Summary of Doctoral Thesis of Philosophy in Biology: Research on the composition of essential oil-bearing plant species in Vu Quang National Park, Ha Tinh Province

Objectives: Assess the diversity of essential oil-bearing plants and the chemical composition of essential oils extracted from some oil-bearing species in Vu Quang National Park, Ha Tinh province

MINISTRY OF EDUCATION AND TRAINING

VINH UNIVERSITY

LÊ DUY LINH

RESEARCH ON THE COMPOSITION OF ESSENTIAL BEARING PLANTS SPECIES IN VU QUANG NATIONAL

OIL-PARK, HA TINH PROVINCE

Major: Botany Code: 9 42 01 11

ABSTRACT OF A THESIS DOCTOR OF PHILOSOPHY IN BIOLOGY

VINH, 2020

This thesis was fulfilled at Vinh University

Supervisors: 1 Assoc Prof Dr Pham Hong Ban

2 Assoc Prof Dr Tran Minh Hoi

Reviewer 1:

Reviewer 2:

Reviewer 3:

The thesis will be defended at Vinh University

The thesis can be found at: The National Library of Vietnam

Library of Nguyen Thuc Hao

INTRODUCTION

1 Rationale

Located in the tropical monsoon region, Vietnam is ranked in the sixteenth

in the world in terms of biodiversity According to the record and description in the “Indochinese General Plant” (Flore Générale de l’Indochine) and its subsequent collections, there are over 240 families with over 7,000 vascular plant species In recent years, many botanists have predicted that the figure could reach over 12,000 species Currently, there have been about 10,500 species of vascular plants, of which about 657 species belonging to 357 genera and 114 families containing essential oils (accounting for about 6.3% of total species; 15.8% of total genera and 37.8% of total families)

Along with the development of the society, the need to learn and to use compounds of natural origin is increasing Among the plants that are considered as

an important resource, those plants containing essential oils play an important part This is an essential source of many industries such as cosmetics, food and pharmaceuticals, etc Most essential oil-bearing plants are in the two branches of Pinus (Pinophyta) and the Magnolia (Magnoliophyta) The family which have many species of essential oil plants include Cúc (Asteraceae), Cam (Rutaceae), Na (Annonaceae), Long não (Lauraceae), Bạc hà (Lamiaceae), Hoa tán (Apiaceae), Hoa hồng (Rosaceae), Dầu (Dipterocarpaceae), Nhài (Oleaceae), Thông (Pinaceae), Hoàng đàn (Cupressaceae)

Today, essential oils are widely used in many industries, especially the food, pharmaceutical and cosmetic industries Especially in the field of cosmetics, essential oil is an indispensable material Although there is an increase in synthetic aromatics, these artificial products cannot replace natural oils, because they do not create a cool aroma and especially synthetic fragrances are often too expensive and highly toxic

Vu Quang National Park was established under the Prime Minister’s Decision No 102/2002 / QD-TTg dated July 30, 2002, with a total area of about 54,000 ha, including 76% of the natural forest area with 5 types of main forest divided by different elevations This is the place of growth for many valuable species: Dalbergia oliveri (Cẩm lai), Chukrasia tabularis (Lát hoa), Erythrophleum fordii (Lim), Magnolia hypolampra (Giổi), Aquilaria crassna (Trầm hương), etc and many precious medicinal plants There have been a number of studies on vascular plant diversity in Vu Quang National Park, but there is a lack of in-depth

studies on plant resources Meanwhile, there exists a huge potential of essential oil

resources in there Therefore, the author selected the topic: “Research on the

composition of essential oil-bearing plant species in Vu Quang National Park, Ha Tinh Province” as the content of the thesis

2 Objectives

Assess the diversity of essential oil-bearing plants and the chemical composition of essential oils extracted from some oil-bearing species in Vu Quang National Park, Ha Tinh province

3 Scientific and practical significance

The research results of the thesis are the survey data that are basic researches on the diversity of essential oil species in Vu Quang National Park, Ha Tinh province, and provide new data on The chemical composition of some essential oils of some families in the flora of Vu Quang National Park

4 New findings

- Providing new data including 366 species of essential oil plants in Vu Quang National Park This is a relatively comprehensive and systematic data on essential oil plant resources in Vu Quang National Park, Ha Tinh Province

