Study on extraction, chemical composition and antioxidant capacity of cinnamomum burmannii essential oil

Nguyen Thuong Tuan Supervisor’s Signature Abstract: The aims of this study was to develop a suitable technological process for extracting essential oil from Cinnamomum burmannii leaves b

THAI NGUYEN UNIVERSITY

UNIVERSITY OF AGRICULTURE AND FORESTRY

Study mode : Full time

THAI NGUYEN, 2022

ABTRACT

Thai Nguyen University of Agriculture and Forestry

Degree Program Bachelor of Food Technology

Student name Vu Thi Thuy

Thesis Title Study on extraction, chemical composition and antioxidant

capacity of Cinnamomum burmannii essential oil

Supervisor(s) MSc Dinh Thi Kim Hoa MSc Nguyen Thuong Tuan

Supervisor’s

Signature

Abstract: The aims of this study was to develop a suitable technological process

for extracting essential oil from Cinnamomum burmannii leaves by steam

distillation method In addition, the quality, chemical compositions and antioxidant capacity of the essential oil also were evaluated The most suitable parameters for extraction process were the wilting time of 84 hours; the ratio between material and distill water of 5/50 (w/v); 50 minutes for extraction The amount of obtained essential oil was 0.6 - 0.7% The chemical ingredient of essential oil consisted of 23 compounds that were reported for chemical

structures The antioxidant activity of the essential oil from Cinnamomum

burmannii leaves was revealed with the IC50 of 41.10 (µg/ml)

Keywords Extraction, Chemical composition, Antioxidant activity,

Cinnamomum burmannii essential oil

Number of pages 64

Date of Submission June 1st, 2022

ACKNOWLEDGEMENT

During my studies and internship at Thai Nguyên University of agriculture and forestry, I have now completed my graduation thesis In order to complete this thesis, I have been guided by the devoted guidance of my supervisor, along with the help of Thai Nguyen University of Agriculture and Forestry, the Institute of Life Sciences, and the Advanced Program Office I also received enthusiastic cooperation from colleagues, help, and encouragement from my family members In response to that sentiment, though here I would like to express my deep gratitude and respect to all collectives and individuals who created conditions to help me throughout the internship process

First of all, I would like to express my sincere thanks to the school management board, the Dean of the Faculty of Food Technology, and the collective of teachers in the Faculty of Food Technology, Thai Nguyen University of Agriculture and Forestry that taught and mentored me during these time, as well as my graduation internship

In particular, I sincerely thank for the attention and guidance of the instructing supervisor MSc Dinh Thi Kim Hoa and MSc Nguyen Thuong Tuan, who directly guided me to implement this thesis successfully

Through this, I would also like to express my gratitude to family, relatives, and friends for helping and encouraging me during my study and practice at school

Finally, I would like to respectfully send my sincere thanks and best wishes

to the teachers and teachers on the evaluation committee

Thank you sincerely!

Thai Nguyen, May 23, 2022

Student

VU THI THUY

TABLE OF CONTENT

LIST OF FIGURES 1

LIST OF TABLE 2

ABBREVIATIONS 3

PART I INTRODUCTION 4

1.1 Research rationale 4

1.2 Research objective 5

1.3 Detail goals 5

1.4 Limitations 5

PART II LITERATURE REVIEW 6

2.1 Overview of Cinnamomum burmannii tree 6

2.1.1 Characteristic of Cinnamomum burmannii 7

2.1.2 Distribution, ecological characteristic and growth 8

2.1.3 Uses of Cinnamomum burmannii leaves 9

2.2 Overview of essential oil 10

2.2.1 Definition of essential oil 10

2.2.2 Physical and chemical properties of essential oil 11

2.2.3 The role of essential oils in plant life 12

2.2.4 Biological activities and uses of essential oils 14

2.2.5 Cinnamomum burmannii essential oil 15

2.3 Methods extraction of essential oil 17

2.3.1 Hydrodistillation 18

2.3.2 Solvent extraction 19

2.3.3 Enfleurage 21

2.3.4 Maceration 21

2.3.5 Cold pressing extraction 21

PART III METHODOLOGY 23

3.1 Material and research scope 23

3.1.1 Material 23

3.1.2 Research scope 23

3.2 Work place and time to proceed 23

3.3 Equipment and chemicals 24

3.4 Research content 25

3.4.1 The research on factors affecting the content of Cinnamomum burmannii

essential oil 25

3.4.2 Determination of the chemical composition of Cinnamomum burmannii essential oil by GC-MS method 25

3.4.3 Investigation of antioxidant capacity of essential oils 26

3.5 Research methods 26

3.5.1 The process of extracting essential oil from Cinnamomum burmannii leaves 26

3.5.2 Experimental design method 27

3.5.3 Determination of the chemical composition of Cinnamomum burmannii essential oil by GC-MS method 29

3.5.4 Investigation of antioxidant capacity of essential oil 29

3.5.5 Determination of moisture and ash content in Cinnamomum burmannii leaves 31

3.5.6 Determination of some physicochemical parameters of Cinnamomum burmannii essential oil 33

3.6 Data satistical analysis methods 36

PART IV RESULT AND DICUSSION 37

4.1 Moisture and ash content in Cinnamomum burmannii leaves 37

4.2 The result of studying factors affecting the distillation process of essential oils 37

4.2.1 Effect of extraction time 37

4.2.2 Effect of the material/distilled water ratio on essential oil content 38

4.2.3 Effect of wilting time of raw material on essential oil content 39

4.4 Result for determining the chemical composition of Cinnamomum burmannii essential oil by GC-MS method 41

4.5 Result for determining the antioxidant capacity of Cinnamomum burmannii essential oil 42

4.6 Result for determining some physicochemical of essential oils 44

4.7 Completing the extraction process of Cinnamomum burmannii essential oils 46

PART V CONCLUSION 48

5.1 Conclusion 48

5.2 Recommendation 48

REFERENCES 50

APPENDIXES 55

LIST OF FIGURES

Figure 2.1 Cinnamomum burmannii Tree 8

Figure 2.2 Main molecules of essential oils 10

Figure 3.1 Cinnamomum burmannii leaves 23

Figure 3.2 The process of extracting from Cinnamomum burmannii leaves 26

Figure 3.3 Reaction mechanism of 2,2-diphenyl-1-picrylhydrazyl (DPPH) with antioxidant 30

