera Utara Initiation step is the first step of free radical formation. Propagation steps are prolongation of radical chain reaction, when a free radical reacts to another molecule resulting a new free radical, then this new free radical reacts as before and chain reaction begins. Termination step is the final step, when a free radical reacts to a free radical or reacts to an antioxidant compound resulting a stable free radical or free radical does not more reactive. If this process happens, so the chain reaction will not occur anymore (Kumalaningsih, 2006).
2.4 Antioxidant
Antioxidants are molecules that can slow or prevent the oxidation of other molecules. Oxidation reactions can produce free radicals, which start chain reactions that damage cells. Antioxidants terminate these chain reactions by removing radical and inhibiting other oxidation reactions by being oxidized themselves. The antioxidant defense systems function through blocking the initial production of free radicals, scavenging the oxidants, converting the oxidants to less toxic compounds, blocking the secondary production of toxic metabolites or inflammatory mediators, blocking the chain propagation of the secondary oxidants, repairing the molecular injury induced by free radicals or enhancing the endogenous antioxidant defense system of the target. These defense mechanisms act cooperatively to protect the body from oxidative stress (Kabel, 2014).
Depending on the action mechanism, antioxidant can be categorized as primary, secondary and tertiary antioxidant (Winarsi, 2007). a. Primary antioxidant (endogenous antioxidant)
Primary antioxidant is referred to enzymatic antioxidant. It can donor hydrogen atom to radical then an radical-antioxidant formed will be turn into a
12 Universitas Sumatera Utara more stable compound. Primary antioxidant is generally called chain-breaking- antioxidant, which inhibits the formation of free radical by breaking chain reaction, then changing into a more stable product or less reactive molecule.
Primary antioxidant such as enzymes produced in body including Superoxide dismutase (SOD), Catalase (CAT), Glutathione peroxidase (GPx) and glutathione reductase (GRx) (Winarsi, 2007). b. Secondary antioxidant (exogenous antioxidant)
Secondary antioxidant is called exogenous antioxidant or non-enzymatic. In this group, secondary antioxidant is referred to preventive defense system. In the defense system, the formation of reactive oxygen is inhibited by metal chelating or destroyed its formation. Non-enzymatic can be non-nutritional and nutritional component of vegetables and fruits. Non-enzymatic antioxidant works by cutting the chain-oxidative reaction of free radical or by capturing the free radical. In other words, free radical will not reactive to cellular component (Winarsi, 2007).
Non-enzymatic antioxidant can be either natural or synthetic antioxidant.
Natural antioxidant commonly found in plants such as fruits, grains and vegetables. Some natural antioxidant compounds are derivatives of phenol, coumarin, tocopherol, hydroxycinnamic, flavonoids, diphenol, dihydroflavon, catechines and ascorbic acid. Synthetic antioxidants include butyl hydroxylanisole
(BHA), butyl hydroxytoluene (BHT), propyl gallate, ethoxyquin and others
(Mustarichie et al., 2017). For example, ascorbic acid (vitamin C) is widely known for its antioxidant activity and is therefore used in cosmetics and degenerative disease treatments. Vitamin C has many physiological functions, among them a highly antioxidant power to recycle vitamin E in membrane and lipoprotein lipid peroxidation (Rao, 2012). Flavonoid group have antioxidant
13 Universitas Sumatera Utara activities including flavones, flavonols (quercetin), isoflavones, catechin and calcon (Kumalaningsih, 2006).
Figure 2.2 Vitamin C Structure (Ascorbic acid) (Rao, 2012) c. Tertiary antioxidant
Tertiary antioxidant includes DNA-repair enzyme system such as methionine sulfoxide reductases. The function of the enzyme is repair damaged biomolecular because of the reactivity of free radical (Winarsi, 2007).
Antioxidant activity is an important and fundamental function in life systems. Antioxidant has many other biological functions such as anti-mutagenic, anti-carcinogenic and anti-aging responses. In general, antioxidants are substances present in low concentration which significantly delay or inhibit oxidation. The radical formed from antioxidants do not propagate an oxidative chain reaction, but are neutralized by reaction with other radicals to form stable products or recycled by other antioxidants (Rao, 2012).
Phenolic compounds are commonly found in both edible and non-edible plants, and they have been reported to have multiple biological effects, including antioxidant activity. Crude extracts of fruit, herbs, vegetables, cereals and other plant materials rich in phenolics are increasingly being used in the food industry because they retard oxidative degradation of lipid and improve the quality and nutritional value of food. Because of phenolic compounds have antioxidant activities, they have specific health effects in functional food for the maintenance of health, protection from coronary heart disease, and cancer. Phenolic plant
14 Universitas Sumatera Utara compounds fall into several categories: simple phenolics, phenolic acids
(derivates of cinnamic and benzoic acids), coumarins, flavonoids, stilbenes, tannins, lignans and lignins. Chief among these are the flavonoid which have potent antioxidant activities (Rao, 2012).
2.4.1 Flavonoid
Flavonoids (the term is derived from the Latin word“flavus”, meaning yellow) are ubiquitous plant secondary products that are best known as the characteristic red, blue and purple anthocyanin pigments of plant tissues. Apart from their physiological roles in the plants, flavonoids are important components of the human diet, although they are not considered as nutrients. Flavonoids can prevent injury caused by free radicals by the following mechanisms: direct scavenging of reactive oxygen species (ROS),activation of antioxidant enzymes, metal chelating activity, inhibition of oxidizes, alleviation of oxidative stress caused by nitric oxide, increase in uric acid levels (Prochazkov et al., 2011).
Flavonoid is polyphenolic molecule containing 15 carbon atoms and is soluble in water. They consist of two benzene rings connected by a short three carbon chain. One of the carbons in this chain is connected to a carbon in one of the benzene ring, either through an oxygen bridge or directly, which gives a third middle ring (Shah et al., 2016).
Figure 2.3 Flavonoid Structure (Shah et al., 2016)
Flavonoids are an important class of phenolic compounds and have potent antioxidant activity. The antioxidant property of flavonoids is the first mechanism
15 Universitas Sumatera Utara of action studied with regard to their protective effect against cardiovascular disease (Rao, 2012). These molecules are found in a variety of fruits and vegetables. The flavonoids can be divided into six major subtypes, which include chalcones, flavones, isoflavonoids, flavanones, anthoxanthins and anthocyanins
(Shah et al., 2016).
2.4.2 Quercetin
The International Union of Pure and Applied Chemistry (IUPAC) nomenclature for quercetin is 3,3’,4’,5,7-pentahydroxyflavanone (or) 3,3’,4’,5,7- pentahydroxy-2-phenylchromen-4-one with a molecular formula C15H10O7.
Quercetin have five hydroxyl groups, at positions 3, 5, 7, 3’, and 4’ as shown in
Figure 2 .4.
Figure 2.4. Quercetin Structure (Kumar et al., 2017)
Quercetin is flavonol type of flavonoid and found in a variety of foods including apples, berries, vegetables, capers, grapes, onions, shallots, tea and tomatoes, as well as many seeds, nuts, flowers, barks and leaves. It is a brilliant citron yellow colour and is entirely insoluble in cold water, poorly soluble in hot water, but quite soluble in alcohol (Shah et al., 2016).
Quercetin is widely known for its antioxidant property that is for its ability to scavenge free radicals and bind transition metal ions (Kumar et al., 2017).
