Nghiên cứu đánh giá nguồn tài nguyên thực vật có tinh dàu ở vườn quốc gia bến én, tỉnh thanh hóa, đề xuất các biện pháp bảo tồn và khai thác hợp lý tt tiếng anh

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 stateowned 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 (); 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
𝑋 (%) =

𝑎
× 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

Vietnam(2)

Ben En
Division

Number
of species

Ratio
(%)

Number
of species

Ratio
(%)

Ben
En/Vietnam
ratio
9,52
64,15
0,0048
62,41

Pinophyta
2
0,49
21
3,20

Magnoliophyta
408
99,51
636
96,80
2
Area (km )
16
330.000
Sum
410
100
657
100
(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
No

Species

Number of
stem Amount
identified
parts
(%)
compounds
16

Percentage of main
essential oil
components (%)


1

Cinnamomum
glaucescens

Leaf

0.42

43

2

Cinnamomum
verum

Leaf

0.45

49

0.18

54

0.12

44

0.45

37

Leaf

0.21

57

Leaf

0.18

31

Branch

0.15

37

Vỏ

0.25

46

Leaf

Branch

3

Litsea
glutinosa
Fruit

4

5

Lindera
racemosa

Phoebe
tavoyana

geraniol
(36.2%),
terpinen-4-ol (19.7%), αpinen (6.0%), sabinen
(6.0%) limonen (5.2%)
linalool(22.0%),
bicyclogermacren
(11.2%),β-bisabolen
(7.7%), caryophyllen oxit
(5.6%)
-caryophyllen (26.4%),
limonen
(12.6%),
germacren D (5.1%), pinen (4.6%)
limonen (16.8%), -pinen

(11.6%), caryophyllen oxit
(10.0%), -caryophyllen
(7.2%)
-caryophyllen
(21.3%),
(E)--ocimen
(14.7%),
limonen
(12.1%),
caryophyllen oxit (8.0%).
linalool (20.9%), 5-epineointermedol (11.2%), βselinen
(7.0%),
caryophyllenol (5.1%)
geraniol (34.2%), z-citral
(25.6%), geranyl acetat
(7.7%), α-pinene (6.5%)
geraniol (20.2%), z-citral
(14.5%), β-pinen 10.4%), muurolol (9.5%), α-cadinol
(9.5%)
linalool (19.2%), 1,8-cineol
(17.1%), α-pinen (6.8%), Ecitral (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
No
1

Species
Piper acre

Number of
Percentage of main
stem Amount
identified essential oil components
parts (%)
compounds
(%)
Leaf
0.20
46
(E)-nerolidol
(22.7%),
sabinen (19.5%), δ-cadinen
(12.4%)
E)-nerolidol
(15.6%),
Stem 0.16
52
sabinen (19.9%), δ-cadinen

(13.5%), benzyl benzoat
(7.0%)

2

3

4

Piper
minutistigmum
C. DC.

Piper
laosanum

Piper saxicola

Leaf

0.22

43

Stem

0.17

48

Leaf

0.15

57

Stem

0.21

45

Leaf

0.20

40

18

spathoulenol (12.4%), βpinen (11.3%), germacren
D (10.1%), isoterpinolen
(8.9%), α-pinen (8.6%)
β-caryophyllen (14.6%),
bicyclogermacree (12.8%),
germacren D (12.3%), 7hydroxy-2methylisoflavon (11.6%),
bicycloelemen
(8.9%),
apiol (8.4%)
-curcumen

(12.0%),
germacren D (6.3%),
sabinen
(6.1%),
spathoulenol (5.1%), αcadinol (4.9%)
Sabinen (14.9%), benzyl
salicylat (14.3%), (E)nerolidol (9.3%), cis copaen-8-ol (4.5%)
trans calamen (16.4%), βcaryophyllen
(14.8%),


caryophyllene
oxit
(13.0%), eucarvon (7.5%)
3.2.2.3. Rutaceae
Results of 5 essential oil samples in leaves and fruits of 4 species of
Rutaceae were summarized in Table 3.26. The concentration of essential oil
varied from 0.12% - 1.22% of fresh weight. Essential oils were light yellow,
lighter than water and had pleasant fragrance. The determined chemical
composition accounted for 89.2% - 98.7% of the total oil contents.
Monoterpen and sesquiterpen were main compounds in essential oils.
Table 3.26. Major components of essential oils in different parts of some
species of Rutaceae at Ben En National Park, Thanh Hoa province
Number of Percentage of main
stem Amount
No.
Species
identified
essential oil
forms

