A summary of the PhD thesis: Researching and evaluating on the resources of plant containg essential oils at Ben En national park, Thanh Hoa province; proposing the conservation measures and rational exploitation

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Researching objectives: Evaluating the diversity and using values of essential oil plants at Ben En National Park, Thanh Hoa province; determining the content and composition of essential oils of some plant species; determinating the anti-mosquito and anti-microbial activities of essential oils of Zingiber zerumbet (L.) Smith.

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Nội dung Text: A summary of the PhD thesis: Researching and evaluating on the resources of plant containg essential oils at Ben En national park, Thanh Hoa province; proposing the conservation measures and rational exploitation

MINISTRY OF EDUCATION VIETNAM ACADEMY OF AND TRAINING SCIENCE AND TECHNOLOGY GRADUATE UNIVERSITY OF SCIENCE AND TECHNOLOGY HOANG VAN CHINH RESEARCHING AND EVALUATING ON THE RESOURCES OF PLANT CONTAING ESSENTIAL OILS AT BEN EN NATIONAL PARK, THANH HOA PROVINCE; PROPOSING THE CONSERVATION MEASURES AND RATIONAL EXPLOITATION SUMMARY OF PHD DISSERTATION Major: Botany Code: 9.42.01.11 Supervisors: 1. Assoc. Prof. Dr. Tran Minh Hoi 2. Dr. Do Ngoc Dai HANOI – 2019
This dissertation has been carried out at The Graduate University of Science and Technology - Vietnam Academy of Science and Technology Supervisors: Assoc. Prof. Dr. Tran Minh Hoi Dr. Do Ngoc Dai The 1st Reviewer: The 2nd Reviewer: The 3rd Reviewer: The dissertation will be defended at the Board of Examiners of Graduate University of Science and Technology, Vietnam Academy of Science and Technology at …. on ... The thesis can be found at the National Library of Vietnam and Library of Graduate University of Science and Technology
INTRODUCTION 1. The urgency of the thesis topic Vietnam is located in the monsoon tropical areas, with a great range of terrains, soil types, landscapes, and different climate characteristics among regions, leading to favorable basis for biodiversity of species composition and species richness. Vietnam is ranked 16th in the world in terms of diversity of biological resources. In the "General Flora of Indochina" and subsequent additional episodes, about 240 families with more than 7,000 vascular plants were described and recorded. In recent years, that number is predicted could reach 15,000 species by many botanists. Currently, about 13,000 species of vascular plants have been recorded, of which about 660 essential oil plants species (accounting for about 6.3% of the total number of known species) are. The known essential oil plants belong to 357 genera (accounting for about 15.8% of total genera) and 114 families of vascular plants (accounting for 37.8% of the total) in the flora of Vietnam. The families which have plenty of genera and species containing essential oils are: Asteraceae, Zingiberaceae, Rutaceae, Lamiaceae, Lauraceae, Apiaceae, Myrtaceae .... The more developed societies are, the higher in the demand for learning and using natural origin compounds. The essential oil plants play an important role in the groups of resourcing plants. Those are essential materials for cosmetic, food and pharmaceutical industries. Ben En National Park is located in northwestern Nhu Thanh district, about 46 km southwest of Thanh Hoa city, with geographical coordinating from 19º28 'to 19º39' north latitude; 105º20' to 105º35' East longitude. The natural area of the park is 16,634 ha, including 16 sub-zones, Song Muc lake and Da Van mountain area, Song Chang. Ben En National Park consists of mixing types of hills, mountains, rivers and lakes. The center of National Park is Song Muc lake with a system of floating islands covering by forests and many spreading branches surrounded by mixing mountains and land rocky terrain. The highest mountain is Dam, with 497 m height. The other mountains are 300-350 m, with average slope from 250 – 300 m, even above 350 m. This terrain is quite rugged, with steep slopes, with many limestone mountains containing caves and forests inside. At Ben En National Park, there are plenty of precious essential oils species such as Cinnamomum parthenoxylon Meisn., C. balansae H. Lecomte, C. loureirii (L.) Presl, Citronella spp., Ardisia silvestris Pitard, Artemisia annua L., Plectranthus aromaticum Benth.... 1
