(Đồ án hcmute) influence of drying methods on the content of bioactive compounds and antioxidant activities of curculigo orchioides

Effect of drying methods on the drying time and moisture content of the COR slices with different thickness .... Effect of different drying methods on total phenolic content of the dried

MINISTRY OF EDUCATION AND TRAINING

HO CHI MINH CITY UNIVERSITY OF TECHNOLOGY AND EDUCATION

FACULTY FOR HIGH QUALITY TRAINING

CAPSTONE PROJECT FOOD TECHNOLOGY

LECTURER: PhD HOANG VAN CHUYEN STUDENT: PHAM TRUONG TIEU LAM

INFLUENCE OF DRYING METHODS ON THE CONTENT

OF BIOACTIVE COMPOUNDS AND ANTIOXIDANT ACTIVITIES OF CURCULIGO ORCHIOIDES

Ho Chi Minh City, December, 2021

SKL 0 0 8 4 4 9

HO CHI MINH CITY UNIVERSITY OF TECHNOLOGY AND EDUCATION

FACULTY FOR HIGH QUALITY TRAINING

GRADUATION PROJECT

CODE: 2021-17116015

INFLUENCE OF DRYING METHODS ON THE

CONTENT OF BIOACTIVE COMPOUNDS AND

ANTIOXIDANT ACTIVITIES OF CURCULIGO

ORCHIOIDES

Major: FOOD TECHNOLOGY

Advisor: HOANG VAN CHUYEN, PhD

PHAM TRUONG TIEU LAM

Student ID: 17116015

Ho Chi Minh City, December 2021

GRADUATION PROJECT ASSIGNMENT

DISCLAIMER

ACKNOWLEDGEMENTS

I sincerely thank the supervisor Hoang Van Chuyen who has taught me during the process of studying.He guided me enthusiastically during my study, he also trained me for theorical knowledge and laboraty skills He was also carefully told us when receiving samples and how to prepare it; moreover, he supported us with the experimental supplies and materials to help us complete the thesis better.

Besides, I would like to thank Ms Tran Thi Thu Trang, who supported me in terms of the facilities system and regulations in the laboratories Moreover, I would like to express

my sincerest thanks to the lecturers, who have trained me step by step not only in specialized knowledge but also self-study skills during my time at the university

Finally, I would like to express my deep gratitude to my family, who have always encouraged me from the time I worked on the research project until the time I finished the experiments