Quercetin seems to be the most powerful flavonoids for protecting the body against reactive oxygen species (ROS) which produced during the normal oxygen
16 Universitas Sumatera Utara metabolism or are induced by exogenous damage (Shah et al., 2016). Beside as antioxidant activity, quercetin also have other pharmacological properties including neurological, antiviral, anticancer, cardiovascular, antimicrobial, anti- inflammatory, hepatoprotective, protective of the reproductive system and anti- obesity agent (Maalik et al., 2014).
2.5 Ultraviolet-Visible Spectrophotometer
Ultraviolet-visible spectrophotometer is a measurement of wavelength and intensity of ultraviolet and visible light that is absorbed by a substance or sample.
Ultraviolet-visible spectrophotometer is consist of two beams of light: Ultraviolet
(200-400 nm) and visible (400-800 nm) (Muchlisyam and Pardede, 2017). This spectrophotometer is one of the most commonly used methods because it is quite easy, fast, reasonably specific and appropriate for small amount of compounds. In addition, it can perform qualitative (identification of given compound) and quantitative analysis (measurement of quantity of given molecule).
Spectrophotometer can be applied for quantification of a substance by preparing its solution in blank/transparent solvent and then measuring its absorbance at appropriate wavelength. This wavelength is actually the maximum absorption wavelength which is expressed as λmax (Mehmood et al., 2015).
2.6 Method of Antioxidant Capacity Assessment
According to Pisoschi and Negulescu (2011), antioxidant activity of sample by in vitro can be examined with several method that basically prevents, inhibits or scavenges free radicals by single electron transfer (SET) or hydrogen atom transfer (HAT).
17 Universitas Sumatera Utara a. ORAC (Oxygen Radical Absorbance Capacity)
b. HORAC (Hydroxyl Radical Averting Capacity)
c. TRAP (Total Radical Trapping Antioxidant Parameter)
d. FRAP (Ferric Reducing/Antioxidant Power)
e. PFRAP (Potassium Ferricyanide Reducing Power)
f. CUPRAC (Cupric Reducing Antioxidant power)
g. DPPH (1,1-diphenyl-2-picrylhidrazyl)
h. ABTS (3-ethylbenzthiazoline-6-sulphonic acid)
2.6.1 DPPH free radical scavenging
In 1922, Goldschmidt and Renn discovered the violet-coloured free stable radical 2,2-diphenyl-1-picrylhydrazyl (DPPH), which now is used as colorimetric reagent for redox processes. Because DPPH can be kept indefinitely with little decomposition and because it neither dimerizes nor reacts with oxygen, it proved to be quite useful in a variety of investigations, such as polymerization inhibition or radical chemistry, the determination of antioxidant properties of amines, phenols or natural compounds (vitamins, plant extracts, medicinal drugs) and for inhibiting homolytic reactions. DPPH is intensely violet like KMnO4 and its reduced 2,2-diphenyl-1- picrylhydrazine (DPPH-H) is orange-yellow (Ionita,
2005). DPPH• is a stable free radical, due to the delocalization of the spare electron on the whole molecule. The delocalization on the DPPH• molecule determines the occurrence of a purple color, with an absorption band with a maximum around 520nm (Pisoschi and Negulescu, 2011). When DPPH• reacts to antioxidant or substance that can donor a hydrogen atom, the reduced form
(DPPH-H) is generated, followed by the disappearance of the violet color to yellow color (Shekar and Anju, 2011).
18 Universitas Sumatera Utara
Figure 2.5 DPPH and Antioxidant Reaction (Marinova and Batchvarov, 2011)
One parameter that has been used recently for the interpretation of the results from the DPPH method is the “efficient concentration” or EC50 values
(generally expressed as IC50 values or “inhibition concentration). This is defined as the concentration of sample that causes 50% loss of the DPPH activity. The
IC50 values of sample is lower indicates that sample has the higher antioxidant activity (Molyneux, 2004).
2.6.2 Maximum absorption wavelength
Maximum absorption wavelength (λmax) is used to the absorbance measurement that is given variously wavelength at 515-520 nm. This wavelength is usually obtained to DPPH solution. In measuring the solution, a curve will come out, the “peak” that is round topped indicates a maximum absorption wavelength where the absorbance is the highest (Molyneux, 2004).
2.6.3 Reaction time
The duration of the reaction of radical scavenging activity between DPPH solution and sample varied from 5 minutes, 10 minutes, 15 minutes, 20 minutes,
30 minutes, 60 minutes, 90 minutes, and 120 minutes (Marinova and Batchvarov,
2011). For this experiment, the author chooses the reaction time is 60 minutes according to Rosidah et al. (2008), because many references recommend or use that time to incubate the DPPH and sample test in methanol solution.
19 Universitas Sumatera Utara CHAPTER III
METHODOLOGY
This research was done based on an experimental method which includes several steps such collection of lakoocha leaves, identification, simplicia preparation, extract preparation, phytochemical screening of extract and simplicia, standardization of simplicia and also antioxidant activity assay using DPPH (1,1- diphenyl-2-picrylhydrazyl) radical. This research was conducted in
Pharmacognosy Laboratory and Research Laboratory (LP) in Faculty of
Pharmacy, Universitas Sumatera Utara, Medan.
3.1 Apparatus and Materials
3.1.1 Apparatus
The apparatus that used in this research were laboratory glassware such volumetric flask 5 ml, 25 mL and 50 ml (Pyrex), spatula, filter paper, ashless filter paper, dropper, TLC plate, chamber, vial, porcelain crucible, analytical balance
(Mettler Toledo), sonicator, aluminium foil, blender (Philips), water bath, oven
(Memmert), rotary evaporator (Stuart), muffle furnace, light microscope, cuvette,
Uv-visible spectrophototometer (Shimadzu UV-1800).
3.1.2 Materials
A plant that used in this research was lakoocha leaves (Artocarpus lacucha
Buch.-Ham). Chemical materials that used in this research were DPPH (1,1- diphenyl-2-picrylhydrazyl) (Sigma), quercetin (Sigma), n-hexane, ethyl acetate, ethanol 96%, aquadest, E-Merck production: amyl alcohol, hydrochloricacid, benzene, iron (III) chloride, mercuric (II) chloride, acetic anhydride, aluminium
20 Universitas Sumatera Utara (III) chloride, iodine, potassium iodide, chloral hydrate crystal, toluene, lead (II) acetate, bismuth (III) nitrate, glacial acetic acid, magnesium powder, chloroform, sulphuric acid, ammonia, nitric acid, n-hexane, ethyl acetate, methanol.
3.2 Sample Preparation
Sample preparations include sample collection, sample identification, simplicia preparation and extract preparation
3.2.1 Sample collection
The lakoocha leaves were collected on November 2017 from Hutatinggi village, Laguboti sub-district, Toba Samosir district, North Sumatera.
3.2.2 Sample identification
Sample identification was conducted in Herbarium Bogoriense, Research
Centre for Biology, Indonesian Institute of Sciences (LIPI), Bogor.
3.2.3 Simplicia preparation
The fresh leaves of lakoocha collected that were washed using water until clean from dirt and dried under a fan overnight. The leaves were dried in a drying cabinet at 40oC-50oC temperature until the leaves dried and fragile. After drying, the leaves were powdered with a blender into powder. The powdered leaves can be stored in a clean container before using (Kemenkes RI, 2013).