- For the first time to provide data on essential oils of 9 species (Bời lời lá

thuôn (Litsea elongata (Wall Ex Nees) Hook.f.), Bời lời phiến lá thon (Litsea

lancilimba Merr.), Bời lời biến thiên (Litsea variabilis Hemsl.), Kháo vàng thơm

(Machilus bonii Lecomte), Kháo nhậm (Machilus odoratissima Nees), Nô vàng(Neolitsea aurata (Hayata) Koidz.), Nô bui san (Neolitsea buisanensis Yam

& Kam.), Vàng tâm (Manglietia dandyi (Gagnep.) Dandy), Sa nhân lông hung (Amomum velutinum X E Ye, Skprnick & N H Xia)

5 The structure of the thesis

The thesis consists of 128 pages, 30 tables, 7 pictures, 39 photos structured into the following main sections: Introduction (3 pages); Chapter 1: Literature review (28 pages); Chapter 2: Objects, content and research methods (8 pages); Chapter 3: Research findings (86 pages); Conclusions and recommendations (2 pages); List of published works related to the thesis (2 pages); References (14 pages) and appendix

(including 3 appendices, 114 photos)

CHAPTER 1 LITERATURE REVIEW

1.1 Some concepts of essential oils

1.1.1 General concept of an essential oil-bearing plant

there was no exact definition of an essential oil-bearing plant before When some oil compounds were found that included in the body of all organisms (animals’ bile acids, carotene in most plants, etc) As such, there is no clear boundary between essential oil-bearing plants and other plants From this perspective, Nicolaev (1968) defined: “Essential oil-bearing plants are those different from other plants in that their essential oils can be obtained”

Later, when studying the structure and functioning of secretory organs, it became clear about the difference in the nature of essential oil-bearing plants From that, it is possible to define that Essential oil-bearing plants are plants which contain specialized structures for the secretion and accumulation of essential oils

1.1.2 Concept of essential oils

The French Standard (1987), gives out the definition of essential oils as follows: “Products obtained from plant-derived materials, by steam distillation or

by mechanical methods for fruit peels of Citrus genus Essential oils are separated

from the water by physical methods ” This definition has the limitation that is it excludes products obtained by solvent extraction as well as products obtained by other methods

Essential oils are understood as blends of organic compounds, complex molecular structures and different physical and chemical properties Essential oils have several properties:

- All essential oils are liquid, viscous, with optical properties, causing the rotation of light

- Most essential oils have less density than water (d<1), some have a greater density than water (d>1), insoluble or very rarely soluble in water, but soluble in organic solvents

- Essential oils have scent since the composition of essential oil is composed from free-form constituents

- Essential oils are evaporable

Based on the chemical molecular structure of essential oils, they are organized into 4 main groups:

- Aliphatic compounds

- Terpenes and their derivatives

- Benzene derivatives

- Other ingredients

1.2 Studying essential oil-bearing plants

1.2.1 Research on essential oil-bearing plants in the world

So far there are not enough documents to visualize the history of studies on essential oil-bearing plants in the world The earliest available document is the

“Medicinal Plants” found in Japan, written in 890 In this document, there are nearly

100 species of essential oil-bearing plants, and it describes the process of producing and using these plants

However, research on essential oils and essential oils plants started to attract a lot of attention from researchers in the early twentieth century The most remarkable works are those published by Charabot and his students in 1903, 1904, and 1907 At later times, there was a boom in the number of researches varying in many fields

According to Brian M Lawrence in “Progress in essential oils” (1992-1994) and “Essential oils” (1995-2005), the author has estimated about 1,000 species of essential oil-bearing plants that have been analyzed for chemical composition in the world According to LPA Oyen and Nguyen Xuan Dung (1999) in the project

“Essential oil plants in South-East Asia”, in Southeast Asian countries, over 70 species of essential oil plants have been analyzed for chemical composition, in which about 30 species have been studied quite comprehensively from biological characteristics, ecology, distribution, ability to grow, develop, use, yield and be harm by pest, traded for commercial purposes

1.2.2 Research on essential oil plants in Vietnam

The research works on essential oil in Vietnam started after 1956 During this time a series of researches on peppermint, lemongrass, etc were published According to La Dinh Mo and Luu Dam Cu (2001), until now, we have exploited from nature and planted about 20 species of essential oil-bearing plants out of over

600 known species (only 3% of the total) The aforementioned species are commonly planted species such as Lemongrass, Peppermint, Fragrance, Camphor, Melaleuca, Cinnamon, Basil, Anise, Patchouli, etc