ADVISOR’S EVALUATION SHEET

PRE-DENFENSE EVALUATION SHEET

EVALUATION SHEET OF DEFENSE COMMITTEE

MEMBER

TABLE OF CONTENTS

GRADUATION PROJECT ASSIGNMENT i

DISCLAIMER ii

ACKNOWLEDGEMENTS iii

ADVISOR’S EVALUATION SHEET iv

PRE-DENFENSE EVALUATION SHEET vi

EVALUATION SHEET OF DEFENSE COMMITTEE MEMBER vii

TABLE OF CONTENTS x

LIST OF FIGURES xii

LIST OF TABLES xiii

LIST OF ACRONYMS xiv

ABSTRACT xv

CHAPTER 1: INTRODUCTION 1

1.1 Research rationale 1

1.2 Research objectives 2

1.3 Research contents 2

1.4 Significance of the research results 2

CHAPTER 2: LITERATURE REVIEW 3

2.1 Overview of Curculigo orchioides 3

2.2 Overview of Polyphenol 6

2.3 Overview of drying methods 10

2.4 Literature review of of the studies on Curculigo orchioides 12

CHAPTER 3: MATERIALS AND METHODS 14

3.1 Materials, Chemicals and Equipments 14

3.2 Experimental design 18

3.3 Extraction of samples 20

3.4 Analytical methods 22

3.4.1 Determination of moisture content 22

3.4.2 Analysis total phenolic content 23

3.4.3 Determination of DPPH free radical scavenging activity 25

3.4.4 Determination of ABTS free radical scavenging activity 27

3.4.5 Determination of Ferric reducing antioxidant power (FRAP) 29

3.5 Statistical analysis 30

CHAPTER 4: RESULTS AND DISCUSSION 31

4.1 Moisture content, TPC and antioxidant activities of the fresh Curculigo orchioides rhizome (COR) 31

4.2 Effect of drying methods on the drying time and moisture content of the COR slices with different thickness 31

4.3 Effect of drying methods on total phenolic content of the dried COR slices 35

4.4 Effect of drying methods on antioxidant activities 38

4.4.1 Effect of drying methods on DPPH free radical scavenging capacity of the dried COR slices 38

4.4.2 Effect of drying methods on ABTS free radical scavenging activity 40

4.4.3 Effect of drying methods on Ferric reducing antioxidant power 42

CHAPTER 5: CONCLUSION AND RECOMMENDATIONS 45

5.1 Conclusion 45

5.2 Recommendations 45

REFERENCES 46

APPENDIX 54

LIST OF FIGURES

Figure 2.1 Curculigo orchioides plant [10] 3

Figure 2.2 Curculigo orchioides tuberous root [11] 4

Figure 2.3 Cinamic acid and Benzoic acid derivatives 7

Figure 2.4 Chemical structure of Flavonoids 7

Figure 2.5 Chemical structure of Stilbenes 8

Figure 2.6 Lignan biosynthesis 9

Figure 2.7 Examples of various lignan skeletal units 9

Figure 2.8 An example of Lignan molecule 10

Figure 3.1 Convective dryer (Memmert, Germany) 15

Figure 3.2 Vacuum dryer (Memmert, Germany) 15

Figure 3.3 Infrared drying (DS.IR – 03, HCMC University of Technology and Education, Vietnam) 16

Figure 3.4 Heat pump drying (cold drying system) (DSL – P – L – T – 02, HCMC University of Technology and Education, Vietnam) 16

Figure 3.5 Analytical balance 10mg (LT 2200C, Precisa, Switzerland) 17

Figure 3.6 Analytical balance 1mg (LS3200C and LS320, Precisa, Switzerland) 17

Figure 3.7 Powder grinder machine (China) 17

Figure 3.8 Spectrophotometers UV-Vis (UH 5300, Hitachi, Japan) 17

Figure 3.9 Diagram of the experimental design 18

Figure 3.10 Curculigo orchioides after cutting hairy roots 19

Figure 3.11 Curculigo orchioides in size 2mm and size 5mm 19

Figure 3.12 Diagram of extraction of samples 21

Figure 3.13 Reaction of evaluation of total Polyphenol content 23

Figure 3.14 The experiment of determining total Polyphenol content 24

Figure 3.15 Reaction of DPPH free radicals scavenging 26

Figure 3.16 The experiment of DPPH free radical scavenging 26

Figure 3.17 Reaction of ABTS free radical scavenging 28

Figure 3.18 The experiment of ABTS free radical scavenging 28

Figure 3.19 Reaction of Ferric reducing antioxidant power 29

Figure 3.20 The experiment of Ferric reducing anitoxidant power 30

Figure 4.1 Effect of different drying methods on the moisture content of the dried COR slices with the thickness of 2 mm (a) and 5mm (b) 33

Figure 4.2 Effect of different drying methods on total phenolic content of the dried COR slices with the thickness of 2 mm (a) and 5mm (b) 36

Figure 4.3 Effect of different drying methods on DPPH free radical scavenging capacity of the dried COR slices with the thickness of 2 mm (a) and 5mm (b) 39

Figure 4.4 Effect of different drying methods on ABTS radical scavenging capacity of the dried COR slices with the thickness of 2 mm (a) and 5mm (b) 42

Figure 4.5 Effect of different drying methods on FRAP antioxidant capacity of the dried COR slices with the thickness of 2 mm (a) and 5mm (b) 44

IRD: infrared drying

HPD: heat pump drying

SD: sun drying

TPC: total phenolic content

DPPH, ABTS: free radicals

FRAP: Ferric reducing antioxidant power

ABSTRACT

Curculigo orchioides is one of the precious medicinal plants in mountainous areas of

the Northern Vietnam, its rhizome contains many crucial chemical compounds, especially polyphenols and brings a variety of health benefits to the consumers Currently, sun drying

is the most common method used for long-term preservation of Curculigo orchioides but

this drying method may cause severe loss of its bioactive ingredients In this study, effects

of different drying methods on moisture content, total phenolic content and antioxidant

activity of Curculigo orchioides were investigated to determine the most suitable drying

method for this material The results showed that the drying processes and material thickness greatly impacted on total phenolic content and antioxidant powers of the dried

Curculigo orchioides In particular, the infrared drying samples had the highest values of

