Summary of biology doctoral thesis a study on biological characteristics and essential oil composition of zingiber boehm and hedychium koen (zingiberaceae lindl ) in the north central of vietnam

MINISTRY OF EDUCATION AND TRAINING VIETNAM ACADEMY OF SCIENCE AND TECHNOLOGY GRADUATE UNIVERSITY SCIENCE AND TECHNOLOGY TRINH THI HUONG A STUDY ON BIOLOGICAL CHARACTERISTICS AND ESSENTIAL OIL COMPOSIT[.]

MINISTRY OF EDUCATION AND

TRAINING

VIETNAM ACADEMY

OF SCIENCE AND TECHNOLOGY

GRADUATE UNIVERSITY SCIENCE AND TECHNOLOGY

-

TRINH THI HUONG

A STUDY ON BIOLOGICAL CHARACTERISTICS

AND ESSENTIAL OIL COMPOSITION OF Zingiber Boehm AND

Hedychium Koen (ZINGIBERACEAE Lindl.)

IN THE NORTH CENTRAL OF VIETNAM

Major: Botany Code: 9.42.01.11

SUMMARY OF BIOLOGY DOCTORAL THESIS

Supervisors: 1 Dr Do Ngoc Dai

2 Dr Nguyen Thi Thanh Huong

HANOI - 2021

This dissertation has been carried out at The Graduate University of Science and Technology - Vietnam Academy of Science and Technology

Supervisors: 1 Dr Do Ngoc Dai

2 Dr Nguyen Thi Thanh Huong

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 dissertation can be found at the National Library of Vietnam and

Library of Graduate University of Science and Technology

INTRODUCTION

1 The urgency of the dissertation topic

Zingiberaceae Lindl., consists of perennial herbaceous plants with transverse

rhizomes or tubers, mainly distributed in tropical and subtropical countries Zingiber Boehm and Hedychium Koen are commonly used in traditional medicine and in food

processing

Nowadays, the demand for using essential oil products of plants is increasing day

by day Furthermore, essential oils from species in the Zingiber and Hedychium have

shown antimicrobial, antifungal, antioxidant and insecticidal activities Thus, they are likely to be Used as safer alternatives to synthetic antibiotics, antifungals, mosquito repellents, pesticides, and in beauty treatments

The North Central region is the intersection of the flora stream from the North to the South, and is also home to many special-use forests, so the flora is very rich and diverse However, many species in the ginger genus Zingiber (Zingiber) and the genus Wormwood (Hedychium) have not been fully studied for the plant and essential oil

chemistry Therefore, the author chose the topic: "A study on biological

characteristics and essential oil composition of Zingiber Boehm and Hedychium

Koen (Zingiberaceae Lindl.) in the North Central of Vietnam”

2 Researching objectives

- Describing some biological characteristics and distribution of Zingiber and

Hedychium species from north-central Vietnam

- Determining the essential oil chemical composition; antimicrobial and

anti-mosquito larvae activities of essential oils of some Zingiber and Hedychium species

from north-central Vietnam

3 Scientific and practical significances of the study

- Scientific significances

+ Updating, supplement and systematic data on the diversity of Zingiber and

Hedychium from north-central Vietnam;

+ Providing new data on the content and chemical composition of essential oils

of thirty nine samples of twelve Zingiber and Hedychium species from north-central

Vietnam;

+ Providing new data on the antimicrobial activities of thirteen essential oil

samples of five Zingiber and Hedychium species; and anti-larvae activity of six essential oil samples of four Zingiber species from north-central Vietnam

