Ethnobotanical study on the traditional knowledge of medicinal plants in bac huong hoa nature reserve, quang tri province, viet nam Ethnobotanical study on the traditional knowledge of medicinal plants in bac huong hoa nature reserve, quang tri province, viet nam
The Importance of the Topic
Plant resources have a long history of being used as medicinal materials It is often cited that 80% of the world‟s population still relies on traditional medicines to meet their primary health care needs (WHO, 2008) and nearly 25% of modern medicines are derived from nature, many of which were derived from traditional uses ( De et al., 2012 ) The use of traditional medicines is generally affected by accessibility, availability and acceptability of health care services (Gaitonde & Kurup, 2005; Sato, 2012) Especially in remote areas of developing countries, medicinal plants may form the only available source of health care (Kasparek et al., 1996; Van, 2000)
Vietnam possesses a vast wealth of historical, cultural, and natural richness that includes big deltas, huge limestone towers, beautiful sand dunes, lush green forests and grass-lands The richness can also be seen in the diversity of flora which is evident with an estimated number of 13,747 vascular plant species (MONRE, 2014) To preserve the rich biodiversity of Vietnam and their sustainable development, the government has demarcated
164 special use forest areas including 30 National Parks, 58 Natural Reserves, 11 Species Conservation Areas, 45 Forest Landscape Protection and 20 Experimental Forests of Scientific Research (MONRE, 2014) The use of traditional medicine in Vietnam has a vibrant history spanning over thousands of years Today, around 75% of Vietnamese people use traditional medicine as their primary source of treatment to meet their health care needs These users mostly include inhabitants of rural or mountainous areas which have less accessibility to hospitals or community health care centers (Sam et al., 2008)
The Van Kieu ethnic minority is one of the 54 officially recognized ethnic minorities of Vietnam, whose population is around 74,500 as of 2009 (GSO, 2010), of which 55,079 (73.9%) live in Quang Tri province They are largely residing around two protected areas, Dakrong and Bac Huong Hoa Nature Reserves (Birdlife, 2013) The people belonging to the Van Kieu ethnic group are extremely poor and heavily depend on forest resources such as wood and non-timber forest products Their major livelihood activities include collection of forest products such as wild honey, bamboo shoots, animals, and mushrooms for consumption (Nguyen et al., 2015)
Contrary to the growing demand of medicinal plants all over the world, traditional knowledge is declining rapidly, especially in the developing countries (Hassan et al., 2005) Considering the vast floristic wealth of the Nature Reserve and the rich knowledge of the local community, an urgent need was felt to take over this work which could help the researchers, forest officials as well as the future generation people involved in ethno-botany research It is worth mentioning that the knowledge of Van Kieu ethnic people about the medicinal plants is vast but poorly documented.
Objectives
The need for the conservation of ethnobotanical knowledge was the core motivation for the conduct of this research with the use of quantitative techniques This method is the first ever study which documents the valuable ethnobotanical information on the medicinal plants used by local Van Kieu ethnic people of Bac Huong Hoa Nature Reserve of Vietnam
Hence, the objectives of this thesis are as follows:
- Document the medicinal plants and their use in traditional knowledge
- Compare the generated data by this study with previously published Dictionary of Vietnam Medicinal plants
- Provide information on the antioxidant, anti-inflammatory and anticancer activity via thirty methanol extracts taken from BHHNR medicinal plants.
The new points of this dissertation
3.1 First-ever study to document the traditional medicinal knowledge of the Van Kieu ethnic people from Bac Huong Hoa Nature Reserve, Vietnam
3.2 Eight medicinal plants used by Van Kieu ethnic people have not been previously reported in DVM
3.3 Provide detailed information to illuminate the complete chloroplast genome structure of Dalbergia tonkinensis and clarify the phylogenetic relationships within Papilionoideae
3.4 Adding new data on the potential treatment of anti cancer, anti in- flammation and anti oxidant of some species.
Literature review
Overview of Ethnobotany and Medicinal plant
Ethnobotany is part of the discipline ethnobiology, which studies the dynamic relationships among peoples, biota and environments (EWG (2003) The term ethnobotany was first suggested by John Harshberger in 1896 to delimit a specific field of botany and describe plant uses It was defined as
"the use of plants by aboriginal people" (Cotton, 1996) Worth noting is the concept of Mahishi et al., (2005) who depicted ethnobotany as the renaissance of traditional herbal medicine In that case, medicinal plants are botanical remedies derived from trees, shrubs, or herbaceous plants that are useful for primary healthcare system and as a remedy for disease and injury including plants used traditionally for foods and drinks that are also believed good for health (Dawit & Ahadu, 1993; Fikadu, 2001; Dawit et al., 2003) Medicinal plants play multipurpose roles such as spices and condiments (Jansen, 1981), apiculture (Fitchtl & Admsu, 1994), ecological services, source of wood and wood products as well as soil conservation in addition to their medicinal value (Legesse, 1995; Azene, 2007)
In particular, the use of medicinal plants based on traditional knowledge is one of the most widely known traditional ecosystem services, as it provides primary healthcare, contributes to subsistence livelihoods, and for its potential value as a source of novel pharmaceuticals People living in close contact with their surroundings for many generations are hypothesized to have developed, through trial-and-error, in-depth knowledge of ecosystems, biodiversity, and their management and utility (Boer et al., 2012)
Along with the definition, the discipline of ethnobotany also changed a lot during the past years and has grown from its initial focus on the use and management of plants, to the addition of socio-cultural and economic aspects and the perceptions, concepts, views and values of the local people (Balick & Cox, 1996; Cruz-Garcisa, 2014) This has led to the more multidisciplinary nature of ethnobotany that not only includes anthropology and botany, but also ecology, economics, linguistics, geography, agriculture, pharmacology, etc (Cruz-Garcisa, 2014; Martin 1995) However, ethnobotanical studies based largely on qualitative methods had all too often been just academic exercises or have served only external interests, with the results of benefiting neither local people nor conservation (Hamilton et al., 2003)
Hence, the interest in quantitative ethnobotany has developed and continuously grown steadily in the last two decades Researchers have developed and applied quantitative methods to ethnobotanical data to test different hypotheses about the relation between plant species and humans (Reyes et al., 2006) As a result, early authors (Prance et al., 1987; Phillips & Gentry, 1993) have developed indices that measure cultural significance and importance of plants quantitatively Quantitative ethnobotanical indices were also utilized to measure plant uses whether as food (Pieroni, 2001), veterinary medicine (Upadhyay, 2011), particular human body system disease remedies (Kim & Song, 2013), or economic value of goods derived from plants (Reyes et al., 2006) Although varying in subject and scope, one common purpose of these quantitative ethnobotanical studies was to determine how important these plants are to ethnic and indigenous cultures
According to the World Health Organization (1977) "a medicinal plant" is any plant, which in one or more of its organs contains substances that can be used for the therapeutic purposes or which, are precursors for the synthesis of useful drugs This definition distinguishes those plants whose therapeutic properties and constituents have been established scientifically and plants that are regarded as medicinal, but which have not yet been subjected to thorough investigation (WHO, 1977) Medicinal plants are the main ingredients of herbal products/traditional medicine It is based on the hereditary experience and made of plants materials, animals or minerals, not in the form of pure substance (Dewoto, 2007) According to Setiawan, (1990), medicinal plants are considered as parts of the plant such as leaves, stems or roots which have efficacy as a drug and is used as a raw material in the manufacture of modern either traditional medicine
