UNIVERSITY OF GONDAR
COLLEGE OF NATURAL AND COMPUTATIONAL SCIENCE
DEPARTMENT OF CHEMISTRY
DETERMINATION OF ESSENTIAL METAL, POLYPHENOL CONTENT, ANTIOXIDANT AND ANTIBACTERIAL ACTIVITIES OF METHANOLIC ROOT EXTRACTS OF CROTON MACROSTACHYUS AND PHYTOLACCA DODECANDRA
A THESIS SUBMITTED TO DEPARTMENT OF CHEMISTRY IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE IN CHEMISTRY
BY: ABEBE TSEGA
ADVISOR: DESSIE TIBEBE (PhD, ASSOCIATE PROFESSOR)
CO-ADVISOR: MR. MULUGETA LEGESSE (M.Sc., ASSISTANT PROFESSOR)
OCTOBER, 2020
GONDAR, ETHIOPIA
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UNIVERSITY OF GONDAR
COLLEGE OF NATURAL AND COMPUTATIONAL SCIENCE
DEPARTMENT OF CHEMISTRY
DETERMINATION OF ESSENTIAL METAL, POLYPHENOL CONTENT, ANTIOXIDANT AND ANTIBACTERIAL ACTIVITIES OF METHANOLIC ROOT EXTRACTS OF CROTON MACROSTACHYUS AND PHYTOLACCA DODECANDRA
BY: ABEBE TSEGA
APPROVED BY THE EXAMINING BOARD:
Name Signature Date
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Advisor
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Co advisor
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External Examiner
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Internal examiner
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Chairman
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DECLARATION
First of all, I declare that this thesis was an outcome of my genuine effort and I submit it to the University of Gondar in partial fulfillment for the requirement of a Degree of Master of Science. The thesis will be placed at the university library to be made accessible to borrowers for reference. I seriously declare that I have not so far submitted this thesis to any other institution anywhere for the reward of any academic degree, diploma, or certificate.
Name of the researcher: Abebe Tsega Signature ______
Place: University of Gondar
Date of submission: ______
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ACKNOWLEDGEMENTS
First and foremost, I would like to thank the almighty God for giving me strength to overcome a number of difficulties I faced when I conducted this study.
I appreciatively acknowledge my advisor, Dr. Dessie Tibebe, for his unremitting supervision, intelligent guidance and dedication of his time for correction of draft copies. His appreciated advice, encouragement and productive comments that helped me a lot to shape the study in its present form. His approach and regular follow up also played a key role for the successful completion of the experimental work thesis and thesis write-up. Without his help, this research work would have not been accomplished timely.
My sincere gratitude also goes to my co-advisor, Mr. Mulugeta Legesse for his excellent technical support during the experimental work, for his valuable comments, guidance on data analysis and important advice towards the completion of this thesis. In addition, I would like to thank laboratory assistants, Mr. Mulat Tiruneh, Mr. Amogne Wondu, Mr. Legesse Terefe and Mrs. Zemenay Alebel for their unreserved support and respectful cooperation during my experimental work.
I am very much indebted to my wife, S/r Haregitu Derso for her moral support and encouragement in the course of my study.
Finally, my deepest thanks go to the Department of Chemistry, University of Gondar for providing me with the necessary laboratory facilities to conduct my Master’s thesis research.
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TABLE OF CONTENT
Titles Page
DECLARATION ...... ii
ACKNOWLEDGEMENTS ...... iii
LIST OF TABLES ...... vii
LIST OF FIGURES ...... viii
LIST OF ABBREVIATIONS ...... ix
ABSTRACT ...... x
1. INTRODUCTION ...... 1
1.1. Background of the Study ...... 1
1.2. Statement of the Problem ...... 3
1.3. Objectives of the Study ...... 4
1.3.1. General Objective ...... 4
1.3.2. Specific Objectives ...... 4
1.4. Significance and Scope of the Study ...... 4
2. LITERATURE REVIEW ...... 6
2.1. Traditional Medicinal Plants ...... 6
2.2. Taxonomy Species and Family of Croton Macrostachyus and Phytolacca Dodecandra ... 7
2.3. The Importance of Medicinal Plants for the Development of Modern Drugs ...... 8
2.4. Traditional Medicinal Plants in Ethiopia ...... 9
2.5. Elemental Composition of Traditional Medicinal Plants ...... 11
2.6. Essential Macro and Trace Metals ...... 11
2.7. Phenolic Contents of Traditional Medicinal Plants...... 14
2.8. Antioxidants ...... 17
2.9. Medicinal Use of Croton Macrostachyus and Phytolacca Dodecandra...... 18
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2.10. Analytical Techniques for Analysis of Metals in Plant Samples ...... 19
2.11. Principle of FAAS ...... 19
2.12. Principle of UV-Vis Spectroscopy ...... 20
2.13. Principles of Optimization of Digestion Procedures ...... 21
3. MATERIALS AND METHODS ...... 22
3.1. Description of the Study Area ...... 22
3.2. Apparatus and Reagents ...... 23
3.2.1. Instruments and Apparatus ...... 23
3.2.2. Reagents and Chemicals ...... 23
3.3. Plant Sampling and Sample Collection Procedures ...... 24
3.4. Sample Pretreatment ...... 25
3.5. Extraction of Medicinal Plants for Total Polyphenolic Content Analysis ...... 25
3.6. Preparation of Standard Solutions ...... 26
3.7. Total Polyphenolic Content Analysis ...... 27
3.8. Flavonoid Content Analysis ...... 27
3.9. Determination of Antioxidant Activity ...... 28
3.10. Antibacterial activities of the root extracts of medicinal plants ...... 28
3.11. Optimization of Digestion Procedure ...... 29
3.12. Digestion of Croton Macrostachyus and Phytolacca Dodecandra Root Samples ...... 34
3.13. Metal Levels Analysis of Traditional Medicinal plant Root Samples ...... 34
3.14. FAAS Instrumentation ...... 35
3.15. Instrument Calibration...... 36
3.16. Method Performance and Validation ...... 37
4. RESULTS AND DISCUSSION ...... 40
4.1. Recovery...... 40
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4.2. Optimal Conditions for Sample Digestion ...... 42
4.3. Method performance and method validation ...... 42
4.4. Concentration of Metals in Croton Macrostachyus and Phytolacca Dodecandra ...... 43
4.5. Distribution Pattern of the Metals in the Traditional Medicinal Plant Root Samples...... 47
4.6. Comparison with Literature Values ...... 49
4.6. Comparison with Other Medicinal Plants ...... 49
4.7. Determination of Total polyphenolic Content, Flavonoid and Antioxidant activities ...... 52
4.7.1. Optimization of Extraction Parameters for polyphenolic Content Determination ...... 52
4.7.2. Total Polyphenolic Content ...... 53
4.7.3. Flavonoid Content Determination ...... 55
4.7.4. Antioxidant Activity Determination ...... 57
4.8. Comparison of the Present Study with Results from Other Countries ...... 61
4.9. Antibacterial Activities of Methanol Water Extracts of Medicinal Plants...... 63
4.10. Statistical Analysis ...... 64
5. CONCLUSIONS AND RECOMMENDATIONS ...... 66
REFERENCES ...... 67
ANNEXES ...... 76
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LIST OF TABLES
Table 1: Some Ethiopian medicinal plants used for various diseases treatments with their parts and ecological/habitat ...... 10 Table 2: Optimization of time for the digestion of Phytolacca dodecandra and Croton macrostachyus root ...... 30 Table 3: Optimization of temperature for the digestion of Phytolacca dodecandra and Croton macrostachyus root ...... 31 Table 4: Optimization of digestion mass of the Phytolacca dodecandra and Croton macrostachyus root ...... 32 Table 5: Optimization of the reagent volumes for the digestion of the Phytolacca dodecandra and Croton macrostachyus root ...... 33 Table 6: Instrument operating conditions used for the determination of metals using flame atomic absorption spectrophotometer ...... 36 Table 7: Working standard concentration, correlation coefficient and equation of the calibration curves for determination of metals using FAAS for both Croton macrostachyus and Phytolacca dodecandra root samples ...... 37 Table 8: Recovery results for Croton macrostachyus root samples ...... 41 Table 9: Recovery results for Phytolacca dodecandra root samples ...... 41 Table 10: Instrumental detection limit (IDL), limits of detection (LOD) and limit of quantification (LOQ) for the determination of metals in the roots of Phytolacca dodecandra and Croton macrostachyus samples using flame atomic absorption spectrophotometer (FAAS) ...... 43 Table 11: Average concentration of metals in selected medicinal plant root samples ...... 44 Table 12 : Comparison of metal concentration (mg/kg) in Croton macrostachyus and Phytolacca dodecandra root with other medicinal plant root in worldwide ...... 51 Table 13: Comparison of total polyphenol, flavonoid, and antioxidant activities in Croton macrostachyus and Phytolacca dodecandra with other medicinal plants in worldwide ...... 62 Table 14: The result of antibacterial activities of the crude extracts, standard drug and zone of inhibition ...... 64 Table 15: Pearson correlation of metals between Croton macrostachyus and Phytolacca dodecandra ...... 65
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LIST OF FIGURES
Figure 1: Structure of simple phenol ...... 15 Figure 2: Basic structures of phenolic acids and flavonoids...... 17 Figure 3: Map of the Study Area ...... 22 Figure 4: Medicinal Plants ...... 25 Figure 5: Distribution pattern of metals in Croton macrostachyus and Phytolacca dodecandra root samples ...... 48
Figure 6: MeOH:H2O and extraction time (h) on the extraction efficiency of TPP from Croton macrostachyus and Phytolacca dodecandra ...... 53 Figure 7: Comparison of total polyphenolic content between Croton macrostachyus and Phytolacca dodecandra ...... 55 Figure 8: Comparison of flavonoid content between Croton macrostachyus and Phytolacca dodecandra ...... 56 Figure 9 : Comparison of total polyphenols and flavonoid between Croton macrostachyus and Phytolacca dodecandra in different sampling sites ...... 57 Figure 10 : Standard curve of ascorbic acid ...... 58 Figure 11: Inhibition of methanol water extracts of Croton macrostachyus ...... 59 Figure 12: Inhibition of methanol water extracts of Phytolacca dodecandra ...... 59 Figure 13: Comparison of antioxidant activities between Croton macrostachyus and Phytolacca dodecandra in different sampling sites ...... 60 Figure 14: Zone of inhibitions of extracts in comparison with Gentamicin (positive control) against the four bacteria: Staphylococcus aurous, Staphylococcus pneumonia, Escherichia coli and Klebsiella pneumonia ...... 63
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LIST OF ABBREVIATIONS
ADP Adenosine Diphosphate
ANOVA Analysis of Variance
ATP Adenosine Triphosphate
DNA Deoxyribonucleic Acid
DPPH 1,1- Diphenyl -2-picryl-hydrazyl
FAAS Flame Atomic Absorption Spectroscopy
FAO Food and Agriculture Organization
FC Folin – Ciocalteu
FDA Food and Drug Administration
IDL Instrumental Detection Limit
ITM Improved Traditional Medicines
LOD Limit of Detection
LOQ Limit of Quantification
MDL Method Detection Limit
MP Medicinal Plants
RNA Ribonucleic Acid
ROS Reactive Oxygen Species
RSD Relative Standard Deviation
TM Traditional Medicine
UV-Vis Ultraviolet Visible
WHO World Health Organization
WM Western Medicine
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ABSTRACT
Croton macrostachyus and Phytolacca dodecandra plant roots are commonly used as traditional medication to treat various diseases. The present study aimed at investigating elemental composition, polyphenol content, antioxidant and antibacterial activities of methanolic root extracts of both plants collected from six sites in Chiliga district, Central Gondar Zone, Ethiopia. For the analyses of metals, an optimized wet-digestion procedure was applied with 0.5 g of root sample using HNO3:HCl:H2O2 (v/v) in the ratio of 8:2:1 at 240 °C for 3:00 h, and 5:2:2 at 300 °C for 3:00 h for Phytolacca dodecandra and Croton macrostachyus, respectively. Then, concentrations of metals were determined in the digested samples using flame atomic absorption spectrophotometer; validity of the optimized procedure was evaluated by spiking experiments. An optimized procedure was also employed for the extraction of total polyphenols with 80% aqueous methanol for 24 h maceration. The Folin-Ciocalteu, Aluminium Chloride and DPPH radical-scavenging spectrophotometric assays were used to quantify, respectively, the total Polyphenol and flavonoid contents, and antioxidant activities of root extracts. Finally, the antibacterial activities of both plant root samples were evaluated by disk diffusion method. Results of metals revealed good accuracy and repeatability of the method, with recovery rates ranging from 87% to 102% for Croton macrostachyus and 85% to 103% for Phytolacca dodecandra. The concentrations of metals (mg/kg) in both plant root samples ranged from: Cu (5–12), Zn (17–196), Mn (62–479), Fe (182–1455), Cr (0.1–3), Ca (550–1407), and Mg (1019– 1318). The total polyphenol contents were in the range of 802 ± 53 – 1557 ± 75 and 950 ± 38 – 4214 ± 45 mg GAE/100 g, respectively, for Croton macrostachyus and Phytolacca dodecandra, whereas the flavonoid content ranged 342 ± 25.60 – 745± 32.00 and 451 ± 25.60 – 828 ± 16.11 mg CE/100 g. Similarly, the antioxidant activities varied from 3.53 ± 0.38 to 6.38 ± 0.62 and 3.80 ± 0.41 to 14.29 ± 0.99 mg AAE/g sample. The root extracts showed inhibition zones of 6.4, 7.6, 6.2 and 8.1 mm (Phytolacca dodecandra) and 5.8, 6.2, 5.9 and 6.0 mm (Croton macrostachyus), respectively, for Staphylococcus aurous, Staphylococcus pneumonia, Escherichia coli and Klebsiella pneumonia, which are more potent than Gentamicin antibiotic. One-way ANOVA revealed significant differences (p < 0.05) among the mean concentrations of all metals in both plant root samples. The result of Pearson correlation showed a strong association between total polyphenol concentrations and their respective antioxidant activities (r = 0.996). To summarize, the root extracts of both medicinal plants contained highest levels of
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Mg followed by Ca and Fe, but lowest levels of Cr. Moreover, the root extracts of Phytolacca dodecandra in general contained relatively higher amounts of total polyphenols and furnished higher antioxidant and antibacterial activities compared to its Croton macrostachyus counterpart.
Keywords: Antioxidant capacity, Croton macrostachyus, Chiliga, metals, Phytolacca dodecandra, total polyphenol
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1. INTRODUCTION
1.1. Background of the Study
Plants are natural resources of nutritional and biologically active ingredients, synthesized during their growth and accumulated from the environment. Despite the use of many plants as everyday food, some of them, known as medicinal plants (MP), have been used in traditional medicine (TM) as therapeutic resources as well as in variable pharmaceutical preparations (Bardarov and Mihaylova 2014).
Traditional medicine is defined as the sum total of all knowledge and practice, whether explainable or not, used in the diagnosis, prevention and elimination of physical, mental or social imbalances and relying exclusively on practical experience and observation handed down from generation to generation, whether verbally or in writing (Abebe 2016). According to World Health Organization (WHO), traditional medicine is a health practices, approaches, knowledge and beliefs integrating plant, animal and mineral based medicines, spiritual therapies, manual techniques and exercises, applied singularly or in combination to treat, diagnose and prevent illnesses and maintain well-being (Kassaye et al. 2007).
Traditional medicine is an ancient medical practice which existed in the communities before the advent of modern health sciences. It is based on indigenous theories, beliefs and experiences that are preserved down from generations. Moreover, it is taking inspiration from nature to solve human problems (Mohammadi et al. 2016). As a result, several countries have realized the need and importance to develop improved traditional medicines (ITM) from native and endemic plants that are traditionally used at various places for various ailments (Tsegaye and Elias 2015).
The contributions of medicinal plants since ancient times have been vital sources of both preventive and curative traditional medicine preparations for human beings and livestock. Historical accounts of traditionally used medicinal plants describe that different medicinal plants were in use as early as 5000 to 4000 BC in China, and 1600 BC by Syrians, Babylonians, Hebrews and Egyptians (Tigist et al. 2014).