TPC analysis (76.82% and 80.04%), DPPH radical scavenging activity (48.52% and 50.9%), ABTS antioxidant activity (67.31% and 67.94%), FRAP assay (47.02% and 50.45%) in order of layers 2mm and 5mm, while the convective drying at 100oC of DPPH and ABTS tests, also the vacuum drying at 40oC of TPC and FRAP tests, were given to be the weakest antioxidant capacity in this study Overall, the infrared drying were found to produce the finished medicinal products in good quality than the other thermal drying techniques

CHAPTER 1: INTRODUCTION

1.1 Research rationale

Curculigo orchioides is a medicinal plant commonly grown in the mountainous

provinces in the North of Vietnam According to the traditional experiences recorded in

the book “Vietnamese medicinal plants and remedies” [1] Curculigo orchioides (COR),

also known as Sam Cau and Tien Mao, is a kidney tonic, a physiological enhancer, which can be used as a decoction to drink or soak in wine Additionally, it also has a number of other benificial effects such as: anti-inflammatory activity, antiasthmatic activity, hepatoprotective activity, anti-chronic fatigue syndrome… [2] Curculigo orchioides has

been reported to be rich in bioactive ingredients through the research works [3], [4], [5] The most bioactive group of COR is polyphenols, especially the phenolic glycosides, which act as antioxidant agents to prevent the pro-oxidation process or biological oxidative damages in the living cells [6]

Drying is one of the most convenient methods for preserving products that has appeared for a long time, to prolong the shelf-life and retain the bioactive compositions and beneficial nutrients of the raw materials for use in a long period [7] Many drying methods have been investigated and applied to preserve plant materials with different structures, shapes and sizes in order to make sure the color, chemical compounds and nutrients of the food products The drying system for agricultural products is currently being favored by many customers, playing an important role in the industries of food processing, agricultural goods production, biological products, medicines, and even exportation

Although COR has been reported to be rich in valuable bioactive ingredients, it is currently sun dried has been reported to be rich in bioactive ingredients for the long-term preservation However, sun drying may cause significant loss of its bioactive ingredients and medicinal quality Therefore in this study, the effects of different drying methods including sun drying, convective drying, infrared drying, vacuum drying and heat pump drying on drying time, moisture content, total phenolic content and antioxidant activities

of COR were investigated to determine the most suitable drying method for this medicinal material In addition, the influence of the thickness of the COR slices on the drying time, physicochemical properties and antioxidant activities of the dried products was also investigated

1.2 Research objectives

- Investigate the effect of thickness of the drying material on the drying time, physicochemical properties and antioxidant activities of the dried products

- Determine the most suitable drying method for drying of Curculigo orchioides rhizome

for maximizing the retaining of bioactive ingredients and antioxidant activities of the dried COR

1.3 Research contents

- Analysis of the moisture content, physicochemical properties and antioxidant activities

of the fresh COR

- Investigate effects sun drying and the thickness of the COR slices on the drying time, moisture content, total phenolic content and antioxidant activities of the dried COR

- Investigate effects convective drying and the thickness of the COR slices on the drying time, moisture content, total phenolic content and antioxidant activities of the dried COR

- Investigate effects vacuum drying and the thickness of the COR slices on the drying time, moisture content, total phenolic content and antioxidant activities of the dried COR

- Investigate effects infrared drying and the thickness of the COR slices on the drying time, moisture content, total phenolic content and antioxidant activities of the dried COR

- Investigate effects heat pump drying and the thickness of the COR slices on the drying

time, moisture content, total phenolic content and antioxidant activities of the dried COR

1.4 Significance of the research results

- The experimental results from the study of drying processes on the COR may be used as the scientific basis for further studies on the drying processes at an industrial scale for COR and other parts of this herbal plant as well as other herbs

- The results from drying methods for COR on retaining beneficial bioactive characteristics

are useful knowledge for the development of functional foods and healthy pharmaceutical products

CHAPTER 2: LITERATURE REVIEW

2.1 Overview of Curculigo orchioides

2.1.1 General introduction

- Curculigo orchioides rhizome has skin in black or brown color, white flesh and has a

distinctive aroma when be exposed to the sunlight Ethnic minorities at the Northern mountainous areas of Vietnam usually use this rootstock as a useful medicine so that it called Sam in Vietnam, and the leaves are the same as Cau leaves in Vietnam and this plant

is named Sam Cau In addition, it is also known as Tien Mao, Ngai Cau, Co Noc Lan [8]