The use of plants as traditional medicine is not novel but has been widely known to the public since thousands of years ago Nowadays the use of drugs derived from plants has been increasing in the community, even on the other hands there are still many synthetic drugs available from chemical processing This evidence reflected the tendency of global community back to nature (Lange, 2002) According to Zuhud (1991) medicinal plants are classified into three groups, namely:
+ Traditional medicinal plant, which is described as the plant species that are known or believed to have been used as medicine and traditional medicine
+ Modern medicinal plants which are defined as plant species that has been scientifically proven to contain bioactive compounds or medicinal ingredient and its use can be justified medically
+ Potential medicinal plant, which is related to the plant species suspected to contain or have medicinal properties but has not been proven medically
The term "Alternative Medicine" became very common in western culture It focuses on the idea of using the plants for medicinal purposes However, people currently believe that the medicines which come in capsules or pills are the only medicines that we can trust and use even that most of pills and capsules we take and use during our daily life came from plants Medicinal plant frequently used as raw materials for extraction of active ingredients The ingredients used in the synthesis of different drugs for example, in the case of laxatives, blood thinners, antibiotics, and medications is contained in plants Moreover, the active compounds of taxol, vincristine, and morphine are isolated from foxglove, periwinkle, yew, and opium poppy, respectively (Kombrink & Somssich, 1995)
World Health Organization (2007) reported that medicinal plants have a promising future because there are about half million plants that have been already identified and classified in around the world Most of them have medical activities which have not been investigated yet Furthermore, their medical activities could be decisive in the treatment of present or future studies
Vietnam‟s territory stretches over 15 latitudes from North to South, with a length of about 1,650 km on the Indochina Peninsula and a total natural land area of 330,591 km 2 Due to the bisected topography with a relatively high annual average rainfall, the network of rivers and streams is relatively dense with the river network density ranging from under 0.5-2 km/km 2 On the continental part, there are 16 major river basins, of which 10 river basins cover an area of more than 10,000 km 2 , accounting for 80% of the total country area The two largest river systems are the Red river in the North and Mekong river in the South, which create two major basins consisting of the Red river Delta and Mekong Delta The majority of Vietnam‟s territory is hilly and mountainous with the highest peak in Hoang Lien Son range being the fansipan at 3,143 m above sea level (MONRE, 2014)
The richness can also be seen in the diversity of flora which is evident with an estimated number of 13,747 vascular plant species, also on land, there are about 10,500 species of animals, including approximately 8,000 species of insects and invertebrates in the soil, nearly 500 species of reptiles and amphibians, 850 species of birds and 312 species of mammals In freshwater there are about 1,500 species of microalgae and seaweed, over 1,000 species of invertebrates and 600 species of fish; and in the sea there are more than 1,200 species of seaweed, grass and microalgae, over 7,000 species of invertebrates, 2,500 fish species and approximately 50 species of sea snakes, turtles and mammals To preserve the rich biodiversity of Vietnam and their sustainable development, the government has demarcated 164 special use forest areas including 30 National Parks, 58 Natural Reserves, 11 Species Conservation Areas, 45 Forest Landscape Protection and 20 Experimental Forests of Scientific Research (MONRE, 2014)
Traditional medicine in Vietnam dates back at least to the 2 nd Century B.C (Le Tran Duc, 1995) It has its foundations in the basis of the yin-yang principle and the interaction between human bodies and their surrounding environments Traditional Vietnamese Medicine (TVM) remedies were documented for the first time in the ten-volume "The Miracle of Southern Medicine" written in the 14 th Century by Tue Tinh, which listed approximately 500 naturally sourced materials forming the basis for over 3,800 traditional remedies, which were used to cure 182 diseases and ailments (Le Tran Duc, 1995; Pham Xuan Sinh & Phung Hoa Binh, 2002)
During the French colonial period (19 th and early 20 th Centuries), French botanists continued the research into the taxonomy of Viet Nam‟s medicinal flora when they collected information for the book "General Flora of Indochina" (Flore Générale de L‟Indochine) (Lecomte, 1907-1952) However, the French regime did not encourage the use of traditional medicine or include it in the formal healthcare system, so western medicine became dominant during this period (Pham Xuan Sinh & Phung Hoa Binh, 2002) Traditional medicine has, however, always remained very popular among Vietnamese people and today it is often used in parallel with, or in replacement of, western medicine
Traditional medicine is typically perceived to produce few or no side effects and is believed to be able to cure certain health problems that western medicine cannot It is also believed that while bacteria and viruses can develop resistance to western medicines, there has been no similar finding from studies on traditional medicines (Le Dien Duc, 1994) Traditional medicines are characterized by the use of crude herbs and prolonged usage A single herb may contain a great many natural constituents and a combination of herbs even more Since the constituents in the plants work better in conjunction with each other, it is believed that the patient is able to achieve significant long term benefits by using traditional medicine remedies (Zhang, 1998)
Today, an estimated 75% percent of Vietnamese people use traditional medicine as their primary source of treatment for common health problems (Nguyen Dao Ngoc Van & Nguyen Tap, 2008) This statistic is in keeping with the WHO, which has estimated that approximately 80% of the population in developing countries still depend on traditional medicines, particularly those made from plants (Chen & Christin, (2006)
Ethnomedicinal plants studies in Vietnam:
Tran Thien An and Ziegler (2001) have documented 432 plant species from Bach Ma National Park with mentions of their medicinal uses Tran Van
On et al., (2001) and Hoang Van Sam (2012) have documented the traditional knowledge on medicinal plants in Ba Vi National Park Van Sam et al.,
(2008) have documented 230 species from Ben En National Park with their medicinal uses Vo Van Minh et al., (2014) have documented 45 species in
Overview biologically activity of plants
Natural products, such as plants extract, either as pure compounds or as standardized extracts, provide unlimited opportunities for new drug discoveries because of the unmatched availability of chemical diversity (Cosa, et al., 2006) According to the World Health Organization, more than 80% of the world's population relies on traditional medicine for their primary healthcare needs The use of herbal medicines in Asia represents a long history of human interactions with the environment Plants used for traditional medicine contain a wide range of substances that can be used to treat chronic as well as infectious diseases (Duraipandiyan, 2006)
Cell viability can be defined as the ability of a maintained sample to show a specific function and expressed a proportion of the same function of an identical fresh untreated sample In vitro assessment of cell viability is essential to numerous aspects of basic and biomedical research Besides, it is also a fundamental tool for screening new drugs and chemicals and provides initial data prior to performing in vivo and clinical studies (Riss et al., 2003) Cell viability assays can roughly be categorized into those which analyze whole populations and those which analyze individual cells The population analysis generates faster result data However, it gives a less detailed result data than analysis on the single cell level (Stoddart, 2011) Viability may be measured as an index on a scale of 0-100% where higher index data indicates higher number of viable cells Various types of assays are available for the detection of cell viability which range from the routine trypan blue dye exclusion assay to highly complex analysis of individuals cells One of the fundamental key to decide before choosing an assay format is the information desired to be obtained at the end of a treatment period Over the years, di Methyl Thiazoldiphenyl-Tetrazoliumbromide (MTT) based assay is one of the most exploited approaches in cancer research for measuring cell viability, cell proliferation and drug cytotoxicity MTT assay involves conversion of MTT tetrazolium salt to purple formazan products by viable proliferating cells (Khor et al., 2017)