3.2.4 Extract preparation
Successive extraction method was carried out using non-polar to polar solvent (n-hexane, ethyl acetate and ethanol 96%) successively. The steps:
About 70 g of the powdered simplicia was macerated with 500 ml of n- hexane in a container, left within 5 days then stirred occasionally and stored in the dark place. The mixture was filtered into another container. The residue was
21 Universitas Sumatera Utara washed with n-hexane until the volume was obtained 700 ml. All macerate were collected and concentrated using rotary evaporator (Sitorus, 2015). After that, the extract was transferred into an evaporating dish and dried on a water bath until the concentrated of n-hexane extract was obtained. The residue was dried in a drying cabinet overnight. The residue was macerated with ethyl acetate and ethanol 96% successively with steps were similar to process like n-hexane extract. So, n- hexane extract (NELL), ethyl acetate extract (EAELL), and ethanol extract
(EELL) were obtained.
3.3 Simplicia Standardization
Simplicia standardizations include the observation of macroscopic and microscopic and the determination of water content, water-soluble extractive, ethanol-soluble extractive, total ash and acid-insoluble ash.
3.3.1 Macroscopic observation
A macroscopic observation was carried out for important characters of lakoocha leaves such as color, shape, size, odor and taste (Qureshi et al., 2017).
3.3.2 Microscopic observation
The lakoocha leaf was washed and peeled thinly. A thin peeled was placed on a glass slide and boiled in chloral hydrate solution then observed under a light microscope (Prashanthi et al., 2016).
3.3.3 Water content determination a. Saturation of toluene
About 200 ml of toluene and 2 ml of aquadest was placed into a round- bottom flask, assembled a graduated receiving tube and a reflux condenser,
22 Universitas Sumatera Utara distilled for 2 hours. Stop distillation and allow it to cool for 30 minutes and read off the volume of water (WHO, 2011). b. Water content measurement
About 5 g of the powdered simplicia was placed into a round-bottom flask that has been filling saturated toluene-water then boiled for 15 minutes. When boiling begins, adjust the temperature to allow the distillation proceed at a rate of
2 drops per second until the water has been completely distilled. Rinse the inside of the condenser with toluene. Continue the distillation for 5 more minutes, then remove the receiving tube away from the heat and allow it to cool at room temperature and dislodge any droplets of water adhering to the walls of the receiving tube by tapping the tube. Allow the water and toluene to separated completely, read off the volume of water, and calculate te content of water as a percentage (WHO, 2011).
3.3.4 Water-soluble extractive determination
About 5 g of the powdered simplicia wasplaced in a conical flask with a stopper. Accurately added 100 ml of saturated aquadest-chloroform (2.5 ml of chloroform in aquadest until 1 L), stirred for the first 6 hours, then allowed to stand for 18 hours. Filtered rapidly and transferred 20 ml of the filtrate to a tared flat bottom evaporating dish and evaporate to dryness on a water bath. The remain was dried to constant weight at 105oC temperatureand then weighed immediately.
Calculate the percentage of water-soluble extractives (Kemenkes RI, 2013).
3.3.5 Ethanol-soluble extractive determination
About 5 g of the powdered simplicia wasplaced in a conical flask with a stopper. Accurately added 100 ml of ethanol 96%,stirredfor the first 6 hours, then allowed to stand for 18 hours. Filtered rapidly and accurately transferred 20 ml of
23 Universitas Sumatera Utara the filtrate to a tared flat bottom evaporating dish and evaporate to dryness on a water bath. The remain was dried to constant weight at 105oC temperature and then weigh immediately. Calculate the percentage of ethanol-soluble extractives
(Kemenkes RI, 2013).
3.3.6 Total ash determination
About 2 g of the powdered simplicia was placedinto previously ignited, dried and tarred a porcelain crucible. The simplicia powder was spread evenly as a thin layer.A porcelain crucible was placed in the muffle furnace and the temperature was adjusted to 600oC and ignited for 3 hours, cooled, and weighed immediately. Calculate the content of total ash (WHO, 2011).
3.3.7 Acid-insoluble ash determination
The porcelain crucible containing the total ash obtained was added with 25 ml of hydrochloric acid, covered with a watch glass and boiled gently for 5 minutes on a hot plate. The watch glass was rinsed with 5 ml of hot water and added this liquid to the crucible. The insoluble matter was collected on an ashless filter paper by filtration. The filter paper was rinsed repeatedly with hot water until the filtrate is neutral. The filter paper containing the insoluble matter was transferred to the original crucible, ignitedin the muffle furnace at 600ºCand weighed. Calculate the content of acid-insoluble ash (WHO, 2011).
3.4 Phytochemical Screening
Phytochemical screening was conducted on simplicia powder, NELL,
EAELL and EELL to screen the secondary metabolites such as alkaloid, tannin, flavonoid, steroid/triterpenoid, saponin and glycoside.
24 Universitas Sumatera Utara 3.4.1 Phytochemical screening using simplicia powder
3.4.1.1 Alkaloid
About 0.5 g of the powdered simplicia was boiled in 1 ml of hydrochloride acid 2N and 9 ml of aquadest for 2 minutes, cooled and filtered. a. About 2 drops of Bouchardate reagent were added in 0.5 ml of filtrate. Brown-
black precipitate was formed b. About 2 drops of Dragendorff reagent were added in 0.5 ml of filtrate. Brown
or brownish red precipitate was formed c. About 2 drops of Mayer reagent were added in 0.5 ml of filtrate. A white or
yellowish precipitate was formed
If 2 of 3 tests above have precipitate according to the color means it is positive alkaloid (Depkes RI, 1995).
3.4.1.2 Glycoside
About 3 g of the powdered simplicia was extracted by heating for 15 minutes under reflux with 30 ml of the mixture of ethanol 96%-aquadest (7:3), few drops of hydrochloride acid 2N until pH 2 was obtained, cooled and filtered.
About 20 ml of filtrate was added in 25 ml of aquadest and 2 ml of lead (II) acetate 0.4M, shaken and left for 5 minutes then filtered. The filtrate was extracted in three times, with 20 ml of the mixture of chloroform-isopropanol
(3:2) respectively, 2 layers will be separated, collected each layer. Upper layer was placed into a test tube and evaporated on a water bath. The remain was added in 2 ml of aquadest, 5 drops of molisch and 2 ml of concentrated sulphuric acid by side of test tube carefully. Blue ring will be formed in the middle of layer. There was glucose bond in this reaction (Depkes RI, 1995).
25 Universitas Sumatera Utara 3.4.1.3 Flavonoid
About 10 g of the powdered simplicia was boiled in 10 ml of aquadest for 5 minutes, filtered while boiling into a test tube. About 5 ml of filtrate was taken in
0.1 g of magnesium powder, 1 ml of concentrated hydrochloride acid and 2 ml of amyl alcohol, shaken and allowed it separate. Positive flavonoid if red, yellow or orange color was present in amyl alcohol layer (Fransworth, 1966).
3.4.1.4 Saponin
About 0.5 g of the powdered simplicia was placed into a test tube and added 10 ml of hot water and filtered. The filtrate was shaken by hand for 10 seconds. Formation of a stable persistent foam on the surface not lost less than 10 minutes with the height range of 1-10 cm and then added few drops of hydrochloride acid 2N, the foam was still not lost. This was indicated the presence of saponin (Depkes RI, 1995).