Figure 4.1 Graph showing the effect of time on essential oil content 38

Figure 4.2 The graph shows the effect of the amount of distilled water on the essential oil content 39

Figure 4.3 Graph showing the effect of wilting time on essential oil content 40

Figure 4.4 Correlation between free radical inhibitory activity and concentration of Cinnamomum burmannii essential oil 43

Figure 4.5 Correlation between free radical inhibitory activity and concentration of Ascorbic acid 43

Figure 4.6 Cinnamomum burmannii essential oil 45

Figure 4.7 The extraction process of Cinnamomum burmannii essential oils 46

LIST OF TABLE

Table 3.1 Experiment Chemicals 24

Table 3.2 Experiment Equipment 24

Table 3.3 Laboratory instruments 25

Table 3.4 The experimental design to determine the effect of extraction time on essential oils 27

Table 3.5 Experimental design to determine the effect of (material/distilled water) ratio on essential oils 28

Table 3.6 The experimental design to determine the effect of wilting time of raw material on essential oils 28

Table 4.1 The moisture and ash content of Cinnamomum burmannii leaves 37

Table 4.2 Results of survey on extraction time of essential oils 37

Table 4.3 Result of effecting the material/distilled water ratio on essential oil content 38

Table 4.4 Results affect the wilting time of raw material on essential oil content 40

Table 4.5 Chemical composition of Cinnamomum burmannii essential oil 41

Table 4.7 Antioxidant activity of Ascorbic acid with DPPH 43

Table 4.8 Some physicochemical of Cinnamomum burmannii essential oils 45

ABBREVIATIONS

ABTS 2,2'-azino-bis

(3-ethylbenzothiazoline-6-sulfonic acid) DPPH 2,2-diphenyl-1-picrylhydrazy

GC-MS Gas Chromatography-Mass Spectrometry

HPLC High-Performance Liquid Chromatography

SDE Simultaneous Distillation Extraction

SFE Supercritical Fluid Extraction

SPME Solid-phase Micro-extraction

PART I INTRODUCTION 1.1 Research rationale

Essential oils are natural ingredients Currently, in the world, people are increasingly interested in medical products derived from essential oils to ensure health and have no negative effects As a result, both domestic and international research on essential oils is rising in number and quality

Vietnam is one of the nations in the tropical monsoon area that has a large and diversified collection of essential oils and aromatic plants, with over 300 essential oil species collected such as Peppermint (1.3%-2%), Gratissimum (0.9%-1.67%), Lemongrass (0.7%-1.5%), Rosemary (0.3%-0.5%), Cinnamon (1%-2.5%), They are used quite commonly in the market due to they mostly contain the main components such as cinnamic aldehyde, geraniol, menthol, borneol, citral, citronellal, menthol, … that pleasant aroma, anti-cold effect, anti-inflammatoryand stress reduction Some essential oils can cure cancer such as Frankincense, Lemongrass, and Orange

Cinnamomum burmanniiis a woody plant in Bao Lac district, Cao Bang

province Cinnamomum burmanniileaves are often used by people as raw

materials for food processing In addition, people often boil the leaves for a bath

or sauna to prevent and treat diseases such as malaria, and bacterial infections, to control blood pressure and headaches Based on the above applications, we can

see that Cinnamomum burmannii leaves are a useful ingredient for health, not

only as a food additive but also as a common healing method for people However, these effects are insufficient to explain the phenomenon clearly from a scientific standpoint, and up to this point, there have been no in-depth studies on

this leaf Therefore, I conducted the topic: “ Study on extraction, chemical composition and antioxidant capacity of Cinnamomum burmannii essential oil.” Thereby contributing to creating more directions and to clarify for the

application of Cinnamomum burmanniiessential oil in the fields of medicine,

pharmaceuticals, and cosmetics Simultaneously, maximizing the potential and

improving the value of Cinnamomum burmannii essential oil on the market

1.2 Research objective

Develop extraction procedure, investigate the chemical composition, and

test the biological activity of the obtained Cinnamomum burmannii essential oil

1.3 Detail goals

- Conduct an experiment to extract Cinnamomum burmannii essential oil by

direct steam distillation method

- Determination of some physicochemical indexes and chemical compositions of essential oils

- Dertermination of antioxidant properties of Cinnamomum burmannii

essential oil

1.4 Limitations

Limitations that are expected to be encountered throughout the study:

- Raw materials: Because Cinnamomum burmannii leaves are available in Bao

Lac town, Cao Bang province, it is difficult to collect and transport raw materials

- There is currently no study on the plant's composition and activities of

Cinnamomum burmannii.

PART II LITERATURE REVIEW

2.1 Overview of Cinnamomum burmannii tree

Cinnamomum burmannii is a woody plant in Bao Lac district, Cao Bang province or known as Phjac Chac, Que Tren or Tren Tren

Cinnamomum burmannii which is one of several species of plants in the

genus Cinnamomum, the family Lauraceae

The flowering plant family Lauraceae, also known as the Laurels, includes the true laurel and its closest relatives This family has over 2850 recognized species in 45 genera worldwide (Christenhusz and Byng, 2016) They are dicotyledons that are found mostly in warm temperate and tropical climates, particularly in Southeast Asia and South America Only six genera account for 66% of the species: Ocotea has about 350 species in tropical America, South Africa, and the Mascarene Islands; Litsea has more than 400 species in Asia, Australasia, and America; Cryptocarya and Cinnamomum (the source of camphor and the spice cinnamon) each have about 350 species; Persea (which includes the avocado plant) has about 200 species; and Beilschmiedia has about 250 species in tropical America, Australia, and New Zealand Many tropical regions have Persea and Cryptocarya, while Cinnamomum is present in all of the main tropical and subtropical climates (F Bruce Sampson and Paul E Berry, 2019)

