CHAPTER 1 INTRODUCTION 1

CHAPTER 1
INTRODUCTION
1.1 Background of the study
There are many types of natural product that have been updated into the medical industry where produce many types of medicine that can treat various kind of treatment. The different kind of natural product includes plants and fruits around people can generate thousands sort of effective and amazing medicines. Hence, natural products are also produced from several source such as a marine organism, bacteria, fungi, and the most popular one is from plants.Basically, the natural products can be classified into two parts which are primary metabolites and secondary metabolites. Primary metabolites are essential for the plant to survive which is important whereas secondary metabolites are non-essential but it is actually unique for certain species of plants. Nowadays, many researchers still using natural products to produce a better product for human life. According to Dr. Josh Axe who was given a certified doctor of natural medicine, doctor of chiropractic and clinical nutritionist in his article named Herbal Medicine Benefits & the top medicinal herbs more people are using stated that many researchers now aware and try to find other opportunity to change the using of conventional
medicine to the natural product based of medicine which are more better for human health.
Propolis is the natural product which derived from stingless bee and sticky honeybee having a long history of application in many countries as a traditional remedy for treating wounds, burns, sore throat, stomach disorders etc (Popova et al., 2013). The composition of propolis depends on the type of plants accessible to the bees. According to Ibrahim et al. in 2016, the more propolis is made by bees, the smaller entrances for the bees to enter their hive. In addition, propolis has been used in biological and pharmacological properties such as antibacterial, anti-inflammatory, antiulcer, antioxidant, hepatoprotective and tumoricidal activities. It showed that the bees collect sticky plant materials that can be produced by different parts of plants. For example, Geniotrigona thoracica sp is one of the largest stingless bees in Malaysia and it has economic potential especially their propolis.
1.2 Problem Statement
The researchers claimed that the propolis can be used for biological and pharmacological activities which are related to its chemical composition. Besides, it is beneficial to human health. The complexity of propolis chemistry is one of the challenges in propolis research. The chemical complexity of propolis has made the effectiveness and quality of the propolis, uncertain. Even they also lack information since there are not much research has been done on the propolis from a stingless bee in Malaysia.

The markets demand for honey production and less attention to propolis. This is due to the lack of studies made on the composition and biological activity a stingless bee propolis to show the true benefit and importance of propolis making it limited for the industry to be developed. In Malaysia, to make a research on stingless propolis were very hard to find (Ibrahim et al., 2016). In addition, a lot of time and money needed to be invested on the basic study which is not proved to be beneficially promising. Even there are many researchers have been done their research about honey but still lacking in research on the propolis.
1.3 Significance of study
This study is conducted to investigate the chemical composition and biological activity of species propolis such as Geniotrigona thoracica from various localities. G. thoracica propolis can give many benefits to human health which has been used to treat various diseases since ancient times. In this research, the chemical composition of G. thoracica inside the propolis was carried out and it reacts as a medicine to treat human health. According to Ibrahim et al. (2016), it can be used as antioxidant, antibacterial, anticancer, antifungal, inflammatory, antiviral and antidiabetic. Besides, the propolis also contains flavonoid and phenolic. While according to Khamsah, the phytochemical screening of the propolis proved that they have phenols and essential oil in G. thoracica. The flavonoid and other phenolic substances contents may also prohibit the development of cancer and heart diseases (Liyana et al., 2016). G. thoracica can be easily found in many localities. So, that is why it has high potential to fulfill the market demand.
1.4 Scope of research
In this research, the scope was focused on the extraction and isolation of chemical constituents from Geniotrigona thoracica sp propolis. Besides, there was focusing on the separation the major and minor is separated from the species G. thoracica. Hence, the different chromatographic and spectroscopic methods were used to make a clear confirmation.

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1.5 Objectives of the research
The specific objectives of this research are as below:
To conduct phytochemical screening on Geniotrigona thoracica propolis extract
To isolate the chemical constituents from Geniotrigona thoracica sp propolisTo elucidate and identify the structures of the isolated compounds using a spectroscopic methods such as ultravisible (UV), infrared (IR), and nuclear magnetic resonance (NMR)
CHAPTER 2
LITERATURE REVIEW
2.1 Propolis and its function
Propolis is one of the natural product which is a sticky material produced by the bees by collecting some pieces from the plants around it, buds and exudates. Besides, propolis contains more than 160 constituents. Basically, it was coming from several plants mixed with waxes and other bee extractions (Melliou et al., 2013). It smells is pleasant because of the presence of honey inside it. The sweetness of the honey made the properties of the propolis become sticky and it looks like a gummy. Even the color of the propolis is looked brown color but it still depends on the species of the propolis whether dark brown or yellowish-brown.