- The scientific name of this plant is Curculigo orchioides, which belongs to

Amaryllidaceae family, with the English name Golden eye grass or Black Musale, the Hindi name is Kali Musli [2], [9]

Table 2.1 Table of Taxonomical hierarchy is presented in [2]

Kingdom Plantae Division Magnoliophyta Class Monocotyledon Order Liliales

Family Amaryllidacae Genus Curculigo Species Orchioides

Figure 2.1 Curculigo orchioides plant as illustrated in [10]

- Parts used as medicine: Tuberous root or rhizome

- Plant type: Herbal medicine

2.1.2 Morphology

- Curculigo orchioides, is a plant which stays a long time, the height of 20 – 30 cm The

rhizome is cylindrical and long, straight, slender at the ends, with accessory roots which are short or long, coarse blackish brown bark and white flesh [8]

- The leaves grow in clusters from the rhizome, folded like Cau leaves

- There is a flower cluster growing on the leaves, the leaf sheath is large and long

- Fruit capsule, oblong, about 1.2 - 1.5 cm long Each fruit contains from 1 to 4 capsules

Figure 2.2 Curculigo orchioides tuberous root [11]

Table 2.2 Quality of Curculigo orchioides crude drug samples collected from different places

Place (%) Ash

(%)

Protein (%)

Starch (%)

Fibre (%)

Na (%)

P (%)

K (%)

Ca (%)

Kottayam 2.92 4.90 15.17 19.97 0.784 0.101 0.479 1.521 Muvattupuzha 5.02 4.94 34.08 20.33 0.790 0.110 0.838 1.227 Ernakulam 4.90 4.73 17.41 22.08 0.756 0.076 0.903 1.668 Thrissur 3.93 5.36 23.49 23.62 0.857 0.180 0.725 1.129

Source: M.Asif (2012) [3]

2.1.2 Distribution

Curculigo orchioides distributed mainly in tropical and subtropical Asia such as some

southern provinces of China, Laos, Vietnam and a few other countries in Southeast Asia area

This is a plant that likes light and moisture, especially can tolerate shade, so they are often found in the fertile soils of the valley, along the fields or at the foot of limestone mountains in the northern provinces of Vietnam such as Lai Chau, Tuyen Quang, Cao Bang

This species was listed less likely to be threatened by IUCN (The International Union for Conservation of Nature and Natural Resources) in 1999, which was due to overexploitation and destroy the habitat; moreover, it has also low quality of seed and germination is significantly declined in wild plant density [5]

2.1.3 Medicinal effects and applications [9]

- According to traditional medicine, this herb is spicy and warm

+ Reduce rheumatism, strengthen tendons

+ Treat impotence and aphrodisiac

+ Help with diuresis

+ Treatment of cold in the back, hands and feet

+ Additionally, Curculigo o also cures hemorrhoids, abdominal pain, cough, jaundice

- According to modern medicine, some studies have indicated that Curculigo o has many health benefits:

+ Boost immunity

+ Be able to replace testosterone – male sex hormone because it contains steroids + Support the body's ability to adapt to the environment without oxygen or in high temperature condition

+ Control heart activity, dilate coronary vessels, protect liver

+ Anti-inflammatory, anti-fungal, against blood-related disorders

+ Enhance muscle contraction, treat convulsions, relieve pain

+ Anti-aging, improve skin

+ Prevent diabetes, cancer

- The dosage of the herb:

+ The usage level of adults: 5 - 10 gram

+ From the ages of 5 and 16: one half of adult usage

+ From 1 to 5 years old: a quarter of adult usage

It acts as preservatives, take n-propyl gallate as an example, it was approved by the FDA [15].

Polyphenols can be composed of different groups according to the number of rings that make up a phenol and the structural principles that bind them together The main categories

of polyphenol below are phenolic acids, flavonoids, stilbenes and lignans

Additionally, the food factories have conducted on the analysis on profile and dosage

of phenolic acids as well as their role on fruit repening, enzymatic control and have used them as preservatives [18] [19] [20]

Currently, citations have pointed to high antioxidant content in fruits and vegetables with inhibitory mechanism on diseases caused by oxidative damage such as cancer, cerebral accident, coronary heart disease [21] [22]

Phenolic acids displace two derivatives: cinnamic and benzoic acid [23]