The economic significance of this plant family stems from the fact that numerous species are employed in industrial areas such as food, lumber, pharmaceutical, and fragrance Lauraceae plants have been used to treat a variety

of illnesses in terms of ethnobotany The genus Beilschmiedia has a variety of therapeutic purposes, including the treatment of infectious disorders, malaria, analgesia, gastrointestinal infections, female genital infections, and rheumatism (Salleh et al., 2015)

Cinnamomum (Cinnamon) is a genus of the Lauraceae family, many of which are used as spices (Shan et al., 2007) Cinnamomum is a genus comprising 250 tree and shrub species found in Southeast Asia, China, and Australia (Gabriel A, Cardoso-Ugarte et al., 2016) There are two major species:

Cinnamomum zeylanicum Nees (cinnamon) and Cinnamomum cassia Blume (cassia) are the sources of the oldest spices known to people (Vernin, G et al.,1994) Their bark, leaves, and buds (particularly their bark) are used as spices

or condiments There are four commercially utilized cinnamon species: C cassia

(syn Cinnamomum Aromaticum) or Cassia, Cinnamomum burmannii known as

Korintje, Cinnamomum Loureiro or Saigon cinnamon, and Cinnamomum zeylanicum (syn C Verum) known as Ceylon (The Seasoning and Spice Association, 2010) These spices had a favorable effect on several MetS indicators in addition to blood glucose They had antihypertensive action in both spontaneously hypertensive rats (Preuss et al., 2006) and MetS patients (Ziegenfuss, 2006; Graf, 2010)

Cinnamomum burmannii is a species closely related to C cassia (Cinnamomum aromaticum Nees) They are native to Southeast Asia and Indonesia, also known as Indonesian cinnamon, Padang cassia, Batavia cassia, or Korintje In addition, it has been used commercial name is a cinnamon stick (Shan, B et al., 2007) The plant has oblong-elliptical leaves that are 4-14 cm long, glossy green, and oppositely oriented, as well as an ovoid long fruit, and small yellow flowers that bloom in early summer The plant's dried bark is sold

on the market in the shape of rolls and quills, which are used in cooking and

flavoring (Tan, 2005) As a traditional plant, C.burmannii has been cultivated for

everyday requirements (cinnamon spice in food) and illness treatment

(Zhang,2008; Al-Dhuhiab, 2012), and logging residues (e.g., berries and leaves) are created as agricultural waste (Wang et al.,2006) C.burmannii leaves have

high antioxidant and antibacterial characteristics (Chandurkar et al., 2014)

2.1.1 Characteristic of Cinnamomum burmannii

According to the assessment result report of Vietnam National Museum of Nature, Vietnam Academy of Science and Technology in March 2022,

Cinnamomum burmannii is a woody plant, 6-8 meters high, the branches and leaves have the smell of lemongrass Peduncle 8-12 mm long, rounded, slightly rough, leaf blade oval to ovate, 9-12 cm long, 3-4.5 cm wide, the base of blade wedge-shaped, tip-shaped, 10-12 mm long, hairless, dark green on both sides, upper side concave veins, arc-shaped, starting from the base of the leaf blade to

the end of the leaf blade, the veins are light brown when dry Inflorescences panicle, short, weak, flower axis has slit longitudinally when dry, with short soft hairs, bracts spoon-shaped, 6-8 mm long, directed upwards Flowers pale yellow cream, flower stalks as long as bracts, 6-8 mm long, lower part wide funnel-shaped, 1-1.5 mm high, soft hairs, upper split into 6 lobes, divided into 2 rings, each ring 3 lobes, lobes oblong, 7-8 mm long

Figure 2.1 Cinnamomum burmannii Tree

2.1.2 Distribution, ecological characteristic and growth

Cinnamomum burmannii tree is a woody tree, growing on rocky mountains

over 1000 meters above sea level Cinnamomum burmannii tree grows in Bao

Lac district, Cao Bang province, concentrated in Lung Quat and Lung Cham hamlets, Khanh Xuan commune, Ta Lay and Lung Rac hamlets, Xuan Truong commune, Lung Cam hamlet, Huy Giap commune

In terms of ecology, the Cinnamomum burmannii tree prefers a hot and humid environment, rainfall, or enough of water for irrigation Cinnamomum

burmannii thrives in steep places more than 1000 meters above sea level and has

a high cold tolerance The tree has dense foliage and does not shed its leaves on a seasonal basis The shrub blooms in the spring and bears fruit shortly after Cao Bang has a tropical monsoon environment with two different seasons each year: the rainy season and the dry season, which is ideal for the establishment and growth of Phjac Chac

The Cinnamomum burmannii tree thrives in the steep highlands of Cao

Bang's Bao Lac area Mostly concentrated in the communes of Khanh Xuan, Xuan Truong, and Huy Giap Leaves can be utilized as culinary flavorings as well as raw materials in the fragrance and medicinal fields

In addition, the tree grows in green forests at altitudes between 500m and 1500m from Ha Tay, Ninh Binh, Thanh Hoa through Nghe An, Quang Tri, Thua Thien-Hue, to Khanh Hoa, Lam Dong

2.1.3 Uses of Cinnamomum burmannii leaves

Both bark and leaves of Cinnamomum burmannii are fragrant, this aroma

also varies depending on the distribution area of the tree Root bark, stem bark, leaves, and branches are spicy, slightly sweet, and warm It has an effect on fighting colds, headaches, rheumatism, joint pain, and stomach pain

In Malaysia, it is widely used to treat diarrhea, illness, and malaria

In China, Bark bark and the root bark are used to treat stomach discomfort caused by cold sores, anorexia, diarrhea, rheumatism, joint pain, back pain, and externally to cure pain, swelling, swelling, and sores Dosage of peel 6-10g, decoction, or 1.5-3g powder for oral use For external use, pound and add alcohol

to apply or apply powder to the wound

In Indonesia, Cinnamomum burmannii is used as a spice and medicinal

instead of C zeylanicum or Sri Lankan cinnamon

In Vietnam, it has been known since ancient times to utilize aromatic plants

to manufacture spices and flavorings to process foods in order to raise the appeal

and improve the quality dish The wood of Cinnamomum burmannii is used in

construction Crushed bark mixed with agglomerates is used as incense Essential oils in the soap industry and medicine People in the Bao Lac area use