Currently, there are a number of studies on flora in Ben En National Park, such as studies of Do Ngoc Dai et al (2007), Hoang Van Sam (2008), Ben En National Park (2013). Regarding essential oil plants, there are only a few studies on chemical compositions and antibacterial activities in some species of several authors such as Do Ngoc Dai, Nguyen Anh Dung, Ngo Xuan Luong ... However, those authors only concerned about some aspects, and did not focus on essential oil plant resources. Due to those reasons, the research topic "Researching and evaluating on the resources of plant containing essential oil at Ben En National Park, Thanh Hoa province, proposing the conservation measures and rational exploitation" was choosen, with both scientific and practical significances. 2. Researching objectives - Evaluating the diversity and using values of essential oil plants at Ben En National Park, Thanh Hoa province. - Determining the content and composition of essential oils of some plant species. - Determinating the anti-mosquito and anti-microbial activities of essential oils of Zingiber zerumbet (L.) Smith - Proposing the conservation measures and rational exploitation to essential oil species at Ben En National Park, Thanh Hoa province. 3. Scientific and practical significances of the study - Scientific significances + Establishing new and relatively completed data on the variety of essential oil plants at Ben En National Park, Thanh Hoa province. + Providing new data on the content and chemical composition of essential oils in the leaves, stems, roots, shells and fruits of 33 samples of 19 species. For the first time, data on essential oils of 6 species were provided. + Providing new data on anti-mosquito and anti-microbial activities of essential oils in rhizome of Zingiber zerumbet (L.) Smith. - Practical significances + Contributing information for admistrators in developing strategies to conserve essential plant species at Ben En National Park based on the scientific results, as well as proper preserving and exploiting suggestions. + Providing a list of valuable essential oil species which will support the orientation of reasonable management, exploitation and sustainable development in the future. 2
4. Structure of the dissertation The dissertation consists of 168 pages, except for Introduction, Conclusions and recommendations, References, and Appendix parts, the dissertation includes three main chapters: Chapter 1. Literature review: 30 pages Chapter 2. Subjects, contents and study methods: 7 pages Chapter 3. Results and Discussions: 102 pages CHAPTER 1 LITERATURE REVIEW 1.1. General information of essential oils 1.1.1. Essential oil plant definition The essential oil plants are plants containing specialized structures that are responsible for secreting and accumulating essential oils. 1.1.2. Characteristics and chemical compositions of essential oils Essential oils are mixtures of organic compounds, have complex molecular structures, are insoluble in water, volatile and have a characteristic aroma. 1.1.3. Natural state and distribution of essential oils - Essential oils are in latent or free state in plant body, it could present in all parts or concentrated in only one or several parts of plant. - In general, essential oil plants present in the entire plant kingdom but they especially present in some families. 1.1.4. Using values, importance of essential oils and materials containing essential oils For a long time, people have used essential oils in daily life as well as the pharmaceutical, food processing, cosmetic industries... Many plant species containing essential oils have become popular crops. 1.2. Study on essential oil plants in the world and in Vietnam 1.2.1. Study on essential oil plants in the world Up to now, there have not been enough documents to visualize the history of the world of essential oil study. The earliest document about essential oil plants is the "Medicinal Plants" book found in Japan, written in 890. In this book, nearly 100 essential oil species were documented, and processing and using these plants were described as well. It is shown that scientists pay special attentions on essential oils and essential oil plants from the early twentieth century; Oustanding studies were published by Charabot and his colleagues in 1903, 1904, 1907. In later 3
times, this kind of studies increased rapidly and belong to many research aspects. According to Brian M. Lawrence, in the project "Progress in essential oils" (1992-1994) and "Essential oils" (1995-2005), the author documented about 1.000 essential oil plant species which were analyzed for chemical compositions on the world. According to L.P.A. Oyen and Nguyen Xuan Dung (1999), in the "Essential oil plants in South-East Asia", in Southeast Asian countries, there are more than 70 essential oil plant species analyzed for chemical compositions, of which about 30 species were studied comprehensively from biological and ecological characteristics, distribution, cultivation, development, using values, yield and trade and chemical compositions. 