In addition, propolis gives more function to the bees and hive. It can be used as medicine for many years (Ibrahim et al., 2016). The researchers have been investigated that the chemical composition of propolis could be used to treat human diseases such as inflammation, heart disease, cancer, and diabetes. Besides, propolis is not only used as antiseptic, but it can be used as a cicatrizant agent by Greek andRoman physicians ADDIN CSL_CITATION {“citationItems”:{“id”:”ITEM-1″,”itemData”:{“DOI”:”10.1016/j.jep.2010.10.032″,”ISBN”:”0378-8741″,”ISSN”:”03788741″,”PMID”:”20970490″,”abstract”:”Introduction: Propolis has plenty of biological and pharmacological properties and its mechanisms of action have been widely investigated in the last years, using different experimental models in vitro and in vivo. Researchers have been interested in the investigation of isolated compounds responsible for propolis action; however, there is lack of clinical research on the effects of propolis. Strategy and objectives: Since propolis-containing products have been marketed and humans have used propolis for different purposes, the goal of this review is to discuss the potential of propolis for the development of new drugs, by comparing data from the literature that suggest candidate areas for the establishment of drugs against tumors, infections, allergy, diabetes, ulcers and with immunomodulatory action. Conclusions: The efficacy of propolis in different protocols in vitro and in vivo suggests its therapeutic properties, but before establishing a strategy using this bee product, it is necessary to study: (a) the chemical nature of the propolis sample. (b) Propolis efficacy should be compared to well-established parameters, e.g. positive or negative controls in the experiments. Moreover, possible interactions between propolis and other medicines should be investigated in humans as well. (c) Clinical investigation is needed to evaluate propolis potential in patients or healthy individuals, to understand under which conditions propolis may promote health. Data point out the importance of this research field not only for the readers and researchers in the scientific community waiting for further clarification on the potential of propolis but also for the pharmaceutical industry that looks for new drugs. © 2010 Elsevier Ireland Ltd. All rights reserved.”,”author”:{“dropping-particle”:””,”family”:”Sforcin”,”given”:”José Maurício”,”non-dropping-particle”:””,”parse-names”:false,”suffix”:””},{“dropping-particle”:””,”family”:”Bankova”,”given”:”Vassya”,”non-dropping-particle”:””,”parse-names”:false,”suffix”:””},”container-title”:”Journal of Ethnopharmacology”,”id”:”ITEM-1″,”issued”:{“date-parts”:”2011″},”title”:”Propolis: Is there a potential for the development of new drugs?”,”type”:”article-journal”},”uris”:”http://www.mendeley.com/documents/?uuid=def1b626-6ab4-3ddd-a6c4-baf31258ed3b”},”mendeley”:{“formattedCitation”:”(Sforcin & Bankova, 2011)”,”plainTextFormattedCitation”:”(Sforcin & Bankova, 2011)”,”previouslyFormattedCitation”:”(Sforcin & Bankova, 2011)”},”properties”:{“noteIndex”:0},”schema”:”https://github.com/citation-style-language/schema/raw/master/csl-citation.json”}(Sforcin and Bankova, 2011). Propolis also used in the construction and repair the nest of the bees. This is because to cover up the flaws, crack or hole inside the nest, so propolis can produce the interior wall that is fully cover and safe for them to shield under it (Shruthi et al., 2012). However, the bees can produce propolis as much as they can to protect themselves from bad weather or situation like rain or wind (Wagh, 2013). If the bees should not cover their nest properly with propolis, they can be easily destroyed. So that was the reason why some of the farmers always keep the propolis over the hive with transparent plastic and zinc.