Cinnamomum burmannii leaves as a condiment because the leaves have a subtle perfume comparable to lemongrass and have a very excellent impact on meridians In addition, on the days leading up to the Lunar New Year, people

frequently use Cinnamomum burmannii leaves to boil water for bathing When

bathing, this plant will provide a sense of rejuvenation and relaxation, as well as erase any weariness

2.2 Overview of essential oil

2.2.1 Definition of essential oil

Essential oils are compounds extracted from plants with a distinct odor that varies depending on the essential oils' fundamental material sourse

Essential oils are found in nature in a free state, with just a few in a latent state, which means they are not present in the raw materials and only manifest under specified processing conditions before separation extracted or mechanically actuated Essential oils may be extracted in their natural state from raw materials under normal conditions Essential oils are classified into two types: pure essential oils and non-pure essential oils

Pure essential oils are those that have not been tainted with other chemicals Extracted entirely from natural plants Except for some essential oils produced from non-edible plants, they are normally edible, extremely delicious, and safe for health in little amounts (in raw forms - such as wintergreen, cypress ) So, if

an essential oil is derived from edible plants in their raw state (orange, lemon, cinnamon, mint, ginger, lemongrass, pepper, etc.), the essential oil will be edible

in a certain amount when extracted into pure essential oils; if not edible, these oils are frequently not pure from nature

Non-pure essential oil: An essential oil that has been blended with pure essential oil but keeps the flavor of essential oils or essential oils produced from plants but it has not achieved fully pure purity Herbal compounds with natural health advantages

Figure 2.2 Main molecules of essential oils

Compounds in essential oils are classified into the following groups:

- Monoterpenes with an open circuit (myrcene, ocimene)

- Monoterpenes with a closed circuit (cyclic monoterpenes) (p-cymene, pinene, sabinene)

- Open-circuit oxidized monoterpenes (farnesol, linalool, neral)

- Cyclic monoterpenes oxidized (terpineol, geraniol)

- Cyclic sesquiterpenes (farnesene)

- Open-circuit sesquiterpenes (copaen, humulen)

- Open-circuit oxidized sesquiterpenes (nerolidol)

- Cyclic sesquiterpenes that have been oxidized (nootkatone, spathulenol)

- Aromatic compounds (indol)

- Long-chain hydrocarbons (tetradecane, dodecanal)

2.2.2 Physical and chemical properties of essential oil

2.2.2.1 Physical properties

To measure the physical qualities of essential oils, markers such as density, refractive index, solubility in alcohol 90°C at 25°C, boiling point, polar rotational yield, and color are commonly used

Most essential oils have a weight of less than one However, a few essential oils have a greater density than water

Although essential oils are insoluble or just slightly soluble in water, they dissolve easily in most organic solvents such as ether, alcohol, etc Although the chemical composition of each essential oil is different, they all have a boiling point of around 100°C - 200°C, are volatile, and have a pleasant scent

Essential oils are often colorless or light yellow in appearance Because of the existence of colored molecules that are attracted to the essential oil during extraction, a few essential oils, such as cinnamon essential oil have a brown color, wormwood essential oil has a yellow or blue color (yellow due to carotenoids, blue due to chlorophyll, ), orange, lemongrass essential oil has a yellow or amber color The oxidized components of essential oils are primarily responsible for their smell and flavor

2.2.2.2 Chemical properties

The components in essential oils are terpenoid compounds ( unsaturated hydrocarbons) so they are easily hydrolyzed (especially at high temperatures) and decomposed by light into other compounds Therefore, people often store essential oils in dark bottles, with small mouths and tight lids, cooling storage at 5°C

2.2.3 The role of essential oils in plant life

Essential oils, like many other plant chemicals, are biosynthesized from the same precursor, acetic acid, according to traced carbon and other techniques This acid breaks down into Geranylpyrophosphate and Farnesylpyrophosphate, which biosynthesize a variety of compounds to suit the demands and growth of the plant body Essential oils have also been demonstrated to have a role in a variety of metabolic processes in plants Essential oils are found in plants not only in free form in essential oil vesicles, but also in connection with diverse substances in plant cell tissues, and so play a role in metabolic, physiological, and biochemical processes Some individuals believe that the free essential oils in essential oil sacs are secretions from plants' secretory organs, but many others believe that they are a reserve, a protective substance Metabolic essential oils and functional essential oils are essential oils that are connected with other compounds in different tissues and organs of plants

Essential oils in plants play the following roles, according to P.X Tanaxienco (1985):

- Protection: against pests, fungus, and the effects of the outside environment

- Alter the surface tension of water in plants, stimulate water transport, and improve enzyme reaction efficiency Essential oils, particularly the light, odorous components, have recently been proposed as chemical messengers between living creatures in the plant kingdom and may have a function in plant survival and growth, particularly in plants Bees, butterflies, and insects are drawn to the scent

of essential oils of flowers, and it is because of this that the transition from one bloom to another, from male to female, carrying Pollination is more fruitful and plants develop faster when the pistils are followed

