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

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. 7 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 ( 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). 8 + 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: 𝑋 (%) = 𝑎 × 0.9 𝑏 × 100 (when d < 1) 9 Or according to this formula 𝑋 (%) = 𝑎 𝑏 × 100 (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 10 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. 2nd 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 11 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 Division Family Genus Species Number of family Ratio (%) Number of genera Ratio (%) Number of species Ratio (%) 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 12 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 Division Ben En Pu Mat(1) Ben En/Pu Mat ratio (%) Number of species Ratio (%) Number of species Ratio (%) 13 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 1Nguyễn 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 Division Ben En Vietnam(2) Ben En/Vietnam ratio Number of species Ratio (%) Number of species Ratio (%) Pinophyta 2 0,49 21 3,20 9,52 Magnoliophyta 408 99,51 636 96,80 64,15 Area (km2) 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 14 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 species Ratio (%) 1 Large woody tree (over 16 m high) GOL 83 20,24 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 15 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) 16 - 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 No Species stem parts Amount (%) Number of identified compounds Percentage of main essential oil components (%) 17 1 Cinnamomum glaucescens Leaf 0.42 43 geraniol (36.2%), terpinen-4-ol (19.7%), α- pinen (6.0%), sabinen (6.0%) limonen (5.2%) 2 Cinnamomum verum Leaf 0.45 49 linalool(22.0%), bicyclogermacren (11.2%),β-bisabolen (7.7%), caryophyllen oxit (5.6%) 3 Litsea glutinosa Leaf 0.18 54 -caryophyllen (26.4%), limonen (12.6%), germacren D (5.1%), - pinen (4.6%) Branch 0.12 44 limonen (16.8%), -pinen (11.6%), caryophyllen oxit (10.0%), -caryophyllen (7.2%) Fruit 0.45 37 -caryophyllen (21.3%), (E)--ocimen (14.7%), limonen (12.1%), caryophyllen oxit (8.0%). 4 Lindera racemosa Leaf 0.21 57 linalool (20.9%), 5-epi- neointermedol (11.2%), β- selinen (7.0%), caryophyllenol (5.1%) 5 Phoebe tavoyana 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 (14.5%), β-pinen 10.4%), - 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 18 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 No Species stem parts Amount (%) Number of identified compounds Percentage of main essential oil components (%) 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 minutistigmum C. DC. Leaf 0.22 43 spathoulenol (1