2.2Nutrients Composition and Uses of the propolis
The propolis has given many uses and benefits to human health especially for treating diseases. Propolis can act as anti-bacterial, anti-inflammatory, antiulcer, antioxidant, hepatoprotective and tumoricidal ADDIN CSL_CITATION {“citationItems”:{“id”:”ITEM-1″,”itemData”:{“DOI”:”10.1016/j.arabjc.2015.11.003″,”ISSN”:”18785352″,”abstract”:”Pandanus tectorius fruits have been used as major food in Micronesia, but not fully exploited in South East Asia. In Malaysia and Indonesia, P. tectorius fruit is wasted and still not utilized either as a source of food as well as for research. The aims of the present study were to determine the phytochemical content, antibacterial and antioxidant activities, total phenolic content (TPC) and cytotoxicity properties of cores and keys parts of P. tectorius fruits extracts against some normal (RAW and L-6) and cancer cell lines (HeLa, HepG2 and MCF-7). Samples were collected from Setiu Wetland, Terengganu, Malaysia. Extracts were obtained by successive extraction using hexane, ethyl acetate and methanol. The antibacterial activity was performed by disk diffusion method against gram positive (Bacillus subtilis and Staphylococcus aureus) and gram negative (Escherichia coli, and Pseudomonas aeruginosa) bacteria. Actives extract was continued for antibacterial kinetic study. The TPC and antioxidant were analyzed by Folin-Ciocalteu and DPPH free radical scavenging assay, respectively. Qualitative phytochemical analysis result confirmed the presence of phenolics, flavonoids, terpenoid, steroids, saponins and glycosides as chemical constituents in P. tectorius fruits extract. It was supported by high TPC content in this extracts. The ethyl acetate extract from cores part (PEC) showed the highest antioxidant capacity (IC50=0.8±0.20mg/mL), while ethyl acetate extract from keys part (PEK) displayed highest antibacterial activity with inhibition zone of 10-15mm, but less strong inhibition as compared to the investigated commercial antibiotics (ampicillin, penicillin, gentamycin, and tetracycline). Antibacterial kinetic study was further proven that PEK has good antibacterial activity. Moreover, cytotoxicity study revealed that all of extracts did not have any cytotoxic activity against all selected cell lines (IC50value>30?g/mL). Result founded that both keys and core parts of P. tectorius fruits were found to be rich by phenolics content and potent as antioxidant and antibacterial agents. Since it is not toxic against selected cell lines, the mechanism study on antiinflammatory, antidiabetic, antiatherosclerosis could be carried out.”,”author”:{“dropping-particle”:””,”family”:”Andriani”,”given”:”Yosie”,”non-dropping-particle”:””,”parse-names”:false,”suffix”:””},{“dropping-particle”:””,”family”:”Ramli”,”given”:”Nadiah Madihah”,”non-dropping-particle”:””,”parse-names”:false,”suffix”:””},{“dropping-particle”:””,”family”:”Syamsumir”,”given”:”Desy Fitrya”,”non-dropping-particle”:””,”parse-names”:false,”suffix”:””},{“dropping-particle”:””,”family”:”Kassim”,”given”:”Murni Nur Islamiah”,”non-dropping-particle”:””,”parse-names”:false,”suffix”:””},{“dropping-particle”:””,”family”:”Jaafar”,”given”:”Jasmin”,”non-dropping-particle”:””,”parse-names”:false,”suffix”:””},{“dropping-particle”:””,”family”:”Aziz”,”given”:”Nur Asniza”,”non-dropping-particle”:””,”parse-names”:false,”suffix”:””},{“dropping-particle”:””,”family”:”Marlina”,”given”:”Leni”,”non-dropping-particle”:””,”parse-names”:false,”suffix”:””},{“dropping-particle”:””,”family”:”Musa”,”given”:”Noor Suryani”,”non-dropping-particle”:””,”parse-names”:false,”suffix”:””},{“dropping-particle”:””,”family”:”Mohamad”,”given”:”Habsah”,”non-dropping-particle”:””,”parse-names”:false,”suffix”:””},”container-title”:”Arabian Journal of Chemistry”,”id”:”ITEM-1″,”issued”:{“date-parts”:”2015″},”publisher”:”King Saud University”,”title”:”Phytochemical analysis, antioxidant, antibacterial and cytotoxicity properties of keys and cores part of Pandanus tectorius fruits”,”type”:”article-journal”},”uris”:”http://www.mendeley.com/documents/?uuid=9c43190f-74cc-4424-8624-3d80df29edda”},”mendeley”:{“formattedCitation”:”(Andriani et al., 2015)”,”plainTextFormattedCitation”:”(Andriani et al., 2015)”,”previouslyFormattedCitation”:”(Andriani et al., 2015)”},”properties”:{“noteIndex”:0},”schema”:”https://github.com/citation-style-language/schema/raw/master/csl-citation.json”}(Andriani et al., 2015). Propolis has been studied by many researchers where it has a high phenolic content. Based on its properties, propolis exhibits hepatoprotective in acute liver damage induced in rats by carbon tetrachloride and in mice by paracetamol and allyl alcohol. Besides, it also exhibits anti-inflammatory effects against acute and chronic models of inflammation (Castaldo et al., 2002). This is because there is an active compound inside the propolis such as flavonoids and phenolic acid. Propolis also shows the various kind of effect towards bacteria, fungi, and virus where propolis can reduce the blood pressure and cholesterol level on a human. But, it must need clinical studied to be confirmed.
However, propolis is popular as an antioxidant. By using diphenyl picrylhydrazyl, DPPH as an antioxidant assay to determine whether there is an antioxidant potential of the propolis extract ADDIN CSL_CITATION {“citationItems”:{“id”:”ITEM-1″,”itemData”:{“DOI”:”10.17576/mjas-2016-2002-26″,”ISSN”:”13942506″,”abstract”:”The aim of this study is to evaluate chemical and biological profile of methanol extracts from Malaysian propolis produced by two commonly found stingless bee species, Heterotrigona itama (MHI) and Geniotrigona thoracica (MGT). Test samples were analyzed for physicochemical parameters such as moisture, fat, crude fibre, crude protein, carbohydrate and ash content. Tests for phytochemical screening by thin layer chromatography of both extracts revealed that presence of terpenoids, flavonoids, phenols and essential oils but steroids, saponin and coumarins only occur in MHI. Both extracts displayed a characteristic profile and vary from each other. Accordingly, MHI possess higher antioxidant activity with an IC50 of 15.0 ± 0.21 ug/mL compared to MGT with IC50 of 270.0 ± 0.19 ug/mL. MHI showed moderate nitric oxide scavenging activity, while MGT only showed mild inhibition. Antidiabetic activity was determined by ?-glucosidase inhibition and found significantly better than that of acarbose (positive control). In conclusion, data gathered in this study revealed that bee species play role in determining the chemical and biological profile of particular propolis and should put into account in decision of further development for propolis”,”author”:{“dropping-particle”:””,”family”:”Ibrahim”,”given”:”Nurhamizah”,”non-dropping-particle”:””,”parse-names”:false,”suffix”:””},{“dropping-particle”:””,”family”:”Mohd Niza”,”given”:”Nurul Farah Shakila”,”non-dropping-particle”:””,”parse-names”:false,”suffix”:””},{“dropping-particle”:””,”family”:”Mohd Rodi”,”given”:”Muhammad Muslim”,”non-dropping-particle”:””,”parse-names”:false,”suffix”:””},{“dropping-particle”:””,”family”:”Zakaria”,”given”:”Abdul Jamil”,”non-dropping-particle”:””,”parse-names”:false,”suffix”:””},{“dropping-particle”:””,”family”:”Ismail”,”given”:”Zhari”,”non-dropping-particle”:””,”parse-names”:false,”suffix”:””},{“dropping-particle”:””,”family”:”Mohd”,”given”:”Khamsah Suryati”,”non-dropping-particle”:””,”parse-names”:false,”suffix”:””},”container-title”:”Malaysian Journal of Analytical Science”,”id”:”ITEM-1″,”issue”:”2″,”issued”:{“date-parts”:”2016″},”page”:”413-422″,”title”:”Chemical and Biological Analyses of Malaysian Stingless Bee Propolis Extracts”,”type”:”article-journal”,”volume”:”20″},”uris”:”http://www.mendeley.com/documents/?uuid=e62a6950-58b0-4524-bd7c-6d550ebd7122″},”mendeley”:{“formattedCitation”:”(Ibrahim et al., 2016)”,”plainTextFormattedCitation”:”(Ibrahim et al., 2016)”,”previouslyFormattedCitation”:”(Ibrahim et al., 2016)”},”properties”:{“noteIndex”:0},”schema”:”https://github.com/citation-style-language/schema/raw/master/csl-citation.json”}(Ibrahim et al., 2016). DPPH has a major application and one of them is as an antioxidant assay in the aboratory test. As a result, there is a free radical of DPPH that make Heterotrigona Itama and Geniotrigona thoracica to scavenge. The two species which H. Itama and G. thoracica are studied by Ibrahim and his partners to analyze these two species with seven strains of an organism using the method of disc diffusion by using three different concentrations. So, both of these species can stop Staphylococcus aureus from growing. The reason because of the phenolic content inside a sample of propolis (Ibrahim et al., 2016). In addition, propolis also can be used as anticancer. This was proved by Millind K. Choudhari and his partners in their research about an anticancer activity of Indian Stingless Bee Propolis; an in vitro study on 2013. They already test Indian stingless bee propolis against four different types of cancer cells such as human breast cancer, human colon adenocarcinoma, human epithelial colorectal adenocarcinoma, and murine melanoma. So, they clarify that Indian stingless bee propolis can be used as anticancer after a test.