In Ethiopia, up to 80% of the population uses traditional medicine. The major reasons why medicinal plants are demanded in Ethiopia are due to culturally linked traditions, the trust the communities have in medicinal values of traditional medicine, relatively low cost in using them
1 and difficulty in accessing modern health facilities. A common misconception is that medicinal plants are pure and natural and that this equates to harmless. Based on their long history of use, users of traditional medicines deem them safe for human consumption. However, the absence of their regulation provides no such assurance (Atinafu et al. 2015). There are many species of plants which are used by traditional healers (Tsegaye and Elias 2015). However, the researcher was conducting research on only two plants, namely; Croton macrostachyus and Phytolacca dodecandra, because the traditional healer usually uses the two plants for treating different diseases in the study area.
Croton macrostachyus is commonly known as rush foil (English) and Bisana (Amharic). The name of Croton comes from a Greek word Kroton which means ticks, because of the seeds’ resemblance to ticks. The specific name macrostachyus is a contraction of two words, the Greek word macro meaning large and stachyus relating to the spike, hence, a species characterized by large spikes (Negash 2010). It is a deciduous, pioneer tree that belongs to the Euphorbiacea family which has 300 genera and 8,000 to 10,000 species and most abundant plants in the tropics. It is native to Ethiopia, Eritrea, Kenya, Tanzania, Uganda and Nigeria (Abdisa 2019). It has a number of medicinal values, and is used for the treatment of malaria, rabies, gonorrhea, wound, diarrhea, hepatitis, jaundice, scabies, toothache, abdominal pain, cancer, typhoid, pneumonia and gastrointestinal disorders and as ethno veterinary medicine (Abdisa 2019). It is used as purgative, epilepsy, insomnia, and typhoid (Maroyi 2017).
Phytolacca dodecandra, which is commonly called in Amharic as Endod in Ethiopia, is amongst the most important plants in the country. It produces a series of triterpenoid saponins which possess very potent and useful biological properties, including antifungal, antiprotozoan, antiviral, spermicidal and insecticidal activities. It is one of the most hopeful plant pesticides because of its high toxicity to the snails, and low toxicity to mammals. It is also used to treat human rabies (Zeleke et al. 2017).
So far, a number of studies have been conducted to determine the levels of metals, polyphenols and antioxidant activities of Croton macrostachyus and Phytolacca dodecandra leaves. To the best of our knowledge, only very limited studies are currently available on the levels of metals in the leaves of Croton macrostachyus from Ethiopia (Dubale et al. 2015; Malede et al. 2019). However, data on the above-mentioned parameters, especially on the total polyphenolic contents
2 and antioxidant activities, in the root samples of both plants are currently fragmentary and scarce. Therefore, the objectives of this study are to: determine the contents of metals, total polyphenols and flavonoid; and evaluate the antioxidant and antibacterial activities of both plant root samples from Chiliga district in the Central Gondar Administrative Zone, Ethiopia.
1.2. Statement of the Problem
Traditional medicines are very important to cure various diseases; and most traditional medicines are obtained from plants, leaves, stems and roots. The roots of Croton macrostachyus and Phytolacca dodecandra are known to be very important in curing a number of diseases, and widely used by most residents in Chiliga district on a regular basis. Generally, traditional medicinal plants have greater potential for medicinal use and are the major sources of traditional drugs. While taking these medicinal plants, even if they are not used as food, users get essential trace metals which might have different biological functions in our body. In traditional medicines, the chemical compositions (concentration of macro- and trace metals) of the drugs, their total polyphenolic content, antioxidant and antibacterial activities are not precisely known. Therefore, evaluation of total polyphenolic content, antioxidant capacity, antibacterial activities and levels of some macro-and trace metals of traditional medicinal plants are hot areas of research. Thus, this research was initiated to determine the total polyphenolic content, antioxidant capacity, antibacterial activities and levels of selected essential macro- and trace metals of Croton macrostachyus and Phytolacca dodecandra root, which are well-known in curing a number of diseases. It is also known that the source of mineral nutrients for human beings is plant materials consumed in the form of food or medicine. Thus, it is very important to assess total polyphenolic content, the antioxidant capacity, antibacterial activities and levels of selected essential macro- and trace metals.
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1.3. Objectives of the Study
1.3.1. General Objective
The main objective of the present study was to determine the total polyphenolic content, flavonoid, antibacterial activity, antioxidant capacity and some selected essential macro- and trace metals of the root extracts of Croton macrostachyus (Bisana) and Phytolacca dodecandra (Endod).
1.3.2. Specific Objectives
The specific objectives of the present study were:
To develop a working procedure for the digestion and analysis of Croton macrostachyus and Phytolacca dodecandra for their essential metal contents by FAAS; To determine the concentrations of some selected essential macro- and trace metals (Cu, Zn, Mn, Fe, Cr, Ca and Mg) of Croton macrostachyus and Phytolacca dodecandra using FAAS; To optimize the working conditions of different parameters in the extraction of Croton macrostachyus and Phytolacca dodecandra root samples for total polyphenolic content analysis; To determine the total polyphenolic contents of Croton macrostachyus and Phytolacca dodecandra using UV-Visible Spectrophotometer; To determine the flavonoid contents of Croton macrostachyus and Phytolacca dodecandra using UV-Visible Spectrophotometer; and To determine the antioxidant capacities and antibacterial activities of Croton macrostachyus and Phytolacca dodecandra
1.4. Significance and Scope of the Study
Seven selected macro- (Mg and Ca) and trace (Mn, Fe, Cu, Zn and Cr) essential metals, total polyphenolic content, antibacterial activity and antioxidant capacity were analyzed from the roots of two traditional medicinal plants, commonly used to treat different diseases in Chiliga district. However, only a little has been studied on the above-mentioned traditional medicinal plants in the district. Therefore, the present study aimed at determining the levels of the selected
4 essential metals, total polyphenolic content, antibacterial activity and antioxidant capacity in the root extracts of the two medicinal plants collected from Chiliga district.
Therefore, the finding of this study will:
Provide information on the antioxidant activities of traditional medicinal plants such as Croton macrostachyus and Phytolacca dodecandra while using these plants as traditional medicine; Help to create awareness of the user in the levels of total phenolic content and the selected essential macro- and trace metals in the tested medicinal plants; and Provide information for other researchers on the elemental composition, total polyphenolic contents and flavonoid as well as; antibacterial activities.
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2. LITERATURE REVIEW
2.1. Traditional Medicinal Plants
Medicinal plants are plants, either growing wild or cultivated and used for medicinal purposes. They include herbal medicines composed of herbs, herbal materials, herbal preparations and finished herbal products, that contain as active ingredients parts of plants, or other plant materials, or combinations of all ( Hailemariam and Bibiso 2019). They are plants that contain in one or more of their parts substances that can be used for therapeutic purposes (Adie and Adekunle 2017).
Traditional medicine plays a significant role in the healthcare of the majority of the people in developing countries, including Ethiopia and medicinal plants provide valuable contribution to this practice (Abebe 2016). Since olden times, medicinal plants have been used to relieve and cure human diseases. In fact, their therapeutic properties are due to the presence of hundreds or even thousands of natural bioactive compounds called secondary metabolites (Bekomo iteku et al. 2019). It is a method of healing founded on its own concept of health and disease which comprises unscientific knowledge systems that developed over generations within various societies before the era of western medicine (WM) (Antwi-Baffour 2014).
Medicinal plants have been used in diagnosis, treatment or prevention of various ailments since past decades in the name of traditional systems of medicine such as Ayurvedic medicine, Chinese traditional medicine, Tibetan medicine, Unani medicine, Japanese medicine, African traditional medicine etc (Admasu and Yohannes 2018). They are plants that contain substances that could be used by man because of their ability to exert a modifying benefit to the physiology of sick animals or plants that have component parts which are used as natural therapies (Tembeni et al. 2016).
According to WHO 1978, traditional medicine is the sum total of knowledge and practices, whether applicable or not, used in diagnosis, prevention and elimination of physical, mental or social imbalance and relying exclusively on practical experience and observation handed down from generation to generation whether verbally or in writing. It is said that the use of medicinal plant species as a medicine is as old as man and this makes traditional medicine an integral part
6 of the different cultures of people who are especially vulnerable to underserved health facilities (Beyi 2019).
2.2. Taxonomy Species and Family of Croton Macrostachyus and Phytolacca Dodecandra
Croton Macrostachyus
The genus Croton belongs to the Euphorbiaceae family, and contains approximately 1300 species of trees, shrubs, and herbs, which are widely distributed throughout tropical and subtropical regions of the world. The genus name Croton was derived from a Greek word kroton, a tick, referring to thick smooth seeds, a common feature of most Croton species which belong to the Croto-noideae subfamily of the Euphorbiaceae family. The specific name macrostachyus is a contraction of two words, the Greek word macro meaning large and stachyus relating to the spike, hence, a species characterized by large spikes (Hui et al. 2018).
Croton macrostachyus is commonly known as broad-leaved croton or rush foil in English, Bisana in Amharic in Ethiopia. Croton macrostachyus is widely distributed in tropical Africa, from Guinea east to Ethiopia and Somalia, south to Angola, Mozambique, and Madagascar. The species has been reported to occur in Angola, Burundi, Cameroon, Central African Republic, Democratic Republic of Congo, Ethiopia, Ghana, Guinea, Ivory Coast, Kenya, Madagascar, Malawi, Mozambique, Nigeria, Rwanda, Somalia, South Sudan, Sudan, Tanzania, Uganda, and Zambia (Maroyi 2017)
Croton macrostachyus is commonly known as rush foil or broad-leaved Croton (English). A deciduous tree belongs to the family Euphorbiaceae, a very large family with 300 genera and 8,000 to 10,000 species. The name of the genus Croton comes from a Greek word Kroton, which means ticks, because of the seeds' resemblance to ticks. The specific epithet is from the Greek macro (large) and stachyus (relating to a spike) hence with a large spike. The genus contains over 1,200 species, which are distributed throughout the world. Eight of these species (Croton dichogamus, Croton zambesicus, Croton menyhartii, Croton somalense, Croton schimperianus, Croton sylvaticus and Croton lobatusand Croton macrostachyus) are found in Ethiopia (Alemayehu 2018).
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Phytolacca Dodecandra
Phytolacca dodecandra (L'Herit) (synonyms: Phytolacca abyssinica Hoffm, Pircunia abyssinica Moq), a member of the Phytolaccaceae, is known in Ethiopia as endod and elsewhere may be referred to as soapberry. The distribution of this plant is East, West, Central and South Africa and parts of South America and Asia. Endod has small berries which when dried, powdered and placed in water, yield a foaming detergent solution. In Ethiopia, endod exists as two main varieties, arabe with pinkish, and ahiyo with greyish, berries. Arabe (possibly meaning from Arabia) has more powerful detergent properties than ahiyo (meaning donkey and implying that it is less active than the other type). The plant is a climber with hanging branches; it grows very rapidly, reaching a height of up to 10 m but the average height is 2 m to 3 m (Lemma 1970).
2.3. The Importance of Medicinal Plants for the Development of Modern Drugs
The annual global medicine market is pricing about 1.1 trillion US dollars. About 35 percent of these medicines made directly or indirectly from natural products including: plants (25%), microorganisms (13%) and animals (about 3%): natural derived products constitute an extremely important resource for global pharmaceutical companies working on the development of new medicines (Calixto 2019).
Medicinal plants have been playing an essential role in the development of human culture. As a source of medicine, Medicinal plants have always been at the forefront of virtually all cultures of civilizations. Medicinal plants are regarded as rich resources of traditional medicines and from these plants many of the modern medicines are produced (Ahmad et al. 2017).
Herbal medicine is broadly trained worldwide. For centuries, people have twisted to natural medications to cure common ailments such as colds, allergy, upset stomachs and toothaches and the trend is constantly increasing. Nature has been a source of medicinal plants. Nature has been a source of medicinal plants (Shakya 2016).
Medicinal plants are directly used as pharmaceuticals and/or as building blocks or starting materials for the production of semi synthetic drugs and hence, have been playing a crucial role in the design and development of potent therapeutic agents. Therefore, medicinal plants are key resources in the creation of new drugs (Tembeni et al. 2016).
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2.4. Traditional Medicinal Plants in Ethiopia
Ethiopia is considered the home of some of the most diverse plant species in Africa that serve as sources of many traditional medicinal plants. Most of these plants are obtained from local sources in the wild by knowledgeable traditional practitioners. It has been reported that approximately 800 species of the medicinal plants grown in Ethiopia are used for treating about 300 medical conditions (Tesfahuneygn and Gebreegziabher 2019).
Ethiopia is one of the six centers of biodiversity in the world with several topographies, climatic conditions and various ethnic cultures. Ethiopia is endowed with a diverse biological resource including about 6, 500 species of higher plants, with approximately 12% endemic, hence making it one of the six plant biodiversity rich regions. Of these, more than 62.5% of the forest areas are found in the southwest region of Ethiopia where most of the medicinal plants are confined and have been used as a source of traditional medicine to treat different human and livestock ailments (Abera 2014).
In Ethiopia, the use of medicinal plants has been practiced since ancient times and become an important source of the health care system. Medicinal plants are the main sources of traditional medicine for the rural population. Healers play an important role in the primary health care of the rural people and are of high demand on the population who could not afford the cost of modern medication (Assefa et al. 2020).
In regions of Ethiopia where modern public health services are limited or not accessible, several of the population relies on traditional medicine for primary health care. Traditional medical services are also sought in urban areas of Ethiopia, where allopathic services are more readily available, and contribute considerably to the public health care system in Addis Ababa, the capital city. It is estimated that at least 25% of all modern medicines are derived, either directly or indirectly, from medicinal plants, primarily through the application of modern technology to traditional knowledge (Tesfahuneygn and Gebreegziabher 2019).
Ethiopia has a long history of using traditional medicines from plants and has developed ways to fight diseases through it. Although a significant number of people in Ethiopian societies use traditional medicinal plants for their primary health care (Beyi 2019).
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Table 1: Some Ethiopian medicinal plants used for various diseases treatments with their parts and ecological/habitat (Admasu and Yohannes 2018)
Scientific name Local Habit Habitat Part used Use name
Phytolacca Endod S Dry or Root Treatment of ascariasis, gonorrhea, moist malaria, rabies, jaundice, eczema, Dodecandra forest and abortifacient
Croton Bisana T Evergreen Root and Treatment of malaria, rabies, forest stem bark gonorrhea, wound, diarrhea, macrostachyus hepatitis, jaundice, scabies, toothache, abdominal pain, cancer, typhoid and pneumonia
Acacia nilotica Girar T Dry bush Latex Latex from the stem pounded is land taken with honey for curing amebiasis; for treating fire wound
Aerva Nech S Dry sandy Root For treating cancer shinkurt plains, javanica dried river Schultes course
Bersama Azamir T Riverine Leaves- For treating wound by squeezing the forest, stem leaves and creaming on the wound abyssinica rainforest Fresen
Brucea anti Abalo S/T Montane, Leaves For treating cancer, skin problem, dysenterica evergreen leprosy, and external parasites Fresen forest margins
*T= Tree, S= Shrub
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2.5. Elemental Composition of Traditional Medicinal Plants
The elemental composition of many plants’ is known. It will be very interesting to determine their trace elements status. It is surprising to note that many curative effects of medicinal plants used in the traditional system of medicines are due to the presence of very minute quantities of trace elements. Important constituents of the body such as enzymes are intimately associated with the chemical elements. Elements, particularly essential trace elements play both curative and preventive roles in fighting diseases. The plant was selected for elemental analysis mainly due to its availability and use against a variety of ailments (Shirin et al. 2010).
Trace elements are nutrients that are needed for plant, animal growth and health (Silva et al. 2016). A number of elements essential to human nutrition are accumulated in the different parts of plants. Most of the medicinal plants are found to be rich in one or more individual elements, thereby providing a possible link to the therapeutic action of the medicine (Rajan et al. 2014).
2.6. Essential Macro and Trace Metals
The word trace elements are used for elements presented in natural and perturbed environments in small amounts, with excess bioavailability having a toxic effect on the living organism. Trace elements are naturally occurring inorganic substances required in humans in amounts <100 mg/day (Al-fartusie and Mohssan 2017). Trace elements are chemical micronutrients which are required rather in minute quantities but play a vital role in maintaining integrity of various physiological and metabolic processes occurring within living tissues. The term trace metal includes both essential and nonessential trace metals, which may be toxic to the organisms depending on their own properties, availability (chemical speciation), and concentration levels (Bhattacharya et al. 2016).