Inflammation or flogose is a reaction of the tissue blood vessels against aggressor agent characterized by access of liquids and of cells to interstice (Lope et al., 1987) The inflammatory reaction is characterized by blush, heat, tumor, pain and lost function (Dassoler et al., 2004)
There are many causes for inflammations, but the mechanisms are common to all The inflammatory agent acts in the cell membranes inducing the activation of phospholipase A2 and consequently, liberates arachidonic acid and metabolites According to Dassoler et al., (2004), the inflammatory mediators such as cytokine, histamine, serotonin, leukotrienes and prostaglandin increase the vascular permeability to all on the migration leukocytes cells to act on the site of inflamed tissue Any interruption of this sequence of events results in the reduction of the liberation of the mediators causing the microcirculation to come back to normal hemodynamic state (Lope et al., 1987)
Chronic inflammation plays an important role in the development of atherosclerosis, a progressive disease characterized by the accumulation of lipids and fibrous elements in the large arteries This inflammation is the mechanism by which the body responds to the interactions between modified lipoproteins, monocytes, macrophages, T-cells, and arterial endothelial cells (Libby, 2008) Activated leukocytes, endothelial cells, and macrophages produce proinflammatory cytokines including interleukin (IL)-1b, IL-6, as well as tumor necrosis factor-alpha (TNF-α) and anti-inflammatory cytokines, like the cytokine IL-10 (Li et al., 2005) These cells also produce proinflammatory enzymes, the inducible forms of nitric oxide sintase (iNOS) and cyclooxygenase (COX), which are responsible for increasing the levels of nitric oxide (NO) and prostaglandins (PEG2) and are known to be involved in various chronic diseases, including multiple sclerosis and colon cancer (Yan et al., 2007) The NF-κB transcription factor also plays an important role in the inflammatory response by regulating the expression of various genes encoding proinflammatory cytokines, adhesion molecules, chemokines, growth factors, and inducible enzymes, such as COX-2 (Hanada & Yoshimura, 2002) The use of plants with pharmaceutical properties has received increased interest nowadays from both the homeopathic and allopathic branches Besides, these medicinal plants play an important role in public health, especially in developing countries (Rubio et al., 2013)
Although inflammation is primarily a protective response (against microorganisms, toxins, or allergens, for example), chronic and uncontrolled inflammation becomes detrimental to tissues Since ancient times, in various cultures worldwide, inflammatory disorders and related diseases have been treated with plants or plant-derived formulations (Krishnaswamy, 2008)
Antioxidants are compounds that can delay or inhibit the oxidation of lipids or other molecules by inhibiting the initiation or propagation of oxidative chain reactions They prevent damage that can be caused by free radicals to cellular components (Javanmardi et al., 2003)
They are therefore critical for maintaining optimal cellular and systemic health and well-being The over production of free radicals can trigger chain reactions which may cause oxidative damage to sensitive biological structures, such as DNA lipids, proteins (Wang et al., 2000; Dimitrios, 2006; Naithani et al., 2006) This damage has been associated with an increased risk of cardiovascular diseases, cancer, and other chronic diseases
Spices, like vegetables, fruit, and medicinal herbs, are known to possess a variety of antioxidant effects and properties (Zheng & Wang, 2001) The antioxidant effect of phenolic compounds is mainly due to their redox properties and their capacity to block the production of ROS formed in several in vitro and in vivo systems This is the result of various possible mechanisms: free-radical scavenging activity, transition-metal-chelating activity, and/or singlet oxygen quenching capacity It has been demonstrated in in vitro and in vivo models, that the antioxidant activity of these bioactive compounds found in herbs and spices could play an important role in suppressing cell growth, viral replication, inhibiting allergy and arthritis, preventing cancer and heart diseases, and abrogating several other pathological conditions responsible for the early stages of multiple pathologies (Aggarwal et al., 2002)
DPPH is one of a few stable and commercially available organic nitrogen radicals bearing no similarity to the highly reactive and transient peroxyl radicals involved in various oxidative reactions in vivo (Huang et al., 2005; Wu et al.,
2004) This assay is based on the measurement of the reducing ability of antioxidants towards DPPH The ability can be evaluated by electron spin resonance or by measuring the decrease of its absorbance The measurement of the loss of DPPH color at 517 nm following the reaction with test compounds is what the antioxidant assays are based on Prior (Prior et al., 2005) DPPH radical, with a deep violet color, receives a hydrogen atom from the antioxidant and is converted to a colorless molecule Using this reagent, the free radical scavenging ability of the antioxidant can be determined by spectrophotometric methods (Yen & Duh, 1994)
Chen and Ho (Chen & Ho, 1995) have reported that inhibition of free radical formation by different antioxidants can be measured using very stable free radicals such as 2,2-diphenyl-1-picrylhydrazyl Using the DPPH assay has its advantages It is simple, rapid and needs only a UV vis spectrophotometer to carry out the experiment
Cancer is the second largest single cause of death claiming over six million lives every year worldwide (Loizzo et al., 2008) There are more than
100 different diseases can be considered as cancer, in all cases, certain body cells multiply in an abnormal and unregulated manner When normal cells begin to grow abnormally and reproduce too rapidly, resulting in a mass of abnormal cells eventually develops, which is called a tumor If the tumor cells localize at the original site in body and slowly multiply, that tumor is called benign Benign tumors such as cysts, warts, moles and polyps do not spread to other parts of the body and are not life threatening Benign tumors can be removed by surgery and will not regrow after surgical excision; thus the patients will recover fully from the tumors In contrast, malignant tumors (called cancers) are consisted of cells that grow rapidly and invade other normal tissues (Johnson, 2003)
There has been a recent upsurge in the use of natural products to supersede current treatment in patients that develop multidrug resistance Scientific studies of plants used in various types of ethnic medicine has led to the discovery of many valuable drugs, including taxol, camptothecin, vincristine and vinblastine (Heinrich & Bremner, 2006; Newman & Cragg, 2007) Plant products have been used by many individuals globally either to prevent or cure cancer, or to alleviate the accompanying symptoms In Vietnam, the best example of a traditional medicine plant used for treatment of cancer would probably be Momordica cochinchinesis seeds and fruits which are developed a drug, Gacavit The drug is said to reinforce the immune system, repair DNA and used as cancer medicine (Beer, 1993) Moreover, Momordica cochinchinesis fruits have also exhibited significant effects on prostate cancer, human cervical epithelial, human embryonic kidney and human small cell, lung cancer cell lines (Chuethong et al., 2007) Various chemical compounds isolated from plants have led to the development of effective anticancer agents used today in the clinical setting Amongst them, polyphenols have received much more attention for health benefits including anticancer activities Many research carried out in different cell lines, animal models and human epidemiological trials showed that polyphenols can use for prevention of different types of cancer (Wenzel et al., 2000; Yang et al., 2000)
Previous researches showed that anthocyanins suppressed the growth of colon cancer cells in vitro (Kamei et al., 1995) Quercetin, kaempferol and myricetin inhibited chemically induced cancers of the lung, tongue, colon, mammary glands and mouth in rats and mice (Hannum, 2004); human prostate and breast cancer cells (Kampa et al., 2000; Damianaki et al., 2000) Ellagic acid inhibited the development of chemically induced cancers of the lung, esophagus, skin and liver of rats, mice and human breast cells (Smith et al., 2001) Glucosinolate induced phase 2 enzymes activity that detoxified carcinogens (Fahey & Stephenson, 1999).