1.2.2. Study on essential oil plant species in Vietnam The studies on essential oil plants in Vietnam began after 1956. During that time, a series of studies on Mentha piperita L., Citronella spp., Litsea cubeba, ... were reported. According to La Dinh Moi and Luu Dam Cu (2001), about 20 essential oil plant species were recently natural exploited and planted in more or less 600 known species (only 3% of total known essential oil plant species). Those species are popular cultivated species such as Citronella spp., Mentha piperita, Ocimum tenuiflorum, Cinnamomum camphora, Melaleuca sp., C. loureirii (L.) Presl, Pogostemon cablin (Blanco) Blanco... 1.2.3. Study on essential oil plant species in Thanh Hoa and at Ben En National Park Essential oil plant studies were only scattered in some species, and were not systematic in this area. 1.3. Study on chemical composition and biological activity of some plant families in the world and in Vietnam 1.3.1. Study on chemical composition and biological activity of some plant families in the world 1.3.1.1. Lauraceae In the world, studies on essential oil of Lauraceae mainly focused on the perfume, pharmaceutical, cosmetic and antifungal, antibacterial pratical applications. The studied species often belong to the genera Cinnamomum, Litsea, Machilus ... 1.3.1.2. Rutaceae Most of the species in the Rutaceae have essential oils or aromas, and there are many studies in the world on essential oils of Rutaceae. The author has summaried main studies until 2018. 4
1.3.1.3. Piperaceae Currently, there are many studies in the world on the chemical compositions of essential oils and biological activities of the species in Piperaceae. Those studies focused mainly on the genus Piper. 1.3.1.4. Zingiberaceae Studies on Zingiberaceae in the world, mainly focused on the genus Curcuma, Zingiber, Alpinia, Amomum ... 1.3.2. Study on chemical composition of and biological activity of some plant families Vietnam 1.3.2.1. Lauraceae There are 21 genera, 273 species belonging to Lauraceae in Vietnam The studies on essential oils are mainly focused on Cinnamomum, Litsea, Machilus, Phoebe ... 1.3.2.2. Rutaceae There are more than 15 essential oil plant species of Rutaceae in Vietnam. Studies on essential oil plant species of Rutaceae maily focused on some genera such as Citrus, Clausena, Zanthoxylum, Euodia, Glycosmis... 1.3.2.3. Piperaceae In Vietnam, studies on essential oil plant species of Piperaceae began 3 decades ago and mainly focused on the genus Piper. 1.3.2.4. Zingiberaceae In Vietnam, there are about 21 genera with more than 140 species in Zingiberaceae; although it is not huge family, but most of its species have essential oils. Until now, there are more than 40 essential oil species studied. 1.4. Natural, economic and social conditions of the study area 1.4.1. Geographical location Ben En National Park is located in northwestern Nhu Thanh district, with geographical coordinating from 19º28' to 19º39' 'North latitude, from 105º20' to 105º35' East longitude. 1.4.2. Geology and soil The main types of soil of Ben En National Park are: Alluvial soil and streams, yellow red feralit soil, developed on the group of clay, pale yellow feralit soil developed on sand and weathered groups on limestone mountains. 1.4.3. Topographic This area has different types of hills, mountains, rivers and lakes with quite rugged terrain. 1.4.4. River 5
There are two main river systems in this ares namely Muc river and Chang river and Ben En lake with a water capacity of 250 - 400 million m3. 1.4.5. Climate Ben En has a subtropical climate: cold and dry winter; hot and humid summer. 1.4.6. Status of forest land at Ben En National Park The area of forested land in Ben En National Park is 11,738.07 ha, accounting for 79.66 %. 1.4.7. Social conditions At Ben En National Park area, there are 1 town, 16 communes, 7 state- owned units; Total population is 41,672 citizens, with complex ethnic composition. CHAPTER 2 SUBJECTS, CONTENTS AND METHODS 2.1. Study subjects Essential oil plant species distributing in Ben En National Park, Thanh Hoa province. 2.2. Study timing The project was conducted from March 2014 to September 2018. 2.3. Study contents - Making a list of essential oil plant species and evaluating the diversity of essential oil species. - Investigating on the using values of essential oil plant species. - Determining the content and chemical compositions of some essential oil plant species. - Testing the activity against Aedes albopictus adult mosquitoes, Culex quinquefasciatus mosquito larvae and anti-microbial activities of Zingiber zerumbet. - Proposing the conservation measures and rational exploitation. 2.4. Study methods 2.4.1. Method of inheriting documents Inheriting the research results on natural and social conditions in the study area, plant samples stored in domestic and foreign museums, published studies relating to the PhD topic. 2.4.2. Field investigation method Based on the map, the main survey lines were selected, including 6 main routes, namely Song Chang; Xuan Thai-Yen Bai; lake road (Plant Island and other islands); Binh Luong; Xuan Hoa - Xuan Quy and Hai Van - Tan Binh. 6