According to Charabot, essential oils operate as storage compounds in the plant, with the potential to transfer to other areas of the plant where the essential oil

is employed as a source of energy or to make new structures near to it Unlike Charabot, Tschirch (1925) proposed that essential oils are occasionally maintained

in essential oil reservoirs and do not participate in subsequent reactions

Mutxtiatse (1985) proved in subsequent years while employing tracer carbon to analyze the metabolism of essential oils in vivo, that the essential oil components stored in the secretory glands are not permanent secretions However, they also actively participate in the plant's metabolism, ensuring that the chemical makeup of the essential oils in the plant is constantly regenerated Many chemical components of essential oils, such as low-molecular-weight acids, alcohols, cyclic aldehydes, and so on, have been proven to be the starting ingredients for the synthesis of a diverse variety of physiologically active chemicals via experimental evidence A number of the aforementioned precursors can be found among the essential oil components The most prevalent organic acids include acetic acid, valerianic acid, isovaleric acid, and their corresponding alcohols, as well as aldehydes, esters, and several terpenoids They are substances with several distinct sorts of chemical structures that engage in many anabolic processes Aromatic molecules such as acetone, pheniletilnol, vanillin, and even nitrogen and sulfur compounds are frequently present in the composition of essential oils As a result, explaining the role of essential oils in general or focusing on a few particular theories is impossible To correctly estimate the significance of essential oils in plant life, investigations of individual components of essential oils or molecules with similar structures are required Currently, the relevant information focuses mostly on the involvement of essential oil components in metabolism, i.e., essential oils participate in physiological and biochemical processes within cells Many recent studies have shown that various terpenoid types of essential oils, such as geraniols, linalool, farnesol, etc., are found in the majority of living creatures at all evolutionary stages microbes, lower plants, higher plants, animals, and humans Terpenes are made up of two, three, or more isoprene molecules, and isoprene is one of the building blocks of carotenoids, steroids, and rubber Subsequent research has

verified that biosynthesis in all plants begins with acetic acid and progresses via intermediate molecules such as mevalonic acid, isopentenyl, and pyrophosphate

to geraniol and geranyl, farnesyl phosphate It has been demonstrated experimentally that the carbon chains in the molecules geraniol, linalool, farnesol, and nerolidol are the major intermediates in the biosynthesis of biologically active terpenoids such as phytons, hormones, steroids, bile acids, vitamins D, K, E, carotenoids, gibberellin group growth stimulants Some typical molecules in essential oil composition, such as linalool, farnesol, nerolidol are constantly present in most plant life activities

2.2.4 Biological activities and uses of essential oils

The biological activities of essential oils, such as antibacterial, antifungal, and antioxidant, killing cancer cells have been studied extensively in different essential oils

In the culinary, pharmaceutical, and cosmetic sectors, essential oils are utilized as flavorings Menthol, for example, is used as a pain reliever, to treat colds and fevers, as an anti-inflammatory, as a powerful disinfectant, as an air cleaner, and as an insect repellant Rose essential oil is used in cosmetics to heal brain-damaged skin, decrease wrinkles, and moisturize dry skin Triterpene chemicals from the Dinh Lang plant are used as a tonic (Vo Van Chi, Dictionary

of Vietnamese medicinal plants, 2018) Citronella is an essence extracted from the lemongrass tree This essence can bring many uses such as preventing bacteria, and infections, supporting the treatment of depression, reducing fever, and killing insects Eucalyptol (1,8-cineole) makes up 90% of eucalyptus oil, used in flavorings, fragrances, and cosmetics Eucalyptol is an ingredient in commercial mouthwashes and has been used in traditional medicine as a cough suppressant (Boland, D J, 1991) Citral (Geranial and Neral) is present in the oils

of several plants, including lemon myrtle (90–98%), Litsea citrata (90%), Litsea cubeba (70–85%), lemongrass (65–85%), lemon tea-tree (70–80%), used as an aroma compound in perfumery or used in the synthesis of vitamin A, lycopene, ionone, and methylionone, to mask the smell of smoke or used as a food additive ingredient Eugenol is extracted from certain essential oils, especially from clove, nutmeg, cinnamon, basil, and bay leaf, used as a flavor or aroma ingredient in

teas, meats, cakes, perfumes, flavorings, and essential oils It is also used as a local antiseptic and anaesthetic, etc ( The Aromatic Plant Project, 2012)

Essential oils are widely used to treat colds and respiratory illnesses since they contain vasodilator and antibacterial qualities Terpinhidrate, for example, is used as a cough suppressant, while Terpin-codeine is utilized to treat cough and expectoration in chronic bronchitis patients Lupeol, which is present in popular fruit trees such as Olives, Mangoes, Grapes, and others, has antibacterial and anti-arthritic qualities It has therapeutic and preventative actions for a number of illnesses, dissolving blocks of malignant tumors in the head and neck Essential oils include compounds that have unique properties, such as anesthetic, digestive help, insect attraction, anti-cancer, etc (Vo Van Chi, Dictionary of Vietnamese medicinal plants, 2018)

Cinnamon essential oil, for example, has nourishing, energizing, antibacterial, and detoxifying properties In addition, it is also used as an anti-fungal, anti-allergic, stomach cancer, anti-oxidant, and anti- H2O2 agent that destroys cells

Because of their cytotoxic, anticancer, and antibacterial properties, sesquiterpene lactone compounds in essential oils play an important role in research Herbivores and insects dislike them because they have a bitter taste and

an unpleasant odor

2.2.5 Cinnamomum burmannii essential oil

The composition and properties of Cinnamomum burmannii essential oil

depend on the variety, soil, and climate of each region

According to Kuspradini, Harlinda, et al.(2016), research the essential oils

of Cinnamomum burmannii extracted from the leaves by steam distillation

resulted in a clear, yellow liquid, and C burmannii appears to be richer in oil

(1.01 %) than the other plant species, besides The essential oils of Cinnamomum

burmannii also have the potency to inhibit the free radicals at concentration 25–

100 ppm Rowaan (1936), explored the existence of essential oil in Cinnamomum

burmannii leaves They discovered that the leaves of Cinnamomum burmannii

contain essential oil (0.4 %) and that the primary ingredients are cinnamaldehyde (45–62 %) and eugenol (10%) Chen et al.(1992), investigated the essential oil

extracted from the leaves of Cinnamomum burmannii using steam distillation The oil production ranged between 0.54 and 0.85%, with Cinnamomum

burmannii having the highest safrole level of any Cinnamomum species studied

By steam distillation, Liu et al.(2007), investigated the essential oils of

Cinnamomum burmannii fruits, shoots, and leaves in Yuebei GC-MS was used

to describe 41 components, the most important of which was borneol (68.5%–73.8%) Linalool, caryophyllene, nerolidol, elemene, citral, camphene, fenchol, guanine, myrcene, Sylvestre, terpineol, and pinene were among the other compounds found