2.3 Recent study of propolis2.3.1 Overview of propolis study
Many research has been done that propolis can be used to treat many of disease. In many years, propolis has become popular because it can give many benefits to human. They also generate some product from the propolis. But there are still limitations on the study about the stingless bee propolis as propolis of honey bee is more potent and attractive to people. There are some popular types of propolis that have been studied by other countries such as green stingless bee propolis which is conducted (Millind K. Choudhari et al., 2016). According to Millind K. Choudhari, the green propolis was produced by a stingless bee can be used as anticancer. Besides, propolis has been widely studied at the Brazil which is to use green Brazilian propolis and proved that the green propolis was produced by a stingless bee can act as addictive in animal nutrition. In Malaysia, the two species that are widely studied is Heterotrigona Itama and Geniotrigona thoracica (Ibrahim et al., 2016). In Malaysia, there are some people still lack studied are conducted on propolis because the researcher is more attracted to the honey compared to the propolis. This is because propolis needs more money and much time to complete this study.Nowadays, the most interesting topic of research is to make comparisons between species of stingless bees because they want to prove that the different species may contain come chemical composition when they live in the same habitat. The propolis made by the stingless depends on the plant growth around them. There is one research that conducted by Malaysian researchers about chemical and biological analyses of Malaysian stingless bee propolis (Nurhamizah et al., 2015). They try to make a comparative study about to compare two different species which is H. Itama and G. thoracica. They compared one of them has a higher potential to the antioxidant activity. Other than that, the researchers from Malaysia make a comparative study between these two species about antibacterial and phenolic content. 2.3.2 Studies on Stingless Bee PropolisThe stingless bees are from the tribe of Meliponini having more than one of genera like Melipona, Scaptotrigona and Trigona (Total phenolic content). In Malaysia, there are two common species that are widely grown in a certain place which are Heterotrigona Itama and Geniotrigona thoracica. But there still a lack study of propolis that is produced by stingless bee propolis. The researchers have investigated the chemical and biological analyses of Malaysian stingless bee propolis extract. Based on the phytochemical screening test by thin layer chromatography that has been done, H. Itama and G. thoracica extract have many pure compounds such as terpenoids, flavonoid, phenol, tannin, saponin etc. For an antioxidant test, they also know that itama are scavenged better compare to thoracica because thoracica only shows a mild inhibition. As the result, propolis from itama species has higher antioxidant activity (Nurhamizah et al., 2015).Table 2.1 Phytochemical tests of the chemical methanol extract propolis produced by stingless bees, H. Itama(MHI) and G. thoracica(MGT) (Nurhamizah et al., 2015)
16510234950
20320292100ConstituentsColour detectedMHIMGTChemical/Spray reagent
TerpenoidsPink + +AnisaldehydeFlavonoidsBlue fluorescence + +Anisaldehyde
SaponinsDark bluish + -Vanillin-sulphuric acid
SteroidsBluish green + -AnisaldehydeCoumarinsLight blue + -UV 365 nm
Essential oilsRed and brown + +Vanillin-sulphuric acid
All compoundsYellow brown + +Iodine
1143010160
Based on another Malaysian researcher who study on the antibacterial and phenolic content of propolis are produced by two Malaysian stingless bees which is H. Itama and G. thoracica (Ibrahim et al., 2016). At the end of this research, they stated that both of the species inhibit the growth of bacteria which is Staphylococcus aureus bacteria. They stop the growth of that species of bacteria better compare to the bacteria with species Escherichia coli and salmonella typhi. From the IR spectrum, they obtain the same IR spectra but differ in intensities of both extracts. This is because of the existence of compounds like phenolic and flavonoids. So, it proved the phenolic and flavonoids content for itama are much better to compare to thoracica.
2.3.3 Propolis and botanical origin
Propolis is started to be famous in the medicinal industry have risen the number of interest researchers to study more and more about the composition of propolis and its botanical origin. This is because there are many research on the propolis that showed that propolis contains polyphenol compounds collected by the Apis mellifera. According to Toreti (2013), one type of polyphenol which is widely found in propolis is flavonoid. Flavonoids are known as secondary metabolites which contain inside the plants or natural product. Typically, the chemical composition contains inside propolis have 50% of resin and balsam, 30% of wax, 10% of essential and aromatic oils, 5% of pollen and another 5% of the other natural compound. The propolis depends on the fauna that growth all around their hive, so that is why all the composition for every propolis is varying. This is because there are so many different plants that grow around them and it different for every place. The bees will be attracted to the plants around their hive to collects the buds and exudates, then the buds and exudates are mixed with the bees saliva and producing the propolis. They make the propolis to protect their hive and also themselves (Toreti, 2013).
Generally, there is various kind of compound that can be found from propolis because it was depended on the plants around them where they are attracted to. Different sort of plants that the bees are attracted to will led to different compounds. This is because the same plant species that grow in different places can produce a different type of compounds. So, the sample and places where they grow around is a very important role to the researchers. The chemicals composition of propolis is varied according to the geographical area and climatic characteristic (Veronique et al., 2015). The propolis will differ based on the bee preferences on botanical sources. The plants around the hive of the propolis secrete resins, buds and also exudates for the bees. The bees will be attracted to a few different species of flora and will collect some of the resins, buds, and exudates to make the propolis inside their hive. The collected parts of the fauna actually will be combined with the saliva of the bees to produce the propolis. Hence, different plants that the bees attracted will lead to various kind of chemical composition.