1.3 Research on chemical composition of essential oil in the world and in Vietnam

1.3.1 Research on the chemical composition of essential oil in the world

1.3.1.1 Camphor family (Lauraceae)

Research on essential oils in the Camphor family (Lauraceae) has typical works such as XD Ji et al (1991), L Zhu et al (1993), Choudhury, S N et al

(1995), Sriramavaratharajan, V et al (2019), etc

1.3.1.2 Magnoliaceae

Many scientific works have been studied on the chemical composition of

essential oils of Magnolia genus such as Theo Oyen L.P.A., Nguyen Xuan Dung

(1999), Grag, SN & Sushil Kumar (1999);Zheng et al (2015), Scharf et al (2016)

1.3.1.3 Ginger (Zingiberaceae)

In the Ginger family (Zingiberaceae), the study of chemical composition of

essential oils is concentrated in the main genera such as Nghệ (Curcuma), Riềng (Alpinia),Gừng (Zingiber), Sa nhân (Amomum) KC Wong et al (2005), B Sabulal

et al (2006), Zhenyang Chen et al (2019), Marliani L et al (2016), S Thubthimthed et al (2005), Wang et al (2015)

1.3.2 Research on chemical composition of essential oil in Vietnam

1.3.2.1 Study on chemical composition of essential oil of Camphor

(Lauraceae)

Research on Camphor’s essential oil (Lauraceae) focuses mainly on the

genera Cinnamomum, Litsea, Machilus, Phoebe There are some typical works such

as: Nguyen Xuan Dung (1996), Tran Dinh Thang et al (2005), Le Cong Son (2013),

Do Ngoc Dai et al (2019)

1.3.2.2 Study on chemical composition of Magnoliaceae family

Many studies have been done on essential oil-bearing plants of Magnoliaceae family by Dung et al (1997), Lesueur et al (2007), Do et al (2016a), Bui Van Huong et al (2014), Do et al (2016b),

1.3.2.3 Study on chemical composition of essential oil-bearing plants of Ginger family (Zingiberaceae)

In Vietnam, the study of the chemical composition of their essential bearing plants of ginger family (Zingberaceae) is typical of the works of Nguyen Xuan Dung et al (1996), Nguyen Xuan Dung et al (2005), Phan Minh Giang et al

oil-(2007), Le Thi My Chau et al (2015) , Le Thi Huong (2016), Le Thi Huong, et al (2019), Nguyen Thanh Hung et al (2018), etc

1.4 The natural, socio-economic conditions of Vu Quang National Park

This section outlines geographical location, terrain, climatic characteristics, socioeconomic characteristics, flora and fauna of the research area

CHAPTER 2 OBJECTIVES AND RESEARCH METHODS

2.1 Subjects and Research locations

The subject of research is essential oil-bearing plant species distributed in Vu Quang National Park, Ha Tinh

2.2 Time conducted for the research

The thesis was conducted from 2015 to 2019

Each year, the sample is collected into 2 batch, each happens in the interval of 7-10 days

Research was carried out at Vu Quang National Park, Ha Tinh Province

The total number of samples obtained was 1,200 samples A total of 1061 templates is collected and stored in the Plant Exhibition room, at the Center of Laboratory Practice, Vinh University

The chemical composition of essential oils is analyzed at the Chemical Institute of Natural Compounds, the Academy of Science and Technology of Vietnam

2.3 Research content

- Determining the composition of essential oil-bearing plant species

-Analyze the diversity of essential oil-bearing plants from these following aspects:

+ Diversity in terms of branches, families, genera and species

+ Diversity in terms of body form

+ Diversity in terms of usage values of essential oil-bearing plants

+ Diversity in terms of geographical factors of essential oil-bearing plants + Diversity in terms of gene sources that are rare and precious, as well as, conservation status

- Analyze the chemical composition of essential oils of some important plants

of the families

2.4 Research methodology

2.4.1 Researching from secondary survey

Data on natural and socio-economic conditions in the study area, plant samples kept in domestic and foreign museums, published works related to the topic

2.4.2 Methodology of field investigations and surveys

Based on the map, some main areas were chosen to survey There are 05 main typical areas which are representative for Vu Quang National Park The first route is Park Center to Sao La Station – Po Mu The second route is Park Center –

Co station (Trạm Cò) – Phan fortress (Thành Cụ Phan) – De slope (Dốc Dẻ) The third route is Son Kim I The fourth route is Son Kim II.The fith route is Huong Khe The process and fieldwork survey were applied following the method

introduced by Nguyen Nghia Thin in “Plant research methods” (2007)