Materials and Methods
General description of the study area
The study was conducted in Bac Huong Hoa Nature Reserve (BHHNR), located in Central Vietnam The study area covers 25,200 hectares, including 20,646.2 hectares of natural forest and is located in the north of Huong Hoa
District of Quang Tri Province, 50 km north-west of Khe San town, and 120 km west of Dong Hai town (16°43'22″ N to 16°59'55″ N latitude and 106°33'00″E to 106°47′03″ E longitude) (Figure 2.1) The Nature Reserve is bisected by the Ho Chi Minh highway which encompasses the territory of five communities, namely Huong Linh, Huong Son, Huong Phung, Huong Viet and Huong Lap To the north, it is bordered by Quang Binh Province and to the east, it is bordered by three districts, Vinh Linh, Gio Linh and Dakrong (Mahood & Tran, 2008)
Figure 2.1 Location of the study area
Bac Huong Hoa Nature Reserve encompasses low lying land to the south of the northern section of the Annamite Range, and a ridge of 1,000 m which runs in a northwest-southeast direction along the boundary between the provinces of Quang Binh and Quang Tri (Mahood & Tran, 2008) The forest on the Quang Binh side of this ridge is dominated by low lying areas with slopes of 15 to 25 degrees, although there are some steeper areas On the other side of the ridge in BHH NR, low-lying land and a number of limestone peaks are present, such as Sa Mu Cave at 1,550 m and Lying-down Elephant Mountain, so named due to its shape, at 1,771 m Although predominately low in elevation, the land is hilly and slopes are steep Another limestone ridge runs in a west-east direction, on the boundary between Huong Lap and Huong Viet communes and near the center of Huong Viet Commune and a further run in a south-north direction
Bac Huong Hoa Nature Reserve is located in a region with a tropical monsoon climate It is hot in summer and cooler in winter Since the central Annamite range which runs along the western edge of BHH NR is relatively high and orientated almost perpendicular to the north-east and south-west monsoons, the nature reserve receives a considerable amount of rain There is rain in summer and autumn-winter; the dryer period lasts only 2-3 months Most rain falls during August, September, and October The nature reserve also receives the "Phon", a warm dry wind originating in Laos, during the months of March-June The average annual temperature is 24-25°C, however monthly temperature variation is considerable December and January are the coolest months when temperatures drop to 20 and sometimes as low as 15 in areas above 500 m In contrast, in June and July temperatures average 29 and regularly reach 39 when the Phon is blowing (Mahood & Tran, 2008)
Annual rainfall totals for BHHNR are between 2400 and 2800 mm There are two main rainy seasons, the first lasts from August to November and accounts for nearly 50% of the annual rainfall The second rainy season lasts from April to August and is characterised by less heavy rain Between February and March there is light rain and December to January is the driest period High levels of rainfall on unstable slopes lead to frequent landslides in BHHNR, particularly along the Ho Chi Minh Highway Low lying areas also receive occasional partial flooding In the context of humidity, there are two seasons The humid season lasts from August to May, during these months the humidity is 85-90% In June and July, the Phon causes the humidity to decrease, sometimes to less than 30%
Rivers in BHHNR are mostly short and relatively steep They flow from the Central annamites to the east sea in an east or northeast direction The following are the main rivers of the area (Mahood & Tran, 2008)
The Ben Hai river is located in the northeast of BHHNR from where it flows into the sea at the Cua Tung estuary All streams which have their origins in the eastern side of the nature reserve flow into the Ben Hai river
- In the north-east and south of the nature reserve is the Xe Pang Hieng river It flows into Laos and down to the Mekong river
- The Cam Lo river has its source on the northern slope of Lying-down Elephant Mountain It flows into the sea at the Cua Viet estuary
- The Rao Quan river flows from the southern part of the nature reserve out to the Thach Han river A hydropower plant is being constructed on the Rao Quan river
The Van Kieu ethnic minority is one of the 54 officially recognized ethnic minorities of Vietnam, whose population is around 74,500 as of 2009 (GSO, 2010), of which 55,079 (73.9%) live in Quang Tri province They are largely residing around two protected areas, Dakrong and Bac Huong Hoa Nature Reserves (Birdlife international) The people belonging to the Van Kieu ethnic group are extremely poor and heavily depend on forest resources such as wood and non-timber forest products Their major livelihood activities include collection of forest products such as wild honey, bamboo shoots, animals and mushrooms for consumption (Nguyen et al., 2015).