2.3.4 Chemical Isolation from propolisResearchers studied about propolis because the chemical composition that contains inside the propolis. Until 2000, there are more than 300 compounds are already identified in this propolis which belongs to a class of flavonoids, terpenes, phenolic and many other organic compounds (Huang et al., 2014). Besides, there are also has about 100 compounds in one sample ADDIN CSL_CITATION {“citationItems”:{“id”:”ITEM-1″,”itemData”:{“author”:{“dropping-particle”:””,”family”:”Marcucci”,”given”:”MC”,”non-dropping-particle”:””,”parse-names”:false,”suffix”:””},{“dropping-particle”:””,”family”:”Ferreres”,”given”:”F”,”non-dropping-particle”:””,”parse-names”:false,”suffix”:””},{“dropping-particle”:””,”family”:”Garc?a-Viguera”,”given”:”C”,”non-dropping-particle”:””,”parse-names”:false,”suffix”:””},”container-title”:”Journal of Ethnopharmacolocy?”,”id”:”ITEM-1″,”issued”:{“date-parts”:”2001″},”page”:”105-112″,”title”:”Phenolic compounds from Brazilian propolis with pharmacological activities?”,”type”:”article-journal”,”volume”:”74″},”uris”:”http://www.mendeley.com/documents/?uuid=ae5e552c-b2f0-4099-9ac9-dcf0573257eb”},”mendeley”:{“formattedCitation”:”(Marcucci et al., 2001)”,”plainTextFormattedCitation”:”(Marcucci et al., 2001)”,”previouslyFormattedCitation”:”(Marcucci et al., 2001)”},”properties”:{“noteIndex”:0},”schema”:”https://github.com/citation-style-language/schema/raw/master/csl-citation.json”}(Marcucci et al., 2001). The chemical compound can be identified from the propolis are vary because it depends on the location of the sample, climatic zones and seasons (Huang et al., 2014). This study reports the chemical isolation from Geniotrigona thoracica propolis collected from Dungun, Terengganu. There is some representative chemical compound are summarized in propolis.

Table 2.2 Representative chemical compound in propolis (Huang et al., 2014)

Caffeic acid phenethyl ester
Atrepillin C

Pinocembrin
Pinobanksin
Caffeic acid
Cinnamic acid

Ferulic acid
p-coumarin acid

Galangin
Chrysin2.4 The species: Geniotrigona thoracica
Figure 2.3 Stingless bee species of Geniotrigona thoracicaGeniotrigona thoracica are one of the most common species for stingless bee which found in Malaysia. The popularity type of species propolis has been increasing since last time. In addition, G. thoracica can be found easily involving many localities. They can be found with the same species at different places because it grows everywhere. Based on the journal characterization of nest structure and foraging activity of stingless bee, G. thoracica (hymenopetra: apidae, meloponini), stingless bee with species G. thoracica are one of the larger species that can be found in Malaysia and have potential in meliponiculture. The body of the stingless bee with this species is in the range of 6.67 mm to 10.80 mm. if we can compare with other species such as Trigona irridipennis, it has a body length of 3.92 mm to 4.1 mm. As we can see, there are big differences.
The study on this journal showed that the body weight that high like G. thoracica, the wing is and the wing use more energy to fly. Other than that, the result can be compared to two other species which led to the same result which is Trigona canifron and Trigona fimbriata. The nest of the stingless bees is differing for every species (Farisya et al., 2015). The structure of the hive for thoracica are a sticky entrance with round shape, have horizontal brood combs inside the nest which have a part for them to store their food such as honey or pollen pots. Based on the characteristic of the nest, beekeepers can know this species well. G. thoracica also to produce more honey compare to a production of bee bread. It was very productive when the dry season is coming (Farisya et al., 2015).
2.4.1 Chemical Compounds of Geniotrigona thoracica propolis
In Malaysia, propolis produced by Geniotrigona thoracica species commonly known as “Kelulut”, is known to acquire various medical values (Nazir et al., 2018). There are 30 compounds are identified from 48 individual compounds. According to Nazir et al. (2018), the main class group of compounds is phenolic acids and terpenoids such as IH-Pyrole-2-carboxylic acid, 1-(2 hyroxy-2-phenylethyl) and fren-9(11)-en-2. alpha-ol. The table 2.4 below shows another example of major class chemical compounds in G. thoracica.

Table 2.4 List major class chemical compounds of Geniotrigona thoracicaCompound names
Phenolic compounds

1H-Pyrrole-2-carboxylic acid
1-(2-hydroxy-2-phenylethyl)

Resorcinol
Benzoic acid trimethylsilyl ester

1-(2-Methoxyphenyl)-2,5-dihydro-1H-pyrrole-2,5-dione
Phenol
Terpenoids

Delta-cadinene
NootkatoneBasically, propolis contains a resin that comprised flavonoids, phenolic acids, fatty acids, terpenoids, aromatic acids, pollen, and mineral. However, chemical composition of propolis is very complex (Bees, M. S. Antibacterial and Phenolic Content of Propolis Produced by Two Malaysian Stingless Bees, Heterotrigona itama and Geniotrigona thoracica.). The major plant in the area of Geniotrigona thoracica is Acacia sp.

2.5 Study of Acacia species
Certain Acacia species are too important economically. Acacia is also known as genus Acacia of the family Leguminosae where it has about 160 species of trees at native Australia. The rest occurs mainly in tropical and subtropical regions of Africa, Asia, and America. Many acacia species have important uses in traditional medicine. For example, plants of genus Acacia are widely used in traditional medicine for diarrhea, urinary tract infections, headaches, sore throat, gastritis, tuberculosis, and bronchial asthma.
In Malaysia, there are several species of acacia, such as Acacia mangium and Acacia auriculiformis. However, from the previously studied show, there are four species of Acacia currently found in Brunei Darussalam such as Acacia mangium, Acacia auriculiformis, Acacia cincinnata and Acacia holosericea (Ahmed et al., 2017).