To identify essential oil-bearing plants, it is often necessary to look up published documents about the chemical composition of essential oils and rely on photosensitivity (using nose to smell for fragrance) during field investigation

2.4.3 Methods of sampling and classification

Sampling: For each type of tree, 1-3 specimens are usually collected from

the same place After collecting the sample, the sample is then numbered When collecting samples, the researchers make a record of some characteristics that are easily faded away when the samples are dried or soaked in alcohol or hormones such as colors, latex, properties of flowers, leaves, etc Also photos of trees are taken with Canon digital cameras

Classification: The research method used to classify is a comparative

morphological method This is a traditional research method that is being used today This method is done based on studying some characteristics of vegetative organs and reproductive organs Among the two, reproductive organs are the focus, such as inflorescence position, flower structure (characteristics of bracts, calyx, corolla, stamens, fruits, seeds, etc)

In the process of identifying scientific names of species, the research relies on the classified work about Vietnamese plants (Pham Hoang Ho, 1999-2000)

The following documents are also used: A handbook for searching and identifying families of angiosperms in Vietnam (Nguyen Tien Ban, 1997); Vietnamese timber trees (Forest Inventory and Planning Institute, Ministry of Forestry) (1971-1989); Indochinese General journal plants (1907); Chinese

plantology (1994-2002); Common plants found in Vietnam; Vietnamese plantology: Annonaceae family, Mint Family (Lamiaceae), Ginger family (Zingiberaceae), Camphor family (Lauraceae)], Asteraceae and some other specialized documents

Rectification of scientific names and list development: Revision of

scientific names according to Vietnam’s Plant Species List, RK Brummitt (1992) and http://www.theplantlist.org

2.4.4 Methods to assess the diversity of the flora

-Diversity of taxon of flora: According to “Handbook of biodiversity

research” by Nguyen Nghia Thin (1997):

- Diversity of the trunk types: Based on the records of field surveys as well

as related documents (Specifically, the names of Vietnamese forest trees to collect and evaluate the different forms of trunks of essential oil-bearing trees

- Diversity in the application of the flora: The application value of essential

oil bearning plants is explored from documents such as: “Dictionary of Vietnamese medicinal plants” (Vo Van Chi, 2012), “1900 species of useful plants

in Vietnam” (Tran Dinh Ly et al, 1993), “List of Vietnamese plant species” (Nguyen Tien Ban (Editor) et al, 2003, 2005), “External forest products Vietnamese timber “(Trieu Van Hung et al, 2007),” Vietnamese medicinal plants and herbs “(Do Tat Loi, 2003),” Vietnamese medicinal plants, how to grow, pick, process, and treat” (Le Tran Duc, 1997), “Medicinal plants and animals in Vietnam” (Do Huy Bich et al, 2004), etc

- Geographical diversity

Establish the spectrum of geographical factors, and apply the division of the authors Póc Tamás (1965), Le Tran Chan (1999) and Nguyen Nghia Thin (2007)

- Diversity of rare and precious plant species and conservation issues: Based

on the Vietnam Red Book (2007), including extinct species (EX), extinct species in nature (EW), critically endangered species (CR), endangered species (EN), vulnerable species (VU), low risk species (LR)

2.4.5 Collect samples and distill essential oil

Specimens for distillation for essential oils are separate parts of plants (leaves, branches, bark, stems, rhizomes, flowers, fruits) Each sample collected are of between 0.5-3 kg in fresh condition The sample is inscribed with the serial number (this number coincides with the model number for classification) and the time of collection After collection, the sample was chopped and distilled by steam

extraction method with reflux in Clevengert equipment for 2-4 hours at normal pressure according to Vietnam Pharmacopoeia IV standard (2009)

2.4.6 Methods of quantifying essential oils

Essential oils of various parts are quantified according to method I of Vietnam Pharmacopoeia II (2002) The content of fresh essential oil is calculated by the formula

Or follow the formula X(%) = (when d>1)

In which: a is the volume of essential oil, measured in ml

b is the mass of the sample, measured in grams

Essential oils are dried with anhydrous Na2SO4, stored in standard sealed jars, stored at 0-5oC before being analyzed

2.4.7 Methods of analyzing chemical composition of essential oils

Preparation of analytical sample for gas chromatography: Dissolve 1.5 mg of dried essential oil with anhydrous Na2SO4 in 1 ml of pure hexane of the type used for chromatographic analysis