3.4.1.5 Tannin
About 0.5 g of the powdered simplicia were boiled in 10 ml of aquadest for
3 minutes and filtered into a test tube. The filtrate was diluted with aquadest until colorless. Few drops of iron (III) chloride 10% was added to the filtrate. A blue or green-black was indicated the presence of tannins (Depkes RI, 1995).
3.4.1.6 Steroid/triterpenoid
About 1 g of the powdered simplicia were extracted in 20 ml of n-hexane for 2 hours, filtered into an evaporating dish. The filtrate was evaporated on a water bath until dried. Few drops ofLiebermann-Burchard reagent was added. A green-blue or red-violet was indicated the presence of steroid/triperpenoid
(Depkes RI, 1995).
26 Universitas Sumatera Utara 3.4.2 Phytochemical screening using extract
Thin Layer Chromatography was carried out to screen secondary metabolites from the extract. About 10 mg of NELL, EAELL and EELL respectively were dissolved in 1 ml of n-hexane, ethyl acetate and methanol respectively (Yuda et al., 2017). Each extract was spotted on a silica plate sized 2 cmx10 cm by the upper limit of 0.5 cm and the lower limit of 1.5 cm so that the mobile phase within 8 cm. The mobile phase was prepared and put in a chamber and allowed to saturate. Once saturated, silica plate inserted and allowed mobile phase reach the upper line. Silica plate was taken out, sprayed with a spray reagent and observed the specific color (Mustarichie et al., 2017).
3.4.2.1 Alkaloid
A mobile phase that used was 10 ml of chloroform-methanol-ammonia
(80:40:15) and Dragendroff as a spray reagent. Positive alkaloid was confirmed if brown or orange-brown zones was observed after spraying (Wagner and Bladt,
1996).
3.4.2.2 Glycoside
A mobile phase that used was 10 of ml ethyl acetate-methanol-aquadest
(100:13.5:10) and sulphuric acid 2N as spray reagent. The TLC plate was sprayed and then heated for 1-3 minutes. Positive glycoside was confirmed if blue, brown, green and yellowish zones was observed after spraying (Wagner and Bladt, 1996).
3.4.2.3 Flavonoid
A mobile phase that used was butanol-glacial acetic acid-aquadest(4:1:5) and Aluminium chloride 5% as spray reagent. The mobile phase was placed into separating filter, shaken, placed the upper layer into a chamber. Positive flavonoid was shown if yellow-brown zone was observed after spraying (Yuda et al., 2017).
27 Universitas Sumatera Utara 3.4.2.4 Saponin
A mobile phase that used was chloroform-glacial acetic acid-methanol- aquadest (64:32:12:8) and vanillin-sulphuric acid as spray reagent. The TLC plate was sprayed with 10 ml of solution I and followed by solution II immediately, heated for 5-10 minutes. Positive saponin was confirmed if blue, blue-violet, red, sometimes yellow-brown zone was observed after heating (Wagner and Bladt,
1996).
3.4.2.5 Tannin
A mobile phase that used was aquadest-methanol (6:4) and iron (III) chloride 10% as spray reagent. Positive tannin was confirmed if blue-green or black zone was observed after spraying (Yuda et al., 2017).
3.4.2.6 Steroid/Triterpenoid
A mobile phase that used was glacial acetic acid-n-hexane (2:8) and
Liebermann-Burchard as spray reagent. The sprayed plate was heated for 5-10 minutes. The positive steroid was confirmed if purple-pink, green-blue zone was observed after warming (Wagner and Bladt, 1996).
3.5 Reagent Preparation
3.5.1 Dragendorff reagent
About 8 g of bismuth (III) nitrate was dissolved in 20 ml of concentrated nitric acid. In the other container, 27.2 g of potassium iodide was dissolved in 50 ml of aquadest. Both of solution were mixed and left it until separated perfectly.
The clear solution was taken and diluted in aquadest until up to 100 ml of volume
(Depkes RI, 1995).
28 Universitas Sumatera Utara 3.5.2 Liebermann-Burchard reagent
About 5 ml of acetic anhydride was mixed with 5 ml of concentrated sulphuric acid carefully and added ethanol up to 50 ml of volume (Depkes RI,
1995).
3.5.3 Meyer Reagent
About 1.359 g of mercuric (II) chloride was dissolved in aquadest until 60 ml of volume. About 5 g of potassium iodide was dissolved in 10 ml of aquadest in a different container. Both of solution were mixed into one container and added up with aquadest until 100 ml of volume (Depkes RI, 1995).
3.5.4 Bouchardate reagent
About 4 g of potassium iodide was dissolved in aquadest, then 2 g of iodine was dissolved in potassium iodide solution and added up with aquadest until 100 ml of volume (Depkes RI, 1995).
3.5.5 Molisch reagent
About 3 g of α-naphthol was dissolved in nitrate acid 0.5N until 100 ml of the volume was obtained (Depkes RI, 1995).
3.5.6 Hydrochloric acid 2N reagent
About 17 ml of hydrochloric acid was added in aquadest until 100 ml of volume (Depkes RI, 1995).
3.5.7 Iron (III) chloride 10% reagent
About 10 gr of iron (III) chloride was dissolved in aquadest until 100 ml of volume (Depkes RI, 1995).
3.5.8 Aluminium (III) chloride 5% reagent
About 5 g of aluminium (III) chloride was dissolved in 100 ml of aquadest
(Depkes RI, 1995).
29 Universitas Sumatera Utara 3.5.9 Sulphuric acid 2N reagent
About 5.5 ml of concentrated sulphuric acid was diluted in aquadest up to
100 ml of volume (Depkes RI, 1995).
3.5.10 Vanillin-sulphuric acid reagent
To vanillin 1% (Solution I): 1 g of vanillin was dissolved in 100 ml of methanol. To sulphuric acid 10% (Solution II): 10 g of sulphuric acid was dissolved in 100 ml of methanol (Wagner et al., 1996).
3.5.11 Lead (II) acetate 0.4M reagent
About 15.17 gr of lead (II) acetate was dissolved in aquadest free of C02 until 100 ml of volume (Depkes RI, 1995).
3.5.12 Chloral hydrate solution
About 50 g of chloral hydrate crystal was dissolved in 100 ml of aquadest
(Depkes RI, 1995).
3.5.13 Nitrate acid 0.5N reagent
About 4.2 ml of concentrated nitrate acid was diluted in aquadest up to 100 ml of volume (Depkes RI, 1995).
3.6 Preparation of Stock Solution
3.6.1 Extract stock solution
About 25 mg of NELL, EAELL and EELL respectively was accurately weighed and dissolved in 25 ml methanol (1000 µg/ml) (Wachidah, 2013).
3.6.2 Quercetin stock solution
About 5 mg of quercetin was accurately weighed and dissolved in 25 ml of methanol (200 µg/ml) (Wachidah, 2013).
30 Universitas Sumatera Utara 3.6.3 DPPH 0.5mM stock solution
About 9.8 mg of DPPH was accurately weighed and dissolved in 50 ml of methanol (200 µg/ml) (Wachidah, 2013).