In another study by Su J et al.(2010), the essential oil of Cinnamomum

burmannii leaves was examined using GC-MS and found to have 40 volatile components, accounting for 99.4 percent of the total oil D-borneol (78.6%), bornyl acetate (3.26%), (-)-spathulenol (2.60%), and eucalyptol (1.92%) were shown to be the primary ingredients Deng et al.(2010), studied the chemical

components and antioxidant activity of the essential oils of Cinnamomum

burmannii leaves: there were 61 components detected, accounting for 93.58% of the oil The maximum DPPH and ABTS+ radical scavenging rates were 21.71% and 58.89%, respectively On the DPPH and ABTS+ radicals, maximal scavenging rates were 21.71% and 58.89%, respectively In comparison to BHT, the oils had a poor reductive capacity The major constituents identified were caryophyllene (21.71%), eucalyptol (18.22%), guaiol (7.52%), (+)-α-terpineol (7.06%), (-)-0β-pinene (3.57%), γ-eudesmol (3.33%), bulnesol (3.16%), (Z)-nerolidol (3.16%), elemol (2.67%), α-caryophyllene (2.22%), (1S)- β- pinene (1.9%), (-)-terpinen-4-ol (1.8%), (+)-ledene (1.35%), caryophyllene oxide (1.29%), and γ-terpinen (1.05%) While the antioxidant activities of these oils were concentration dependant, they showed high scavenging activities on the ABTS+ radical and poor activities on the DPPH radical

Zhang J et al.(2009), the effects of temperature, light, and pH on the anthocyanin's radical scavenging activity which was extracted from the fruit

extract of Cinnamomum burmannii using semi-preparative HPLC The

anthocyanin's IC50 was determined to be 4.6 g/mL, and its antioxidant activity was shown to be drastically reduced after heating it for 5 hours at 100°C or 30

minutes at 130°C The DPPH radical scavenging activity was not altered by increasing the pH, however exposure to fluorescence radiation for 1 hour lowered the DPPH radical scavenging activity dramatically, and sunlight intensity also influenced the anthocyanin's DPPH radical scavenging activity

In another attempt, Shan et al.(2007), used the agar well diffusion technique

to test extracts of 46 dietary spices and medicinal plants, including Cinnamomum

burmannii, for antibacterial activity Bacillus cereus, Listeria monocytogenes, Staphylococcus aureus, Escherichia coli, and Salmonellaanatum were among the microorganisms used The majority of the extracts had substantial phenolic content and good antibacterial activity The strains with the highest activity against were S aureus and the lowest activity against were E coli

2.3 Methods extraction of essential oil

Essential oils are natural odorant and aroma agents that are created in hundreds of tons per year from the bark, leaves, and flowers Studies on the physical properties, chemical composition, and biological activity of essential oils obtained by modern extraction techniques have received much attention in recent years

In the world, there have been many research works on essential oils such as lemon, grapefruit, lemongrass, cinnamon, anise, etc For example, Lin and Rouseff (2001) and Napapor Thavanapong of the Faculty of Pharmacy, Silpakorn University, Thailand, investigated grapefruit essential oil using the cold pressing method; Atti-Santos, Serafini, Moyna, et al (2005) investigated the best conditions for extracting lemon essential oil using steam distillation (HD) and supercritical CO2 extraction (CO2-SFC); Wang, R., Wang, R., & Yang, B (2009) have extracted essential oils from five cinnamon leaves and identification of their volatile compound compositions; B Uysal et al (2011) investigated the essential oil content and antibacterial activity of grapefruit peel essential oils extracted without using solvents

In the country, there have been researched projects such as Tran Thien Hien

et al (2019) studied the extraction of lemongrass by Microwave-assisted distillation (MAHD); Phat, Dao Tan, et al (2019) "Study on the kinetic model of the extraction process and chemical composition of the essential oil of orange

hydro-peel (Citrus sinensis)." ; Phuc, Nguyen Dinh, et al (2020) "Extraction of

Rosemary Essential Oil by Steam Distillation." Currently, the following extraction methods are still commonly used to extract essential oils:

2.3.1 Hydrodistillation

Hydrodistillation has long been used to extract essential oils and bioactive substances from plant sources Water distillation, water and steam distillation, and direct steam distillation are the three basic forms of steam distillation used in industry (Rasul, M G.,2018)

a, Water distillation

Ingredients and water are combined in a single device When water boils, the steam that escapes will carry the essential oil, while the condensed steam will include a combination of water and essential oil Because these two components

do not dissolve, they may be readily separated

This process is straightforward, and the equipment is inexpensive and simple to construct, making it ideal for small production facilities with limited resources However, this process has drawbacks such as low efficiency, low essential oil quality, and difficulty adjusting technical factors such as speed and temperature since raw materials are in direct contact with the equipment distillation

b, Water and steam distillation

Ingredients and water are combined in the same apparatus, but a pot griddle separates them Steam rises through the material mass as it boils, pushing the essential oil out of the condenser Depending on the type of material, one or more layers of bags can be placed on the blister to prevent items from dropping into the water portion This approach is appropriate for medium-sized manufacturing plants This process has more benefits than direct water distillation; the material is less burned since it is not in direct contact with the bottom of the device, and other shortcomings have not been solved For materials that cannot survive high temperatures, this approach is appropriate (Azmir, J et al.,2013)

c, Direct steam distillation

In this method, steam is produced in a separate boiler and directed into the distillation unit in this approach This technology is appropriate for large

manufacturing operations This approach has the benefit of being able to employ multiple distillation equipment at the same time, having lighter working conditions for workers, being easy to mechanize and automate the production stages, and having strong control than technological characteristics, reducing production time Furthermore, this process eliminates the need for raw material combustion and allows for the distillation of superheated steam in accordance with technological specifications However, certain essential oils can degrade and lose quality when exposed to high temperatures and pressure during distillation Furthermore, the equipment required for this procedure is both complicated and costly (Azmir, J et al.,2013)

Advantages and disadvantages of the distillation method

Advantages:

- The technical procedure is straightforward

- Compact, easy-to-manufacture equipment

- Auxiliary materials, such as impregnation and absorption processes, are not required

- Distillation time is relatively short

Disadvantages:

- Ineffective with low-essential-oil-content ingredients

- If there are components in essential oils that are easily degraded, the quality of the essential oil might be impacted

- Raw materials comprise resins and waxes that cannot be obtained (which are valuable natural flavoring agents)

- There is usually a significant percentage of essential oil in distilled water

- Essential oils with high boiling points frequently function poorly

2.3.2 Solvent extraction

When subjected to organic solvents, the approach is based on the phenomena

of osmosis, diffusion, and dissolution of essential oils found in raw tissues Extraction is frequently employed to separate volatile flavorings for which distillation has been found to be ineffective The raw ingredients are soaked in an organic solvent, then vacuum distilled to recover the solvent, leaving a combination

of essential oils and waxes known as essential oils To get pure essential oil, dissolve

"concrete oil" in absolute alcohol and cool The wax will harden and be separated, and the remaining liquid will be steam-distilled There are two main product forms

of aromatics currently circulating on the market

Requirements of extraction solvent

The quality and character of the extraction solvent is the most essential aspect of the method's success, hence it must match the following requirements:

- The aromatic components in the raw materials must be thoroughly and promptly dissolved

- Other insoluble substances are found in raw materials, such as wax and oleoresin

- Essential oils have no chemical impact

- When used repeatedly, it does not deteriorate

- It is fully odorless, non-toxic, and non-corrosive to equipment It does not produce explosive combinations with air and has a low viscosity

- The boiling point is low because a high boiling point will damage the quality of the essential oil when distilling the extraction solution to recover the solvent The solvent's boiling point should be lower than the boiling point of the essential oil's most volatile component

- In addition, other criteria such as low-cost, easy-to-find materials are required There is usually no solvent that meets all of the following criteria People mix water-insoluble solvents like diethyl ether, kerosene ether, hexane, chloroform, and others with water-soluble solvents like ethanol, acetone, and others In some circumstances, people merely utilize a solvent combination (Bottu, H M et al., 2022)

Advantages and disadvantages of the method

Advantages: The scent of the products acquired using this process is typically natural The yield of the product is generally higher than with other procedures

Disadvantages: High equipment requirements, solvent loss, and a very difficult procedure (Rasul, M G.,2018)

2.3.3 Enfleurage

The approach is based on the aromatics' adsorption by refined solid or liquid fats (vaseline, paraffin, olive oil, or tallow) People utilize trays that are 8cm deep, 60cm broad, and 100cm long, with a fat-coated glass bottom to absorb aromatics The trays may be layered on top of each other, and the components are equally distributed on the tray's surface to extract the incense After about 12-72 hours, depending on the kind of material, replace the layer of fresh ingredients and let them adsorb 10-15 times Aromatic fat is produced at the end To extract the essential oil, saturate the fat with high-speed ethanol, cool to -10°C to eliminate all the fat, and then concentrate the ethanol under low pressure to collect the essential oil This method is frequently used to extract the aroma of flowers in order to create a concentrated essential oil that may be utilized in cosmetics ( Pensuk, W et al.,2007)

2.3.4 Maceration

The absorption of aromatics by fatty oils is the basis for this approach Flowers are steeped in refined fatty oil for 1-2 hours in a device heated by steam at 45-60°C Remove the flowers after each bath and place them in a centrifugal flaker to collect the oil before adding further ingredients Each batch of solvent can be used 10-15 times before it has to be replaced Aromatic saturated fatty oils are mixed with ethanol and condensed under low pressure to produce a concentrated essential oil comparable to that obtained by marinating(Rasul, M G.,2018)

2.3.5 Cold pressing extraction

This approach can be used on medicinal species with a high essential oil concentration as well as essential oil-containing cells in the bark of Citrus species Essential oil is produced from removed materials by pressing them at room temperature using endless screw presses or presses The end result is a combination of essential oils and pulp that may be readily separated by adding saturated salt to the mixture; the essential oil will float to the top, followed by wax and resin Dissolve the essential oil in 75-80°C ethanol, then chill to remove the wax We have an essential oil product with a tiny bit of resin mixed in after concentrating under low pressure to eliminate ethanol When compared to the

measured essential oil content in the laboratory, this approach can provide an efficiency of above 90% (Johner, J C et al., 2018)

In addition, novel technologies for extracting essential oils are being used nowadays, such as distillation or extraction with the use of microwaves, ultrasound, solid-phase micro-extraction (SPME) is a solid phase extraction sampling technique that includes the employment of a fiber coated with an extracting phase, which may be a liquid (polymer) or a solid (sorbent) (Spietelun, Agata, et al., 2010) which extracts various analytes (volatile and nonvolatile) from various media, which can be in liquid or gas phase The amount of analyte extracted by the fiber is proportional to its concentration in the sample as long as equilibrium is attained or, in the event of short-term pre-equilibrium, with the assistance of convection or agitation (Mitra, Somenath, ed., 2003).; supercritical fluid extraction (SFE) uses a supercritical fluid, most typically CO2 Because of the inherent low viscosity and high diffusivity of supercritical CO2, SFE is a quicker and more efficient extraction process as compared to standard liquid extraction This allows for quicker flow rates and, as a result, faster extraction times without the need for a higher pressure system The addition of a co-solvent

to the CO2 flow can aid in fine-tuning the strength even more Temperatures in the extraction oven can reach 100°C Following the extraction, the vessel is the backpressure regulator, which provides the backpressure required to maintain the CO2 supercritical and is an essential aspect of the extraction's performance; simultaneous distillation extraction (SDE), etc However, the approaches described above are costly More costly and more complicated technology is less typical in practice