Trace elements take part in an important role in chemical, biological, biochemical, metabolic, catabolic and enzymatic reactions in the living cells of plants, animals and human beings. More than sixty (60) elements are found in the human body in various forms among which twenty-five (25) are considered essential to human health out of which fourteen (14) exist usually less than 1 mg/Kg of tissue and so termed trace (Dubale et al. 2015).
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Most studies on medicinal plants have been focused on their organic contents, such as essential oils, alkaloids and phenolic compounds. Medical plants also contain various trace elements, the excess or deficiency of which can affect human health. Trace elements refer to elements that occur in small amounts in nature and that when present in higher concentrations are toxic to living organisms (Malede et al. 2019).
In recent decades, to guarantee the quality of traditional medicines, phytopharmaceuticals and traditional medicines have been extensively analyzed by laboratories around the world for their metal content. Moreover, the World Health Organization (WHO) and the US Food and Drug Administration (FDA) have standardized safe limits for the occurrence of certain metals in herbal drugs. However, the WHO has not yet decided what the permissible limits in medicinal plants are for all metals, because many of these metals are also essential dietary micronutrients for humans (Sarma et al. 2011).
According to WHO, essential elements are needed for growth, normal physiological functioning and maintaining of plants’ and human life, and their content in the plants and plants’ products is important for composition of balanced diet. Some elements are cofactors of enzymes and by activating them influence in essential manner the biochemical processes in living cells. Whereas some are macronutrients such as Ca, K, Mg and Na others occur in trace quantities. Cu, Fe, Ni, Zn, and Mn are at the top end of this trace scale and play an important role in biological systems. Also, Cr, Co and Se are essential for normal development and function of human cells, but only in defined quantitative limits. Content of toxic elements like Cd, Pb, As, Hg etc., because of possible intake by food and/or plants’ preparations, should be regulated (Bardarov and Mihaylova 2014).
According to the classification proposed, essential trace elements are elements that are required below 100 mg/ day by an adult person and their deficiency can lead to disorders and may prove fatal. These elements include copper (Cu), iron (Fe), manganese (Mn) and zinc (Zn) (Bhattacharya et al. 2016).
Copper (Cu) is essential for a variety of biochemical processes and is needed for enzymes in the body. It is also involved in the functioning of the nervous system, and in maintaining the balance of other useful trace metals in the body such as Zn. Cu is an essential micronutrient necessary for
12 the hematologic and neurologic systems. It is necessary for the growth and formation of bone, formation of myelin sheaths in the nervous systems, helps in the incorporation of Fe in hemoglobin, assists in the absorption of Fe from the gastrointestinal tract, and in the transfer of Fe from tissues to the plasma (Malede et al. 2019).
Zinc (Zn) is an essential trace element that functions as a cofactor for certain enzymes involved in metabolism and cell growth. As a component of many enzymes, zinc is involved in the metabolism of proteins, carbohydrates, lipids, and energy. Zinc is vital for the healthy working of many of the body’s systems; it plays an essential role in numerous biochemical pathways. It is particularly important for healthy skin and is essential for a healthy immune system and resistance to infection. Zn plays a crucial role in growth and cell division where it is required for protein and DNA synthesis, in insulin activity, in the metabolism of the ovaries and testes, and in liver function. Zinc is found in several enzymes and genetic material transcription (Fartusie and Mohssan 2017).
Manganese (Mn) is a trace mineral that is present in tiny amounts in the body. It is one of the most important nutrients for human health. The average human body contains about 12 mg of Mn. Manganese helps the body to form connective tissue, bones, blood-clotting factors and sex hormones. It also plays a role in fat and carbohydrate metabolism, calcium absorption and blood sugar regulation. It is a component of the antioxidant, and is also necessary for normal brain and nerve function. In addition, Manganese is a key component of enzyme systems, including oxygen-handling enzymes (Fartusie and Mohssan 2017).
Iron (Fe) is the most abundant essential trace element in the human body. The total content of iron in the body is about 3–5 g with most of it in the blood and the rest in the liver, bone marrow, and muscles in the form of heme. Heme is the major iron containing substance in ferrous or ferric state which is present in hemoglobin, myoglobin and cytochrome. Heme forms covalent bonds with the globin protein to form hemoglobin which is the major oxygen carrying pigment in red blood cells of mammalians. Apart from participation in maintaining innumerable physiological and metabolic processes, it is also necessary for DNA, RNA, collagen, antibody synthesis and so forth (Bhattacharya et al. 2016).
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Chromium (Cr) is a trace element that humans require in trace amounts. It is found primarily in two forms: Trivalent (chromium III), which is biologically active and found in food and hexavalent (chromium VI), a toxic form that results from industrial pollution. Chromium produces significant increases in enzyme activity and serves an important function in carbohydrate metabolism, stimulation of fatty acid and cholesterol synthesis from acetate in the liver and improved sugar metabolism through the activation of insulin. Chromium renders the body’s tissues more sensitive to insulin. It is a critical cofactor in the action of insulin (Fartusie and Mohssan 2017).
Calcium (Ca) is the most abundant inorganic constituent of the human body, accounting for about 1 kg of the body weight. Ca is a cofactor for numerous enzymes and is also important for intracellular functions as a messenger in cascade signaling reactions, for example, muscle and nerve function and impulses, cell division and for blood coagulation, keeping blood pH stable. Ca is a major component of normal bone and teeth (Zoroddu et al. 2019).
Magnesium (Mg) is used in so many biological functions, where it functions as a cofactor in more than 300 enzyme systems that regulate diverse biochemical reactions in the body, including protein synthesis, muscle and nerve function, blood glucose control, and blood pressure regulation. Mg is needed for energy production, oxidative phosphorylation, and glycolysis. It contributes to the structural development of bone and is required for the synthesis of DNA, RNA, and the antioxidant glutathione (Fartusie and Mohssan 2017).
The presence and distribution of macro- and trace elements in plant species and plant parts is diverse. Plants are rich in one or more trace elements, which contribute to their therapeutic actions, however can be toxic at high concentrations (Malede et al. 2019). Macro- and trace metals (Cu, Zn, Mn, Fe, Cr, Ca and Mg) can be present in the traditional medicinal plants (Marcovecchio et al. 2007). This study was evaluated the concentrations of copper (Cu), zinc (Zn), manganese (Mn), iron (Fe), Chromium (Cr), Calcium (Ca) and Magnesium (Mg) in 6 samples of two different medicinal plants obtained from farm lands in Chiliga district.
2.7. Phenolic Contents of Traditional Medicinal Plants
The potency of different medicinal plants is related to their individual mechanisms of action in different disorders. Humans consume and use a variety of vegetable materials in the form of
14 leaves, roots, seeds and fruits. Although medicinal plants are widely considered to be of lower risk compared with synthetic drugs, they are not completely free from the possibility of toxicity or other adverse effects. However, there is considerable interest in identifying natural antioxidants from plants that protect against free radical damage as an alternative to synthetic medicines. Phenolic compounds from plants belong to a class of bioactive components with antioxidant activities (Spiridon et al. 2011). Phenolic compounds are commonly known as plant secondary metabolites that hold an aromatic ring bearing at least one hydroxyl group. More than 8000 phenolic compounds as naturally occurring substances from plants have been reported. These phytochemical substances are presented in nutrients and herbal medicines, both flavonoids and many other phenolic components have been reported on their effective antioxidants, anticancer, antibacterial, antiviral, cardio protective agents, anti-inflammation, immune system promoting and interesting candidate for pharmaceutical and medical application (Duangjai Tungmunnithum 2018).
OH
Figure 1: Structure of simple phenol (Liaudanskas et al. 2017) Phenolic compounds widely distributed in the medicinal plants, spices, vegetables, fruits, grains, pulses and other seeds are an important group of natural antioxidants with possible beneficial effects on human health. They can participate in protection against the harmful action of reactive oxygen species, mainly oxygen free radicals (Al-Shemari 2017). They are secondary metabolites of plants and are generally involved in the defense against ultraviolet radiation or aggression by pathogens. They have been associated with diverse functions, including nutrient uptake, protein synthesis, enzyme activity, photosynthesis and structural components (Nour et al. 2013).
Plants are furnished with various phytochemical molecules such as terpenoids, phenolic acids, vitamins, lignins, stilbenes, tannins, amines, flavonoids, quinones, coumarins, alkaloids, and
15 other metabolites, which are rich in antioxidant activity. Studies have revealed that a lot of these antioxidant compounds have anti-inflammatory, antimutagenic, anticarcinogenic, antibacterial and antiviral activities (Meresa et al. 2019). Plant phenolic is important compounds related to antioxidative (preventing damage from reactive oxygen species (ROS) and preventing the formation of these species), antimicrobial, antiviral, anti-inflammatory, analgesic, antipyretic and vasodilatory effects (Bittner et al. 2013). Traditional plants, as a rich source of natural antioxidants, can complement endogenous antioxidant systems to a point where the levels are sufficient. While natural antioxidants occur in all parts of all higher plants, those with medicinal or culinary uses are valuable sources of antioxidants, such as vitamins A, E and C and phenolic compounds including phenolic acids, flavonoids, lignin, stilbenes and tannins. These secondary metabolites also exhibit antiinflammatory, antibacterial, antiviral, anticancer and immune- stimulating activities (Magulska and Wesolowski 2019).
Phenolic compounds are a class of chemical compounds consisting of a hydroxyl group (-OH) bonded directly to an aromatic hydrocarbon group. They play an important role in plant driven antioxidant activity which, due to their redox properties, permit them to act as reducing agents, hydrogen donors, singlet oxygen quenchers or metal chelators (Rao and Ahmed 2014).
Polyphenols are antioxidants with redox properties, which allow them to act as reducing agents, hydrogen donators and singlet oxygen quenchers. A great number of aromatic plants have been reported as having anti-inflammatory, antiallergic, antimutagenic, antiviral, antithrombotic, and vasodilatory actions (Proestos et al. 2013).
Phenolic compounds are an important group of plant based biologically active compounds that strengthen the organism and prevent disease. They have a particularly strong antioxidant effect, which is closely related to the anti-inflammatory and anticancer effect. Multiple epidemiologic studies have proven a significant correlation between the consumption of phenolic compound- rich plants and a decreased risk for developing cardiovascular and neurodegenerative diseases. Phenolic compounds have been proven to have a strong antimicrobial and antiviral effect (Liaudanskas et al. 2017).
Phenolic compounds comprise a wide variety of molecules that have a polyphenol structure (i.e. several hydroxyl groups on aromatic rings), but also molecules with one phenol ring, such as
16 phenolic acids and phenolic alcohols. Polyphenols are divided into several classes according to the number of phenolic rings that they contain and to the structure elements that bind these rings to one another. The main groups of polyphenols are: Flavonoids, phenolic acids, tannins (Khan et al. 2014).
Phenolic Acids
Phenolic acids constitute about one third of the dietary phenols which may be present in plants. Phenolic acids consist of two sub groups, the hydroxybenzoic and hydroxycinnamic acids. Hydroxybenzoic acid includes: Gallic, p-hydroxybeznoic, protocatechuic, vanilli and syringic acid, which have in common the C6 – C1 structure (Zadernowski et al. 2009).
COOH
COOH + O C
A B
Hdroxybenzoic Acid Hydroxycinnamic Acids Flavonoids
Figure 2: Basic structures of phenolic acids and flavonoids (Zadernowski et al. 2009).
Flavonoids
Flavonoids are the largest group of polyphenol compounds found in plants and the most potent antioxidative compounds due to scavenging ability conferred by their hydroxyl groups. Chemically, flavonoids and bioflavonoids are one-electron donors. They are derivatives of conjugated ring structures and hydroxyl groups that have the potential function as antioxidants (Darchivio et al. 2007).
2.8. Antioxidants
Antioxidants are defined as compounds that when present in low concentration in relation to the oxidant prevent or delay the oxidation of the substrate. Their importance in the safeguarding of health, and the protection from coronary heart disease and cancer, has recently been established, thus constituting them as functional food preservatives (Proestos et al. 2013). Antioxidants are
17 the chemical substances that reduce or prevent oxidation stress and have the ability to counteract the damaging effects of free radicals in tissues. Antioxidants can be natural or synthetic various substances with different chemical characteristics and they offer protection against lipid oxidation, react with free radicals, reduce oxidative stress, protect both the biologically important cellular components and bad cholesterol from being oxidized (Radha and Kusum 2019). Antioxidants are necessary to prevent oxidative cell damages, which related many diseases and to maintain cell components in reduced state. Antioxidant constituents can protect the human body from free radicals and reactive oxygen species (Rababah et al. 2004).
Types of Antioxidants
Based on their occurrence, antioxidants are categorized as natural and synthetic.
Natural Antioxidants: They are the chain breaking antioxidants which react with lipid radicals and convert them into more stable products. They are mainly phenolic in structure and include antioxidant minerals, antioxidant vitamins and phytochemical (Atta et al. 2017).
Synthetic Antioxidants: These are phenolic compounds that carry the role of capturing free radicals and stopping the chain reaction. These compounds include butylated hydroxylanisole (BHA), butylated hydroxytoluene (BHT), propyl gallate (PG), metal chelating agent (EDTA), tertiary butyl hydroquinone (TBHQ) and nordihydroguaiaretic (NDGA) (Atta et al. 2017).
2.9. Medicinal Use of Croton Macrostachyus and Phytolacca Dodecandra
The bark, fruits, leaves, roots, and seeds of Croton macrostachyus are reported to possess diverse medicinal properties and cure various human and animal diseases and ailments throughout the distributional range of the species. Croton species have function for the treatment of various diseases such as rabies, malaria, hypertension, cancer, constipation, diabetes, digestive problems, inflammation, dysentery, external wounds, fever, leukemia, balsamic, narcotic, rheumatism, leprosy, bronchitis, diarrhea, intestinal worms, psoriasis, urtcaria, hypercholesterolemia, weight loss and ulcers (Meresa et al. 2017). Also, people are generally familiar with the use of Endod for medicinal purposes. Berries, leaves, and roots are used for various human and animal ailments. The use of Endod against abortion and skin itching (including ringworm) is most
18 common, followed by treatment of gonorrhea, leeches, intestinal worms, anthrax and rabies (Esser et al. 2003).
2.10. Analytical Techniques for Analysis of Metals in Plant Samples
Many instrumental analytical methods may be employed to measure the concentration level of metals in various samples. The most predominant techniques are atomic absorption spectrometry (AAS), atomic emission/fluorescence spectrometry (AES/AFS), inductively coupled plasma mass spectrometry (ICP-MS); inductively coupled plasma optical emission spectrometry (ICP- OES), neutron activation analysis (NAA), X-ray fluorescence (XRF) and anodic striping voltammeters (ASV). Flame atomic absorption spectrometry (FAAS) is the most widely used technique for the analysis of trace metals at ppm concentration levels with good precision for many elements in plant samples. Furthermore, AAS is cheap and its usage is easier than other instruments (Khalid et al. 2016).
2.11. Principle of FAAS
Flame atomic absorption spectrometry is often accepted as a suitable instrumental technique for the measurement of heavy metals because of its speed and ease of operation (Shakerian et al. 2013). Flame Atomic Absorption Spectrometry (FAAS) is one of the most successfully implemented analytical techniques. Its main characteristics are the versatility and low cost (Dionisio et al. 2011).
FAAS is a suitable technique for determining metals at part per million (ppm) concentration levels with good precision for many elements. FAAS offers air-acetylene and/or nitrous oxide flame atomizer. Samples are introduced into the atomizer as an aerosol by the nebulizer. FAAS technique provides fast analysis of 10 to 15 s per sample, with very good precision (repeatability), moderate interferences that can be easily corrected, and relatively low cost (Helaluddin et al. 2016).
This principle has been based on the absorption of energy by valence electrons of the ground state atom. In short, the electrons of the atoms in the atomizer can be promoted to higher orbitals (excited state) for the short period of time (nanoseconds) by absorbing a defined quantity of energy (radiation of a given wavelength). This amount of energy; that is the wavelength is
19 specific electron transition in a particular element. In general, each wavelength corresponds to only one element and the width of an absorption line is only of the order of a few picometers 30 (pm) which gives the technique its elemental selectivity. The radiation flux without a sample and with a sample in the atomizer is measured using a detector and the ratio between two values (the absorbance) is converted to analyte concentration or mass using the Beer-Lambert law. In order to analyze samples for its atomic constituents, it has to be atomized. The atomizer most commonly used flames and electro thermal (graphite tube) atomizer. The atom should then be eradicated by optical radiation and the radiation source could be an element specific line radiation source or a continuum radiation source. The radiation then passes through a monochromator in order to separate the element specific radiation from any other radiation emitted by the radiation source which is finally measured by a detector (Emumejaye 2014).