They are discovered in many species of plants: Cinnamic and benzoic acid derivatives appear in most of edible plants (examples in fruits, vegetables, and grains) and are spread

in kernels, leaves, stems and roots of the plant [24] [18]

Phenolic acids are relatively resistant biochemical species; they undergo metabolism in the soil for some microorganisms can consume them as carbon sources [25]

Besides, the natural presence of phenolic acids usually occurs in dried fruit, horse gram plants, mushroom Basidiomycetes and soil humus [26]

Figure 2.3 Cinamic acid and Benzoic acid derivatives

2.2.2 Flavonoids

Chemically, flavonoids are formed on 15 carbon frames, containing 2 aromatic rings,

A and B, connected through a pyran called C as an illustration below [27]

Figure 2.4 Chemical structure of Flavonoids

Normally, flavonoids are synthesized at specific parts in plants and contribute to the color and fragrance of flowers For fruis, flavonoids play a role to attract pollinators and thus helping with dispersal during seed and spore germination and seedling growth and

development [28] Flavonoids include many subgroups such as: flavones, flavonols,

flavanones, flavanonols, flavanols or catechins, anthocyanins and chalcones [29]

2.2.3 Stilbenes

Stilbenes have the structure of a 1,2-diphenylethylene nucleus with hydroxyls attached

to the aromatic hydrocarbons and appear as monomers or oligomers; beside that, most of the compounds studied are trans-resveratrol [30]

Stilbenesare phytoalexins generated by plants in response to disease, injury or stress

symptoms [31]

Figure 2.5.Chemical structure of Stilbenes

Stilbene compounds were formed in many crops including sorghum (Sorghum bicolor), peanut (Arachis hypogaea), grape wine (Vitis vinifera) and many trees such as

Pinus and Picea [32]

Commercially, sources of stilbenes include a variety of flora grown in parts of Asia as folk drugs, are Polygonum cuspidatum, Rhodomyrtus tomentosa, Rheum undulatum, Melaleuca leucadendron, and Euphorbia lagascae For pterostilbene, which is mostly

discovered in blueberries, bilberries and other kinds in grapes and juice pulp [33]

2.2.4 Lignans

The lignans are constituted of two or three mono-lignols at the C position of 6-3 and the catalyst of the reaction is an oxidizing enzyme At units of 6-3, a lignan is formed by the dimerization, which is derived from cynamyl groups [34]

Figure 2.6 Lignan biosynthesis

Neolignans, another kind of lignans, are quite widespread in the plant kingdom Neolignans are also dimers of cinnamyl units, but their structures are assembled by

coupling of mesomeric radicals other than the ꞵ–ꞵ link typical of lignans [35]

Figure 2.7 Examples of various lignan skeletal units

Figure 2.8 An example of Lignan molecule 2.3 Overview of drying methods

 Purpose:

The quantification of the moisture content in medicinal herbs or food is the determination of the percentage of water to check whether these products meet the moisture standard and more importantly, effects of moisture on the stability and quality of foods In addition, moisture determination is an essential for quantitative calculation or extraction efficiency of bioactive compounds in the medicinal herb [36]

The moisture content of harvested medicinal herbs is often very high, about 60 – 80% [37] The final moisture content of the medicinal herbs are then stored usually from 10 to 14% [38] Particularly, the moisture content of the fresh gingsen roots is about 72% (wet basis) [39] and the dried ones with good quality need have between 5.5 and 7.5% of the final moisture content, which is dependent on the size of the roots as mentioned in the analysis[40]

 Determination of moisture content by drying method:

Drying is the process of using thermal energy to take water out of a solid or liquid material For the purpose of reducing the volume of materials, enhancing the durability and helping preserve foods for a long time [41], [42] Drying can be divided into two types: natural drying and equipment drying (or artificial drying)

Natural drying that means the process of drying materials outdoors, without the use of

a drying device

Artificial drying, based on the method of heat supply that can be classified as convective drying, radiation drying, rotary drying, drying with sublimation, microwave drying,

fluidized bed drying

This is the drying technique to let materials directly contact with hot air or smoke to act

as a drying agent at the suitable temperature, humidity and velocity, the flow movement covers the drying material, causing the moisture in this drying object to evaporate and then follow the drying agent out of the drying chamber The air agent can move in the same direction, in the opposite direction of the product flow Convection drying can be done in batches or continuously [7]

There are two processes occurring at the same time in convective drying of the solid material One is the evaporation of the surrounding environment, the other is the transfer

of moisture inside the solid [44]

2.3.3 Infrared drying

The infrared drying operate by under exposing the raw product to electromagnetic radiation with the wavelength between 0.8 and 1000 µm The energy of infrared radiant is transmitted from the heating power to the surface of the product and no heated air surrounding the product simultaneously In addition, it is supposed to generate heat quickly and efficiently to yield better products in terms of sensory and nutritional qualities with uniform heat and lower final costs [45]

The infrared drying method are designed to be aimed at utilizing radiant heat as the primary source [46].