2.5 Major structure of flavonoids found in Acacia mangium and Acacia auriculiformis
2,3-cis-3,4′,7,8-tetrahyroxyflavanone

Teracacidin
4′,7,8-trihydroxyflavanone
According to Berry et al. (2005), there have been few studies on the chemistry for Acacia mangium and Acacia auriculiformis heartwood extractives. Besides, there are only two major flavonoids have been reported in Acacia mangium healthy heartwood. For examples are 2,3-cis-3,4′,7,8-tetrahyroxyflavanone and tetracacidin. While in Acacia auriculiformis heartwood, having 4′,7,8-trihydroxyflavanone even there are eight flavonoids have been identified. Based on the journal renewable and sustainable energy reviews, the thermochemical conversion of Acacia such as Acacia auriculiformis and Acacia mangium can also to produce biofuels and bio-products.

2.5.1 Acacia auriculiformis
Figure 2.1 Forest and leaves of Acacia auriculiformisAcacia auriculiformis is a vigorously growing and native trees, possibly reaching 30m height belongs to the sub-family Fabaceae. According to Kaur et al. (2014), it has secondary metabolites include phenolics, tannins, and terpenoids mainly. In the plant, it is found to be rich in methylglucuronic acid, glucuronic acid, galactose, arabinose and rhamnose (Singh et al., 2007). Besides, some of the medicinal properties are including central nervous system-depressant, spermicidal and filaricidal activities (Subbaraj et al., 2017).

In addition, the component with the highest composition was furfural 35.3% followed by free fatty acid 34.796%. The others including, furan alcohol 11.412%, Nitrogen compound 8.532%, alkanal 4.94%, glucose 4.02%, alkanone 0.272%, ether 0.272% and alkane 0.228% ADDIN CSL_CITATION {“citationItems”:{“id”:”ITEM-1″,”itemData”:{“author”:{“dropping-particle”:””,”family”:”Ba”,”given”:”A M”,”non-dropping-particle”:””,”parse-names”:false,”suffix”:””},{“dropping-particle”:””,”family”:”Shuaibu”,”given”:”H”,”non-dropping-particle”:””,”parse-names”:false,”suffix”:””},”id”:”ITEM-1″,”issue”:”4″,”issued”:{“date-parts”:”2015″},”page”:”462-467″,”title”:”Potential of Earleaf Acacia ( Acacia auriculiformis ) Leaves for Industrial Raw Materials”,”type”:”article-journal”},”uris”:”http://www.mendeley.com/documents/?uuid=d84f475c-fdd9-4ce4-a3a6-43fb57a0368e”},”mendeley”:{“formattedCitation”:”(Ba & Shuaibu, 2015)”,”plainTextFormattedCitation”:”(Ba & Shuaibu, 2015)”,”previouslyFormattedCitation”:”(Ba & Shuaibu, 2015)”},”properties”:{“noteIndex”:0},”schema”:”https://github.com/citation-style-language/schema/raw/master/csl-citation.json”}(Ba & Shuaibu, 2015). Besides, the wood componets can be composed of cellulose, hemicelluloses, lignin, and extractives.
2.5.2 Acacia mangium
Figure 2.2 Forest and leaves of Acacia mangiumAcacia mangium is identified as a promising fast-growing tree species on Peninsular Malaysia, Sabah, and Indonesia. This is because it has been planted on a large scale. A. mangium is an essential hardwood plantation trees grown in many fields of Asia (Barry et al., 2005). It has a medium-size to fairly large tree measuring up to 35 m tall. Its bole is branchless for up to 15m. It also can measure up to 90 cm in diameter. A. mangium is known as a nitrogen-fixing tree and can be used for land rehabilitation, particularly in eroded and nitrogen-deficient soils (Paula et al., 2005).
According to Yamato el al. (2006), A. mangium have been promoted for pulp production, and a large amount of wood waste, mainly bark, is being discharged from pulp mills. In chemical composition, the gum of A. mangium have 5.4% ash, 0.98% Nitrogen, 1.49% methoxy, and by calculation, 32.2% uronic acid. While the sugar composition after hydrolysis contains 9.0% 4-0-methylglucuronic acid, 23.2% glucuronic acid, 56% galactose, 10% arabinose and 2% rhamnose. According to Pinta et al. (2005), the chemical composition and chemical structure of wood components especially lignin and polysaccharide.

CHAPTER 3
METHODOLOGY
In brief, propolis of Geniotrigona thoracica will be collected, dried and ground until it become fine powder. Then, the sample powder will be successively soaked with a solvent like methanol to get the crude. Basically, the methanol extract will be partitioned using three different organic solvents with various polarities namely hexane, ethyl acetate, and dichloromethane to obtain the respective crude fractions. Then, the phytochemical screening will be conducted to identify the presence of phenol, tannins, flavonoids, steroids, terpenoids, alkaloids and saponins. Next, the crude fractions will be subjected to isolation and purification process using thin layer chromatography and column chromatography. Finally, the pure compounds will be subjected to infrared spectroscopy (IR) and nuclear magnetic resonance spectroscopy (NMR) for structural elucidation.