2.4.3. Methods of sampling and classification - Each sample must have all the parts, especially: branches, leaves, flowers and even fruit as well (for large trees) or even whole plant body for herbaceous plants. - From 3-5 samples per each species, and for herbaceous plants, similar samples were collected with the same number above to study and discuss about the deformations of the species. - Samples collected on the same individual together a common label. - All samples were taken photos by Canon digital camera. Samples were pretreated in the field, and afterwards, they were dried in the Herbarium at Hong Duc University. Samples collected during the field trips were brought back to the laboratory for further analysis and processing. Squeezing: Before pressing on the dried, flat and thick newspaper, all the leaves are fully stretched, not curled edges, parts of flowers or fruits are opened or cut out to facilitate analysis, pressing and drying samples. Sample drying: Samples after pressing are dried immediately. Specimens were identified by comparative morphological method. For difficult specimens, expert method was used. A total of more than 1,000 samples were collected and used for analysis and scientific identification. The sample is currently stored at the Herbarium, Faculty of Natural Sciences, Hong Duc University. The main documents used in scientific identification are: - Vietnamese herbs (Pham Hoang Ho, 1999 - 2003); - Handbook to Reference and identification of the families of Angiospermae Plants in Vietnam (Nguyen Tien Ban, 1997); - General flora of Indochina (1907); - Flora of China (1994-2002); - Set of Vietnamese botanicals (Annonaceae, Verbenaceae, Myrsinaceae, Lamiaceae, Lauraceae, Zingiberaceae) and some other specialized materials. Adjusting the scientific name and developing the list: Adjusting the scientific name according to the List of Vietnamese plant species and The plant list website (http://www.theplantlist.org); Sort the list by R. K. Brummitt and colleagues (1992). 2.4.4. Methods to assess the diversity of flora - Diversity of taxa of flora: According to Nguyen Nghia Thin's method (1997). 7
+ Evaluating the diversity of taxa in division (reporting the number of species, genera and families according to each division from low to high based on the list of flora, calculating the percentage of each taxon to avaluate their diversity level). + Assessing the species diversity of each family (identifying the species richness of family, calculating the percentage (%) of species of those families compared to the whole of the flora). + Evaluating species diversity of genera (identifying species richness of genera, calculating the percentage (%) of species of those genera to the whole species of the whole flora). - Diversity in stem forms: Based on the record of the field survey, án related documents and divided according to "Vietnam Forest Plants" for reporting and assessing stem forms of essential oil plants. - Diversity of using values of flora: Reporting using valuable species from the list of plants with specialized documents, such as: Vietnamese medicinal plant dictionary (2012), 1900 useful plant species in Vietnam (1993), Checklist of plants species of Vietnam (2003, 2005),"Vietnamese plants (1999-2003), Medicinal plants and medicinal herbs in Vietnam (2003), Medicinal plants and medicinal animals in Vietnam, ... - Diversification of rare and precious plant species and conservation issues: Based on Vietnam Red Data Book (2007) and IUCN red list, reporting rare species and conservation status. 2.4.5. Methods of identifying the chemical compositions of essential oils 2.4.5.1. Sample collection and essential oil distillation Samples for essential oil distillation included separate parts of the plant (leaves, branches, shells, pneumatophores, rhizomes, flowers, fruits). 0.5-3 kg fresh sample was collected. Samples were labeled with identified number (similar to species identified number) and the collected time. After collecting, samples were chopped and distilled by the method of attracting refluxed water in Clevenger equipment in 2 - 4 hours at standard pressure according to Vietnam Pharmacopoeia (2009). 2.4.5.2. Quantitative method of essential oils Essential oils of different parts were quantified according to the First method of Vietnam Pharmacopoeia (2009). The content of fresh essential oils (X (%)) were calculated according to the formula: 𝑎 × 0.9 𝑋 (%) = × 100 𝑏 (when d < 1) 8