The absorbance of a known standard and that of the sample is usually compared and calculated or results are plotted graphically to obtain the concentration of the sample. Absorbance is directly proportional to the concentration. For a linear calibration curve, it is assumed that the instrument response A is linearly related to the standard concentration C for a limited range of concentration. It can be expressed in a model such as; A = mc + b. This is a technique that makes the use of absorption of spectrometry to assess the concentration of an analyte in a sample. It requires standards with known analyte concentration to establish the relation between the measured absorbance and the analyte concentration and relies, therefore on the Beer-Lambert law (Tilahun and Zemene 2015).
2.12. Principle of UV-Vis Spectroscopy
The basic principle behind the UV spectroscopy is absorption of visible and UV radiation (200– 400 nm) is associated with excitation of electrons, in both atoms and molecules, from lower to higher energy levels. Since the energy levels of matter are quantized, only light with the precise amount of energy can cause transitions from one level to another will be absorbed. Among other analytical techniques, UV-visible spectroscopy appears to be suitable for the quantification of phenolic compounds. This is due to two main reasons. First of all, phenolic substances have the ability to strongly absorb UV light and secondly, certain compounds due to the colored nature can lead to absorption features in the visible range. Polyphenols are biological compounds containing π conjugated systems with hydroxyl-phenolic groups. The π type molecular orbital’s
20 electronic transitions provide the UV-visible spectrum of this group of compounds (Atole and Rajput 2018).
2.13. Principles of Optimization of Digestion Procedures
In any scientific experiments especially in analytical chemistry creating an optimum working condition before starting analysis of the actual samples is a common practice. That means before preparing the samples for analysis the temperature, the volumes of reagents to be used and the duration of the preparation should be optimized by varying one parameter at a time and keeping the others constant. These optimum conditions were selected based on clarity of digests, minimum reagent volume consumption, minimum digestion time and minimum temperature applied for complete digestion of sample. Wet acid digestion is one of the methods that are involved to get free metal ions in dissolved form from a complex organic matrix based on changing different digestion parameters like volume ratio of reagents to be added, digestion temperature and duration of time (Abebe and Chandravanshi 2017).
Adding the strong mineral acids or their combinations (i.e., commonly nitric and perchloric acids) as they are strong oxidizing agents and form soluble salts with metals(Tilahun and Zemene 2015). During digestion organic components are assumed to be decomposed in the form of different gaseous forms and other metallic elements are left in the solution except those easily volatile metals like Hg (Abebe and Chandravanshi 2017).
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3. MATERIALS AND METHODS
3.1. Description of the Study Area
The study was conducted on selected traditional plants collected from Chiliga district, which is situated in the Central Gondar Zone of the Amhara regional state. The district is located 12 55 N and 37 06 (Figure 3). It encompasses 47 administrative kebeles. The elevation of the district ranged from 900 to 2,267 meters a.s.l. The temperature of the district ranged from 11 to 32 °C with average annual rainfall of 995 to 1,175 mm. It has an estimated total population of 241,712 and an area of 318 kilometer square. The income of the local people is mainly based on sustenance mixed agriculture (crop-livestock production). The study area (Chiliga) was selected based on the large abundance of the two plants and dependence of majority of its population on such traditional medicinal plants; and high prevalence of patients infected with rabies who took the root extracts of these plants as traditional medicine could be another reason to mention.
Figure 3: Map of the Study Area (prepared in ArcGIS software using Ethiogis data)
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3.2. Apparatus and Reagents
3.2.1. Instruments and Apparatus
A flame atomic absorption spectrometer (Buck Scientific, 210VGP, USA) (FAAS) equipped with deuterium background corrector and hollow cathode lamps of Cu, Zn, Mn, Fe, Cr, Ca and Mg with air-acetylene flame was used for the analysis of metals in both Croton macrostachyus and Phytolacca dodecandra root samples. Microprocessor double beam UV–Visible spectrometer (Abron, India) was used to determine total polyphenolic content, flavonoid and antioxidant activity of the plant samples. Polyethylene bags were used for sample collection, sample drying, preserving the grinded and homogenized samples. Electrical ground (IAK – WERKE, Germany) was used for grinding the samples. Digital analytical balance (± 0.0001 precision) was used to weight the sample. Refrigerator (Hitach LR902T, England) was used to keep the digested and filtered samples until analysis. Borosilicate flask (100 mL) and hot plate (SH3, STERILINE LTD, UK) to digest the dried and powdered Croton macrostachyus and Phytolacca dodecandra root samples. Whatman filter paper no. 42, flasks (25, 50 and 100 mL), Cuvette, magnetic stirrer and micropipettes were used. Rotary evaporator was used to remove methanol from the sample extracts to get crude for antibacterial activity test. Lyophilizer was used to remove water from sample extracts. The apparatuses used throughout the experiment were soaked with a mixture of
H2SO4 and K2Cr2O7 for 24 h, followed by rinsing with distilled water and all the measurements were carried out at room temperature.
3.2.2. Reagents and Chemicals
The chemicals and reagents used in this study were all analytical grade. That was (30%) H2O2
(Okhlaindustrial area, Newdelhi, India), (69%) HNO3 (Bluluxlaboratories, Haryana, India), and (37%) HCl (Blulux, India) used for the digestion of Croton macrostachyus (Bisana) and Phytolacca dodecandra (Endod) root samples. 1% Lanthanum chloride hydrate (Blulux laboratories, Haryana, India) was used to decrease the precipitation of Ca and Mg ions in the form of phosphates and sulfates. Standard solutions of concentration (1000 mg/L) of metals Cu, Zn, Mn, Fe, Cr, Ca and Mg (Merck, Germany) were used for preparation of calibration standards and in the spiking experiments. Distilled water was used throughout the experiment for sample preparation, dilution and rinsing apparatus prior to analysis. H2SO4 (98%) and K2Cr2O7 were
23 used to prepare chromic acid solution for soaking and washing digestion flasks and other glassware before starting digestion to remove metals and other greasy contaminants left on the surface of the apparatuses. Methanol (99%, Fine chemicals, India) was used for maceration of the Croton macrostachyus (Bisana) and Phytolacca dodecandra (Endod) root samples with distilled water by 80:20 (v/v). Folin-Ciocalteu reagent (Fine chemicals, Mumbai, India), gallic acid (Fine chemicals, Mumbai, India), standard, ascorbic acid (Blulux laboratories), standard, sodium carbonate (Blulux laboratories) and DPPH (Fine chemicals, Mumbai, India) were used for the determination of total polyphenolic content and antioxidant activities of the selected medicinal plant samples. Sodium hydroxide (Blulux laboratories), aluminum chloride (Fine chemicals, Mumbai, India), sodium nitrite (Blulux laboratories) and catechin (Fine chemicals, Mumbai, India), standard were used for the determination of flavonoids in the plant extracts.
3.3. Plant Sampling and Sample Collection Procedures
Samples of two traditional medicinal plants usually used by the local people for their medicinal value were nominated. Representative samples of Croton macrostachyus (Bisana) and Phytolacca dodecandra (Endod) root were composed from 6 kebeles (Eyaho Seraba, Teber Serako, Nara Awurarda, Aykel Town, Laza Buladigie and Bezaho Mekenet) in which the local people used them as traditional medicine. From each site, 3 sampling points were selected and from each sampling point 2 aged plants were selected, that is per-site 6 plants were taken. For each selected medicinal plant, a total of 36 matured plants were selected from 6 sites and per plant 2 roots were taken starting from the bottommost to the tops. Each selected traditional medicinal plant root collected from each sampling point was homogenized to get a composite sample of each of the six sites. The collected plant root samples were packed into cleaned polyethylene plastic bags, labeled according to their root type and transported to the Department of Chemistry laboratory, University of Gondar for further treatment and analyses (Figure 4a & b).
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b a
Figure 4: Medicinal Plants (a) Phytolacca dodecandra (Endod) (b) Croton macrostachyus (Bisana)
3.4. Sample Pretreatment
Surface contaminants of the plant’s roots were washed with tap water, followed by distilled water. The root was then dried at ambient temperature for three weeks, powdered using an electrical grinder, sieved through a 100 μm pore size sieve, then homogenized, and kept in clean polyethylene plastic bags until digestion and extraction.
3.5. Extraction of Medicinal Plants for Total Polyphenolic Content Analysis
To efficiently determine the total polyphenolic contents, flavonoids and antioxidant activities in Croton macrostachyus and Phytolacca dodecandra samples, different working parameters: extraction time and solvent composition, were carried out. The one that gave the highest yield of TPP with minimum possible extraction time and solvent composition were selected for the routine extraction of the samples.
Extract Preparation
A known amount (0.5 g) of each sample was macerated at room temperature in 50 mL flasks of 50%, 60%, 70%, 80%, 90% and 100% extraction solvent (methanol in water) for 12, 18, 24 and
25
30 h. The extracts were filtered using Whatman filter paper no. 42, and analyzed for their polyphenolic contents, flavonoids and antioxidant activities. The optimal solvent composition and time for extraction of plant samples were 80% (v/v) methanol:water and 24 h, respectively.
3.6. Preparation of Standard Solutions
Preparation of Gallic Acid
A standard stock solution of gallic acid was prepared by dissolving 0.075 g gallic acid in 125 mL methanol water (v/v) 80:20) (600 mg/L of final stock). For the calibration curves, five additional standards of 25, 50, 100, 200 and 300 mg/L solutions were prepared (in 50 mL volumetric flask) by serial dilution of the stock solution. Then, standard solutions were stored at 4 °C until they were analysed.
Preparation of Sodium Carbonate, Aluminum Chloride, Sodium Nitrite and Sodium Hydroxide
For the determination of TPP, 7.5 % Na2CO3 was prepared by dissolving 7.5 g Na2CO3 with a small amount of distilled water, transferring the solution to a 100 mL volumetric flask, and adjusting the volume with distilled water up to the mark. Finally, it was stored at room temperature.
For the determination of total flavonoid content, 5% NaNO2 was prepared by dissolving 5 g
NaNO2 with a small amount of distilled water, transferring the solution to a 100 mL volumetric flask, and adjusting the volume with distilled water up to the mark. Finally, it was stored at room temperature. 10% ACl3 was prepared by dissolving 10 g ACl3 with a small amount of distilled water, transferring the solution to a 100 mL volumetric flask, and adjusting the volume with distilled water up to the mark. It was stored at room temperature.1 M NaOH was also prepared by dissolving 2 g of NaOH with a small amount of distilled water, transferring the solution to a 100 mL volumetric flask, and adjusting the volume with distilled water up to the mark.
Preparation of Ascorbic Acid Solution
A standard stock solution of ascorbic acid (500 mg/L) was prepared by dissolving 50 mg of ascorbic acid in 100 mL of methanol keeping the ratio stated in the method (Kirby and Schmidt
26
1997). Then, different concentrations of ascorbic acid calibration standards (1, 2.5, 5, 10, 15, 20, 25, 50, 100, 150 and 200 mg/L) were prepared from the stock solution.
Preparation of Catechin Solution
It was prepared by dissolving 50 mg of catechin in 100 mL of methanol (a stock of 500 mg/L). The catechin was diluted to different concentrations; 5, 10, 25, 50, 150, 300 and 400 mg/L.
3.7. Total Polyphenolic Content Analysis
Total polyphenol in the plant extract was spectrophotometrically determined by the Folin– Ciocalteu assay using gallic acid as a standard (Haile et al. 2016). Methanol water extracts of Croton macrostachyus and Phytolacca dodecandra were used for this analysis. The reaction mixture was prepared by mixing 0.5 mL of each extract, 3 mL of distilled water and 0.25 mL of
Folin-Ciocalteu reagent. After 5 min in the dark, 1 mL of 7.5% Na2CO3 was added and incubated at room temperature for 90 minutes in the dark. Blank was parallelly prepared containing 0.5 mL of each solvent, 0.25 mL Folin-Ciocalteu reagent and 1 mL of Na2CO3. The absorbance was measured using a double beam UV-Vis spectrophotometer at 760 nm. The samples were prepared in triplicate for each analysis and the mean value of absorbance was obtained. The same procedure was repeated for the standard solution of gallic acid and the calibration curve was constructed. From the measured absorbance, the concentration of phenolic was calculated (as mg/L) from the calibration graph. The total polyphenolic content of the medicinal plant extracts was expressed in terms of milligram gallic acid equivalent per gram sample (mg GAE/g of extract).
3.8. Flavonoid Content Analysis
The flavonoid content in the plant extracts was determined using aluminum chloride assay. Briefly, an aliquot (0.5 mL) of the extract was added to a 10 mL test tube containing 2.0 mL of distilled water. To each test tube, 0.15 mL of 5% NaNO2 was added. After 5 min of incubation,
0.15 mL 10% AlCl3 was added. After 1 min, 1.0 mL of 1.0 M of NaOH was added and the volume was adjusted to 5 mL with distilled water. After 10 min, the absorbance of the resulting solution was measured at 510 nm. Catechin was used as standard to express total flavonoids contents of samples as mg Catechin equivalent per gram of sample (mg CE/g of sample).
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3.9. Determination of Antioxidant Activity
The antioxidant activity of the Croton macrostachyus and Phytolacca dodecandra extracts were evaluated using the DPPH method reported by Haile et al. 2016 with slight modification. A mass of 0.02 g of DPPH was dissolved with a small amount of methanol in a 500 mL volumetric flask. After the DPPH was fully dissolved, the flask was filled up to 500 mL using methanol. The control was measured using 3 mL of methanol and 2 mL of DPPH solution. Besides, a stock solution of ascorbic acid was prepared by dissolving 50 mg of ascorbic acid in 100 mL of methanol. Generation of calibration curve was achieved by preparing different concentrations, 1, 2.5, 5, 10, 15, 20, 25, 50, 100, 150 and 200 mg/L, from the stock solution. To each test tube, 3 mL of methanol and 2 mL of DPPH solution was added; then the test tubes were covered by aluminum foil and kept in the dark for 30 min. For the samples, a 0.1, 0.2, 0.4, 0.8, 1.6 and 2.4 mL portion of the extract was mixed with 1.6 mL of DPPH and the final volume of each solution was adjusted to 4.0 mL with 80% aqueous methanol. The mixture was kept in the dark for 30 min. Finally, the absorbance was measured at 517 nm. The results are expressed as mg ascorbic acid equivalent per gram of sample. The percentage inhibition of the DPPH radical was also calculated. Each sample was analyzed in triplicate.
3.10. Antibacterial activities of the root extracts of medicinal plants
The antibacterial activities of the crude extracts of methanol: water (80%) of the plant were done by disk diffusion method (Ogutu et al. 2012) using Gentamicin disc as standard drug and evaluated by using four bacteria strains, namely; gram positive bacteria species (Staphylococcus aurous and Staphylococcus pneumonia) and gram negative bacteria species (Escherichia coli and Klebsiella pneumonia). These microorganisms were cultured at the molecular Biology laboratory, Department of Biology, University of Gondar.
The media were prepared according to the manufacturer’s instruction as follows: 38 g of the Muller Hinton agar powder was dissolved in 1000 mL of distilled water, then heated, shacked well and allowed to boil and completely dissolved. Then, it was placed in an autoclave at 121 °C for about 15 min to sterilize the media. After that, the media was allowed to cool and poured in 12 plates and put on a leveled surface. Lastly, the media were allowed to solidify, kept in the upright position in the incubator avoiding contamination from the hood.
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Procedures for Performing the Disc Diffusion Test
After the preparation of the media plates as per the manufacturer’s instructions, the test culture bacteria were swabbed on the top of the pre-leveled media and allowed to dry. The sterilized cork borer was used to bore holes on plates and 100 μL of each extract was applied to the holes in triplicate by using Gentamicin drug as positive control. The petri dish was incubated at 37 °C for 24 h. At the end of the incubation period, the antibacterial of each sample (Croton macrostachyus, Phytolacca dodecandra and mixtures of the two extracts) were determined by measuring the average inhibition zones of each extract and positive control in radius millimeter.
