Summary of PHD dissertation: Researching and evaluating on the resources of plant containing essential oil at Ben En National Park, Thanh Hoa province, proposing the conservation measures

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.

MINISTRY OF EDUCATION

AND TRAINING

VIETNAM ACADEMY OF 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

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

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

+ 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

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

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

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,

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

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 owned units; Total population is 41,672 citizens, with complex ethnic composition

state-CHAPTER 2 SUBJECTS, CONTENTS AND METHODS 2.1 Study subjects

Essential oil plant species distributing in Ben En National Park, Thanh Hoa province

- 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

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)

+ 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)

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

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

1 st 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

2 nd 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

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

Number

of genera

Ratio (%)

Number

of species

Ratio (%)

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