3.7 Antioxidant Activity Assay byUsing Uv-vis Spectrophotometer
3.7.1 Principle of DPPH radical scavenging assay
When DPPH (1,1-diphenyl-2-picrylhydrazyl) radical react to extract that has radical scavenging activity or antioxidant compound, DPPH will be decolorized
(the changes color from purple to yellow) which can be quantitatively measured the absorbance with Uv-vis spectrophotometer. Antioxidant activity is expressed as IC50 (the concentration of extract required to scavenge 50% of DPPH activity)
(Hasan et al., 2009).
3.7.2 DPPH blank solution
About 1 ml of DPPH stock solution was placed into 5 ml of volumetric flask, diluted with methanol up to the line mark (40 µg/ml) (Molyneux, 2004).
3.7.3 Determination of maximum absorption wavelength
DPPH blank solution was homogenized and the absorbance was measured at ultraviolet and visible region of 200-400 nm and 400–800 nm wavelength
(Muchlisyam and Pardede, 2017).
3.8 Preparationof Test Samples
3.8.1 Test sample of extract
About 0.25 ml; 0.5 ml; 1 ml; and 2 ml of NELL stock solution were taken and placed into 5 ml of volumetric flask. Then, 1 ml of DPPH stock solution was added, diluted with methanol up to the line mark, shaken vigorously and
31 Universitas Sumatera Utara incubated for 60 minutes at room temperature in the dark place (50 µg/ml, 100
µg/ml, 200 µg/ml, 400 µg/ml concentration respectively was obtained). The absorbance was measured at 516 nm by Uv-Vis spectrophotometer. This experiment was done in triplicate. These steps were also carried out to EAELL and EELL, but for EELL can be made the concentration from 25 µg/ml, 50 µg/ml,
100 µg/ml, 200 µg/ml (Kumar et al., 2010).
3.8.2 Test sample of quercetin
About 0.0625 ml; 0.125 ml; 0.25 ml; and 0.5 ml of quercetin stock solution was placed into 5 ml of volumetric flask. Then, 1 ml of DPPH stock solution was added, diluted with methanol up to the line mark, shaken vigorously and incubated for 60 minutes at room temperature in the dark (0.625 µg/ml, 1.25
µg/ml, 2.5 µg/ml, 5 µg/ml concentration was obtained). The absorbance was measured at 516 nm by Uv-Vis spectrophotometer. This experiment was done in triplicate (Mustarichie et al., 2016).
3.8.3 The percentage of radical scavenging assay
The percentage of radical scavenging activity of NELL, EAELL and EELL respectively were determined by DPPH free radical method. Quercetin was used as positive control (Rosidah et al., 2008).
Radical scavenging assay (RSA) was calculated using the following formula:
Acontrol−Asample RSA (%) = x 100% Acontrol
Where: Acontrol = the absorbance of blank (without sample test)
Asampel = the absorbance of DPPH and sample test
32 Universitas Sumatera Utara 3.8.4 IC50 values
In testing the antioxidant activity with DPPH method, an parameter was used for interpretation antioxidant activity was IC50 (Inhibitory Concentration
50%). The IC50 values were calculated from the linear regression equation that stated the relationship between the concentration of the sample test (X-axis) and the percent inhibition (Y-axis) showing a 50% reduction activity of DPPH concentration (Mustarichie et al., 2017).
The formula to determine IC50 values : 푌 = 푎푥 + 푏
Where : Y = independent variable (IC50)
X = dependent variable (concentration of extract)
a = intercept
b = slope
Antioxidant activity was categorized as a very powerful when IC50<50
µg/ml, strong if IC50 values of 50-100 µg/ml, moderate at 101-150 µg/ml and weak when IC50 151-200 µg/ml (Mardawati et al., 2008).
33 Universitas Sumatera Utara CHAPTER IV
RESULT AND DISCUSSION
4.1 Plant Identification
Plant identification that was conducted in Herbarium Bogoriense,
Indonesian Institute of Sciences (LIPI), Bogor stating that this plant belongs to
Artocarpus lacucha Buch.-Ham. as species and Moraceae as a family. The result of plant identification was carried out by Pesta Sihombing (2017) which can be seen in Appendix 1.
4.2 Simplicia Standardization
Standardization in pharmaceutical is a set of parameters and procedures, which the result is related to the quality, it means having to comply the standard requirements (chemistry, biology and pharmacy), including limit of stability as a general pharmaceutical product. Other things, standardization means the process of ensuring that the final product (drug or extract product) has a certain constant parameter value and determined first. The parameters of standardization consist of specific parameter (identity, organoleptic, ethanol/water-soluble extractive, chemical content in simplicia) and non-specific parameter (water content, ash total, acid insoluble-ash) (Depkes RI, 2000).
The leaf was dark green and the leaf powder was olive green color. The leaf was pinnately compound 33-31-25 x 15-12-14 cm, alternate and oblong to ovate with cordate base and acute apex. The petiole was about 0.4-0.3-0.3 cm and the margin was entire. The odor was characteristic and the taste was slightly bitter.
The result of macroscopic observation can be seen in Appendix 2.
34 Universitas Sumatera Utara Microscopic observation from a fresh leaf which appeared stomata, trichomes, trachea and ca oxalate. The result can be seen in Appendix 3.
Table 4.1 The Result of Standardization of Lakoocha Leaves Simplicia No. Specification Amount (%) 1 Water content 8.00 2 Water-soluble extractive 26.58 3 Ethanol-soluble extractive 10.38 4 Total ash 8.00 5 Acid-insoluble ash 4.29
The water content of the powdered simplicia was found to be 8.00%. This result was fulfilling the requirement of standardization of water content which was not more than 10% (Saifudin et al., 2011). An excess of water in medicinal plant materials will encourage microbial growth, the presence of fungi or insects, and deterioration following hydrolysis (WHO, 2011).
The water-soluble extractive of the powdered simplicia was found to be
26.58% and the ethanol-soluble extractive was found to be 10.38%. This parameter was used to determine the number of active constituents extracted with solvent from a given amount of simplicia (WHO, 2011). The water-soluble extractive values contained glycoside, glucose, gom, protein, enzyme, pigment and organic acids. The ethanol-soluble extractive contained glycoside, antraquinone, steroid, saponin, flavonoid and tanin (Sitorus, 2015).
The total ash of the powdered simplicia was found to be 10.04%. This parameter was conducted to measure the number of inorganic and mineral such as
Mg, K, Ca, Na, Pb, Zn, Cr, Mn, Fe and others (Sitorus, 2015).
The acid-insoluble ash of the powdered simplicia was found to be 5.55%.
Total ash with HCl 2N reacted with mineral to form soluble salts and the residue which consists mainly of silica was acid-insoluble ash (Qureshi et al., 2017).
35 Universitas Sumatera Utara 4.3 Phytochemical Screening
Phytochemical screening profile of all the extracts was developed by the successive extraction method of using the different solvent to show maximum separation of phytoconstituents (Prashanthi et al., 2016). The result of phytochemical screening the powdered simplicia and NELL, EAELL and EELL can be seen in Table 4.2
Table 4.2 The Result of Phytochemical Screening of Simplicia and Extract Extract No. Secondary Metabolites Simplicia Ethyl n-Hexane Ethanol acetate 1. Alkaloid - - - - 2. Steroid/triterpenoid + + + - 3. Tannin + - + + 4. Glycoside + - + + 5. Flavonoid + - + + 6. Saponin + - + + Wheres: (+) contains secondary metabolite compound (-) does not contain secondary metabolite compound From Table 4.2 showed that NELL contained steroid compound, EAELL contained steroid, tannin, glycoside, flavonoid and saponin whereas EELL contained tannin, glycoside, flavonoid and saponin. The powdered simplicia contained steroid, tannin, glycoside, flavonoid and saponin.