Or according to this formula 𝑎 𝑋 (%) = × 100 𝑏 (when d > 1) Where: a is the volume of the essential oil in ml b is the mass of the sample in grams. The essential oils were dried with anhydrous Na2SO4, stored in sealed standard vials at 0 - 5º C before further analysis. 2.4.5.3. Methods of chemical composition analysis of essential oils Preparation of analytical samples for gas chromatography: Dissolving 1.5 mg of dried essential oil by anhydrous Na2SO4 in 1 ml of pure hexane for chromatographic analysis. + Gas chromatography (GC) with FID detector: Made on Agilent Technologies HP 6890N Plus with FID detector, capillary column HP-5MS length of 30 m, internal diameter (ID) = 0.25 mm, layer film 0.25mm thin with carrier gas is hydrogen. The temperature of the sample pump chamber is 250º C. Detect temperature is 260º C. Program temperature 60º C (2 minutes), increase 4º C/minute to 220º C, stop at this temperature for 10 minutes. + Gas chromatography mass spectrometry (GC/MS): It is carried out on Agilent Technologies HP 6890N/HP 5973 MSD conjugated gas chromatography system with separation column and chromatographic operating conditions as mentioned above and with helium as carrier gas. The qualitative determination of essential oil components was done by the following methods: - Based on the values of the Retention Index, determinng with a sequence of n-alkanes in the same chromatographic condition. - Based on mass spectra, comparing with mass spectra found in data banks (NIST 08 and Wiley 9th Version) or comparing with the references. The percentage of components in essential oils were calculated based on the area or height of the chromatographic peak (detector FID) without using any adjustment factors. 2.4.6. Methods of investigation and interview To assess the current status of management, exploitation and use of plant resources containing essential oils, conducting surveys and direct interviews with the managers and people. 2.4.7. Biological activity test methods 9
Test of mosquito resistance: Mosquito resistance activity was determined by Reed-Muench method [164]. Adult mosquitoes: Aedes albopictus and Culex quinquefasciatus were maintained in insect cages (40 x 40 x 40 cm) and fed by 10% of sugar solution and rat blood. Hatching eggs were spread out by tap water. Aedes albopictus larvae were kept on plastic trays (24 × 35 × 5 cm). Their larvae were fed by dog biscuits and yeast flour at a ratio of 3: 1. All steps were held at 25 ± 2°C, 65 - 75% relative humidity, and a dark cycle of 12 : 12 at the Research Center for entomology and parasites, Duy Tan University. The anti-mosquito activity of rhizomes essential oils of Zingiber zerumbet was evaluated according to WHO protocol (2005) with minor changes. For the assay, the essential oil fraction was dissolved in EtOH (1% stock solution) was placed in a 200 ml beaker and was added water containing 20 larvae (fourth instar). For each test, a controller using EtOH was also run for comparison. Mortality rates were recorded after 24 hours and after 48 hours of exposure while not supplemented with nutrients. The experiments were carried out at 25 ± 2° C. Each test was replicated four times in every concentrations (70, 60, 50, 40, 30 and 25 μg/mL). The average lethal concentration (LC50) was determined by the Reed-Muench method. Testing the resistance to test microorganisms Testing the biological activities of essential oils on some Gram (+) strains: Bacillus subtilis (ATCC 6633), Staphylococcus aureus (ATCC 13709); Gram (-) bacteria: Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 15442); Mold: Aspergillus niger, Fusarium oxysporum and yeast: Saccharomyces cerevisiae, Candida albicans. 1st step. Qualitative test by diffusion method on agar plate, using filter paper to impregnate the test substance with standard concentration. The strains of inspection microorganisms include: - Bacteria Gr (-): E. coli, P. aeruginosa. - Bacteria Gr (+): B. subtillis, S. aureus. - Mold: A. niger, F. oxysporum - Yeast: S. cerevisiae, C. albicans. nd 2 step. Samples with positive activities in 1st step were tested continuously in 2nd step to calculate the minimum inhibitory concentration (MIC) according to the method of Vanden Bergher and Vlietlink (1991) conducted on microtitre plates in 96 hours. Types of antibiotics tested were: Ampicilin, Tetracycline, Nystatin 10