Data collection
The permission was obtained from Quang Tri Forest Protection Department (FPD), BHHNR to conduct our research (Permission No.: 857/UBND-NN, 230/SNgV-LSVK) The survey was conducted from April
2016 to March 2018 A total of 93 people were interviewed belonging to Huong Linh, Huong Son, Huong Phung, Huong Viet and Huong Lap Communities (Figure 2-1) Prior informed consent was obtained for all interviews conducted Since all of the interviewees speak Vietnamese, the interviews were conducted in Vietnamese and subsequently translated into English For this survey, the interviewees were selected based on the recommendations and introductions by the authorities of BHHNR and local village leaders The informants were mainly farmers, housewives, healers, labourers (forest) and headmen of villages, who have used or using the traditional medicinal plants of Van Kieu
Purposive stratified sampling was the method used in choosing the informants, who accepted the request for interview in their own free will Tongco, (2007) noted that methods in informant selection need to be actively discussed Purposive sampling is a practical and efficient tool when used properly and can be just as effective as, and even more efficient than, random sampling Moreover, when information is retained by specific members of a community, it is advisable to use an intentional sample, which will save time especially when information is not equally distributed and some potential informants may even be excluded This type of selection could be applied when the parameter being studied is not shared by all of the members of a community
Etkin et al., (1990), explained that knowledge of medicinal plants is carried in varying amounts by almost everyone in the community, and focusing only on specialists, such as herbalists or shamans, may give a biased view of plants utilized by a group of people Limiting respondent population to specialized healers would also negate the fact that people turn to family and friends for medical information before turning to specialized healers
In addition, depending on the research objectives and the financial and time resources available, ethnobiology studies can be oriented towards different types of informants These informants may be chosen according to criteria such as age (e.g children, adolescents and adults), gender, local recognition of expertise (i.e people with increased knowledge about specific subjects) and generalist knowledge (i.e people from the community or region who are non experts) and social roles (e.g shamans and heads of families) when it is not possible or desirable to obtain the participation of the whole population Commonly, researchers opt to conduct interviews with heads of families (one for each family unit) to optimize the efficiency of their fieldwork based on the assumption that these individuals represent the knowledge of their family unit (Arausjo et al., 2012)
2.2.2 In-depth semi-structured interview
Each participant was interviewed in isolation to avoid the possibility of one informant‟s answer influencing another‟s answer The informants were asked about their personal experience or participation in the use and preparation of medicinal plants At times, pictures were shown to informants to make sure that they could identify what plant was being asked about
At first, open interviews were carried out, in which people were interviewed through informal conversation The aim of such interviews was to make them speak freely, without any form of pressure, in order to obtain a list of medicinal species and natural remedies that they use or have used in the past The second round was of semi-structured interviews, to obtain specific quantitative and qualitative information and to gain a wide range of the insights on specific issues When possible, walks in the countryside were organized to collect plant specimens and to complete the list of plant uses known by the informants A written questionnaire was used to record information concerning name, age, gender, address, level of education of the interviewees along with place and date of interview The parameters like medicinal properties mentioned by the informants, the vernacular name, the plant parts used, the gathering period and sites, the frequency of use, the attributed medicinal properties, the efficacy of preparations and the methods of preparation were listed
An essential activity associated with ethnobotanical interviews is the collection of plant voucher specimens The use of herbarium specimens ensures that future researchers and others can verify results and make useful comparisons (Alexiades & Sheldon, 1996) Full botanical documentation is achieved by collecting voucher specimens and depositing them both in an internationally accessible herbarium (like the national herbarium of a country) and for local access also in regional herbaria (Heinrich et al., 2009)
The collected plants were identified using "An Illustrated Flora of Vietnam" (Ho P.H., 2000) and DNA sequencing Scientific names of the plant species were determined according to the Plant List (http://www theplantlist.org) and voucher specimens were deposited at the herbarium of Korea Research Institute of Bioscience and Biotechnology (KRIB) in Republic of Korea and in the herbarium of Vietnam Academy of Science and Technology (HN); Herbarium of Museum of Biology, VNU University of Science (HNU) in Vietnam
2.2.4 DNA extraction, PCR, and sequencing
Specimens which could not be morphologically identified due to unfamiliarity or lack of reproductive parts were determined molecularly by DNA sequencing in Korea, and by comparing the sequences with those found in the GenBank through BLAST search This molecular barcoding method provides consistent and reliable results regardless of the age, plant part, or environmental factors of the sample (Techen et al., 2014) DNA extractions from silica gel-dried leaves were done using QIAGEN DNeasy Plant Mini Kit
PCR amplification of the four fragments including ITS, matK, trnH- psbA and rbcL were selected (Table 2.1) PCR reaction system was optimized and modified based on the recommended protocol (CBOL, 2009) All PCR products were separated by agarose gel electrophoresis and purified using
QIAQuick PCR Purification Kit All steps were done following the manufacturers‟ instructions Product concentration was determined by visual comparison Purified products were then sent to Macrogen Inc., Korea for DNA sequencing reactions (ABI sequencer, Applied Biosystems)
Table 2.1 Information of the four DNA marker
ITS ITS1 TCC GTA GGT GAA CCT GCG G White et al.,
ITS4 TCC TCC GCT TAT TGA TAT GC White et al.,
(1990) matK 390F CGA TCT ATT CAT TCA ATA TTT C Cuenoud et al.,
1326R TCT AGC ACA CGA AAG TCG AAG T Cuenoud et al.,
(2002) trnH- psbA trnH CGC GCA TGG TGG ATT CAC AAT Tate and
Simpson, (2003) psbA GTT ATG CAT GAA CGT AAT GCT C Sang et al (1997) rbcL Z1 ATG TCA CCA CAA ACA GAA ACT
1351R CTT CAC AAG CAG CAG CTA GTT
Data analysis
2.3.1 Chloroplast genome sequencing and assembly
Total genomic DNA was extracted from 20 mg of dried leaves of D tonkinensis were collected from a single individual using DNeasy Plant Mini Kit (QIAGEN, German) following manufacturer‟s instructions The total genomic DNA was sequenced using the Illumina (San Diego) Hiseq 2500 platform in Macrogen (Seoul, Korea) The pair-end library comprised 43,285,230 reads (length 2 x 101 bp) Paired reads were processed using
„Trim using BBDuk' (Bushnell, 2016) The trimmed paired-read set was assembled using the Geneious de novo assembler
De novo assembly generated 565,298 contigs The draft chloroplast genome of 156,086 bp achieved by identifying the repetitive IR sequences at 131,558 bp contig suspected of chloroplast genome with a mean coverage of 287.8 X
The gene annotations of chloroplast genome sequence of D tonkinensis were performed by using software Geneious v 11.1.5 (Biomatters Ltd., Auckland, New Zealand) via comparison with chloroplast genome of D hainanensis Merr et Chun (MF926268) and D odorifera T Chen
The annotated GenBank file was used to draw the circular plastid genome map with the online program Organellar Genome DRAW (OGDRAW) (Lohse et al., 2013) The annotated chloroplast genome sequence was submitted to the GenBank under the accession number of MK599470
The ethnobotanical data were analyzed using Microsoft Office Excel Spreadsheet 2010 The categories suggested for diseases were classified according to International Classification of Primary Care (ICPC; http://www who.int/ classifications/ icd/adaptations/icpc2/en/) system for intercultural comparisons (Soler et al., 2008; Staub et al., 2015)
The categories are (1) Eye, (2) Musculoskeletal, (3) Endocrine/ Metabolic and Nutritional diseases, (4) Pregnancy, Childbearing, Family Planning, (5) Female Genital, (6) Urological, (7) General and Unspecified, (8) Skin, (9) Respiratory, (10) Digestive, (11) Cardiovascular, (12) Neurological, (13) Psychological, (14) Blood, Blood Forming Organs and Immune Mechanism
Informant Consensus Factor (ICF) is used to check an agreement in the use of a plant species in particular ailment category by the users It was calculated by using the following formula
ICF = (Nur - Nt)/(Nur - 1), where Nur is the number of use reports in a particular category and Nt is the number of plant species recorded in the category (Trotter & Logan, 1986)
ICF values range from 0 to 1; values close to 1 indicates relatively few taxa are used by a large population of healers or there is a well defined criterion in the community about the use of particular plant species The low value (close to 0) indicates informants disagree about the use of taxa in the said treatment category (Heinrich et al., 1998; Gazzaneo et al., 2005).