+ Gas chromatography (GC) with FID probe: Made on Agilent Technologies

HP 7890A Plus with FID detector, HP-5MS capillary column at length of 30 m, inner diameter (ID) = 0.25 mm, a layer of 0.25m-film with hydrogen as carrier gas The injection chamber temperature is 250 ° C Detector temperature is 260oC Program the temperature of 60 ° C (2 min), increase by 4 ° C / min until 220 ° C, stop at this temperature for 10 minutes

+ Gas chromatography (GC / MS): This is performed on Agilent Technologies

HP 7890A / HP 5973 MSD combined gas chromatography system with separation column and chromatographic operating conditions as mentioned above and with Helium as carrier gas

2.4.8 Method of data processing

Data are processed using Microsoft Office Excel 2007 software

CHAPTER 3 RESEARCH FINDINGS AND DISCUSSION

3.1 Diversity of essential oil plants in Vu Quang National Park

3.1.1 Diversity in phyla and classes

Results of surveys and identification of essential oil-containing plants in Vu Quang National Park, Ha Tinh There are 366 species, 145 genera and 45 families

of 2 vascular plant phyla (Table 3.1) that have been identified

Table 3.1 Distribution of taxon with essential oils in some phyla of flora in Vu

Quang National Park

Phylum

Number of families

Percentage

%

Number of genera

The analysis of the taxons of two classes in the Magnoliophyta phylum (Table 3.2), there are also some differences Magnoliopsida has a dominant taxon number of over 80% of the total number of families, genera and species; The class

of Onions (Liliopsida) accounts for a low rate with 7 families (accounting for 16.67%); 19 genera (accounting for 13.67%) and 55 species (accounting for 15.32%)

Table 3.2 The ratio of two classes in the Magnolia phylum (Magnoliophyta) Name of class

Number of families

3.1.3 Diversity of genera

Essential oil-bearing plant species in Vu Quang National Park belong to 145 genera In particular, the number of species distributed in the genera is uneven There are 17 genera accounting for 11.72% of the total number of genera, but there are 164 species accounting for 44.81% of the total species The number of genera with the number of species less than 5 is 128 genera (accounting for 88.28%), with a total of

202 species accounting for 55.19% The genera with species which produce more

essential oils are Litsea with 25 species, Cinnamomum with 21 species, followed by

Alpinia – 13 species, Piper and Fissistigma with 11 species and Syzygium – 10

species The remaining genera have the number of species ranging from 5 to 9

Further analysis of the genera having essential oil-bearing species in Vu Quang National Park showed that 17 genera have more than 5 species belonging

to 7 families: Camphor (Lauraceae), Ginger (Zingiberaceae), Annonaceae, Pepper (Piperaceae), Myrtaceae, Magnoliaceae, Euphorbiaceae Of which, the Camphor family has 7 genera (accounting for 41.17%) with 81 species, accounting for 49.39% The results of this analysis show that in Vu Quang National Park, the Camphor family is very diverse and abundant This is also perfectly reasonable, because camphor trees are adapted to the local climatic conditions, … while Vu

Quang National Park is home to tropical monsoon climate, with both low and high belts, therefore, it is very convenient for camphor tree species to grow and

develop Ginger family has 3 genera: Alpinia with 13 species, Amomum with 8 species, Zingiber with 7 species The total number of species of the three genera of

the Ginger family is 28, accounting for 17.07% of the total species The Annonaceae

family also has 3 genera: Fissistigma with 11 species, Desmos and Uvaria with 5

species The total number of species of 3 genera is 21 species, accounting for 12.80%

3.1.4 Comparing composition of essential oil plants in Vu Quang National Park with that of Pu Mat National Park

- Compare with Pu Mat National Park

To see the diversity of essential oil-bearing plants in Vu Quang National Park,

Ha Tinh Province, the results were compared with essential oil species in Pu Mat National Park (2016) by Nguyen Viet Hung (Table 3.5)

Table 3.5: Comparison essential oil plants in Vu Quang with Pu Mat

Phylum

Percentage of Vu Quang to Pu Mat Number

of species

Proportion (%)

Number

of species

Proportion (%)

to that of Pu Mat Thus, the above results show that the number of species of essential oil-bearing plants in Vu Quang National Park is higher than that of Pu Mat even though the area of Pu Mat is 1.8 times higher The number of vascular plant species currently known in Vu Quang National Park is 1.5 times higher than that in Pu Mat National Park The results show that the species of plants bearing