Samples, with a value of MIC ≤ 50 πg /ml, were considered to be antimicrobial positive. 2.4.8. Data analysis Data were analyzed on Microsoft Office Excel 2007 software. CHAPTER 3: RESULTS AND DISCUSSIONS 3.1. Diversity of essential oil plant resources at Ben En National Park, Thanh Hoa province 3.1.1. Division diversity The results showed that there were essential oil 410 species, belonging to 180 genera and 42 families of 2 divisions namely Magnoliophyta and Pinophyta (Table 3.1) at Ben En National Park. Further more, one additional species was recorded for the flora of Vietnam, Ben En Pepper (Piper minutistigmum C. DC.). Table 3.1. The distribution of essential oil plants in division of flora of Ben En National Park Family Genus Species Division Number Ratio Number Ratio Number Ratio of family (%) of genera (%) of species (%) Pinophyta 2 4,44 2 1,11 2 0,49 Magnoliophyta 43 95,56 178 98,89 408 99,51 Magnoliopsida 37 82,22 166 92,22 369 90,00 Liliopsida 6 13,33 12 6,67 39 9,51 Mag./Li Ratio 6,17 13,83 9,46 Sum 45 100 180 100 410 100 The results showed that most of taxa concentrated in the Magnoliophyta with 408 species, accounting for 99.51 % of total species; 178 genera, accounting for 98.89 % and 43 families, accounting for 95.56 % of the total of them; there were only 2 species in Pinophyta, accounting for 0.49 %; 2 genera, accounting for 1.11 % and 2 families, accounting for 4.44 %. Thus, taxa with essential oils mainly concentrated in Magnoliophyta with the number of genera and species accounting for over 95%. This is perfectly reasonable compared to the evolution of plants because the Magnoliophyta is the dominant of higher vacular plants. The uneven distribution of taxa was not only shown between divisions but also among classes in the Magnoliophyta. The Magnoliopsida had a number of taxa that dominate over 80 % of the total number of families, genera and species of division; Liliopsida had 6 families (accounting for 13.33 %); 12 genera (accounting for 6.67 %) and 11
39 species (accounting for 9.51 %). This is reasonable, because Magnoliopsida always dominates over Liliopsida and is consistent with studies of La Dinh Moi et al (2001), Nguyen Nghia Thin (2008) in studied on other flora areas in Vietnam. Comparing with the list of flora of Ben En National Park, Thanh Hoa province, which were recorded 59 essential oil species, it was found that there were only 36 species having essential oils and 23 species having fatty oils (List of flora of Ben En National Park, 2013). In short, our results identified and supplemented 374 species to the list of essential oil species at Ben En National Park, bringing the total number of essential oil species to 410 species. In addition, for every 6.1 families of Magnoliopsida, there was 1 family of Liliopsida; 13.83 genera of Magnoliopsida versus 1 genus of Liliopsida and 9.46 species of Magnoliopsida versus 1 species of Liliopsida. 3.1.2. Family diversity Of the total 45 families having essential oils at Ben En National Park, there were 10 richness families (from 16 to 56 species), accounting for 22.22 % of the total families, 298 species, accounting for 72.68 % of the total species. Typical families are Lauraceae - 56 species, Annonaceae - 46 species, Asteraceae - 35 species, Rutaceae - 33 species, Zingiberaceae - 32 species, Verbenaceae - 24 species and the species having the least species were Lamiaceae and the Scrophulariaceae with 16 species. 3.1.3. Genus diversity With 10 most diverse genera out of 180 genera of essential oil species (from 7 20 species), accounting for 5.56% of total genera, there are 112 species, accounting for 27.32 % of total species, including Piper - 20 species, Litsea - 17 species, Cinnamomum and Alpinia with 12 species, Syzygium - 10 species, Lindernia and Polyalthia with 9 species; Jasminum and Zingiber with 8 species, and Phoebe with 7 species. 3.1.4. Comparing composition of essential oil species at Ben En National Park with the ones at Pu Mat National Park and in Vietnam * Comparing with Pu Mat National Park In order to reveal the diversity of essential oil species at Ben En National Park, the results were compared with essential oil species at Pu Mat National Park by Nguyen Viet Hung (2017) (Table 3.4). Table 3.4. Comparison of essential oil plants in Pu Mat National Park compared to Vietnam's essential oil plants Ben En Pu Mat(1) Ben En/Pu Division Number Ratio Number Ratio Mat ratio (%) of species (%) of species (%) 12
Pinophyta 2 0,49 6 1,66 33,33 Magnoliophyta 408 99,51 355 98,34 114,93 Area (ha) 16.000 94.000 17,02 Sum 410 100 361 100 113,57 1 Nguyễn Viết Hùng (2017). The results in table 3.4 shows that the number of essential oil plant species belonging to Pinophyta at Ben En only accounted for 33.33% compared to Pu Mat ones; however, the total number of species in the Magnoliophyta at Ben En was higher than that of Pu Mat (equal to 114.93 %). Meanwhile, the area of Ben En only accounted for 17.02% compared to Pu Mat and the vegetation in Ben En only distributed in the low belt (from 500 m or less) and in Pu Mat, there were both high and low belts (as high as 1,800 m). Thus, the the number of essential oil plant species at Ben En was higher than that of Pu Mat although the area of Pu Mat was more than 6 times higher and the number of species of plants has 1.6 times higher. Those differences may be due to the differences in investigation and evaluation. * Compare with Vietnam Results on essential oil species at Ben En National Park compared with Vietnam showed in Table 3.5. Table 