3.11. Optimization of Digestion Procedure
The optimization procedures for the sample preparation of determination of essential macro- and trace metal contents in Croton macrostachyus and Phytolacca dodecandra samples were made by hot plate using wet a digestion method.
Optimization of Digestion Procedures for Medicinal Plant Root Samples
The critical necessities of sample preparation for analysis are to get an optimum condition for digestion. In this study, to get ready clear and colorless sample solution which was suitable for analysis using FAAS, different digestion procedures were optimized using HNO3 and HCl acid mixtures and H2O2 reagent by variable parameters such as reagent volume, temperature, time and mass. The optimized procedures were elected based on clearness of digests, minimal reagent volume utilization, minimal digestion time, ease and temperature applied for complete digestion of each sample (Tigist et al. 2014).
In wet acid digestion, the optimized digestion process was attained based on changing various digestion parameters like mass, volume ratio of reagents added, digestion temperature and digestion time. In this method organic components were decomposed in the unlike gaseous forms and other metallic elements were left in the solution except volatile metals. The digestion was assumed to be complete if the solution is clear and colorless. As can be seen from Table 2 to 5, different digestion procedures were optimized using the HNO3: HCl: H2O2 reagent mixtures by varying different parameters listed above.
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Table 2: Optimization of time for the digestion of Phytolacca dodecandra and Croton macrostachyus root (0.5 g). Reagents and temperature: 8 mL HNO3, 2 mL HCl, 1 mL H2O2 at
240℃ for Phytolacca dodecandra and 5 mL HNO3, 2 mL HCl, 2 mL H2O2 at 300 ℃ for Croton macrostachyus root samples
Appearance of Solution
Trial Time (h) Phytolacca dodecandra Croton macrostachyus No.
1 2:00 Yellowish solution with suspension Deep yellow solution
2 2:30 Slightly clear and colorless solution Colorless but turbid
3 3:00 Clear and colorless solution Clear and colorless solution
4 3:30 Clear but light-yellow solution Clear and light yellow
5 4:00 Clear but yellowish Clear and yellow solution
*The bold font shows the optimized time
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Table 3: Optimization of temperature for the digestion of Phytolacca dodecandra and Croton macrostachyus root (0.5 g, 3 h). Reagents: 8 mL HNO3, 2 mL HCl, 1 mL H2O2 for Phytolacca dodecandra and 5 mL HNO3, 2 mL HCl, 2 mL H2O2 for Croton macrostachyus and root samples
Appearance of solution
Trial T Phytolacca dodecandra Croton macrostachyus No. (°C)
1 120 Yellowish solution with more residue Yellowish solution
2 150 Yellowish solution with residue Yellowish solution
3 180 Slightly colorless solution but not Deep yellow solution with residue clear
4 210 Yellowish and turbid Yellowish with no turbidity
5 240 Clear and colorless solution Colorless but turbidity
6 270 Clear and colorless solution Slightly clear and colorless solution
7 300 Clear and colorless solution Clear and colorless solution
*The bold font shows the optimized temperature
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Table 4: Optimization of digestion mass of the Phytolacca dodecandra and Croton macrostachyus root (3 h). Reagents and temperature: 8 mL HNO3, 2 mL HCl, 1 mL H2O2 at
240℃ for Phytolacca dodecandra and 5 mL HNO3, 2 mL HCl, 2 mL H2O2 at 300 ℃ for Croton macrostachyus root samples
Appearance of solution
Trial Mass Phytolacca dodecandra Croton macrostachyus No. (g)
1 0.1 Yellowish solution with residue yellow turbid solution
2 0.2 Light Yellow with some residue Yellowish solution with large residue
3 0.3 Slightly clear and colorless solution Deep yellow solution with residue
4 0.4 Clear and colorless solution Slightly clear and colorless solution
5 0.5 Clear and colorless solution Clear and colorless solution
6 0.7 Deep yellow solution with residue Yellowish solution with small residue
*The bold font shows the optimized mass
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Table 5: Optimization of the reagent volumes for the digestion of the Phytolacca dodecandra and Croton macrostachyus root (0.5 g, 270℃ and 3 h)
Volume Appearance of solution (mL)
Trial HNO3 HCl H2O2 Total Phytolacca dodecandra Croton macrostachyus No. volume (mL)
1 3 1 0.5 4.5 Pale yellow solution Yellow solution
2 3 2 1 6 Yellow solution Clear and red solution
3 3 1 2 6 Yellow with some Clear and reddish solution residue
4 4 1 1 6 Deep yellow solution Clear and reddish solution
5 4 0.5 1.5 6 Yellowish solution Clear and yellow solution
6 4 2 2 8 yellow turbid solution Clear and yellow solution
7 5 2.5 0.5 8 Yellowish solution Yellow turbid solution
8 5 2 1.5 8.5 Yellowish solution Clear light yellow solution residue
9 5 2 2 9 Yellowish solution with Clear and colorless small residue solution
10 6 2 1 9 Yellowish solution with Clear and colorless solution small suspension
11 8 2 1 11 Clear and colorless yellow turbid solution solution
*The bold font shows the optimized volume ratio
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3.12. Digestion of Croton Macrostachyus and Phytolacca Dodecandra Root Samples
Plant root samples were digested as per acid digestion method, after optimization with respect to the volumes of HNO3 (69%), HCl (37 %) and H2O2 (30 %) in mixtures, digestion time and digestion temperature. Applying the optimized conditions, 0.5 g each of the powdered root, corresponding to the plant species, was transferred into twelve flasks. Then, 11 and 9 mL of
HNO3: HCl: H2O2 acid mixtures were added in ratio by volume of 8:2:1 for Phytolacca dodecandra and 5:2:2 for Croton macrostachyus samples respectively. The samples were digested for 3 h at 240℃ for Phytolacca dodecandra and 300℃ for Croton macrostachyus placed on a hot plate. Digestion was done triplicate. To cool the solutions, 2 to 5 mL portions of distilled water were added and gently swirled to reduce dissolution of the filter paper by digest residue. The cooled digested samples were filtered into a 100 mL standard volumetric flask with a Whatman No.42 filter paper to remove any suspended or turbid matter. Subsequent rinsing of the digestion flask with 5 mL distilled water was followed until the volume reached the mark. To each sample 1% matrix modifier lanthanum chloride hydrate was added so that lanthanum may bind the phosphate and liberate calcium and magnesium in case large phosphates exist in the +2 sample. The main reason why LaCl3.7H2O was added was to prevent the precipitation of Ca +2 -2 -3 and Mg with the SO4 and PO4 if they were present in the samples or the reagents used in the process.
Each site samples were digested and determined separately and their mean value was taken. The digested samples were kept in the refrigerator, and then the total amount of metal in the sample solutions were determined by FAAS. Each site samples were digested and determined separately and their mean value was taken. The digested samples were kept in the refrigerator, and then the total amount of metal in the sample solutions were determined by FAAS. The reagent blank (only reagent but not the addition of sample) were digested by the same amount of volume reagent, the same amount of temperature and time with the sample digestion. Three different blank reagents were prepared. This reagent blank was used to calibrate the instrument.
3.13. Metal Levels Analysis of Traditional Medicinal plant Root Samples
For the analysis of the samples calibration of the instrument with the known concentration of standards were done for each metal of interest. First the intermediate (100 mg/L in 100 ml
34 volumetric flask) standard solutions were prepared from the stock solutions which were 1000 mg/L in concentration. From the intermediate solutions five working standards were prepared by serial dilution for each metal of interest (C1V1= C2V2). The working standards were prepared based on the sensitivity of the instrument towards the particular metals and the level of metals to be present in the samples. All the standards used were AAS grade. Each of the sets of working standards was then aspirated one after the other by their increasing order of concentration into the atomic absorption spectrometry and their absorbance was recorded. Calibration curves were plotted by five points for each of the metal’s standard using absorption/concentration mode, which is then used to determine the analyte concentrations and the calibration curves were given (Figure 17 annex 2).
3.14. FAAS Instrumentation
Many analytical methods including atomic absorption spectrometry for elemental analysis in plant materials required the digestion of the sample. Because of its sensitivity, specificity, simplicity and precision, atomic absorption spectrometry is the most widely recommended instrument utilized in analytical procedure for metal determination (Polkowska-Motrenko et al. 2000). In order to separate the analyte from the sample matrix and avoid organic matter which may react with the metal ions or chemical reagents and interference with the analyte the acid digestion method is a very important step.
In the present study, seven metals were analyzed using flame atomic absorption spectrophotometer by adjusting slit width, lump current, energy and wavelength. Triplicate determinations were carried out on each sample. The concentration of metals in the digested root samples were determined by using flame atomic absorption spectrophotometer (FAAS) equipped with a deuterium arc background corrector and air-acetylene flame at different operating conditions used for FAAS for each of the elements (Table 6).
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Table 6: Instrument operating conditions used for the determination of metals using flame atomic absorption spectrophotometer
Metal λ (nm) SW (nm) I (mA) Energy (J) Flame type
Cu 324.7 0.7 3.5 3.775 Air-acetylene
Zn 213.9 0.7 2.0 3.059 Air-acetylene
Mn 279.5 0.7 3.0 4.054 Air-acetylene
Fe 248.3 0.2 7.0 3.078 Air-acetylene
Cr 357.9 0.7 2.0 2.861 Air-acetylene
Ca 422.7 0.7 2.0 3.885 Air-acetylene
Mg 285.2 0.7 1.0 3.803 Air-acetylene
* λ = wave length, SW = slit width, I = lamp current
3.15. Instrument Calibration
The atomic absorption spectrometer was calibrated using five series of working standards. The working standards of each metal were prepared by diluting the intermediate standard solutions. The solution thus prepared was used to calibrate the instrument before determining the metal concentrations in the sample. Metal standard solutions for calibration were prepared from an intermediate standard solution containing 100 mg/L which was prepared from the atomic absorption spectroscopy standard stock solutions that contained 1000 mg/L for each of the metals. All the metals included in this study were analyzed with FAAS after the instrument was calibrated using five series of working standards. Three replicate determinations were carried out for each metal and the same analytical procedure was employed for the determination of the elements in blank solutions (Gebre and Singh 2012). The correlation coefficients (R2) of the calibration curves were greater than 0.994 which confirmed a very good positive correlation between the change in absorbance and the concentration and were linearly fit (Abebe and
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Chandravanshi 2017). Concentration of working standards and values of correlation coefficient of the calibration graph for each metal are listed in Table 7.
Table 7: Working standard concentration, correlation coefficient and equation of the calibration curves for determination of metals using FAAS for both Croton macrostachyus and Phytolacca dodecandra root samples
Metal Concentration of working standard Correlation Calibration equation (mg/L) coefficient (R2)
Cu 0.5, 1.5, 2.5, 3.5, 4.5 0.999 A= 0.0443C - 0.0086
Zn 0.5, 1.5, 2.5, 3.5, 4.5 0.997 A= 0.095C + 0.0191
Mn 0.5, 1.5, 2.5, 3.5, 4.5 0.999 A= 0.0221C + 0.0091
Fe 15, 20, 25, 30, 35 0.997 A= 0.0011C + 0.003
Cr 0.5, 1.5, 2.5, 3.5, 4.5 0.994 A= 0.0023C - 0.0003
Ca 2, 4, 6, 8, 10 0.999 A= 0.0028C - 0.0017
Mg 2.5, 3.5, 4.5, 5.5, 6.5 0.996 A= 0.1331C + 0.5067
* A=Absorbance C=Concentration
Directly after calibration using the standard solutions, the sample solutions were articulated into the AAS instrument and through readings of the metal absorbance was recorded. The same analytical procedure was done for the determination of elements in digested blank solutions.
3.16. Method Performance and Validation
Method Validation
Method detection limit (MDL) is defined as the minimum concentration of analyte that can be measured and reported with 99% confidence that the analyte concentration is greater than zero. In other words, it is the lowest analyte concentration that can be distinguished from statistical fluctuations in a blank, which usually correspond to three times the standard deviation of the
37 blank (3*δ blank divided by the slope, where δ = standard deviation of the blanks) (Dubale et al. 2015).
Accuracy, precision, sensitivity and limits of detection were determined to assess the validity of the methods used for the digestion and analysis of the root samples. The precision of the method was evaluated from the relative standard deviation of the results obtained from repeated measurements made on a given sample for an element while the accuracy was determined by spiking the samples with known concentrations of standard solutions. The limit of detection of the method was calculated as the average blank signal plus three times its standard deviation divided by the slope of the calibration equation. Method validation was determined from limit of detection (LOD) and limit of quantification (LOQ) (Malede et al. 2019).
Limit of Detection (LOD) and Limit of Quantification (LOQ)
Limit of detection is the minimum amount of concentration that can be detected or the lowest concentration that gives an instrument signal significantly different from the blank (Ziegel 2004). LOD was determined three times of the standard deviation of the blank per slope of the calibration graph. Whereas the limit of quantification is the lowest concentration of analyte that can be determined quantitatively (Ziegel 2004). LOQ was determined ten times the standard deviation of the blank per slope of the calibration graph.
Precision and Accuracy
The precision of a method is a measure of closeness or agreement of individual measurements. Precision is commonly divided into two categories; repeatability and reproducibility. Repeatability is the precision obtained when all measurements are made by the same analyst during a single period of laboratory work, using the same solutions and equipment. Reproducibility, on the other hand, is the precision obtained under any other set of conditions, including that between analysts, or between laboratory sessions for a single analyst. Precision characterizes the random component of the measurement error and, therefore, it does not relate to the true value (Harvey 2004). It can be expressed in terms of standard deviation (SD) or percent standard deviation (%RSD).
The reproducibility of the analytical procedure was checked by carrying out a triplicate analysis and calculating the relative standard deviations for each metal. The % RSD results did not differ
38 by more than 10% of the mean which indicated that the analytical method used is precise and reliable (Gebre and Singh 2012). But accuracy is a measure of how close a measure of central tendency is to the true or expected value. Accuracy is usually expressed as either an absolute error or a percent relative error. Accuracy is a measure that combines the effects of both random and systematic deviations (Harvey 2004).
Instrumental Detection Limit (IDL)
Instrumental detection limit (IDL) is the smallest signal above background noise that an instrument can detect reliably (Addis and Abebaw 2018).
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4. RESULTS AND DISCUSSION
4.1. Recovery
Method validation is the process of providing that analytical method is acceptable for its intended purpose. Because of the absence of certified reference material for the samples in the laboratory, the validity of the optimized digestion procedure was assured by spiking the samples with a standard of known concentration of the analyte metals. Thus, the efficiency of the optimized procedure was checked. The spiked samples were digested in triplicate following the same digestion procedure developed previously for plant samples. The digested spiked samples were analyzed for their respective metals using FAAS. Finally, % recovery was determined (Hailemariam and Bibiso 2019).
To validate the accuracy of the optimized procedure, spiking experiments were used to study recoveries. This was done by adding a known concentration (1000 mg/L) standard solution of each metal after determining the concentration of metals in each sample. The spiking was done in 100 mL borosilicate flasks of each sample. All the spiked and unspiked samples were digested succeeding the procedure designated prior in triplicates. Individual samples were investigated for their metallic contents. The acceptable ranges of percentage recovery for the studied metals will be within 85 to 105%, for metal analysis (Atinafu et al. 2015).
The information shown in Tables 8 and 9 clearly indicated that the recoveries are in the range between 85.2 to 103.1%. These results established that the optimized digestion procedure was valid for both Croton macrostachyus and Phytolacca dodecandra sample analysis. This confirms that the method is of good accuracy.