Screening of ethnomedicinal plants for biological activities
Dried plants were pulverized using a cutting mill, followed by extraction using methanol solvents Briefly, plant powder (10 g) was subjected to extraction for 24 hours with 100 ml of solvent in a glass conical flask on a shaker at room temperature, followed by filtration through filter paper (No 1, Whatman International LTD, Maidstone, England) The residue was then extracted twice more with 100 ml of solvent as described above The combined solvent extracts were concentrated using a rotary evaporator (Heidolph VV 2011-Antrieb, Heidolph Instruments GmbH & Co KG, Schwabach, Germany) at 40°C under a vacuum to obtain dry extracts The extracts were stored at -5°C until use
To evaluate the effects of extracts on the cell viability, a Cytotoxicity assay was performed The cancer cells (AGS, A549, HCT116, MCF7, HepG2) were seeded onto 96-well plate at 1 x 105 cells/ml well then, cultured in 5% CO 2 incubator at 37℃ for 24 hrs and cells were treated with final concentration 40 ug/ml of extracts The cells were incubated for an additional
24 hrs., and the medium was replaced with 180 ㎕ fresh mediums contained
20 ㎕ of 0.5 ㎎ / ㎖ 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) (Sigma Co.) solution in each well The absorbance was measured at 570 ㎚ using microplate reader (Figure 2.2)
Figure 2.2 The general procedure of anti-cancer assay
Cells were cultured in DMEM supplemented with 5% of FBS (Fetal Bovine Serum) incubated 4 hr at 37°C and 5% CO 2 RAW 264.7 macrophages were seeded in 96-well plates at density of 1 × 10 4 cells After 4h of incubation, the adhered cells were treated with 50 ug/ml extract, after 1h treatment, cells were incubation with 0.5 μg/ml of LPS (lipopolysaccharide, Sigma) for 24 h and 100 μL of cell culture medium with an equal volume of Griess reagent (Sigma) in a 96-well plate was incubated at room temperature for 10 min Then the absorbance was measured at 540 nm in a microplate reader (Figure 2.3).
Figure 2.3 The general procedure of nitric oxide assay
Cells were cultured in DMEM supplemented with 5% of FBS (Fetal Bovine Serum) incubated 4 hr at 37°C and 5% CO 2 RAW 264.7 macrophages were seeded in 96-well plates at density of 1 × 10 5 cells/well and 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay was performed to measure the cell viability
Briefly, the cells were washed with PBS and incubated (Thermo Scientific, Waltham, MA, USA) with PE to concentrations of 100 μl/ml at
37°C and 5% CO 2 The RAW264.7 macrophage cells were plated in 96-well plates in growth medium at a density of 1 × 10 5 cells/well and grown for 24 h in serum-free medium in the presence of different sample concentrations Then, 10 μl of MTT (5 mg/ml) was added to phosphate buffer followed by further incubation for 4 h at 37°C After supernatant removal from wells, 100 μl, of buffer was added for dissolution of formazan crystals The absorbance of each well was then read at 570 nm using a Microplate reader (Figure 2.4)
Figure 2.4 The general procedure of MTT assay
The antioxidant activity of the extracts was measured on the basis of the scavenging activity of the stable DPPH free radical according to the method described by Brand-Williams et al., (1995) with slight modifications 1ml of 0.1 mM DPPH solution in methanol was mixed with 1ml of plant extract solution of concentrations 10 àg/ml Corresponding blank sample were prepared and DMSO 10 àg/ml was used as reference standard Mixing of 1 ml methanol and 1 ml DPPH solution was used as control
The reaction was carried out in triplicate and the decrease in absorbance was measured at 517 nm after 15 minutes in dark using UV-Vis spectrophoto-meter (Figure 2.5)
The scavenging effect (%) was calculated using the following formula Scavenging effect (%) = A contol - A sample /A contol ×100
Where: A conto is the absorbance of the control
A sample is the absorbance of the sample
Figure 2.5 The general procedure of DPPH radical assay
Results and Discussion
Data analysis
Total 356 use-reports were documented in this study The species with the high use-reports (UR) were Artocarpus heterophyllus Lam (14 UR), Chromolaena odorata (L.) R.M.King & H.Rob (13 UR), Blumea balsamifera
(L.) DC., Psidium guajava L and Catunaregam spinosa (Thunb.) Tirveng (9 UR), Mussaenda hoaensis Pierre ex Pit., Citrus aurantium L and Jatropha curcas L (8 UR) (Table 3.3)
Artocarpus heterophyllus (Figure 3.5) has long been recognized as an economically important edible fruit i.e Jackfruit (Verheij & Coronel, 1992) The Van Kieu people use this species to treat postpartum depression, lactation symptoms, fever and abdominal pain and as an antidote In Thailand and Laos, Jackfruit appears to be one of the traditionally used plants, of which the fruits, barks and leaves are consumed for its galactagogic properties (Lamxay et al., 2011; Buntuchai et al., 2017) Though the mechanism of galactagogic herbs is not much known, they are supposedly oxytocic in nature that stimulate blood flow to the breasts and further providing a rich source of fatty acids, or directly increasing breast tissues As a result, milk production was increased (Westfall, 2003)
Figure 3.6 Chromolaena odorata (L.) R M King & H Rob
Similarly, Chromolaena odorata (Figure 3.6) is used to treat abdominal pain, diarrhoea, bleeding, irregular menstruation and female genital diseases by the local communities This species seems to be a widely used plant for wound healing and hemostasis by various ethnic groups in the whole Indo-China region (Srithi et al.,
2009; Inta et al., 2013; Chassagne et al., 2016) According to Pandith et al., (2013), the leaf extract of C odorata treatment to Balb/c 3T3 fibroblast cell increased the transcript and protein levels of the wound healing enzyme, heme oxygenase-1 (HO- 1) to accelerate the cell proliferation and migration This study supported the properties of C odorata for hemostatic and wound healing
The Van Kieu people also use the leaves of Blumea balsamifera (Figure 3.7) for the treatment of rheumatism According to physiological activity studies, its crude extracts, as well as its isolated constituents, display numerous biological activities including the anti-inflammatory properties (Pang et al., 2014; Fan et al., 2015) Borneol has been found as one of the major active compounds of B balsamifera (Hao et al., 2000; Du et al., 2009; Chu et al., 2013) which possesses antinociceptive, anti- hyperalgesic and anti-inflammatory activities (Almeida et al., 2013; Jiang et al., 2015) In addition, the essential oils of B balsamifera are found to promote anti- inflammatory activity by reducing levels of IL-1 and TNF-α (Fan et al., 2015).