- Practical significances

On the basis of the scientific arguments obtained, the research results of the dissertation contribute information for admistratorsin developing strategies to

conservation, development and exploitation of valuable species of Zingiber and

Hedychium, Zingiberaceae Lindl., from north-central Vietnam in particular and in

Vietnam in general

4 Structure of the dissertation

The dissertation consists of 141 pages, 24 tables, 5 pictures, and 20 pictures are

structured into the following main sections: Introduction (02 pages); Chapter 1: Literature review (22 pages); Chapter 2: Subjects, contents and study methods (08 pages); Chapter 3: Results and discussion (106 pages); Conclusions and recommendations (02 pages); New contributions of the dissertation; List of published works related to the dissertation; References (170 documents, updated to May 2021); and Appendix parts

CHAPTER 1: LITERATURE REVIEW 1.1 Studying plants on Zingiberaceae Lindl

1.1.1 In the world

C Linnaeus (1753) was the first to classify family Ginger In 1835, Lindley took

the genus Zingiber name as the standard genus to name the family Ginger is

Zingiberaceae Since then, there have been many studies on Zingiberaceae Lindl., typically P Sirirugsa (1998), K Larsen et al (1998), J Kress et al (2002), K Larsen and S.S Larsen (2006), A Lamb et al (2013), J M Christenhusz and J W Byng (2016), According to The Plant List, Zingiberaceae consists of 52 genera with 1587 accepted name species

1.1.2 In Vietnam

J Loureiro (1793), Gagnepain (1908), Lê Khả Kế et al (1975), Phạm Hoàng Hộ (1993, 2000), Nguyễn Quốc Bình (2005, 2017), …

There are no systematic studies on Zingiberaceae in north-central Vietnam, but only individual statistics on this family, such as Nguyễn Nghĩa Thìn and Mai Văn Phô (2003), Nguyễn Nghĩa Thìn and Nguyễn Thanh Nhàn (2004), Đỗ Ngọc Đài and Lê Thị Hương (2010),Đậu Bá Thìn et al (2013), Võ Minh Sơn et al (2015), Lê Thị Hương et

al (2015), Đậu Bá Thìn et al (2017), Nguyễn Danh Hùng et al (2018)

1.2 Studying plants on Zingiber and Hedychium

1.2.1 Zingiber Boehm

1.2.1.1 In the world

Boehm (1760), T L Wu and K Larsen (2000), P Triboun (2006), R Kumar et al (2015), M M Aung and N Tanaka (2019), …

1.2.1.2 In Vietnam

Gagnepain (1908); Phạm Hoàng Hộ (1993); J Škorničková, Nguyễn Quốc Bình

et al (2015); Lý Ngọc Sâm (2017); …

In north-central Vietnam: Some separate statistics on Zingiber in the studies of

Đậu Bá Thìn et al (2013, 2017), Võ Minh Sơn (2015), Nguyễn Danh Hùng et al (2018), …

1.2.2 Hedychium Koen

1.2.1.1 In the world

Koenig (1783), C T L Wu and K Larsen (2000), M F Newman et al (2007),

P Thomas et al (2015), …

1.2.1.2 In Vietnam

Phạm Hoàng Hộ (2000), Nguyễn Quốc Bình (2017), …

In north-central Vietnam: There are some separate statistics on this genus in the

studies of Đậu Bá Thìn et al (2013, 2017), Võ Minh Sơn (2015), Nguyễn Danh Hùng

et al (2018), …

1.3 Using values of Zingiber and Hedychium Species

A lot of Zingiber and Hedychium species are used as medicine, flavoring agent, and flavoring agent In addition, a few Hedychium species are also used in cosmetics,

perfumes and ornamental plants They are used very popularly in China, Japan, Indonesia, Thailand, and Vietnam

1.4 General information of essential oils

1.4.1 Essential oil plant definition

The essential oil plants are plants containing specialized structures thatare responsible for secreting and accumulating essential oils

1.4.2 Define and characteristics of essential oils

Essential oils can be defined as a mixture of volatile and natural substances, characterized by a strong odor and produced by aromatic plants as secondary metabolites

1.4.3 Chemistry of Essential Oils

According to V P S Rao and D Pandey (2006), there are more than 200 components present in the mixture of pure essential oils They can be categorized into two classes:

Volatile fraction: 90 - 95% of total oil weight It contains monoterpenes, sesquiterpenes, and their oxygenated derivatives

Nonvolatile residue: 1 - 10% of of total essential oil in weight It contains fatty acids, hydrocarbons, sterols, waxes, flavonoids, and carotenoids

1.5 Study on Zingiber and Hedychium essential oils

Most Zingiber and Hedychium species contain essential oils Content and chemical composition of essential oils in each species are different and often different

in the same species if collected in different geographical regions

1.6 Natural, economic and social conditions in north-central Vietnam

1.6.1 Geographical location

Including 6 provinces: Thanh Hoá, Nghệ An, Hà Tĩnh, Quảng Bình, Quảng Trị and Thừa Thiên - Huế

1.6.2 Topography, geomorphology

There are 3 main types: Mountain: mostly lowland; Hills: occupies a fairly large area; and Plain: medium size

1.6.3 Climate, rivers

Climate: Both the tropical monsoon climate with cold winters in the northern climate of Vietnam, and the anomalous characteristics of the monsoon tropics with wet and wet seasons of the East Truong Son Rivers: There is an average altitude across the basin reaching 300-700 m, with a great slope (18-22%)

1.6.4 Natural and socio-economic conditions

The total area is 51,180.5 km2, with a total population of 10,500,000 people There are many ethnic groups such as: Kinh, Tày, Thái, Thổ, Mường, Dao, H'mông,

Khơ Mú, Đan Lai, Bru-Vân Kiều, Cơ Tu, Tà Ôi, Chút

1.6.5 Plant conditions

Five National Parks: Bến En, Pù Mát, Vũ Quang, Phong Nha Kẻ Bàng and Bạch

Mã Nine Nature Reserves: Pù Luông, Pù Hu, Xuân Liên, Pù Hoạt, Pù Huống, Kẻ Gỗ, Bắc Hướng Hóa, Đa krông và Phong Điền

CHAPTER 2: SUBJECTS, CONTENTS AND METHODS 2.1 Study subjects

Zingiber and Hedychium species from north-central Vietnam Total number of

samples has been researched over 200 with a total of about 100 sample numbers

2.2 Study timing and placing

The project was conducted from May 2017 to October 2020

2.3 Study contents

- Researching on the plant diversity of Zingiber and Hedychium from

north-central Vietnam

- Studying the bio-ecological characteristics and value using of Zingiber and

Hedychium species from north-central Vietnam

- Determinaing the content and chemical compositions of essential oils of some

Zingiber and Hedychium species from north-central Vietnam

- Testing the antimicrobial and anti-mosquito larvae activities of essential oils

of some Zingiber and Hedychium species from north-central Vietnam

2.4 Study methods

2.4.1 Biological study methods:

2.4.1.1 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

2.4.1.2 Field investigation method

Based on the map and topographic conditions, the main survey lines were selected

so that the lines must pass through the habitats of the study area From the main line, the herringbone-styled sub-glands are opened in both directions and pass through different communities On average, 1.5km of the main line has 2 sub-lines opened Investigate species of two study genus located within 10m on each side of each transect

2.4.1.3 Methods of sampling and classification

Plant samples were collected and processed according to the method of Nguyễn Nghĩa Thìn (2007)

2.4.1.4 Methods to assess the diversity of flora

Evaluating species diversity of genera according to the method of Nguyễn Nghĩa Thìn (2007)

2.4.1.5 Method of learning about the use value of flora

Based on published documents and through interviews with the people

2.4.2 Methods of identifying the chemical compositions of essential oils:

2.4.2.1 Sample collection and essential oil distillation

Samples for essential oil distillation included separate parts of the plant (leaves, stem, 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 (2017)

2.4.2.2 Quantitative method of essential oils

Essential oils of different parts were quantified according to the First method of Vietnam Pharmacopoeia V (2017)