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Table 8: Recovery results (Mean ± SD, n = 3) for Croton macrostachyus (Bisana) root samples
Metals Amount in the Amount added Amount found Recovery (%) sample (mg/kg) (mg/kg) (mg/kg)
Cu 80 ± 4 8 87 ± 6 87.5 ± 1.2
Zn 81 ± 5 41 123 ± 14 102.4 ± 8.2
Mn 91 ± 1 45 135 ± 16 97.8 ± 1.3
Fe 848 ± 52 424 1277 ± 28 101.2 ± 5.8
Cr 45 ± 2 23 65 ± 4 87.0 ± 0.9
Ca 992 ± 62 496 1460 ± 31 94.4 ± 5.1
Mg 800 ± 48 402 1201 ± 15 99.8 ± 4.1
Table 9: Recovery results (Mean ± SD, n = 3) for Phytolacca dodecandra (Endod) root samples
Metals Amount in the Amount added Amount found Recovery (%) sample (mg/kg) (mg/kg) (mg/kg)
Cu 71 ± 3 36 102 ± 11 86.1 ± 2.7
Zn 141 ± 8 71 211 ± 10 98.6 ± 4.1
Mn 63 ± 3 32 96 ± 12 103.1 ± 5.2
Fe 788 ± 45 394 1172 ± 116 97.5 ± 1.7
Cr 48 ± 3 27 71 ± 5 85.2 ± 2.9
Ca 871 ± 51 436 1266 ± 53 90.6 ± 3.1
Mg 818 ± 12 409 1197 ± 113 92.7 ± 8.2
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4.2. Optimal Conditions for Sample Digestion
In the present study, to make a clear and colorless sample solution that is appropriate for the examination of metals using FAAS, different Phytolacca dodecandra and Croton macrostachyus digestion procedures were optimized using nitric acid (HNO3), hydrochloric acid (HCl) and hydrogen peroxide (H2O2) mixtures by altering parameters such as volume of the acid mixture, digestion time, sample mass and digestion temperature. From the optimized procedures, the acid mixture of HNO3 (69%), HCl (37%) and H2O2 (30%) in the ratio 8:1:2 (v/v) with a digestion time of 3 h at a digestion temperature of 240 °C were found to be the optimal conditions for 0.5 g
Phytolacca dodecandra sample; and the acid mixture of HNO3 (69%), HCl (37%) and H2O2 (30%) in the ratio 5:2:2 (v/v) with a digestion time of 3 h at a digestion temperature of 300 °C were found to be the optimal conditions for 0.5 g Croton macrostachyus sample. These optimal conditions were selected based on clarity of digests, minimum reagent volume consumption, minimum digestion time, simplicity, and minimum temperature applied for complete digestion of sample (Tables 2 to 5).
4.3. Method performance and method validation
Instrument and method performance parameters were anticipated for precision, accuracy, linearity, detection limits, and LOQ. The average percentage recoveries of metals in the spiked Phytolacca dodecandra sample were found between 85.2% to 103.1% and 87.0 to 102.4% for Croton macrostachyus samples. Both the recovery values were within the tolerable range of 85% to 105% for metal determination (Tables 8 and 9). The %RSD values obtained for Croton macrostachyus and Phytolacca dodecandra samples ranged from 5.0 to 11.1% and 5.7 to 12.5%, respectively, which was under the dynamic control limits ≤ 10%. These confirm that the method was of good precision and accuracy (Table 11). The determination coefficients (R2) of the calibration curves were greater than 0.996 (except R2 = 0.994 for Cr) which indicated a very good coefficient of determination (Table 7). Moreover, to determine the method detection limit of each metal, three blanks for each sample were digested and analyzed along with Croton macrostachyus and Phytolacca dodecandra samples. Then, the mean concentration of the blank and the standard deviation of the three blank samples were calculated for each metal. Finally, the detection limits were obtained by mean concentration of the blank plus three times of the standard deviation of the reagent blank. As can be seen in Table 10, the method detection limit
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(mg/kg) for each metal under investigation was higher than the corresponding instrument detection limit.
Table 10: Instrumental detection limit (IDL), limits of detection (LOD) and limit of quantification (LOQ) for the determination of metals in the roots of Phytolacca dodecandra and Croton macrostachyus samples using flame atomic absorption spectrophotometer (FAAS)
Croton Phytolacca macrostachyus dodecandra
Metal IDL (mg/L) LOD (mg/kg) LOQ(mg/kg) LOD (mg/kg) LOQ (mg/kg)
Cu 0.014 0.4 1.4 1.2 4.0
Zn 0.005 1.9 6.4 9.2 30.8
Mn 0.004 33.3 111.0 18.7 62.2
Fe 0.012 40.6 135.5 27.6 92.1
Cr 0.001 0.4 1.2 0.04 0.1
Ca 0.003 33.0 110.1 24.2 80.6
Mg 0.002 51.6 172.1 36.0 120.0
*IDL=Instrumental detection limit, LOD=Limit of detection, LOQ=Limit of quantification
Generally, it was found that: LOQ > LOD > IDL, this shows the method was proved and tolerable. The spiking experiments showed good accuracy and repeatability of the method for the analysis of all metals investigated.
4.4. Concentration of Metals in Croton Macrostachyus and Phytolacca Dodecandra
The levels of macro– (Ca and Mg) and trace essential metals (Cu, Zn, Mn, Fe and Cr) were determined by FAAS. All the metals mentioned above were detected in both the Croton macrostachyus (Bisana) and Phytolacca dodecandra (Endod) root samples; and the results obtained for the mean concentrations of metals in both root samples are presented in Table 11.
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Table 11: Average concentration (mg/kg) (Mean ± SD, n=3) of metals in selected medicinal plant root samples Croton Phytolacca dodecandra Macrostachyus
Metals Mean ± SD %RSD Mean ± SD %RSD WHO/FAO safe limit (mg/kg)
Cu 9 ± 1 11.11 8 ± 1 12.5 10
Zn 40 ± 4 10.00 38 ± 3 7.89 100
Mn 157 ± 10 6.37 127 ± 9 6.30 200
Fe 848 ± 52 6.13 788 ± 45 5.71 20
Cr 2 ± 0.1 5.00 1 ± 0.1 10.00 2
Ca 919 ± 62 6.75 871 ± 51 5.86 -
Mg 1217 ± 97 7.97 1204 ± 89 7.39 850
*%RSD=Relative standard deviation, SD= standard deviation
In both plant root samples analysed, highest amounts of Mg followed by Ca and Fe were observed, reflecting their expected abundance in the soils and vital role for plant growth and development. For instance, Mg is a constituent of every chlorophyll molecule, while Ca is an important component of plant cell walls (Adeyeye 2005). In contrast, the concentration of Cr was found to be the lowest. Significant differences (P<0.05) were not observed in the mean concentrations of the seven metals between the two plant root samples.
The results of Mg concentrations (mg/kg) in this study were 1217 and 1204 in Croton macrostachyus and Phytolacca dodecandra root samples, respectively. The maximum FAO/WHO safe limit for Mg concentration in medicinal plant samples is 850 mg/kg. The results obtained for both plant root samples were higher than the recommended limit. Magnesium is essential to cell, e.g. major action in biological compounds such as ATP and DNA (Latif
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Mohammed Raouf et al. 2014). Magnesium is critical to many cell functions. It assists in the operation of more than 300 enzymes, and is required for the release and use of energy from the energy-yielding nutrients, and directly affects the metabolism of potassium, calcium and vitamin D. Magnesium acts in the cells of all the soft tissues, where it is part of the protein-making machinery and is necessary for the release of energy (Dubale et al. 2015).
The concentrations of Ca were 919 and 871 mg/kg in Croton macrostachyus and Phytolacca dodecandra root samples, respectively. Calcium is a major essential metal for both plants and animals. The higher concentrations of Ca are very significant because Ca is known to enhance the qualities of bones and teeth and also of neuromuscular systemic and cardiac functions (Dubale et al. 2015).
The mean Fe concentrations (mg/kg) were found to be 848 and 788 in Croton macrostachyus and Phytolacca dodecandra root samples, respectively. The FAO/WHO recommended limit for Fe concentration in medicinal plants is 20 mg/kg. The results obtained in the present study were beyond the recommended limit. Iron is the most abundant and an essential trace metal for all plants and animals. Iron has several key functions in the human body including oxygen supply, energy production, and immunity. However, Fe overdose is associated with symptoms of dizziness, nausea and vomiting, diarrhea, joint pain, shock, tissue damage and liver damage (Dghaim et al. 2015). Fe was found to have the highest concentration among essential trace elements in all the samples analyzed. The high concentrations of Fe in the roots of plants may be due to higher absorption capacity of plant’s roots or the presence of higher amounts of iron in the respective soil.
With respect to Cu, Croton macrostachyus and Phytolacca dodecandra root samples were found to contain 9 and 8 mg/kg, respectively. The maximum FAO/WHO safe limit for Cu levels in medicinal plant samples is 10 mg/kg. The results obtained in this study were lower than the recommended limit. Copper is an essential component of many enzymes, thereby playing a significant role in a wide range of physiological processes, including iron utilization, free radicals’ elimination, bone and connective tissues development, melanin production, and many others. Nevertheless, excessive intake of copper can cause dermatitis, irritation of the upper respiratory tract, abdominal pain, nausea, diarrhea, vomiting, and liver damage (Dghaim et al. 2015).
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The average Zn concentrations (mg/kg) in this study were 40 and 38 in Croton macrostachyus and Phytolacca dodecandra root samples, respectively. The maximum FAO/WHO safe limit for Zn concentration in medicinal plant samples is 100 mg/kg. The results obtained in this study for both Croton macrostachyus and Phytolacca dodecandra samples were lower than the recommended limit. Zinc is an essential trace element that functions as a cofactor for certain enzymes involved in metabolism and cell growth. Zn is involved in the metabolism of proteins, carbohydrates, lipids, and energy. It plays an essential role in numerous biochemical pathways. Zn plays a crucial role in growth and cell division where it is required for protein and DNA synthesis, in insulin activity, in the metabolism of the ovaries and testes, and in liver function. Zinc is found in several enzymes and genetic material transcription (Fartusie and Mohssan 2017). Zinc is an essential element that plays an important role in growth, and has a recognized action in more than 300 enzymes by participating in their structure or their catalytic and regulatory action. Zinc deficiency causes growth retardation and hypogonadism (Nkansah et al. 2016). Zinc deficiency may also affect the bone metabolism and gonadal function.
The mean Mn concentrations (mg/kg) in this study were 157 and 127 in Croton macrostachyus and Phytolacca dodecandra root samples, respectively. These results are all below the maximum FAO/WHO safe limit for Mn (200 mg/kg) in medicinal plant samples. Manganese is an essential element required for various biochemical processes. Mn is essential for the normal bone structure, reproduction and normal functioning of the central nervous system. Its deficiency causes reproductive failure in both male and females (Bedassa and Desalegn 2017).
Finally, the concentrations of Cr (mg/kg) in Croton macrostachyus and Phytolacca dodecandra root samples were 2 and 1, respectively, which did not surpass the FAO/WHO recommended limit of 2 mg/kg in medicinal plants. Cr (III) is an essential element required for normal sugar and fat metabolism. It is effective in the management of diabetes and it is a cofactor with insulin. Cr (III) and its compounds are not considered a health hazard; while the toxicity and carcinogenic properties of Cr (VI) have been documented for a long time (Wodaje 2015). Its deficiency causes hyperglycemia, elevated body fat, and decreased sperm count; in contrast, it is toxic and carcinogenic at high concentration (Shah et al. 2013).
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4.5. Distribution Pattern of the Metals in the Traditional Medicinal Plant Root Samples
The concentrations of seven metals (Cu, Zn, Mn, Fe, Cr, Ca and Mg) were determined in both plant roots from six different sampling areas by flame atomic absorption spectrophotometer. All the Croton macrostachyus and Phytolacca dodecandra root samples contained higher amounts of Mg, Ca and Fe. The observed higher levels of Mg and Ca could be linked to the fact that nutrient elements such as N, P, K, S, Ca and Mg are highly mobile in the plant tissue and translocated from old plant tissue to new plant tissue. The other probable reason is the application of highly fertilized manure and organic residues in the soil on which both plants are grown (Tilahun and Zemene 2015). Similarly, the high concentration of Fe from trace metals in Croton macrostachyus and Phytolacca dodecandra might be due to the fact that these metals are readily transferred from the soil to plants, and accumulate in plants (Tegegne et al. 2017).
The concentrations of the studied macro- and trace-essential metals could be arranged according to their concentrations (mg/kg) in the Croton macrostachyus and Phytolacca dodecandra samples from all the sampling sites in the following order: Cu (5.0 to 12.0), Zn (17.0 to 196.0), Mn (62.0 to 479.0), Fe (182.0 to 1455.0), Cr (0.1 to 3.0), Ca (550.0 to 1407.0) and Mg (1019.0 to 1318.0) (Table 17 Annex 1).
In summary, we can conveniently say that the concentration pattern of metals in both Croton macrostachyus and Phytolacca dodecandra root samples decreased as: Mg > Ca > Fe > Mn > Zn > Cu > Cr.
47
1400 a Croton macrostachyus
1200 Phytolacca dodecandra
1000
800
600
Concentrattion (mg/kg) Concentrattion 400
200
0 Mn Fe Ca Mg Metals
50.0 b 45.0 Croton macrostachyus 40.0 Phytolacca dodecandra
35.0 30.0 25.0 20.0 15.0
10.0 Concentrattion (mg/kg) Concentrattion 5.0 0.0 Cu Zn Cr Metals
Figure 5(a-b): Distribution pattern of metals in Croton macrostachyus and Phytolacca dodecandra root samples
48
4.6. Comparison with Literature Values
Although there were no previous studies on the determination of metals in the root of Croton macrostachyus and Phytolacca dodecandra, the levels of metals in the present study were compared with their corresponding metal concentrations in the leaves of Croton macrostachyus and Phytolacca dodecandra samples.
Most of the values reported in the literature were lower than the observed values in the present study. For instance, a study conducted on the leaves of Croton macrostachyus, collected from Kenya, showed lower concentrations (mg/kg) of Ca (3.45), Mg (77.17), Fe (2.57), Mn (657.80), Zn (0.90), Cu (0.16) and Cr (0.59) than the present study, except for Mn (Adongo, 2013). In contrast, the leaves of Croton macrostachyus collected from four different regions of Ethiopia (viz., Akaki, Abomsa, Bonga and Dilla) were found to contain higher concentrations (mg/kg) of Ca (8510.25), Mg (2919.30), Fe (342.80), Mn (657.80), Zn (37.10), Cu (10.20) and Cr (6.30) than the present study, except for Fe and Zn (Dubale et al. 2015). Similar study revealed higher contents (mg/kg) of Fe (235), Mn (192), Zn (342) and Cu (6) in the leaves of Phytolacca dodecandra collected from Dembia Woreda than the present study, except Mn and Zn.
4.6. Comparison with Other Medicinal Plants
The results obtained from the analyses of Croton macrostachyus and Phytolacca dodecandra root powders were compared with their corresponding metal levels in root powder of different medicinal plants reported in various literatures. This comparison helps to identify the difference in composition and if there exists a deviation from certain guidelines. Most of the values reported in the literature were comparable with the values in the present study.
The average concentrations (mg/kg) of Cu in Croton macrostachyus and Phytolacca dodecandra root powders were 9 and 8 respectively, which are higher than those recorded from Ethiopia (Hailemariam and Bibiso 2019) and kenya (Adongo 2007), but lower than that reported in India (Sarma et al. 2011) and Pakistan (Shah et al. 2013). On the other hand, the mean concentrations (mg/kg) of Zn were 40 and 38 in Croton macrostachyus and Phytolacca dodecandra root samples, respectively, which were lower than those reported from Pakistan (Shah et al. 2013) and Iraq (Latif Mohammed Raouf et al. 2014), but they were almost comparable with a previous value reported in Ethiopia (Hailemariam and Bibiso 2019).
49
The levels of Mn (mg/kg) in Croton macrostachyus and Phytolacca dodecandra root samples, respectively, obtained in this study were higher than those recorded from India (Sarma et al. 2011) and Ethiopia (Hailemariam and Bibiso 2019), but lower than the values reported in Pakistan (Shah et al. 2013). Similarly, the levels of Fe (mg/kg) in Croton macrostachyus and Phytolacca dodecandra root powders were 848 and 788, respectively, which were higher than those recorded from Ethiopia (Hailemariam and Bibiso 2019) and Kenya (Adongo 2007), but lower than that reported in India (Sarma et al. 2011) and Pakistan (Shah et al. 2013).
The concentrations (mg/kg) of Cr were 2 and 1 in Croton macrostachyus and Phytolacca dodecandra root samples, respectively, which were lower than those recorded from India (Sarma et al. 2011), Pakistan (Shah et al. 2013) and Iraq (Latif Mohammed Raouf et al. 2014), but they were comparable with those recorded in Kenya (Adongo 2007).
The mean concentrations (mg/kg) of Ca in Croton macrostachyus and Phytolacca dodecandra root powders were 910 and 871, respectively, which were higher than those recorded from Kenya (Adongo 2007) and Ethiopia (Hailemariam and Bibiso 2019), but almost comparable with that was recorded in Iraq (Latif Mohammed Raouf et al. 2014).