P guajava L family Myrtaceae (Fig 3.8) is found in tropical and subtropical countries (Ravi & Divyashree, 2014) The Van Kieu people use this species to treat abdominal pain P guajava is an important food crop and medicinal plant in tropical and subtropical countries is widely used like food and in folk medicine around of the world (Gutiesrrez et al., 2008) Many pharmacological studies have demonstrated the ability of this plant to exhibit antioxidant, hepatoprotection, anti- allergy, antimicrobial, antigenotoxic, antiplasmodial, cytotoxic, antispasmodic, cardioactive, anticough, antidiabetic, antiinflamatory and antinociceptive activities, supporting its traditional uses (Gutiesrrez et al., 2008; Morais-Braga et al., 2016)
Figure 3.9 Mussaenda hoaensis Pierre ex Pit
Mussaenda hoaensis Pierre ex Pit (Figure 3.9) belonging to the family
Rubiaceae is a flowering shrub which is distributed in endemic to Vietnam
Mussaenda genus is an important source of pharmacologically active natural products, particularly iridoids, triterpenes and flavonoids Their medicinal activities includes diuretic, antipyretic and effective in laryngopharyngitis, acute gastroenteritis and dysentery and also anti-fertility activity (Gunasekaran et al., 2015) However, no studies have been made on
M hoaensis Van Kieu people use this species to treat ibromyoma uterus, gonorrhoea female, vaginitis and fever, and they are also used as cambine with other species
The ailments reported by informants were grouped into 14 categories (Table 3.5) The highest values of Informant Consensus Factor (ICF) were recorded for Eye diseases (ICF = 1.0, included eye injuries and other), followed by Musculoskeletal disorders (ICF = 0.76, included musculoskeletal and rheumatic disorders), endocrine/metabolic and nutritional disorders (ICF
= 0.67) and Pregnancy problems such as childbearing, family planning (ICF = 0.65)
Eye diseases, endocrine/metabolic and nutritional disorders category had less UR and plant species, so it was exhibited high degree of consensus with ICF values
The highest UR and plant taxa used were recorded for digestive problems (which included abdominal pain, teeth/gum diseases, diarrhoea etc.) viz 94 reports and 43 species The general and unspecified category (fever, bleeding and health maintenance) was represented by 70 reports and 28 species followed by skin related problems (scabies, Acne etc.) which were represented by 57 reports and 23 species
Table 3.5 Informant consensus factor for commonly used medicinal plants
No Category Number of taxa (Nt)
14 BL = Blood, Blood Forming Organs and
3.2.3 Rare plants in IUCN Red List
Out of 112 plants studied from this region, three plant species have been listed in the IUCN Red list (IUCN, 2018) viz Dalbergia tonkinensis
Prain as Vulnerable (VU), Cinnamomum balansae Lecomte as Endangered (EN) and Aquilaria crassna Pierre ex Lecomte as Critically Endangered (CR) (Table 3.3)
Dalbergia tonkinensis (Figure 3.10) was distributed in Vietnam and Hainan Island of China (Nguyen et al., 2018) The plant has dense reddish brown colored heartwood, with a distinct aroma (Nguyen H.N., 2008; Do et al., 2009) The fragrant incense can be distilled for medicinal purposes (Pham et al., 2014) and the wood is very popular for processing into high quality furniture (Nguyen H.N., 2008;
Do et al., 2009; Tran N.H., 2006) The Van Kieu people used D tonkinensis for the treatment of toothache and insect bite There are several studies which include D tonkinensis for isolation and identification of various constituents and their physiological and antimicrobial activities (Nguyen et al., 2017; Ninh et al., 2017; Ninh et al., 2018)
Cinnamomum balansae (Figure 3.11) is endemic to Vietnam The seeds of C balansae contain fatty oils which can be used in food (Hai et al., 2017) The species was also used to treat rheumatism in Vietnamese traditional medicine (Do, 1991) The Van Kieu people used this species against post-partum symptoms
Aquilaria crassna (Fig 3.12) is endemic to Indochina region and known to be used in diverse Southeast Asian traditional medicine systems to treat infectious and inflammatory diseases, arthritis and cardiac disorders (Dahham et al., 2015) The Van Kieu people use the root of this species to treat fever and abdominal pain Studies on A crassna revealed that the extracts from leaves and stems possess antioxidant, antimicrobial, cytotoxic, antipyretic, analgesic, antiischemic, laxative and digestive properties (Hideaki, 2012; Jermsri et al., 2012; Sattayasai et al., 2012; Kamonwannasit et al., 2013)
Figure 3.12 Aquilaria crassna Pierre ex Lecomte
It is necessary to inform the Van Kieu people about medicinal properties of aerial parts of the species which will reduce uprooting of already critically endangered species from this region
3.2.4 Complete Chloroplast genome sequencing of Dalbergia tonkinensis Prain
The complete chloroplast genome of D tonkinensis has a total length of
156,086 bp, with a pair of inverted repeats (IRs) of 25,720 bp that separate a large single copy (LSC) region of 85,761 bp and a small single copy (SSC) region of 18,885 bp (Figure 3.13) The chloroplast genome of D tonkinensis contained 131 genes including 86 protein-coding genes, 36 tRNA genes and 8 ribosomal RNA genes Among these genes, 15 genes (atpF, ndhA, ndhB, petB, petD, rpl2, rpl16, rpoC1, rps12, ycf68, trnA-UGC, trnI-GAU, trnK-UUU, trnL- UAA, trnV-UAC) have one intron and three genes (clpP, rps12, ycf3) have two introns The ycf1 gene was inferred to be pseudogenes Most of the genes occurred as a single copy, however, six protein-coding genes (ndhB, rpl2, rpl23, rps7, rps12, ycf2), seven tRNA genes (trnA-UGC, trnI-CAU, trnI-GAU, trnL- CAA, trnN-GUU, trnR-ACG, trnV-GAC) and four rRNA genes (rrn16, rrn23, rrn4.5, rrn5) in the IR regions were duplicated The overall G+C content of D tonkinensis chloroplast genome is 36.1% and the corresponding values in LSC, SSC and IR regions are 33.5, 29.4 and 42.8%, respectively
Phylogenetic analysis was performed using chloroplast coding sequences (CDS) of D tonkinensis and those of 35 related species of Papilionoideae de
Candolle including Cercis glabra Pamp, as an outgroup These chloroplast genomes were aligned by using MAFFT (Katoh & Standley, 2013) Maximum likelihood tree was generated using IQ-TREE web server (Nguyen et al., 2015) based on TVM +F +R3 model with ML + ultrafast bootstrap (Minh et al., 2013)
Screening of ethnomedicinal plants for biological activities
3.3.1 Screening of ethnomedicinal plants for anti-inflammatory activity
Inflammatory is a tissue response to harmful stimuli, such as pathogens and damaged cells or irritants thereby, inheriting to the pathogenesis of a variety of diseases (Koh et al., 2010; Dudhgaonkar et al., 2009)
Macrophages are known to be one of the critical immune cells in the regulation of inflammatory responses LPS-activated macrophages, have usually been used for evaluating the anti-inflammatory effects induced by various materials which secrete inflammatory mediators such as nitric oxide (NO), prostaglandin (PG)E2 and pro-inflammatory cytokinin including interleukin-6 (IL-6), tumor necrosis factor (TNF-α), etc Lipopolysaccharide (LPS) is a prototypical endotoxin derived from Gram-negative bacterial membrane and can act as initial stimulus to induce septic shock syndrome (Chun et al., 2006; Jo et al., 2008; Chae et al., 2011)
3.3.1.1 Screening of ethnomedicinal plants for cell viability