Among the secondary metabolites, flavanoids had potent antioxidant activities. The antioxidant properties of flavonoids are the first mechanism of action studied with regard to their protective effect against cardiovascular disease
(Rao, 2012). Flavonoids were shown to have a very strong biological effects such antioxidant that can inhibit the clotting of blood cell, induce the production of nitric oxide (NO) that plays a role dilate blood vessel (vasorelactation) and also inhibit the growth of cancer cell (Winarsi, 2007).
36 Universitas Sumatera Utara 4.4 Antioxidant Activity of Lakoocha Leaves Extract using DPPH Method
DPPH (1,1-diphenyl-2-picrylhydrazyl) is widely used to test the ability of extracts to act as free radical scavengers or hydrogen donors and to evaluate antioxidant activity because it is a rapid, simple and inexpensive method (Shekar and Anju, 2014). The result of the antioxidant activities of lakoocha leaves extracts with 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging assay by using UV-vis spectrophotometer that was measured at wavelength of 516 nm.
4.4.1 Determination of maximum absorption wavelength
Maximum absorbance of DPPH 40 ppm solution was measured by using an
UV-visible spectrophotometer. The result of maximum absorption wavelength could be seen in Figure 4.1.
Figure 4.1 The Curve of Maximum Absorption Wavelength of DPPH 40 ppm Solution using an UV-visible Spectrophotometer This measurement showed that DPPH solution resulting maximum absorbance at wavelength of 516 nm. This wavelength was within the range of visible light at 400-800 nm (Muchlisyam and Pardede, 2017).
4.4.2 Antioxidant activity of test samples
Antioxidant activities of lakoocha leaves extracts in various solvents were measured its absorbance at 60 minutes with the concentration of 0 µg/ml, 25
µg/ml, 50 µg/ml, 100 µg/ml, 200 µg/ml, 400 µg/ml with DPPH addition.
Quercetin with concentration 0.625 µg/ml, 1.25 µg/ml, 2.5 µg/ml and 5 µg/ml.
37 Universitas Sumatera Utara The result of absorbance measurement against extract concentration in various solvents and quercetin was expressed as % scavenging (% RSA). The percentage of scavenging could be seen at Table 4.3, Table 4.4, Table 4.5 and Table 4.6.
Table 4.3 The Percentage of Scavenging from NELL Concentration % Scavenging Mean (µg/ml) I II III 0 0.00 0.00 0.00 0.00 50 4.75 4.65 4.55 4.65 100 5.34 5.25 5.25 5.28 200 9.30 9.21 9.21 9.24 400 19.59 19.60 19.60 19.60
Table 4.4 The Percentage of Scavenging from EAELL Concentration % Scavenging Mean (µg/ml) I II III 0 0.00 0.00 0.00 0.00 50 18.38 18.40 18.40 18.39 100 22.33 22.25 22.25 22.28 200 38.69 38.63 38.63 38.65 400 56.69 56.74 56.74 56.72
Table 4.5 The Percentage of Scavenging from EELL Concentration % Scavenging Mean (µg/ml) I II III 0 0.00 0.00 0.00 0.00 25 4.21 4.31 4.21 4.24 50 36.79 36.85 36.79 36.81 100 66.45 66.49 66.34 66.42 200 87.16 87.18 87.16 87.17
Table 4.6 The Percentage of Scavenging from Quercetin Concentration %Scavenging Mean (µg/ml) I II III 0 0.00 0.00 0.00 0.00 0.625 19.75 19.84 19.73 19.77 1.25 33.71 33.78 33.78 33.76 2.5 61.94 62.09 62.09 62.04 5 93.49 93.39 93.49 93.46
From Table 4.3, Table 4.4, Table 4.5 and Table 4.6 showed the percentage of scavenging increased as the concentration of the sample increases. The increase of percent scavenging indicated the greater of antioxidant activity which associated the ability of compound in test sample to act as a free radical scavenger
38 Universitas Sumatera Utara or hydrogen donor. This capability was used to evaluate antioxidant activity.
Interaction of antioxidant compound with DPPH was based on transfer hydrogen atom to DPPH radical (DPPH●) and convert it to DPPH non-radical (DPPH-H).
The result of reduction DPPH radicals caused discoloration from purple color to yellow pale color which indicated the scavenging activity (Shekhar and Anju,
2014).
30 20 10 0 Scavenging 50 100 200 400 % Concentration (ppm)
Figure 4.2 The Percentage of Scavenging of n-Hexane Extract
100
50
0 Scavenging 50 100 200 400 % Concentration (ppm)
Figure 4.3 The Percentage of Scavenging of Ethyl Acetate Extract
100
50
0 Scavenging 25 50 100 200 % Concentration (ppm)
Figure 4.4 The Percentage of Scavenging of Ethanol Extract
39 Universitas Sumatera Utara 150 100 50 Scavenging
% 0 0.625 1.25 2.5 5 Concentration (ppm)
Figure 4.5 The Percentage of Scavenging of Quercetin
Based on Figure 4.2, Figure 4.3, Figure 4.4 and Figure 4.5 could be seen that the concentration increased in test sample solutions as the percentage of scavenging DPPH increases. The increasing concentration was directly proportional to scavenge DPPH activity. So, it showed that was antioxidant activity presented in test sample.
4.4.3 IC50 values
The IC50 value for lakoocha extracts and quercetin were determined using linear regression equation by plotting the concentration of lakoocha leaves extract and quercetin as X-axis and the percent scavenging of lakoocha leaves extract and quercetin as Y-axis. The result of linear regression and IC50 value of lakoocha leaves extracts and quercetin could be seen at Table 4.7.
Table 4.7 The Result of Linear Regression and Mean±SD of Lakoocha Leaves Extracts and Quercetin Test Sample Regression Equation Mean±SD Category NELL Y = 0.04633X+0.8045 1062.03±1.42 Very weak EAELL Y = 0.131595X+7.46875 323.18±0.02 Very weak EELL Y = 0.45698+ 4.6545 99.23±0.07 Strong Quercetin Y = 18.2416X + 7.603 2.32±0.006 Very strong
The IC50 value for NELL was 1062.03 µg/ml, EAELL was 323.18 µg/ml,
EELL was 99.23 µg/ml and quercetin was 2.32 µg/ml. The EELL had the highest antioxidant activities compared with other extracts because it required a concentration of about 99.23 µg/ml to inhibit 50% of DPPH radical activity but
40 Universitas Sumatera Utara still much weaker than quercetin. Compound thought to has antioxidant activity was polyphenolic such as flavonoid. Flavonoid was polar, thus it could dissolve in semi-polar and polar solvent (Mustarichie et al., 2017). In study of lakoocha leaves had the result that the highest antioxidant activities contained in ethanol extract. The greater antioxidant activity could be affected by the amount and position of hydroxyl and methyl group on ring. Compound having many hydroxyl groups would be stronger to capture free radical, because the capable of donating hydrogen atom increases (Hasim et al., 2016). That statement explained why
EAELL had inactive antioxidant although it contained flavonoid in phytochemical screening. Flavonoid contained in EAELL might be had many methyl group than hydroxyl group, so it made flavonoid tend to semi-polar. Addition, flavonoid was widely distributed in glycoside forms which was more extracted in polar solvent.