2.4.2.3 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

2.4.3 Biological activity test methods

2.4.3.1 Testing the resistance to test microorganisms

Antimicrobial activity of Zingiber and Hedychium essential oils was carried out

on three Gram-negative organisms, Salmonella enterica (ATCC 13076), Pseudomonas

aeruginosa (ATCC 27853), and Escherichia coli (ATCC 25922); three Gram-positive

organisms, Bacillus cereus (ATCC 14579), Enterococcus faecalis (ATCC 299212), and Staphylococcus aureus (ATCC 25923); and the pathogenic yeast, Candida

albicans (ATCC 10231), using the microbroth dilution assay (Clinical and Laboratory

Standards Institute, 2009)

The essential oil sample is diluted in DMSO (Dimethyl sulfoxide) in the

decreasing concentration range (µg / ml): 256-128-64-32-16-8-4-2 The antimicrobial assays were carried out in triplicate.Bacteria and fungi were standardized to 5 × 105 CFU/mL for bacteria and 1 × 103 CFU/mL for the yeast After 24 hours of incubation

at 37°C, the MIC value was determined by observation The MIC value was determined

at the well with the lowest reagent concentration that completely inhibited the growth

of microorganisms after 24 hours of culture and accurately determined based on cell turbidity data by Bioteck spectrophotometer and Raw data software The IC50 values were determined by the percentage of microorganisms that were inhibited for growth based on the BioTeK turbidity data from the BioTeK spectrometer and the Raw data computer software using the following equations:

% inhibition = ODcontrol(-) - ODtest agent x 100%

ODcontrol(-) - ODcontrol(+)

IC50 = Highconc - (Highinh% - 50%) x (Highconc - Low conc )

(Highinh% - Lowinh%) Comment: % inhibition: inhibitory concentration; OD: optical density; Control (-): cells with medium but no antimicrobial agent; , test agent is a known concentration of antimicrobial agent; Control (+): culture medium without cells; Highconc/Lowconc: concentration of test agent at high concentration/low concentration; Highinh% / Lowinh%:

% %inhibition at high concentration/% inhibition at low concentration

2.4.3.2 Test of mosquito larvicidal resistance

Mosquito larva resistance activity was determined by Reed-Muench method

Adult mosquitoes: Aedes aegypti, 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 larva activity of rhizomes essential oils of Zingiber and

Hedychium species 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

2.4.4 Data analysis:

Data were analyzed on Microsoft Office Excel 2016 software

CHAPTER 3: RESULTS AND DISCUSSIONS

3.1 Some biological characteristics of Zingiber and Hedychium species from

north-central Vietnam

3.1.1 Morphological characteristics of Zingiber and Hedychium from north-central Vietnam

3.1.1.1 Morphological characteristics of Zingiber Boehm

Herbs perennial; pseudostems formed by leaf sheaths, up to 3 m; rhizomes branched, tuberous, aromatic, lying horizontally below the ground Leaves: spacing, distichous, held in plane parallel to rhizome; petiole absent or very short, swollen, cushionlike; leaf blade oblong, lanceolate, or linear Inflorescences arising from rhizomes on peduncle clothed with scalelike sheaths, rarely breaking through leaf

sheaths without peduncle; bracts closely imbricate, rarely without roofing (Zingiber

tubular, split on 1 side, apex 3-toothed Corolla proximally tubular, distally 3-lobed, central lobe white or cream, usually wider than lateral lobes Lateral staminodes adnate

to labellum, forming a 3-lobed labellum; central lobe retuse or cleft at apex Filament short; connective with elongate appendage wrapped around style; anther 2 cells, parallel Ovary 3-loculed Fruit a capsule Seeds black, covered by aril; aril white, margin irregularly lacerate

3.1.1.2 Morphological characteristics of Hedychium Koen

Herbs perennial; pseudostems 1-2 m, rarely more than 3 m; rhizomes thick, fleshy Leaves: distichous, held in plane parallel to rhizome; ligule conspicuous, petiole absent

or very short, leaf blade usually oblong or lanceolate Inflorescences: breaking through

leaf sheaths without peduncle; bracts imbricate, rarely lax (Hedychium stenopetalum),