(%)
compounds
components (%)
sabinen (36.9%), 3,6dimethylpiperazin-2,5Atalantia
dion
(7.6%),
β1
Leaf
0.35
43
roxburghiana
caryophyllen (6.1%), γterpinen
(3.7%),
bicycloelemen (3.7%)
Leaf
0.43
41
α-pinen (17.5%), βcaryophyllen (15.5%),
caryophyllen oxit (10.6%)
Maclurodendron Fruit
0.71
37
benzyl benzoat (16.8%),
2
oligophlebia
farnesol (8.3%), βcaryophyllen (6.0%),
limonen (4.7%), αcadinol (4.7%)
Leaf
0.12
70

-pinen (17.2%), limonen
Atalantia
3
(9.7%), α-humulen (7.7%)
sessiliflora
và β-caryophyllen (7.6%)

Leaf
4

1.22

36

Tetradium
trichophorum

3.2.4. Zingiberaceae

19

(E)-β-ocimen (24.8%), αpinen (10.4%), (Z)-βocimen (9.4%), βcaryophyllen (8.0%)


Results of 13 essential oil samples in parts of leaves, fake stem,
rhizomes and fruits of 6 species of Zingiberaceae were summarized in table
3.34. The concentration of essential oils varied from 0.14 % - 0.32 % of
fresh weight. Essential oils were light yellow, lighter than water and had a
pleasant fragrance. The determined chemical compositions accounted for
94.1 % -99.5 % of the total oil content. Monoterpen and sesquiterpen were

main compounds in essential oils.
Table 3.34. Major components of essential oils in different parts of some
species of Zingiberaceae at Ben En National Park, Thanh Hoa province
Number of
Percentage of main
stem Amount
No.
Species
identified essential oil components
parts (%)
compounds
(%)
β-pinen
(12.1%),
αgurjunen (10.5%), (Z)-13Alpinia
1
Leaf
0.16
73
docosenamit (9.0%), γglobosa
terpinen (4.8%), farnesol
(4.3%)
β-eudesmol (33.3%), βpinen (22.5%), δ-cadinen
Leaf
0.25
25
(8.9%), α-pinen (5.7%),
camphen (5.3%).
β-pinen
(40.8%),

muurolol
(10.7%),
αStem 0.19
25
phellandren (9.1%), βphellandren (9.1%), α-pinen
Alpinia
(7.2%)
2
malaccensis
β-pinen
(24.3%),
βphellandren (16.7%), benzyl
Root 0.27
42
salicylat (8.9%), farnesol
(8.1%),
α-phellandren
(7.1%)
methyl cinnamat (16.5%),
germacren D (16.4%), δFruit 0.32
26
cadinen (11.8%), benzyl
acetat (7.3%)
1,8-cineol
(23.2%),
Alpinia
3
Root 0.20
32
terpinen (12.5%), terpinennapoensis

4-ol (9.6%), -pinen (9.2%)
20


4

5

6

Alpinia
tonkinensis

Amomum
villosum

Zingiber
zerumbet

Leaf

0.21

51

Leaf

0.22

22

Stem

0.19

35

Root

0.24

51

Leaf

0.14

55

Stem

0.18

26

Root

0.31

27

β-pinen (33.5%), (E)-βocimen (9.6%), γ-terpinen
(9.2%), α-pinen (8.4%)
-pinen (53.6%), -pinen
(24.5%), sabinen (13.6%)
-pinen (38.8%), sabinen
(19.2%), -pinen (18.5%),
-pinen
(30.8%),
caryophylen
(13.0%),
caryophyllen oxit (12.0%),
-pinen (7.8%)
zerumbon
(51.3%),
caryophyllen oxit (5.7%),
camphor (5.2%)
zerumbon
(51.3%),
camphor (6.7%), humulen
epoxit I (6.4%)

3.3. Anti-mosquito and microbial activities of essential oils of Zingiber
zerumbet (L.) Smith)
3.3.1. Mosquito activities
Anti-mosquito and mosquito larvae activities of essential oils in
rhizomes of Zingiber zerumbet revealed that essential oils could kill Aedes
albopictus adults and Culex quinquefasciatus mosquito larvaes. The average
lethal concentration (LC50) after 24h and 48h were shown in Table 3.36.