3.5. Comparison of essential oil plants of Ben En National Park versus essential oil plants of Vietnam Ben En Vietnam(2) Ben Division Number Ratio Number Ratio En/Vietnam of species (%) of species (%) ratio Pinophyta 2 0,49 21 3,20 9,52 Magnoliophyta 408 99,51 636 96,80 64,15 2 Area (km ) 16 330.000 0,0048 Sum 410 100 657 100 62,41 (2) Luu Đam Cu (2000). The data in Table 3.5 show that the number of essential oil species in Ben En National Park accounted for 54.95 % of the total number of known and recorded essential oil plants in Vietnam. Moreover, there were differences in the distribution of essential oils in Pinophyta and Magnoliophyta at Ben En National Park vs. Vietnam. These differences may be due to the fact that Ben En National Park is characterized by a low mountainous area, surrounded by people, with a long-term impact on forest vegetation. In addition, essential oil species in Pinophyta mainly distributed in high belts, therefore there were only 2 species that made up 0.49 % of the 13
total plant species. Moreover, Ben En National Park, locating in the tropical monsoon climate, is very convenient for the species of Magnoliophyta to grow and develop. 3.1.5. Diversity in stem forms Studies on the stem forms of essential oil species in Ben En National Park, based on "Names of Vietnam forest plants" revealed 5 main stem forms namely big large woody tree, small woody tree, bush tree, climbing plant and herbaceous plant (Table 3.6). Table 3.6. Body forms of essential oil plants at Ben En National Park TT Dạng thân ID Number of Ratio (%) species 1 Large woody tree GOL 83 20,24 (over 16 m high) 2 Small woody tree (6-16 m) GON 98 23,90 3 The bush tree BUI 65 15,85 4 Climbing plant GLT 41 10,00 5 Herbaceous plant TH 123 30,00 Sum 410 100 In which, 65 species were shrubs, accounting for 15.85 % and mainly belonging to Verbenaceae, Euphorbiaceae, Araliaceae, Annonaceae, ...; 83 species were large timber plants, accounting for 20.24% of the following families: Podocarpaceae, Magnoliaceae, Lauraceae, Euphorbiaceae, Myrtaceae, ...; 98 species were small timber plants, accounting for 23.90 % with main families such as Annonaceae, Araliaceae, Lauraceae, Myrtaceae, Magnoliaceae, ...; 41 species were climbing plants, accounting for 10.00 % concentrated in Piperaceae, Annonaceae, Rutaceae, ...; Herbaceous plants included 123 species, accounting for 30.00 %. Therefore, herbaceous plants were the most diverse, belonging to Zingiberaceae, Asteraceae, Araceae, Lamiaceae... 3.1.6. Diversity in using values In addition to the essential oils using value, these plant species were listed for other using values such as medicine, edible, spice, wood, ... Report of using values based on documents: Dictionary of medicinal plants, 1,900 useful plant species, Checlist of Vietnamese plant species, .... The other using values of essential oil plants were presented in Table 3.7. Table 3.7. Other using values of essential oil plants at Ben En National Park 14
TT Using values ID Amount Ratio (%) 1 Medicinal plants THU 286 69,76 2 Wood plants LGO 101 24,63 3 Ornamental plants CAN 24 5,85 4 Edible plants ĂNĐ 69 16,83 5 Essential oil plants CTD 410 100 6 Herbs CGV 13 3,17 7 Fatty oil plants CDB 5 1,22 - Essential oil plants: These are plants containing essential oils, and they have been studied extensively, such as the studies of La Dinh Moi et al (2001), Tran Dinh Thang et al (2014), ... In addition, during the study, the chemical compositions of essential oils were distilled and analyzed such as Piper leptostachyum, Michelia alba, Atalantia roburxghiana, Tetradium trichotonum, Macclurodendron oligophlebia), Cinnamomum glaucescens, Cinnamomum verum, and Amomum muricarpum, ... Some genera with large amount of stored essential oils widely distributed at Ben En National Park such as Amomum, Alpinia, Homalomena, Zanthoxylum, Clausena, Euodia, ... - Medicinal plants consisted of 286 species. In addition to the value of essential oils, those species were also medical used by the people in the study areas, mainly improving health, weather and bone pain killer... - Ornamental plants consisted of 24 species, belonging to Podocarpaceae, Annonaceae, Asteraceae, Magnoliaceae, Myrtaceae, ... some species were used as typical ornamental plants such as Artabotrys hongkognensis Hance, Desmos chinensis Lour., Desmos cochinchinensis Lour., ... - Edible plants included 69 species, were also used by people as daily vegetables or fruit, ... Some typical species such as Schefflera heptaphylla (L.) Harms, Zanthoxylum acanthopodium DC., Zanthoxylum myriacanthum Wall. ex Hook.f., ... - Wood plants consisted 101 species, mainly belonging to Magnoliaceae, Lauraceae, Myrtaceae, Euphorbiaceae, 3.1.7. Diversity in values and conservation Based on the Vietnam Red Data Book (2007), 8 species of essential oil species were in danger of extinction. Among them, 01 critically endangered species (CR) - Cinnamomum parthenoxylon Meisn.; 02 endangered species (EN) - Acorus macrospadiceus (Yam.) F. N. Wei & Y. K. Li) and Acanthopanax trifoliatus (L.) Voss; 05 vulnerable species (VU) 15