The levels of Mg (mg/kg) in Croton macrostachyus and Phytolacca dodecandra root powders were 1217 and 1204, respectively, which were higher than those recorded from Kenya (Adongo 2007) and Ethiopia (Hailemariam and Bibiso 2019), but lower than that reported in Iraq (Latif Mohammed Raouf et al. 2014).
To summarize, comparison of concentrations of metals in Croton macrostachyus and Phytolacca dodecandra root with other medicinal plants reported in various parts of the world were summarized in Table 12. The levels of all metals in this study were higher than most of the reported values, except for Cr and Zn, which were comparable with the literature values.
50
Table 12 : Comparison of metal concentration (mg/kg) in Croton macrostachyus and Phytolacca dodecandra root with other medicinal plant root in worldwide
concentration (mg/kg) Plant species Country Cu Zn Mn Fe References Amacylus pyrethrom India 14 39 37 1260 (Sarma et al. 2011) Bergemia liquilata India 1 12 37 27 (Sarma et al. 2011) (Hailemariam and Echinops kebericho Ethiopia 3 1 1 0.1 Bibiso 2019) Capparisspinosa Pakistan 5 11 23 39 (Shah et al. 2013) Tamarix articulata Pakistan 9510 32580 15240 72870 (Shah et al. 2013) (Latif Mohammed Raouf et al. Rosmarinusofficinalis Iraq NR 1920 5550 98000 2014) Carissa edulis Kenya 0.07 9 NR 3 (Adongo 2007) Solonum incanum Kenya 0.02 2 NR 3 (Adongo 2007) Croton macrostachyus Ethiopia 9 40 157 848 This study Phytolacca dodecandra Ethiopia 8 38 127 788 This study *NR is not reported
concentration (mg/kg) Plant species Country Cr Ca Mg References Amacylus pyrethrom India 3 NR NR (Sarma et al. 2011) Bergemia liquilata India 334 NR NR (Sarma et al. 2011) Echinopskebericho Ethiopia NR 2 1 (Hailemariam and Bibiso 2019) Capparisspinosa Pakistan 3 NR NR (Shah et al. 2013) Tamarix articulata Pakistan 783 NR NR (Shah et al. 2013) Rosmarinusofficinalis Iraq 240 638 8495 (Latif Mohammed Raouf et al. 2014) Carissa edulis Kenya 1 8 13 (Adongo 2007) Solonum incanum Kenya 1 2 6 (Adongo 2007) Croton macrostachyus Ethiopia 2 919 1204 This study Phytolacca dodecandra Ethiopia 1 871 1127 This study
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4.7. Determination of Total polyphenolic Content, Flavonoid and Antioxidant activities
4.7.1. Optimization of Extraction Parameters for polyphenolic Content Determination
Among the experimental parameters, optimal conditions for the extraction of phenolic contents in the Croton macrostachyus and Phytolacca dodecandra mostly depend on solvent composition and extraction time to achieve the best overall optimization conditions of the extraction parameters. This is because previous studies showed that both factors have significant effect on the extraction of different plant samples (Spigno et al. 2007). The extraction procedure was established to get the optimum desirable maximum TPP yield.
Effect of Solvent Composition and Extraction Time
The extraction of TPP is influenced by the interaction of functional groups in plant materials and the composition of the solvent used (MeOH:H2O). The present study depicted that the yield of the TPP increased with increasing the composition of methanol as shown in Figure 6a & b. But, at higher methanol level (>80%), the extraction efficiency of TPP gradually decreased.
1800 a
1600
1400
1200
1000 12 h 18 h 800 24 h 600 30 h 400
200 Polyphenolic Polyphenolic content (mg GAE/100 g) 0 0 20 40 60 80 100 120 MeOH:H O (%v/v) 2
52
4500 b
4000
3500
3000 12 h 2500 18 h 2000 24 h 1500 30 h 1000
500 Polyphenolic Polyphenolic content (mg GAE/100 g) 0 0 20 40 60 80 100 120 MeOH:H O (%v/v) 2
Figure 6: MeOH:H2O (%v/v) and extraction time (h) on the extraction efficiency of TPP from (6a) Croton macrostachyus and (6b) Phytolacca dodecandra: mass of plant root sample (0.5 g); volume of solvent (25 mL; MeOH:H2O); and at room temperature
Experimental optimization on TPP extraction efficiency was also conducted as a function of extraction time, and the result is shown in Figure 6a & b. In this study, the extraction efficiency of TPP increases with increasing time and it started to level off as extraction time increases beyond the equilibrium time. This is because the extent of extraction increased rapidly at the initial stages due to the presence of large amount of TPP in the plant root samples. But after a certain time, most TPPs were exhaustively extracted and a negligible amount would be left in the plant root samples. So, the maximum extraction efficiency achieved the equilibrium point at 24 h. This finding is in agreement with other studies (Seifu et al. 2017).
4.7.2. Total Polyphenolic Content
In the present study, total phenolic content was determined spectrophotometrically according to the Folin-Ciocalteu method and calculated as gallic acid equivalent (GAE). Gallic acid was taken as a standard because it is the most known synthetic organic compound possessing maximum amount of total phenol content and natural total phenol content from plant extracts evaluated using it as standard and Folin-Ciocalteu reagent. The Folin-Ciocalteu reagent is sensitive to
53 reduce compounds including polyphenols, thereby producing a blue color upon reaction. This blue color is measured spectrophotometrically and the total phenolic contents can be determined (Mongkolsilp et al. 2004).
The total phenolic contents were determined by plotting a standard calibration curve of different concentrations of gallic acid by spectrophotometer at 760 nm. The standard gallic acid calibration curve (R2 = 0.998) (Figure 15 annex 2) was used to calculate the total phenolic contents of the Croton macrostachyus and Phytolacca dodecandra plant extracts. The values of TPP were calculated as gallic acid equivalents (GAE) per gram of sample.
It has been well recognized that the polyphenol content of plant materials is strongly associated with their antioxidant capacity. Phenolics are the principal plant compounds with antioxidant capacity attributable to their redox properties, which exhibit an important function in neutralizing free radicals (Haile et al. 2016). The total polyphenolic content varied in the range of 802 ± 53 to 1557 ± 75 mg GAE/100 g (Croton macrostachyus) and 950 ± 38 to 4214 ± 45 mg GAE/100 g (Phytolacca dodecandra) of sample across the different sampling sites (Tables 19 & 20 annex 1). The mean polyphenolic contents of Croton macrostachyus and Phytolacca dodecandra root samples were 2402.50 ± 80 and 1104.86 ± 204 mg GAE/100 g of sample, respectively. The Croton macrostachyus samples from Teber Serako and Bezaho Mekenet had higher total polyphenolic contents, while lower values were recorded for samples collected from Laza Buladigie. With regards to Phytolacca dodecandra, root samples from Nara Awurarda and Bezaho Mekenet exhibited higher total polyphenolic contents, followed by Laza Buladigie. On the contrary, lower values were recorded for samples collected from the remaining three sampling sites (Figure 7).
54
5000
4500 Croton macrostachyus 4000 Phytolacca dodecandra 3500 3000 2500 2000 1500 1000
500 TPP (mg GAE/100 g) sample g) GAE/100 (mg TPP 0 Eyaho Teber Aykel Town Laza Bezaho Nara seraba serako Buladgie Mekenet awudarda Sampling site
Figure 7: Comparison of total polyphenolic content between Croton macrostachyus and Phytolacca dodecandra
Generally, as shown above in Figure 7, the total polyphenolic content distribution pattern was in the order of: Teber Serako > Bezaho Mekenet > Nara Awurarda > Aykel Town > Eyaho Seraba > Laza Buladigie (Croton macrostachyus); and Nara Awurarda > Bezaho Mekenet > Laza Buladigie > Aykel Town > Eyaho Seraba > Teber Serako (Phytolacca dodecandra).
4.7.3. Flavonoid Content Determination
The flavonoid content was determined by plotting standard calibration curve of different concentration of catechin by spectrophotometer at 510 nm (Haile et al. 2016). The standard catechin curve and regression equation (R2 = 0.9987) (Figure 16 annex 2) was used to calculate the total flavonoid contents of the Croton macrostachyus and Phytolacca dodecandra plant root extracts. The values of flavonoid contents were calculated as catechin equivalents (CE) per gram of sample based on triplicate measurements.
In the present study, the flavonoid contents of the Croton macrostachyus and Phytolacca dodecandra samples were given as milligram catechin equivalent per 100 g of sample (mg CE/100 g sample). The concentrations of flavonoids in the Croton macrostachyus and
55
Phytolacca dodecandra were found in the range of 342 ± 26 to 745 ± 32 and 463 ± 13 to 873 ± 19 mg CE/100 g sample, respectively (Tables 19 & 20 annex 1).
Among samples of the Croton macrostachyus grown in the different areas, the highest concentration of flavonoids was found in Teber Serako, whereas the lowest was found at Laza Buladigie. The other sites had relatively similar flavnoid concentrations.
1000
Croton macrostachyus
900 Phytolacca dodecandra 800 700 600 500 400 300 200 100
Flavonoid content (mg CE/100sample) g (mg Flavonoid content 0 Eyaho Seraba Teber Serako Aykel Town Laza Bezaho Nara Buladigie Mekenet Awurarda Sampling site
Figure 8: Comparison of flavonoid content between Croton macrostachyus and Phytolacca dodecandra
When it comes to flavonoid contents in Phytolacca dodecandra root samples, relatively higher values were observed for Bezaho Mekenet and Laza Buladigie samples while relatively lower and more or less similar values recorded for samples from the remaining sampling sites (Figure 8). It was also noted that samples with highest polyphenolic contents in the plant root extracts had also highest flavonoid concentration, and so did the same for samples with lowest total polyphenolic contents (Tables 19 & 20 annex 1).
56
5000 4500 Flavonoid(mg CE/100 g) 4000 TPP(mg GAE/100 g) 3500 3000 2500 2000
Concentration 1500 1000 500
0
Aykel Town Aykel Town Aykel
Teber Serako Teber Serako Teber
Eyaho Seraba Eyaho Seraba Eyaho
Laza Buladigie Laza Laza Buladigie Laza
Nara Awurarda Nara Awurarda Nara
Bezaho Mekenet Bezaho Mekenet Bezaho Croton macrostachyus Phytolacca dodecandra
Sampling site and type
Figure 9 : Comparison of total polyphenols and flavonoid between Croton macrostachyus and Phytolacca dodecandra in different sampling sites
Generally, as shown above in Figure 9, the flavonoid content distribution pattern was Teber Serako > Bezaho Mekenet > Eyaho Seraba Nara Awurarda > Aykel Town > Laza Buladigie (Croton macrostachyus) and Bezaho Mekenet > Laza Buladigie > Teber Serako = Eyaho Seraba > Aykel Town > Nara Awurarda (Phytolacca dodecandra).
4.7.4. Antioxidant Activity Determination
Antioxidant activity of plants is mainly attributed to the presence of active compounds in them. This can be not only due to the high percentage of main constituents, but also the presence of other constituents in small quantities (Haile et al. 2016). In the present study, the antioxidant activities of Croton macrostachyus and Phytolacca dodecandra were determined by the method of DPPH radical scavenging assay. Results were compared with ascorbic acid as a reference
57 antioxidant compound. The values are expressed as milligrams of ascorbic acid equivalents per gram sample (mg AAE/g).
Figure 10 : Standard curve of ascorbic acid
From the graph given above (Figure 10), it can be inferred that 7.5 mg/L of standard ascorbic acid solution was required to attain 50% inhibition concentration (IC50), which was later used as a base to calculate the concentrations of both plant root extracts. Accordingly, the DPPH reagent scavenged 50% of the total polyphenols in the root extracts from all the sampling sites and achieved IC50 values in the ranges of 3.5 – 6.4 mg AAE/g (Figure 11; Croton macrostachyus) and 3.8 – 14.3 mg AAE/g (Figure 12; Phytolacca dodecandra).
58
100.0 90.0
80.0 70.0 Croton_2 60.0 Croton_4 50.0 Croton_1 40.0 30.0 Croton_3 20.0 Croton_5 10.0 Croton_6
0.0 % Inhibition Inhibition concentration % 0 5000 10000 15000 20000 25000 Concentration of sample (mg/L)
Figure 11: Inhibition of methanol water (80:20 (v/v)) extracts of Croton macrostachyus (Bisana)
100.0
90.0 80.0 70.0 Phytolacca_2 60.0 Phytolacca_6 50.0 Phytolacca_1 40.0 Phytolacca_3 30.0 Phytolacca_4 20.0
% Inhibition Inhibition concentration % Phytolacca_5 10.0 0.0 0 5000 10000 15000 20000 25000 Concentration of sample (mg/L)
Figure 12: Inhibition of methanol water (80:20 (v/v)) extracts of Phytolacca dodecandra (Endod)
59
18.0
16.0 Croton macrostachyus 14.0 Phytolacca dodecandra 12.0 10.0 8.0 6.0 4.0 2.0
DPPH scavenging activity (mg sample) AAE/g (mg activity scavenging DPPH 0.0 Eyaho Teber Serako Aykel Town Laza Bezaho Nara Seraba Buladigie Mekenet Awurarda Samplig site
Figure 13: Comparison of antioxidant activities between Croton macrostachyus and Phytolacca dodecandra in different sampling sites
Generally, free radical scavenging and antioxidant activity of phenolics (e.g. flavonoids) mainly depends on the number and position of hydrogen-donating hydroxyl groups on the aromatic ring of the phenolic molecules (Haile et al. 2016). As shown in Figure 13, the Phytolacca dodecandra samples had good antioxidant activities, in the range of 3.80 to 14.29 mg AAE/g of sample. These results demonstrated that Phytolacca dodecandra have potentially good antioxidant activity. However, the Croton macrostachyus samples exhibited lower antioxidant activities, ranging from 3.53 to 6.38 mg AAE/g of sample; and DPPH scavenging activities were estimated at 50% inhibition concentration (IC50).
In the present study, the antioxidant activity distribution pattern was Teber Serako > Nara Awurarda > Bezaho Mekenet > Aykel Town > Eyaho Seraba > Laza Buladigie (Croton macrostachyus) and Nara Awurarda > Bezaho Mekenet > Laza Buladigie > Aykel Town > Eyaho Seraba > Teber Serako (Phytolacca dodecandra) (Figure 13)
60
4.8. Comparison of the Present Study with Results from Other Countries
The total polyphenolic content, flavonoid and antioxidant activities of Croton macrostachyus and Phytolacca dodecandra of the present study were compared with literature values of other medicinal plants from different countries.
The mean total polyphenolic contents of Croton macrostachyus (2402.50 ± 0.80 mg GAE/100 g) and Phytolacca dodecandra (1104. 86 ±2.04 mg GAE/100 g sample), root samples obtained in the present study were lower than other medicinal plants recorded in Indonesia (Sembiring et al. 2018), Serbia (Stankovic 2011) and Italy (Phuyal et al. 2020), but were higher than those recorded for Ethiopia (Mekonnen et al. 2018), Sri Lanka (Wanigasekera et al. 2019) and India (Kumari and Sharma 2017) as shown below in Table 13.
In the present study, concentrations of flavonoid in the Croton macrostachyus and Phytolacca dodecandra root samples were 502.83 ± 23.43 and 589.227 ±18.68 mg CE/100 g sample, respectively, which were higher than those reported in Serbia (Stankovic 2011), India (Kumari and Sharma 2017), Sri Lanka (Wanigasekera et al. 2019), and other regions of Ethiopia (Mekonnen et al. 2018). However, they were lower than those reported in Italy (Phuyal et al. 2020), Pakistan (Saeed et al. 2012), Indonesia (Sembiring et al. 2018) (Table 13).
Finally, the mean antioxidant activities of Croton macrostachyus (4.78 ± 0.40 mg AAE/g) and Phytolacca dodecandra (8.6 ± 0.79 mg AAE/g sample) observed in the present study were smaller than those recorded in Indonesia (Sembiring et al. 2018), Serbia (Stankovic 2011), India (Kumari and Sharma 2017) (Table 13).