3.0 Propolis Material
The propolis of Geniotrigona thoracica will be collected at Dungun district of Terengganu, Malaysia. The propolis was dried and ground it into a fine powder.
2178050139700Dried, ground propolisDried, ground propolis
3101975480060
1892300146685Soaked in methanol
Soaked in methanol

3098165141605
247967567310Methanol crude
Methanol crude

309943576835
4770120238760110045523876010826752419353098800160655 Solvent partitioning
2396490264795Ethyl Acetate
Fraction
Ethyl Acetate
Fraction
4132580285750Dichloromethane
Fraction
Dichloromethane
Fraction
536575280670Hexane
Fraction
Hexane
Fraction

477393019685031026101803401079500169545
169354523622040659052362201088390231775
32207208255Phytochemical screening
Phenol and tannin test
Flavonoids test
Steroid test
Terpenoids test
Alkaloids test
Saponin test
Glycoside test
Phytochemical screening
Phenol and tannin test
Flavonoids test
Steroid test
Terpenoids test
Alkaloids test
Saponin test
Glycoside test
8096259525Isolation and purification
CC
TLC
Isolation and purification
CC
TLC

169354542545
915670238125Pure compound
Pure compound

1702435177165
92138519685Structural Elucidation
IR
NMR
Structural Elucidation
IR
NMR

Scheme 3.1: General procedure
3.1Chemical Reagents
In this research, chemicals used for extraction and isolation procedures are methanol, hexane, ethyl acetate, dichloromethane, and acetone. Besides, the same solvent used in extraction such as acetic acid for thin layer chromatography. Next chemicals for screening tests are ferric chloride, sodium hydroxide, sulphuric acid, glacial acetic acid, hydrochloric acid, ammonia, aluminium solution, acetic anhydride, chloroform, and diethyl ether. Table 3.1 shows the list of chemicals.

Table 3.1 List of chemicals
Chemical name Molecular formula
Hexane C6H14
Ethyl acetateC4H8O2
Methanol CH3OH
Dichloromethane CH2Cl2
AcetoneC3H6O
Ferric ChlorideFeCl3
Sodium HydroxideNaOHSulphuric AcidH2SO4
Glacial Acetic AcidCH3COOH
Hydrochloric AcidHClAmmonia NH3
ChloroformCHClAcetic AnhydrideC4H6O3
-577851905
3.2 Apparatus
Apparatus that will be used including conical flask (100mL, 250mL and 1000mL), test tube, vials with cap, spatula, pestle and mortar, filter funnel, separating funnel (500ml), thin layer chromatography tank, beaker (250mL, 500mL and 1000mL), reagent bottle (250ml), pasteur pipette, ear plugs, rubber tits, thin layer chromatography plate that coated with silica, glass funnel, retort stand and clamps, round bottom flask (100mL, 250mL and 500mL), big glass column, small glass column, test tube rack, glass rod, petri dish, measuring cylinder (10mL and 100 mL) and aluminium foil.

3.3Material and method
3.3.1 Collection of a sample of propolisThe sample propolis with species Geniotrigona thoracica was collected at Dungun district of Terengganu, Malaysia. The sample collected must be cleaned and packed before the sealed plastic bag. Next, store the sample in a refrigerator with a temperature -20°C before use in the next process.
3.3.2 Extraction of PropolisFirstly, the sample of propolis will be crushed using a pestle and mortar or grinder to get the powder of sample. Then, the sample put into the 1000 ml of the conical flask for the soaking process. Next, about 700ml of methanol will be added into the conical flask containing a sample. The sample with methanol inside the conical flask will be stirred. The mixture of a sample must be left for 3 days. After 3 days, filter the sample and the remaining residue kept for the next process. The product that obtained from the process of a filter will be dried by using the turbo vapor to get the methanol crude. When extraction became crude, transfer it into the vial and conical flask. Another some of the crudes will be transferred into the conical flask of 250 ml to be used for the next process. Repeat the process of extraction by adding 700 ml of methanol into the remaining residue of a sample. The process will be continued until getting the crude and kept the sample into the vials. Repeat the process extraction of a sample by using methanol until the solution becomes a clear compound.
3.3.3 Solvent partitioning
The methanol extract will be partitioned by using three different solvents with various polarities which are hexane, ethyl acetate, and dichloromethane.

3.4 Isolation and Purification of Compound
Isolation is a process to separate and purify chemical compounds. In this research, the various chromatographic method such as thin layer chromatography and column chromatography will be applied for the isolation process. Chromatographic techniques typically involve the distribution of extract components between two phases, a moving mobile phase over an inmobile stationary phase. The separation depends on the difference in affinity of the components towards the stationary phase and mobile phase.