- Canarium tramdenum Dai et Yakovt., Actinodaphne elliptibacca Kosterm., Cinnamomum balansae H. Lecomte, Michelia balansae Dandy, Ardisia silvestris Pitard. 3.1.8. Characteristics of essential oil chemical composition identified plant species at Ben En National Park In this part, the author presented the characteristics of 19 plant species which were analyzed in chemical composition of essential oils (including species name, synonym, description, biology, ecology, distribution, using values, research sample, drawings and color photos). 3.2. The content and chemical compositions of essential oils of some plants at Ben En National Park, Thanh Hoa 3.2.1. Oil contents of some essential oil plants in Ben En National Park, Thanh Hoa province There were 102 samples collected for extracting essential oils, of which 83 samples belonged to 40 species were identified oil contents, and other samples had low contentes of essential oils. The results showed that the content of essential oils ranged from 0.10 % to 1.22 % of fresh weight; The highest amount of essential oil concentrated in the leaves of Tetradium trichotorum Lour, accounting for 1.22 % fresh weight, followed by the flowers of Michelia alba DC., reaching 1.20 %, .... The average oil content of 83 samples of 40 species was 0.27% of fresh weight. 3.2.2. Chemical composition of essential oils of some plant species in Ben En National Park, Thanh Hoa province 3.2.2.1. Lauraceae Results of 9 essential oil samples in leaves, branches, shells and fruits of 5 species in Lauraceae were summarized in Table 3.16. The content of essential oils varied from 0.12 % - 0.45 % of fresh weight. Essential oils were light yellow, lighter than water and had a pleasant fragrance. The determined chemical compositions accounted for 85.3 % - 95.5 % of the total oil contents. Monoterpen and sesquiterpen were main compounds in essential oils Table 3.16. Major components of essential oils in different parts of some species od Lauraceae at Ben En National Park, Thanh Hoa province Number of Percentage of main stem Amount No Species identified essential oil parts (%) compounds components (%) 16
geraniol (36.2%), Cinnamomum terpinen-4-ol (19.7%), α- 1 Leaf 0.42 43 glaucescens pinen (6.0%), sabinen (6.0%) limonen (5.2%) linalool(22.0%), bicyclogermacren Cinnamomum 2 Leaf 0.45 49 (11.2%),β-bisabolen verum (7.7%), caryophyllen oxit (5.6%) Leaf -caryophyllen (26.4%), limonen (12.6%), 0.18 54 germacren D (5.1%), - pinen (4.6%) Branch limonen (16.8%), -pinen Litsea (11.6%), caryophyllen oxit 3 0.12 44 glutinosa (10.0%), -caryophyllen (7.2%) Fruit -caryophyllen (21.3%), (E)--ocimen (14.7%), 0.45 37 limonen (12.1%), caryophyllen oxit (8.0%). linalool (20.9%), 5-epi- Lindera neointermedol (11.2%), β- 4 Leaf 0.21 57 racemosa selinen (7.0%), caryophyllenol (5.1%) Leaf 0.18 31 geraniol (34.2%), z-citral (25.6%), geranyl acetat (7.7%), α-pinene (6.5%) Branch 0.15 37 geraniol (20.2%), z-citral Phoebe (14.5%), β-pinen 10.4%), - 5 tavoyana muurolol (9.5%), α-cadinol (9.5%) Vỏ 0.25 46 linalool (19.2%), 1,8-cineol (17.1%), α-pinen (6.8%), E- citral (5.9%) 3.2.2.2. Piperaceae Results of 7 essential oil samples in leaf and stem parts of 4 species in Piperaceae were summarized in Table 3.21. The concentration of essential oil varied from 0.15 % - 0.22 % of fresh weight. Essential oils were light yellow, lighter than water and had a pleasant fragrance. The determined 17
chemical compositions accounted for 85.7 % - 99.4 % of the total oil contents. Monoterpen and sesquiterpen were main compounds in essential oils. Table 3.21. Major components of essential oils in different parts of some species of Piperaceae at Ben En National Park, Thanh Hoa province Number of Percentage of main stem Amount No Species identified essential oil components parts (%) compounds (%) 1 Piper acre Leaf 0.20 46 (E)-nerolidol (22.7%), sabinen (19.5%), δ-cadinen (12.4%) Stem 0.16 52 E)-nerolidol (15.6%), sabinen (19.9%), δ-cadinen (13.5%), benzyl benzoat (7.0%) 2 Piper Leaf 0.22 43 spathoulenol (12.4%), β- minutistigmum pinen (11.3%), germacren C. DC. D (10.1%), isoterpinolen (8.9%), α-pinen (8.6%) Stem 0.17 48 β-caryophyllen (14.6%), bicyclogermacree (12.8%), germacren D (12.3%), 7- hydroxy-2- methylisoflavon (11.6%), bicycloelemen (8.9%), apiol (8.4%) 3 Piper Leaf 0.15 57 -curcumen (12.0%), laosanum germacren D (6.3%), sabinen (6.1%), spathoulenol (5.1%), α- cadinol (4.9%) Stem 0.21 45 Sabinen (14.9%), benzyl salicylat (14.3%), (E)- nerolidol (9.3%), cis - copaen-8-ol (4.5%) 4 Piper saxicola Leaf 0.20 40 trans calamen (16.4%), β- caryophyllen (14.8%), 18