Before investigating the NO activities, the effect of the 35 methanol extracts on the viability of RAW 264.7 cells were evaluated
As shown in Table 3-6, Mussaenda hoaensis extract was showed the highest cell viability (132.73 ± 0.14) on RAW 264.7 cells and Catunaregam spinosa (97.64 ± 0.7), Connarus semidecandrus (96.50 ± 0.85), Justicia gendarussa (94.85 ± 2.49), Fibraurea recisa (93.11 ± 9.64) and Citrus aurantium (90.31 ± 1.1) showed significantly high cell viability On the other hand, Eurycoma longifolia (7.49 ± 2.53), Crinum asiaticum (10.65 ± 0.58),
Homonoia riparia (11.55 ± 2.04), Lantana camara (11.94 ± 3.7) and
Elephantopus scaber (19.99 ± 1.46) showed significantly low cell viability
These species are needed to study cytotoxic effect analysis in the near future for the development of anti-cancer drug
Table 3.6 Results of cell viability in RAW 264.7 cells from selected 35 extracts
No Family Scientific name Cell viability in RAW
NO production was determined by Griess reagent assay The NO production was measured in the medium of RAW 264.7 cells cultured with LPS in the presence of methanol extracts Six of the 35 plants screened had anti-inflammatory activity above 80% (Table 3.7)
Homonoia riparia extract was showed the highest NO inhibition activity (104.23 ± 0.25) and Alstonia scholaris, Mussaenda hoaensis and
Gelsemium elegans extracts were showed significantly high NO inhibition activity, corresponding to 99.56 ± 5.47, 98.07 ± 0.13, 96.57 ± 6.83, but reduced the cell viability in RAW 264.7 cells except M hoanesis extract
NO inhibition activity was more than 40%, and cell viability rate of more than 60% were Fibraurea recisa, Blumea balsamifera, Mussaenda hoaensis, Embelia ribes and Catunaregam spinosa medicinal plants So, on the basis the current results, it can be suggested that F recisa, B balsamifera,
M hoaensis, E ribes and C spinosa medicinal plants can be used as a potent anti-inflammatory activity
Fibraurea recisa Pierre has traditionally been used in the treatment of gynaecological inflammation and surgical infection (Xing et al., 2018) The Van Kieu people use this species to treat diarrhoea and abdominal pain
Blumea balsamifera (L.) DC, belonging to the Asteraceae plant family, is a perennial herb or subshrub distributed mainly in Southeast Asia (Perry & Metzger, 1980) The Van Kieu people use the leaves of B balsamifera for the treatment of rheumatism The major constituents of B balsamifera have been reported to be sesquiterpenoids and flavonoids, which displayed antitumor, antifungal, plasmin inhibitory, anti-obesity, free-radical-scavenging, and nitric oxide (NO) inhibitory effects (Ma et al., 2017)
Table 3.7 Results of NO inhibition activity in RAW 264.7 cells from selected 25 extracts
No Family Scientific name NO inhibition activity
3.3.2 Screening of ethnomedicinal plants for antioxidant activity
The 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging assay is one of the most popular antioxidant assays used by almost 90 % of antioxidant studies (Moon & Shibamoto, 2009) The DPPH radical scavenging activity of methanol extracts derived from medicinal plants is shown in Table 3-8 Homonoia riparia extract showed the highest antioxidant activity (75 ± 1.2) and Vernicia fordii
(65 ± 0.8), Psidium guajava (46 ± 0.1) and Macaranga denticulata (41 ± 2.9) showed high antioxidant activity
Homonoia riparia Lour (Figure 3.16) is small shrub which is distributed throughout China, India, Thailand, Philippine, and Vietnam at lower elevations on the river banks of rivers and streams especially among rocks (Shiddamallayya et al., 2010) Van Kieu people use this species to treat abdominal pain Similarly, Xavier et al., (2017), reported that extracts of H riparia was found to be antioxidant activity
Table 3.8 Results of DPPH radical scavenging activity from selected 35 extracts
No Family Scientific name Antioxidant activities
3.3.3 Screening of ethnomedicinal plants for anticancer activity
Cancer, also known as “tumor”, is a multi-step disease induced by various environmental, chemical, physical, metabolic and genetic factors (Ryu, 2011) It is the second largest single cause of death claiming over six million lives every year worldwide (Loizzo et al., 2008) Apoptosis or programmed cell death is an important way not only to maintain cellular homeostasis between cell division and cell death but also, removes potential cancer cells However, the cancer cells when already developed, have an acquired capability to evade apoptosis by various ways Recent research is an effort to find natural resources with anticancer activity because of their safety and low toxicity Many researchers have concentrated on especially therapeutic medicinal plants to screen more effective anticancer agents and to elucidate the mechanisms of cancer prevention and apoptosis (Reddy et al., 2003; Dai & Mumper, 2010)
The cytotoxicity of 29 methanol extracts medicinal plants was undertaken by MTT assay using human cancer cell lines (A549, AGS, HepG2, MCF7).
Table 3.9 Results of anticancer activities on A549, AGS, HepG2 and MCF7 cell lines from selected 29 extracts
3.3.3.1 Anticancer activity on lung cancer cell line
The results relating to cytotoxicity on human lung cancer cells (A549) are shown in Table 3.9 Fibraurea recisa extract was showed the highest anticancer activity (60.86) on A549 cells In addition, stomach cancer activity experiment were showed high activity (35.83) Cissus quadrangularis (54.51), Artocarpus heterophyllus (52.9), Elephantopus scaber (48.1), Spatholobus suberectus (46.64) and Eurycoma longifolia (45.3) were showed significantly high anticancer activities on A549 cells
Fibraurea recisa Pierre (Menispermaceae, Figure 3.17) is an autochthonous medicinal plant distributed in the southeast of Asian, such as China, Vietnam and Laos (JNMC, 1977) Previous phytochemical studies on this plant resulted in obtaining major components including palmatine and other related alkaloids jatrorrhizine, berberine and columbamine, some diterpenoids were isolated as well (Fu et al., 2007) The Van Kieu people use this species to treat diarrhoea and abdominal pain It was also high activity in anti-inflammatory this study experiment
3.3.3.2 Anticancer activity on stomach cancer cell line
The results relating to cytotoxicity on human stomach cancer cells (AGS) are shown in Table 3.8 Lantana camara extract was the showed the strongest cytotoxic activity (51.6, 43.2) on AGS cells and liver cancer cells (HepG2) and Cissus quadrangularis (41.34), Choerospondias axillaris (41.3), Macaranga denticulata
(39.94), Crinum asiaticum (38.29), Fibraurea recisa (35.83) and Scoparia dulcis
(35) were showed high anticancer activities on AGS cells
Lantana camara (Figure 3.18) complex are toxic to small ruminants and this effect has been associated with the types and relative amounts of some triterpene ester metabolites (Sharma et al., 1988) However, L camara also produces a number of metabolites in good yields and some have been shown to possess useful biological activities (Ghisalberti, 2000) It was also found in high activity in Anticancer (stomach cancer, liver cancer) experiments in this study
3.3.3.3 Anticancer activity on liver cancer cell line
The results relating to cytotoxicity on human liver cancer cells (HepG2) are shown in Table 3-8 Lantana camara extract was showed the highest cytotoxic activity (43.2) on HepG2 and Gelsemium elegans (39.7), Crinum asiaticum (37.02), Elephantopus scaber (35.8) and Cissus quadrangularis
(27.66) were showed high anticancer activities on HepG2 cells