3.4.1 Thin Layer Chromatography (TLC)
TLC is a separation technique in which solutes partition between a mobile and stationary phase. Typically, TLC is used to determine the composition of crude extract were to determine the purification of chemical constituents. The apparatus should be used were chromatography tank, measuring cylinder, TLC plate, and dropper. Firstly, prepare a suitable solvent system and pour into chromatography tank. The solvent system should be used about 10ml. Then, prepare a TLC plate and spot the extract in a small spot on the TLC plate on the bottom line.
After that, put the TLC plate into TLC tank until the solvent travel up to the solvent front line. To avoid the surrounding inside the tank become homogenous, close the developing tank by using the lid. Make sure the volume of the solvent must not exceed the spot in the bottom line. The solvent will be drawn up the plate by capillary action. When the solvent nearly reaches the solvent front line, take out the TLC plate from the tank and allow it to dry in air. Then, observe the TLC plate under the UV light. Circle the spot that appears neither under UV light or naked eyes by using the pencil. If the sample is not carried upwards, change a ratio of the polarity of the solvent system.Use the different solvent system where has more polarity by increasing the amount of ethyl acetate. If the sample is moved upwards until it reaches the solvent front, it shows the solvent system are very polar. So, the solvent can be changed to a less polarity by lowering the amount of ethyl acetate in the solvent system. Lastly, select the best crude for column chromatography to get the purified compound.
3.4.2 Column chromatography
Column chromatography is a separation, isolation, fractionation and purification method of chemical constituents from crude extract. The apparatus must be used were a column, conical flask, stirrer, dropper, and beaker. Firstly, insert a small amount of cotton wool into the column and press using the strong iron wire to make sure it close enough the hole of the column. After that, insert the sand and tap the column using the rubber tube to make sure it uniform. Besides, insert the solvent slowly which is hexane by using the dropper. Insert the silica that already mixes with hexane inside the column. Next, pour the sand inside the column. Add solvent followed by the sample inside the column. The sample is prepared by mixing the crude sample and silica powder until it becomes powder and sticky. After preparing the sample, pour it inside the column chromatography. Add the sand once again before adding hexane into the column until 8cm of the column. Start to collect the fraction from the column when the yellow color has been reached the bottom of the column by using the conical flask (100ml). If the solvent almost dry, add the solvent by changing the hexane to the hexane and ethyl acetate. Add 100ml solvent into the column. When adding the solvent, the polarity of the solvent system will be increased by times. 3.4.3 Spraying reagent process for thin layer chromatography
All the thin layer chromatography plate needs to be sprayed with spraying reagent so that the spot is more obvious. The spraying reagent is used in this research is vanillin. Firstly, prepare the spraying reagent (vanillin). The proper way to prepare the vanillin is called as French Formula which is vanillin in H2SO4 solution. Firstly, mix 10g of vanillin and 1L EtOH and 20ml H2SO4… After that, spray the reagent on the thin layer chromatography plate. Then, put the chromatography plate on the hot plate until the spot become colorful. Take the chromatography plate faster if it became colorful since the plate can be suspected if too hot.3.5 Spectroscopic method
A spectroscopic method will be used to analyze the organic compound. While Infrared (IR) and Nuclear Magnetic Resonance (NMR) used to elucidate the functional group and structure of pure compounds.
3.5.1 Infrared Spectroscopy (IR)
Prepare the liquid sample before running the sample. Then, place a few drops of liquid sample on the face of a highly polished salt plate such as sodium chloride and potassium bromide. After that, place the second place on top of the sample. So, it will spread over the place as a thin layer and clamp the plates together like a sandwich plate. Wipe the excess drop of the sample around the plate. Next, the sandwich plate will be clamped onto the sample holder. Lastly, clean all the sandwich plate and sample holder when it was done.
3.5.2 Nuclear magnetic resonance (NMR)
In NMR, it will irradiate the compound with electromagnetic energy of a constant frequency while using different magnetic strength. The magnetic field will reach the correct strength and at that time the nuclei will absorb energy and the resonance will have occurred. The adsorption that occurs lead to tiny electrical current to flow the receiver coil surrounding the sample. The instrument then will give data on a chart paper showing a signal that are calibrated in the unit of frequency (Hz).

3.6 Phytochemical screening
Phytochemical screening test is a qualitative method and the purpose of phytochemical screening are to conduct the presence of alkaloid, flavonoid, steroid, phenolic, tannin, terpenoid, saponin and glycosides were adapted from Yadav and Agarwala (2011) will be used in this research.

3.6.1 Test for Alkaloid
Add some of the methanol crudes into the test tube. Then, add 2ml of 1% of HCl and heat it gently. Wagner’s and Mayer’s reagent will be added into the mixture and stir the mixture. Next, observe the result. Turbidity precipitate formed shows the positive result for the presence of alkaloids. The process was repeated using the other crude which is hexane, ethyl acetate, and dichloromethane crude.
3.6.2 Test for Flavonoid
Add some of the methanol crude samples into the test tube. After that, add 2 ml of 2% of sodium hydroxide solution into the test tube and stir the mixture. Next, observe the result. An intense yellow color will be formed turn to colorless when adding a few drops of diluted acid indicated the presence of flavonoids. The process must be repeated using the other crude, hexane, ethyl acetate, and dichloromethane crude.

3.6.3 Test for Steroids
Add a few methanol crude samples into the test tube. Then, add 2 ml of chloroform and concentrated H2SO4 and stir the mixture. Next, observe the result. The red color produces in the lower chloroform layer will be indicated the presence of steroids. The process will be repeated by using the sample of another crude, hexane, ethyl acetate, and dichloromethane crude.

3.6.4 Test for phenol and tannins
Add some of the methanol crude extract using earplug into the test tube. Then, add 2 ml of 2% ferric chloride solution into the crude extract inside the test tube and stir the mixture. After that, observe the result. The blue-green to black color will be indicated the presence of phenol and tannins. The process will be repeated using hexane, ethyl acetate, and dichloromethane crude.
3.6.5 Test for terpenoidsAdd some of the methanol crudes into the test tube. After that, we will add 2 ml of chloroform and will be left to evaporate until dry. Then, add 2ml of concentrated sulphuric acid and heat for 2 minutes. Observe the result. A grayish color of the mixture indicates the presence of terpenoid. The process must be repeated using another crude, hexane, ethyl acetate, and dichloromethane crude.

3.6.6 Test for saponinAdd some of the methanol crudes into the test tube. After that, dilute the crude using 5 ml of distilled water. The mixture then must shake vigorously. Next, observe the result. The formation of stable foam will be indicated the presence of saponin. The process will be repeated using the other crude, hexane, ethyl acetate, and dichloromethane crude.

3.6.7 Test for glycoside
Add a few of the methanol crude into the test tube. Then, add 2ml of glacial acetic acid that containing 1-2 drops of 2% ferric chloride solution and stir the mixture. Next, the mixture will be poured into another test tube that containing 2ml of concentrated sulphuric acid carefully. Observe a result. A brown ring interphase shows the positive result of the presence of glycoside. The process must be repeated using the other crude sample which hexane, ethyl acetate, and dichloromethane crude.

CHAPTER 4
EXPECTED RESULT
Based on the literature review that has been done, Geniotrigona thoracica species contains two major compounds and followed by the minor compound. From the compound that contain inside the propolis, it can isolate types of flavonoids compound since there are much research shows the stingless bees are rich with the compound based on flavonoids. According to the researchs that have done by researchers before, a phytochemical screening test is expected to shows the positive result for the presence of terpenoids. Flavonoids and essential oil can be confirmed by spraying reagent anisaldehyde and vanillin-sulphuric acid. All the compound that have done be an isolated need to confirm with Infrared Spectroscopy (IR) and Nuclear Magnetic Resonance (NMR) to know their structure of a pure compound.
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