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Table 13: Comparison of total polyphenol, flavonoid, and antioxidant activities in Croton macrostachyus and Phytolacca dodecandra with other medicinal plants in worldwide
Plant species country TPP(mg GAE/ Flavnoid(mg DPPH Reference 100 g) sample CE/100 g) scavenging sample activity (mgAAE/g) Caesalpinia Indonesia 8981 ± 3.00 1255 ± 0.08 135,778 ± 54 (Sembiring Bonduc et al.2018) Marrubium Serbia 4927 ± 0.82 54.77 ± 0.60 89.78 ± 9.17 (Stankovi 2011) peregrinum Ailanthus India 28.56±0.006 1.11±0.008 1493.46 ± 20.03(Kumari and Sharma R Excelsa sa Roxb 2017) Phyllanthus Sri Lanka 1130 ± 0.38 364 ± 0.08 1037 ± 11.00 (Wanigasekera amarus et al. 2019) Torilis Pakistan 12190 ± 3.10 5960±1.50 189 ± 4.00 (Saeed et al. 2012) leptophylla L Piper betle Sri Lanka 14.83 ± 0.06 443 ± 0.03 623 ± 31.20 (Wanigasekera et al. 2019) Zanthoxylum Italy 18,515 ±1.22 9127 ± 3.13 67.82 ± 4.20 (Phuyal armatum et al. 2020) Euclea schimperi Ethiopia 748.78 ± 3.11 3.306 ± 0.14 19.707 ± 0.18 (Mekonnen et al. 2018) Otostegia Ethiopia NR 416.5 ± 0.29 82.91±0.37 (Chekol and integrifolia Desta 2018) Croton Ethiopia 2402.50 ± 0.80 502.83 ± 23.43 4.78 ± 0.40 This study macrostachyus Phytolacca Ethiopia 1104. 86 ± 2.04 589.227 ±18.6 8.60 ± 0.79 This study dodecandra
* NR is not reported
62
4.9. Antibacterial Activities of Methanol Water Extracts of Medicinal Plants
The antibacterial activities of Croton macrostachyus, Phytolacca dodecandra and mixture of the two plant root extracts were tested against the four bacterial species, namely: Staphylococcus aurous, Staphylococcus pneumonia, Escherichia coli and Klebsiella pneumonia, which were done in triplicates using Gentamicin as a positive control (Figure 14).
A B
C
Figure 14: Zone of inhibitions of extracts in comparison with Gentamicin (positive control) against the four bacteria: Staphylococcus aurous, Staphylococcus pneumonia, Escherichia coli and Klebsiella pneumonia; A) Croton macrostachyus B) Phytolacca dodecandra C) mixture of the two extracts
Here, the methanol:water (v/v; 80:20) extracts of Phytolacca dodecandra and Croton macrostachyus, respectively, showed inhibition zones of 6.4, 7.6, 6.2 and 8.1 and 5.8, 6.2, 5.9 and 6.0 mm for Staphylococcus aurous, Staphylococcus pneumonia, Escherichia coli and Klebsiella pneumonia, which were more potent than Gentamicin antibiotic. The antibacterial activities of both plant root mixtures were also evaluated (Table 14). And, their inhibition zones were estimated at 5.7, 5.9, 5.4 and 6.0 mm for Staphylococcus aurous, Staphylococcus
63 pneumonia, Escherichia coli and Klebsiella pneumonia, respectively, which were also more potent than Gentamicin antibiotics. In conclusion, the data given below revealed that Phytolacca dodecandra had more antibacterial potency than Croton macrostachyus root samples.
Table 14: The result of antibacterial activities of the crude extracts, standard drug and zone of inhibition in (mm)
Zone of inhibition in( mm) of methanol water crude extracts Name of Croton Phytolacca microorganisms macrostachyus dodecandra Mixed Gentamicin S. aurous 5.8 6.4 5.7 5.1 S. pneumonia 6.2 7.6 5.9 8.5 E. coli 5.9 6.2 5.4 7.3 K. pneumonia 6.0 8.1 6.0 8.9
* S. aurous = Staphylococcus aurous, S. pneumonia = Staphylococcus pneumonia, E. coli =
Escherichia coli, K. pneumonia = Klebsiella pneumonia
4.10. Statistical Analysis
Analysis of Variance (ANOVA)
Statistically, one-way ANOVA was employed to see whether there existed significant differences among the mean levels of each analyzed parameters between the two plant root samples. The ANOVA results clearly indicated that the difference among means in all the six sampling sites were significant (p < 0.05) for all metals investigated (Table 22 annex 1). Contrary to this, statistically insignificant difference was observed (p > 0.05) in the mean metal values between Croton macrostachyus and Phytolacca dodecandra root samples (Table 23 annex 1). Similarly, there were no significant differences (P > 0.05) in the total polyphenol contents and antioxidant capacities between the two plant root samples (Tables 24 &25 annex 1).
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Correlation of Metals between the Two Traditional Medicinal Plant Root Samples
High correlation coefficient (near +1 or -1) refers a good relation between two variables; and its correlation around zero means no relationship between them at a significant level of 0.05%; it can be strongly correlated, if r > 0.7, whereas r values between 0.5 and 0.7 shows moderate correlation between two different parameters (Addis and Abebaw 2018).
The results of correlations showed strong and moderate associations between the levels of the six metals. A strong positive correlation was observed between Mn to Mg, Fe to Ca, Fe to Mg, Ca to Mg, and Zn to Ca. Moderate positive correlation was also observed between Cr to Mg, Cr to Ca, Fe to Cr, Mn to Fe, Mn to Cr, Mn to Ca, Zn to Fe, Zn to Ca, Zn to Mg, Cu to Zn, and Cu to Fe (Table 15). Similarly, strong positive correlation was observed between total polyphenol and their corresponding antioxidant activities (r = 0.997) (Table 26 annex 1), indicating the expected roles of total polyphenols as good antioxidants.
Table 15: Pearson correlation of metals between Croton macrostachyus and Phytolacca dodecandra
Cu Zn Mn Fe Cr Ca Mg Cu 1 Zn 0.733* 1 Mn -0.160 0.352 1 Fe 0.551* 0.708* 0.698* 1 Cr 0.417 0.856** 0.539* 0.717* 1 Ca 0.436 0.596* 0.691* 0.925** 0.612* 1 Mg 0.153 0.556* 0.929** 0.895** 0.706* 0.890** 1
Note that (*) and (**) refer to moderate and strong correlations
65
5. CONCLUSIONS AND RECOMMENDATIONS
In the present study, an optimized wet-digestion procedure was developed, which was validated and later applied for the analyses of six macro- and trace-essential metals using FAAS. All the metals analysed were detected; and a decreasing trend was noted in the metal levels as: Mg > Ca > Fe > Mn > Zn > Cu > Cr for Croton macrostachyus and Phytolacca dodecandra root samples. Despite the absence of reported values in literature for the levels of metals in both plant root samples, the results obtained in this study were comparable to those reported for leave samples, and all below the WHO/FAO permissible limits, except for Fe and Mg.
Similarly, an optimized procedure was also employed for the extraction of total polyphenols with 80% aqueous methanol for 24 h maceration. The mean values of total polyphenols, flavonoids and antioxidant activities of Croton macrostachyus and Phytolacca dodecandra root extracts were estimated at: 2402.50 ± 0.80 and 1104.86 ± 2.04 mg GAE/100 g (total polyphenols), 502.83 ± 23.433 and 589.227 ± 18.684 mg CE/100 g (flavonoids), and 4.78 ± 0.40 and 8.6 ± 0.79 mg AAE/g (antioxidant activities). These results were, in most instances, lower than those reported in the literature. Generally, the root extracts of Phytolacca dodecandra contained higher amounts of total polyphenols, and furnished higher antioxidant and antibacterial activities compared to its Croton macrostachyus counterpart.
Finally, the root extracts of Phytolacca dodecandra and Croton macrostachyus showed inhibition zones of 6.4, 7.6, 6.2 and 8.1 mm and 5.8, 6.2, 5.9 and 6.0 mm for Staphylococcus aurous, Staphylococcus pneumonia, Escherichia coli and Klebsiella pneumonia, respectively, which were more potent than Gentamicin antibiotic.
The following recommendations are forwarded:
Further studies on antiviral and antimalarial tests should be conducted Investigation must done for identified the chemical structure of the of polyphenolic that used as antibacterial activities; and Further investigations should be made with samples from other study areas using large sample size with the help of high-tech and robust analytical techniques.
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ANNEXES
ANNEX I
Table 16: Summarized optimal parameters for the digestion of two traditional plants root samples
Reagent Optimized volume ratio parameters (mL)
Samples Total HNO3 HCl H2O2 Temperature(℃) Mass(g) Time(hr) volume(mL)
Phytolacca 11 8 2 1 240 0.5 3:00 dodecandra
Croton 9 5 2 2 300 0.5 3:00 macrostachyus
Table 17: Mean and range of metal levels in the two selected traditional medicinal plant root samples
Metal Concentration range (mg/kg) (Mean ± SD, n=3)
Cu (5.0 ± 0.24) __(12.0 ± 0.08)
Zn (17.0 ± 1.29) __(196.0 ± 22.13)
Mn (62 .0 ± 6.89) __ (479.0 ± 39.01)
Fe (182 .0 ± 20.33)__ (1455.0 ± 80.11)
Cr (0.1 ± 0.01)__ (3.0 ± 0.16)
Ca (550.0 ± 32.19)__ (1407.0 ± 72.15)
Mg (1019.0 ± 65.58 __1318.0 ± 69.72)
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Table 18: Concentration and absorbance of working standards for both Croton macrostachyus and Phytolacca dodecandra root samples
Metal Concentration Absorbance Metal Concentration Absorbance 0.5 0.015 0.5 0.001 1.5 0.055 1.5 0.003 Cu 2.5 0.103 Cr 2.5 0.005 3.5 0.148 3.5 0.008 4.5 0.19 4.5 0.010 0.5 0.058 2 0.004 1.5 0.168 4 0.009 Zn 2.5 0.261 Ca 6 0.015 3.5 0.358 8 0.02 4.5 0.438 10 0.026 0.5 0.031 2.5 0.851 1.5 0.054 3.5 0.967 Mn 2.5 0.075 Mg 4.5 1.096 3.5 0.097 5.5 1.228 4.5 0.120 6.5 1.386 15 0.019 20 0.025 Fe 25 0.030 30 0.035 35 0.041
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Table 19: Total Polyphenols (TPP) and Flavonoids of Croton macrostachyus (Mean ± SD, n =3)
TPP (mg GAE/100 g) Sampling site sample Flavnoid (mg CE/100 g) sample Eyaho Seraba 986 ± 98 489 ± 13 Teber Serako 1557 ± 75 745 ± 32 Aykel Town 1063 ± 6 470 ± 19 Laza Buladigie 802 ± 53 342 ± 26 Bezaho Mekenet 1122 ± 98 495 ± 19 Nara Awurarda 1099 ± 86 476 ± 32
Table 20: Total Polyphenols (TPP) and Flavonoids of Phytolacca dodecandra (Mean ± SD, n =3)
TPP (mg GAE/100 g) Sampling site sample Flavnoid (mg CE/100 g) sample
Eyaho Seraba 955 ± 46 463 ± 26
Teber Serako 950 ± 38 463 ± 13
Aykel Town 969 ± 58 457 ± 13
Laza Buladigie 3341 ± 273 828 ± 16
Bezaho Mekenet 3985 ± 361 873 ± 19
Nara Awurarda 4214 ± 45 451 ± 26
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Table 21: Antioxidant activities of Croton macrostachyus and Phytolacca dodecandra (Mean ± SD, n =3)
DPPH Scavenging activities in (mg AAE/g of sample)
Sampling site Croton macrostachyus Phytolacca dodecandra
Eyaho Seraba 4.1 ± 0.4 4.0 ± 0.4
Teber Serako 6.4 ± 0.6 3.8 ± 0.4
Aykel Town 4.6 ± 0.5 4.3 ± 0.5
Laza Buladigie 3.5 ± 0.4 12.0 ± 0.8
Bezaho Mekenet 5.0 ± 0.3 13.0 ± 0.9
Nara Awurarda 5.2 ± 0.2 14.3 ± 1.0
Table 22: One way ANOVA (single factor) among means in all the sampling sites for all metals
Source of SS df MS F P-value F crit Variation Between 3968696.1 6 661449.3 112.4 2.20603E- 2. Groups 21 Within Groups 205920.1 35 5883.4 Total 4174616.2 41
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Table 23: One way ANOVA (single factor) mean metal values between Croton macrostachyus and Phytolacca dodecandra root samples
Source of Variation SS df MS F P-value F crit
Between Groups 1716.071 1 1716.071 0.006548 0.936838 4.747225
Within Groups 3144751 12 262062.6
Total 3146467 13
Table 24: One way ANOVA (single factor) total polyphenol between Croton macrostachyus and Phytolacca dodecandra root samples
Source of Variation SS df MS F P-value F crit Between Groups 5050519 1 5050519 3.814903 0.079329 4.964603 Within Groups 13238918 10 1323892
Total 18289437 11
Table 25: One way ANOVA (single factor) antioxidant between the two medicinal plants
Source of Variation SS df MS F P-value F crit Between Groups 43.047068 1 43.04706802 3.2765 0.1003842 4.9646027 Within Groups 131.381257 10 13.13812565
Total 174.428325 11
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Table 26: Pearson Correlations of polyphenol content and antioxidant activities between the two medicinal plants
polyphenol Antioxidant polyphenol 1
Antioxidant 0.997 1
Table 27: Optimization time and solvent composition for extraction of Phytolacca dodecandra
30h 24h 18h 12h
MeOH:H2O mg/100 g mg/100 g mg/100 g mg/100 g 50 1136.2 985.6 1183.3 1044.5 60 1072.7 802.1 1232.7 1025.6 70 1355.1 1063.3 1310.4 1093.9 80 1496.2 1557.4 1425.6 1352.7 85 1413.9 1122.1 1366.8 1204.5 90 1366.8 1098.6 1350.4 1178.6 100 1251.5 990.4 1331.5 1157.4
Table 28: Optimization time and solvent composition for extraction of Croton macrostachyus
30h 24h 18h 12h
MeOH:H2O mg/100 g mg/100 g mg/100 g mg/100 g 50 1651.5 1679.8 1378.6 686.8 60 1905.6 1900.9 1700.9 846.8 70 2580.9 2616.2 1926.8 1035.1 80 3444.5 4213.9 2437.4 1246.8 85 3112.7 3985.6 2293.9 1176.2 90 3157.4 3790.4 2183.3 1126.8 100 2740.9 3896.2 2075.1 1098.6
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ANNEX II
2 1.8 y = 0.006x - 0.179 R² = 0.998 1.6
1.4 1.2
1 Absorbance 0.8 0.6 0.4 0.2 0 0 50 100 150 200 250 300 350 Concentration (mg/L)
Figure 15: Standard curve of gallic acid
1.2
1 y = 0.0025x - 0.0154 R² = 0.9987
0.8
0.6
Absorbance 0.4
0.2
0 0 100 200 300 400 500
Concentration of Catechin (mg/L)
Figure 16: Standard curve of catechin
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Cu standard a Ca standard b 0.25 0.03 y = 0.0028x - 0.0017 y = 0.0443x - 0.0086 0.025
0.2 R² = 0.999
R² = 0.9993 0.02 0.15 0.015 0.1
0.01
Absorbance Absorbance
0.05 0.005
0 0 0 2 4 6 0 5 10 15 Concentration (mg/L) Concentration (mg/L)
Mg standard c Fe standard e 1.6 0.045 1.4 y = 0.1331x + 0.5067 0.04 y = 0.0011x + 0.003 R² = 0.9968
1.2 0.035 R² = 0.9986
0.03 1 0.025 0.8 0.02 0.015 0.6 Absorbance Absorbance 0.01 0.4 0.005 0.2 0 0 10 20 30 40 0 0 2 4 6 8 Comcentration in mg/L Concentration (mg/L)
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Cr standard f Mn standard g 0.012 0.14 0.12 y = 0.0221x + 0.0091 0.01 y = 0.0023x - 0.0003 R² = 0.9998
R² = 0.9944 0.1 0.008 0.08 0.006
0.06 Absorbance
Absorbance 0.004 0.04
0.002 0.02
0 0 0 1 2 3 4 5 0 2 4 6 Concentration (mg/L) Concentration
Figure 17(a-g): Standard calibration curves of different metals
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Figure 18: During metal analysis using FAAs
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Figure 19: During sample digestion
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