Compared with the above methods, the steam distillation method is easy to apply under laboratory conditions, has low investment costs, is easy to operate, and gives the highest extraction efficiency Therefore, I have applied the direct

steam distillation method to distill essential oil from Cinnamomum burmannii

leaves for the topic

PART III METHODOLOGY 3.1 Material and research scope

3.1.1 Material

The major raw material utilized in the study to extract essential oils is the

leaves of the Cinnamomum burmannii tree, which was collected in the Bao Lac

area of Cao Bang, identified by Mr Nguyen Quoc Binh, Vietnam Academy of

Science and Technology identify the scientific name was Cinnamomum

burmannii (Nees.) Blume, 1826, Lauraceae family

The leaves of Cinnamomum burmannii used to extract essential oil must be

fresh, not moldy, not damaged, bruised, wilted, not infested by pests and illnesses, mature leaves; do not gather leaves that are too young or too old because the leaves are too old That means the essential oil content of the leaves

is minimal

Figure 3.1 Cinnamomum burmannii leaves

3.1.2 Research scope

Research was carried out in the laboratory scale

3.2 Work place and time to proceed

- Location: Laboratory of Institute of Life Sciences, at Thai Nguyen University of Agriculture and Forestry

- Implementation time: January 2022 to May 2022

3.3 Equipment and chemicals

Table 3.1 Experiment Chemicals

7 2,2-diphenyl-1-picrylhydrazyl America

Table 3.2 Experiment Equipment

6 Essential oil distillation set China

7 Gas chromatography–mass

Table 3.3 Laboratory instruments

- Content 1: Determination the effect of extraction time on essential oil content

- Content 2: Determination the effect of (material/distilled water) ratio on

essential oil content

- Content 3: Determination the effect of wilting time of raw material on

essential oil content

3.4.2 Determination of the chemical composition of Cinnamomum burmannii essential oil by GC-MS method

Content 4: Determination of the chemical composition of Cinnamomum

burmannii essential oil

3.4.3 Investigation of antioxidant capacity of essential oils

Content 5: Investigation of the antioxidant capacity of Cinnamomum

burmannii essential oils by using 2,2-diphenyl-1-picrylhydrazyl (DPPH) technique by Radical Scavenging Activity method

Extracted by direct steam distillation method

Antioxidant activity testing

Essential oil

Chemical composition analysis

3.5.2 Experimental design method

Extraction duration, the volume of solvent used for extraction, and wilting time of raw materials are studied as three parameters impacting the quantity of

Cinnamomum burmannii essential oil

The chemical composition of essential oil samples is determined by using Gas Chromatography-Mass Spectrometry (GC/MS) after the three technical parameters were investigated

3.5.2.1 Research on the effect of extraction time on essential oil content

The experiment is carried out with various tests in a totally randomized design, each experiment was repeated three times

Table 3.4 The experimental design to determine the effect of extraction time

on essential oils

Experiment

formula

Extraction time (minutes) Fixed factors Monitored factor

Mass of sample: 5 kilograms

Extraction solvent volume: 15 liters Distillation temperature:

100°C

Amount of essential oil obtained

3.5.2.2 Research on the effect of (material/distilled water) ratio on essential oil

Experiments to determine the effect of (material/distilled water) ratio on essential oil are shown in the Table 3.5 The experiment is carried out with various tests in a totally randomized design, each experiment was repeated three times

Table 3.5 Experimental design to determine the effect of

(material/distilled water) ratio on essential oils

Experiment

formula

Material/distilled water (w/v) ratio Fixed factors

Monitored factor

kilograms

Extraction time will

be selected from experiment 1

Distillation temperature: 100°C

Amount of essential oil obtained

3.5.2.3 Research on the effect of raw material withered time on essential oil content

With the optimal extraction time and solvent volume selected, continue to fix the temperature, and sample weight and investigate the extraction of essential oils with samples with different wilting times: 48 hours, 60 hours, 72 hours, 84 hours, 96 hours The experiment is shown in the Table 3.6

Table 3.6 The experimental design to determine the effect of wilting time

of raw material on essential oils

Experiment

formula

Wilting time of raw material (hours)

Fixed factors Monitored

factor

Mass of sample: 5 kilograms

Extraction time will be selected from experiment 1

The material/distilled water radio will be selected from experiment 2

Distillation temperature: 100°C

Amount of essential oil obtained

Each experiment is repeated three times Based on the amount of essential oil obtained, the most suitable wilting time of raw material will be selected

3.5.3 Determination of the chemical composition of Cinnamomum burmannii essential oil by GC-MS method

The chemical composition of Cinnamomum burmannii essential oil

obtained after extraction by steam distillation is analyzed by Gas Chromatography Mass spectrometry (GC/MS) at Vietnam Academy of Science and Technology, Ha Noi GC-MS instrument: Agilent 7890A gas chromatograph paired with Agilent 5975C Mass Selective Detector, HP-5MS column size (30m×0.25mm×0.25µm) A temperature program with 60°C conditions increases temperature by 4°C /min to 240°C The transition chamber temperature is 270°C, complete fragmentation with a probe potential difference of 70eV, and a spectral range of 35-450Da at 4 scans/sec Components were identified based on their retention coefficients (calculated according to the n-alkane homologous sequence) and compared their mass spectra with standard mass spectrometric data stored in the spectrometric library (HPCH1607, NIST08, Wiley 09) The relative concentrations of the components were calculated based on the peak areas obtained from the chromatogram The mass spectrometry software is Mass Finder 4.0

3.5.4 Investigation of antioxidant capacity of essential oil

Investigation of the antioxidant capacity of Cinnamomum burmannii

essential oils is tested by using a 2,2-diphenyl-1-picrylhydrazyl (DPPH) technique by Radical Scavenging Activity method

The history of hydrazyl free radicals begins around the 1920s when Goldschmidt discovered that oxidation of triphenylhydrazine produces an intense blue color that fades quickly (Goldschmidt,1920) Among numerous triphenylhydrazine congeners, he discovered that the oxidation of 2,2-diphenyl-1-picrylhydrazine, a yellow chemical, also creates an intense violet solution, but in this case, the color is stable Goldschmidt isolated the first stable hydrazyl free radical in high yields in this manner (Goldschmidt, S., & Renn, K., 1922) It was discovered that this free radical does not dimerize or react with oxygen and is stable in both solution and solid-state