The extract could be said that had an active antioxidant, if IC50 value of extract <
200 µg/ml and inactive antioxidant when IC50 value >200 µg/ml (Wachidah,
2013).
The experimental data was expressed as mean±SD. One way analysis of variance (ANOVA) was carried out to determined significant differences (p<
0.05). The result of statistic analysis showing, there was a significant difference between the IC50 value and NELL, EAELL, EELL, quercetin which was 0.00
(p<0.05). The statistic analysis data could be seen in Appendix 11 (Rosidah et al.,
2008).
41 Universitas Sumatera Utara CHAPTER V
CONCLUSION AND SUGGESTION
5.1 Conclusion
Based on the result of experiment and observation which had been carried out, it can be concluded as: a. The result of phytochemical screening of the powdered simplicia contain
flavonoid, tannin, glycoside, steroid, and saponin. For NELL obtains steroid,
EAELL obtain steroid, tannin, glycoside, flavonoid and saponin whereas
EELL obtain tannin, glycoside, flavonoid and saponin. b. The result of the measurement of antioxidant activity in NEEL, EAELL and
EELL with DPPH (1,1-diphenyl-2-picrylhydrazyl) free radical scavenging
assay indicated EELL has antioxidant activity compared to other extracts. c. The results of IC50 value of NELL, EAELL, and EELL were 1062.03 µg/ml,
323.18 µg/ml, and 99.23 µg/ml respectively. The IC50 values of EELL can be
categorized as strong antioxidant activity whereas NELL and EAELL can be
categorized as an inactive antioxidant activities.
5.2 Suggestion
Based on discussion and conclusion, so author recommends to the next researcher to do other antioxidant activity methods such as ABTS (3- ethylbenzthiazoline-6-sulphonic acid) and also can use other part such as fruits.
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46 Universitas Sumatera Utara Appendix 1. The Result of Plant Identification
47 Universitas Sumatera Utara Appendix 2. The Figure of Artocarpus lacuhca Buch.Ham
a. Lakoocha plant
b. In front of fresh leaf c. back of fresh leaf
d. The powdered simplicia of lakoocha leaves
48 Universitas Sumatera Utara Appendix 3. The Result of Microscopic Observation of Lakoocha Leaf
a c
d b
Wheres: 10x40 magnification a. Stomata with anisocytic type b. Trichomes with unicellular c. Ca oxalate with rosette form d. Trachea
49 Universitas Sumatera Utara Appendix 4. The Flowchart of Simplicia Production
Lakoocha leaves
washed from impurities until clean drained and dried under a fan overnight weighed as wet weight dried in a drying cabinet until fragile easily after leaves dry, weighed as dry weight
Lakoocha leaves simplicia
powdered with a blender
stored in a clean container before used
The powdered simplicia of Lakoocha leaves
50 Universitas Sumatera Utara Appendix 5. The Flowchart of the Production of Lakoocha Leaves Extract with a Successive Maceration Method
70 g of simplicia powder
macerated with 500 ml of n-hexane within5 days in dark place stirred occasionally filtered
residue macerate I
washed with n-hexane until 700 ml of volume was obtained stirred occasionally filtered
macerate II
collected all macerates concentrated using rotary evaporator placed into evaporating dish evaporated on water bath
residue n-hexane extract
dried in a drying cabinet overnight until n-hexane disappear macerated with 500 ml of ethyl acetate within 5 days in dark place stirred occasionally filtered
residue macerate I
washed with ethyl acetate until 700 ml of volume was obtained stirred occasionally filtered
macerate II
collected all macerates
51 Universitas Sumatera Utara Appendix 5. Continued concentrated using rotary evaporator placed into evaporating dish evaporated on water bath
residue Ethyl acetate extract
dried in a drying cabinet overnight until ethyl acetate disappear macerated with 500 ml of ethanol 96%within 5 days in dark place stirred occasionally filtered
residue macerate I
washed with ethanol 96% until 700 ml of volume was obtained stirred occasionally filtered
residue macerate II
collected all macerates concentrated using rotary evaporator placed into evaporating dish evaporated on water bath
Ethanol extract
52 Universitas Sumatera Utara Appendix 6. The Flowchart of Antioxidant Activity Assay
n-hexane extract weighed about 25 mg dissolved in 25 ml of n-hexane
stock solution of n-hexane extract (1000 ppm)
taken about 0 ml; 0.25 ml; 0.5 ml; 1 ml; and 2 ml (with concentration 0 µg/ml; 50 µg/ml; 100 µg/ml; 200 µg/ml; and 400 µg/ml respectively) added 1 ml of DPPH 0.5 mM stock solution diluted with methanol until 5 ml of volume and shaken vigorously incubated for 60 minutes at room temperature in the dark place
sample test of n-hexane extract
examined using spectrophotometer uv- visible at wavelength of 156 nm recorded the absorbance calculated inhibition percent
IC50 values of n-hexane extract
53 Universitas Sumatera Utara Appendix 7. The Calculation of Lakoocha Leaves Standardization
1. Calculation of water content
Water volume (ml) %Water content= x100% Sample weight (g)
No. Sample weight (g) Initial volume (ml) Last volume(ml) 1. 5.0036 0.85 1.20 2. 5.0025 1.20 1.60 3. 5.0012 1.60 2.05 0 1.20-0.85 1. %Water content= x100%= 7% 5.0036
1.60-1.20 2. %Water content= x100%= 8% 5.0025
2.05-1.60 3. %Water content= x100%= 9% 5.0012 7%+8%+9% Mean of Water content= = 8% 3
2. Calculation of water-soluble extractive
Extractive weight (g) 100 %Water-soluble extractive= x x100% Sample weight (g) 20
No. Sample weight (g) Extractive weight(g) 1. 5.0103 0.2472 2. 5.0106 0.2704 3. 5.0108 Type0.2814 equation here.
0.2472 100 654277 1. %Water-soluble extractive= x x100%= 24.67% 5.0103 20 78ik72 0.2704 100 2. %Water-soluble extractive= x x100%= 26.98% 5.0106 20 72 0.2814 100 3. %Water-soluble extractive= x x100%=28.08% 5.0108 20 24.67%+26.98%+28.08% Mean of water-soluble extractive= =26.58% 3
54 Universitas Sumatera Utara Appendix 7. Continued
3. Calculation of ethanol-soluble extractive
Extractive weight (g) 100 %Ethanol-soluble extractive= x x100% Sample weight (g) 20
No. Sample weight (g) Extractive weight(g) 1. 5.0105 0.1084 2. 5.0064 0.0802 3. 5.0185 Type0.1236 equation here.
0.1084 100 654277 1. %Ethanol-soluble extractive= x x100%= 10.82% 5.0105 20 78ik72 0.0802 100 2. %Ethanol-soluble extractive= x x100%= 8.01% 5.0064 20 72
0.1236 100 3. %Ethanol-soluble extractive= x x100%=12.31% 5.0185 20 10.82%+8.01%+12.31% Mean of ethanol-soluble extractive= =10.38% 3
4. Calculation of ash total
Ash weight (g) %Ash total= x100% Sample weight (g)
No. Sample weight (g) Ash weight(g) 1. 2.0085 0.1592 2. 2.0105 0.1621 3. 2.0093 Type0.2027 equation here.