1- or more flowered; bracteoles tubular Calyx tubular, usually split on 1 side, apex truncate or 3-toothed Corolla tube long, slender; lobes reflexed at anthesis, linear Lateral staminodes petaloid, larger than corolla lobes Labellum suborbicular, large, apex usually 2-cleft; claw long or short Filament usually long, 2-cell anther, connective appendage absent Ovary 3-loculed Capsule globose, 3-valved Seeds numerous; aril lacerate

3.1.2 Ecological characteristics, distribution of Zingiber and Hedychium from north-central Vietnam

3.1.2.1 Ecological characteristics, distribution of Zingiber Boehm

Habitat: grows in primary forest, secondary forest; under the canopy of humid

forests, wet humus, along streams, moist limestone mountain cavities, planted in

gardens Usually at 100-900 m, rarely less than 100 m (Zingiber mekongense) or less than 300 m (Zingiber collinsii, Zingiber cornubracteatum)

Phenology: Flowering usually begins in spring (Mar-May), rarely in summer

(Zingiber montanum, July) or autumn (Zingiber cornubracteatum, Sep) Flowering time lasts from 2-9 months, 2 months: Zingiber vuquangense (Apr-May), Zingiber

montanum (July-Aug); 3 months: Zingiber castaneum (May-July), Zingiber nitens

(May-July), Zingiber cornubracteatum (Sep-Nov); 4 months: Zingiber castaneum

(Apr-July), Zingiber officinale (May-Aug), Zingiber zerumbet (May-Aug); 6 months:

Zingiber nudicarpum (Apr-Sep), Zingiber collinsii Oct), Zingiber ottensii

(May-Oct); 9 months: Zingiber mekongense and Zingiber neotruncatum (Apr-Dec) Fruit season: after flowering 1-2 months; from May to Mar next year: Zingiber

neotruncatum; Jun-Sep: Zingiber vuquangense; Jun-Nov: Zingiber castaneum, Zingiber nitens; Jun-Feb next year: Zingiber mekongense; Oct-Dec: Zingiber cornubracteatum; Oct-Jan of next year: Zingiber zerumbet

3.1.2.2 Ecological characteristics, distribution of Hedychium Koen

Habitat: grows in primary forest, secondary forest; wet humus, wet soil, along

streams, under thin forest canopy, humus-covered rock recesses, planted in gardens;

prefer less (Hedychium coronarium) or more shade (Hedychium stenopetalum,

Hedychium gardnerianum), Hedychium flavum prefers less shade to light shade

Phenology: Flowering usually begins in summer (July-Aug), rarely winter

(Hedychium villosum, Feb) The flowering period lasts for 2-6 months; 2 months:

Hedychium stenopetalum (July-Aug); 3 months: Hedychium villosum (Feb-Apr), Hedychium flavum (Aug-Oct), Hedychium gardnerianum (Aug-Oct); 6 months: Hedychium coronarium (July-Dec) Fruit season: 1-2 months after flowering; from

April-June: Hedychium villosum; Oct-Dec: Hedychium flavum, Hedychium

stenopetalum, Hedychium gardnerianum; Sept-Feb next year: Hedychium coronarium

3.1.3 Diversity of Zingiber and Hedychium from north-central Vietnam

Twenty-four species have been identified in north-central Vietnam Including a new

species for science Zingiber vuquangense N.S.Lý, T.H.Lê, T.H.Trịnh, V.H.Nguyễn & N.Đ.Đỗ and two new record for the flora of Vietnam, namely Zingiber mekongense Gagnep and Zingiber cornubracteatum Triboun (Table 3.1)

Table 3.1 List of Zingiber and Hedychium species from north-central Vietnam

Larsen & Turland

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