Table 3.36. Anti-mosquito and mosquito larvae activities of rhizome
essential oils of Zingiber zerumbet
Activities
LC50 (24 h)
LC50 (48 h)
Aedes Albopictus
55.94 ± 1,3 μg/mL
40.48 ± 0,71 μg/mL
Culex quinquefasciatus
38.63 ± 2,9 μg/mL
25.42 ± 2,2 μg/mL
3.3.2. Anti-microbial activities
The anti-microbial activity results of essential oils on 8 strains of tested
microorganisms, Escherichia coli, Pseudomonas aeruginosa, Bacillus
subtillis, Staphylococcus aureus, Aspergillus niger, Fusarium oxysporum,
21


Saccharomyces cerevisiae, Candida albicans, showed that oils in leaf and
aero-stem could not resist to the above 8 strains. However, essential oils in
rhizomes could resist to Aspergillus niger with MIC = 50 μg / mL. Thus,
essential oils of rhizomes could apply in mold control.
3.4. Proposing solutions to conserve and rationally exploit essential oil
resources at Ben En National Park, Thanh Hoa province
3.4.1. Current status of management, exploitation and use of essential
oils in Ben En National Park, Thanh Hoa province
Interview results of 28 managers and 76 people showed that the
management of forests in Ben En has been strictly implemented, however,
the management efficiency was not very high but the reason is due to lack
of forces and equipments, coordination between the National Park

Management Board and other units and organizations was not good; The
understanding of essential oils was limited; Many types of essential oil
plants are now declining compared to the past.
3.4.2. Solutions to conserve and rationally exploit essential oil resources
in Ben En National Park
Essential oil plant resources play important roles in forest resources
conservation and exploitation. Therefore, in parallel with conserving plant
resources, it is neccessary to proper develope and effective exploit. Combine
the essential oil plants conservation with the development in bufer zones (ex
situ) are important to create benefits, improve income and living standards
for the community.
Within the scope of PhD study, 3 groups of solutions were initially
proposed to conserve and rationally exploit essential oil plant resources at
Ben En National Park: Raising awareness for the community; Solutions for
management; Technical solutions.

CONCLUSIONS AND RECOMMENDATIONS
1. Conclusions
1. Initial identifying 410 essential oil plant species of 180 genera, 45
families, 02 higher plant divisions Magnoliophyta and Pinophyta at Ben En
National Park. In which, one additional species was recorded for the flora
of Vietnam, Ben En Pepper (Piper minutistigmum C. DC.).
2. Species with essential oils belong to 5 main stem types, of which
the most common are herbaceous plants of 123 species (30%), followed by
small woody tree of 98 species (23.9%) and 83 species of big large woody
22


tree (20.24%), bush tree 65 species (15.85%) and climbing plant 41 species
(10%).

3. In addition to essential oil using value, many species of 410 species
also give other using values such as medicine, 286 species (69.76 %),
ornamental, 24 species (5.85 %), food 69 species (16.83 %), wood, 101
species (24.63%), spices 13 species (3.17 %) and the lowest was the fatty
oil-bearing plant with 5 species (1.22 %).
4. Among endangered essential oil species at Ben En National Park,
Thanh Hoa province, according to Vietnam Red Data Book (2007), there
are 8 species, of which 01 species is critically endangered (CR), 02
endangered species ( EN) and 05 species vulnerable endangered (VU).
These are species with a small number of individuals, so it is necessary to
have further research to measures proper conservation and development.
5. Determining of oil contents of 83 samples of 40 species and
chemical compositions of 33 samples of essential oils of 19 species
belonging to 4 plant families, Lauraceae, Piperaceae, Rutaceae, and
Zingiberaceae. For the first time, determining the content and chemical
composition of essential oils of 6 species, Lindera racemosa Lecomte,
Phoebe tavoyana (Meisn.) Hook. f., Piper acre Blume), Piper
minutistigmum C. DC., Piper saxicola C. DC., Tetradium trichophorum
Lour..
6. Essential oils of a number of studied species contain valuable
chemical components such as oxygen-containing monoterpenes and
oxygen-containing sesquiterpen in species of Lindera racemosa Lecomte),
Piper laosanum C . DC., Piper saxicola C. DC., Macclurodendron
oligophlebia (Merr.) Hartl.), Atalantia sessiliflora Guillaum., Alpinia
malaccensis (Burm . f.) Rosc., Zingiber zerumbet (L.) Smith.
7. Testing bioactive properties of rhizome essential oil of Zingiber
zerumbet (L.) Smith; results after 24 hours and 48 hours showed resistance
to Aedes albopictus adult with LC50 = 55.94 μg / mL and 40.48 μg / mL,
resistance to Culex quinquefasciatus mosquito larvae with LC50 = 38.63 μg
/ mL and 25.42 μg / mL. In addition, rhizomes essential oils are also resistant

to mold with MIC: 50 μg / mL.
8. Proposing 03 measyres of solutions to conserve and rationally
exploit essential oil resources at Ben En National Park, Thanh Hoa province.
2. Recommendations
Studies on essential oil species at Ben En National Park, Thanh Hoa
province in particular and Vietnam in general are still very defficient
compared to their diversity. Therefore, further research is needed to fully
23