0.1592 65427 1. %Ash total= x100%= 7.93% 2.0085 778ik7 0.1521 2. %Ash total= x100%= 8.06% 2.0105 272
0.1607 3. %Ash total= x100%=8% 2.0093
7.93%+8.06%+8% Mean of ash total= =8% 3
55 Universitas Sumatera Utara Appendix 7. Continued
5. Calculation of acid-insoluble ash
Ash weight (g) %Ash total= x100% Sample weight (g)
No. Sample weight (g) Ash weight(g) 1. 2.0085 0.0849 2. 2.0105 0.0883 3. 2.0093 Type0.0861 equation here.
0.0849 65427 1. %Acid-insoluble ash= x100%= 4.23% 2.0085 778ik7 0.0883 2. %Acid-insoluble ash= x100%= 4.39% 272 2.0105
0.0861 3. %Acid-insoluble ash= x100%= 4.28% 2.0093
4.23%+4.39%+4.28% Mean of acid-insoluble ash= = 4.29% 3
56 Universitas Sumatera Utara Appendix 8. The Result of Phytochemical Screening of Simplicia Powder of Lakoocha Leaves
1. Alkaloid 2. Flavoinoid 3. Tannin
4. Saponin 5. Steroid 6. Glikosida
57 Universitas Sumatera Utara Appendix 9. The Result of Phytochemical Screening of Lakoocha Leaves Extract
(a) (b) (c)
1. Alkaloid 2. Flavonoid 3. Tannin
(a) (b) (c)
4. Saponin 5. Steroid 6. Glikosida
Wheres : (a) n-hexane extract, (b) Ethyl acetate extract, (c) Ethanol extract
58 Universitas Sumatera Utara Appendix 10. The Calculation of Scavenging Percent and IC50Values
The Calculation of Scavenging Percent of NELL
• Data I No. Concentration (µg/ml) Absorbance 1. 0 1.011 2. 50 0.963 3. 100 0.957 4. 200 0.917 5. 400 0.813
Acontrol-Asample %RSA= x100% Acontrol
Wheres : Acontrol =Absorbance of does not contain sample
Asample =Absorbance of sample
- 0 µg/ml
1.011-1.011 %RSA= x100%= 0% 1.011
- 50 µg/ml
1.011-0.963 %RSA= x100%= 4.75% 1.011
- 100 µg/ml
1.011-0.957 %RSA= x100%= 5.34% 1.011
- 200 µg/ml
1.011-0.917 %RSA= x100%= 9.30% 1.011
- 400 µg/ml
1.011-0.813 %RSA= x100%= 19.59% 1.011
59 Universitas Sumatera Utara Appendix 10. Continued
Calculation of IC50 values
X Y XY X2 0 0.00 0.00 0 50 4.75 237.5 2,500 100 5.34 534 10,000 200 9.30 1,860 40,000 400 19.59 7,836 160,000 ƩX =750 ƩY = 38.98 ∑XY= 10,467.5 2 x̅ =150 y̅ =7.796 ∑X =212,500
Wheres : X = Concentration (µg/ml) Y = % Scavenging
∑XY-(∑Xx∑Y)/n a = ∑X2-(∑X)2/n
10,467.5-(750x38.98)/5 a = 212,500-(750)2/5
4,620.5 a = 100,000 a = 0.046205 b = y̅-ax̅ b = 7.796-(0.046205)150 b = 0.86525
So, linear regression equation for IC50 is
Y=0.046205X+0.86525
50=0.046205X+0.86525
X =1063.41 µg/ml
60 Universitas Sumatera Utara Appendix 10. Continued
• Data II 2 X Y XY X IC50 0 0.00 0.00 0 50 4.65 232.5 2,500 a = 0.04633 100 5.25 525 10,000 200 9.21 1,842 40,000 1062.11 400 19.60 7,840 160,000 µg/ml ƩX = 750 ƩY = 38.71 ƩXY = ƩX2 = b = 0.7925 x̅= 150 y̅ = 7.742 10,439.5 212,500
linear regression equation Y = 0.04633X + 0.7925
• Data III 2 X Y XY X IC50 0 0.00 0.00 0 50 4.55 227.5 2,500 a = 0.04643 100 5.25 525 10,000 200 9.21 1,842 40,000 1060.58 400 19.60 7,840 160,000 µg/ml Ʃx = 750 Ʃy = 38.61 Ʃxy = Ʃx2 = b = 0.7575 10,434.5 212,500 x̅= 150 y̅ = 7.722 linear regression equation Y = 0.04643X + 0.7575
1063.41 +1062.11 + 1060.58 The mean of IC50 = = 1,062.033 µg/ml 3
25 y = 0.0462x + 0.8653 20 R² = 0.9818 15
10
% scavenging % 5
0 0 100 200 300 400 500 Concentration (ppm)
Graph of The concentration (µg/ml) vs % scavenging of NELL data I
61 Universitas Sumatera Utara Appendix 10. Continued
Sample Concentration % Scavenging IC50 Mean test (µg/ml) I II III I II III 0 0.00 0.00 0.00 0.00 Ethyl 50 18.38 18.40 18.40 18.39 acetate 100 22.33 22.25 22.25 323.21 323.17 323.17 22.28 extract 200 38.69 38.63 38.63 38.65 400 56.69 56.74 56.74 56.72 0 0.00 0.00 0.00 0.00 25 4.21 4.31 4.21 4.24 Ethanol 50 36.79 36.85 36.79 99.24 99.15 99.29 36.81 extract 100 66.45 66.49 66.34 66.42 200 87.16 87.18 87.16 87.17 0 0.00 0.00 0.00 0.00 0.625 19.75 19.84 19.73 19.77 Querce- 1.25 33.71 33.78 33.78 2.33 2.32 2.32 33.76 tin 2.5 61.94 62.09 62.09 62.04 5 93.49 93.39 93.49 93.46
62 Universitas Sumatera Utara Appendix 11. The Result of Statistical Analysis a. Normality test
Hypothesis : H0 = data is normally distributed
H1 = data is not normally distributed
Conclusion : Sig. >0.05, H0 is accepted so data is normally distributed b. ANOVA test
Hypothesis : H0 = There is no significant difference between the IC50 value and NELL, EAELL, EALL, quercetin H1 = There is a significant difference between the IC50 value and NELL, EAELL, EALL, quercetin
Conclusion : There is a significant difference between the IC50 value and NELL, EAELL, EALL, quercetin [F(589494.817)= 1370804.878, P= 0.000] with probability <0.05. Therefore, H0 is rejected and H1 is accepted
63 Universitas Sumatera Utara
From Post-Hoc test using Turkey can be concluded as:
1. The mean IC50 value of NELL(1062.03±0.82) has a significant difference
with the mean IC50 values of EAELL, EELL and quercetin
2. The mean IC50 value of EAELL(323.18±0.01) has a significant difference
with the mean IC50 values of NELL, EELL and quercetin
3. The mean IC50 value of EELL(99.23±0.04) has a significant difference
with the mean IC50 values of NELL, EAELL and quercetin
4. The mean IC50 value of quercetin (2.32±0.00) has a significant difference
with the mean IC50 values of NELL, EAELL and EELL
64 Universitas Sumatera Utara