2.3.4 Vacuum drying

This process is applied in the reduction of the atmosphere pressure around the product When the boiling point of water or other solvents is near and the moisture begins to evaporate parallel to the temperature reduced [47]

2.3.5 Heat pump drying (or Cold drying)

This drying method uses the sensible heat recovered from the latent heat of the air stream passing through the condenser to dehydrate [48]

At the heat pump evaporator, the airflow is condensed in the purpose of using the latent heat again for reheating the drying gas [45]

The drying temperature and the humidity can be modified [49]; thereby, helping the heat pumpers can be widely applied in agricultural and pharmaceutical products [50]

Another finding mentioned that the heat pump dryer usually uses low energy [51], this dryer can be operated at low temperature and still retain intact of colour and bioactive substances [52], [53]

2.4 Literature review of of the studies on Curculigo orchioides

Currently, there are many articles about the composition, biological compounds as well

as the uses of this precious medicinal herb, such as: A survey of M Asif on Phytochemical

and Ethnopharmacological Activities of Curculigo orchioides in 2012 [3], Jitendra Mehta

and Krishnendra Singh Nama (2014) mentioned a report of ethnic medicine originating from COR [2], or the experiments by Praveen Kumar and SK Shukla (2019) on the functions of COR in liver toxicity in roosters [54] There are also some domestic studies such as A research on the genetic diversity of the endangered herb in Vietnam, CORs by Quang and his companers [55], Ngoc and her associates (2015) were sucessful in the

isolation of three phenolic compounds from the rootstock of Curculigo orchioides [4], or

antioxidant activity and the content of bioactive compounds were carried out on in vitro

Curculigo orchioides rhizomes by Hieu et al (2020) [56]

Moreover, many research have reported the impacts of drying technology on the components and biological activities of many plants such as Results of drying method on total polyphenolic content and antioxidant capacity of mint carried out by Orphanides et.al (2013) [57], Investigations on drying temperature to physico-chemical features and antioxidant capacity of yellow berries of Jessica et al (2013) [58], or an effect of

dehydration of roselle calyxes in total phenolic and its antioxidant activities by Nguyen Quang Vinh et al (2014) [59]

Because of the characteristics and roles of this plant should be produced the dried products long-term and its wide application, this research is considered the contribution of

thermal processing and phytonutrient content of Curculigo orchioides were determined at

different temperatures. we have conducted a research project about “Influence of drying

methods on the content of bioactive compounds and antioxidant activities of Curculigo

orchioides”

CHAPTER 3: MATERIALS AND METHODS

3.1 Materials, Chemicals and Equipments

3.1.1 Materials

The fresh Curculigo orchioides rhizomes were brought from Lao Cai province and then

transported to the laboratory of the Department of Chemistry and Food Technology, Ho Chi Minh University of Technology and Education within 3 days after being harvested The rhizomes were then washed with tap water and the hairy roots are removed to obtain the central tuberous rhizomes The tuberous rhizomes were then cut into slices for drying

3.1.2 Chemicals

The chemicals used for the study and their corresponding suppliers/manufactures are shown below:

- CH3OH (Methanol, Xilong Scientific Co., Ltd, Shantou City, China)

- Folin-Ciocalteu’s phenol reagent (Merck KgaA, Germany)

- Na2CO3 (Sodium carbonate, Xilong Scientific Co., Ltd, Shantou City, China)

- Gallic acid (3,4,5-trihydroxybenzoic acid, Sigma-Aldrich, U.S.A)

- DPPH (2,2-diphenyl-1-picrylhydrazyl, Thermo Scientific™, Alfa Aesar, U.S.A)

- ABTS (2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid, Sigma-Aldrich Pty Ltd, Castle Hill, NSW, Australia)

- K2S2O8 (Potassium persulfate, Xilong Scientific Co., Ltd, Shantou City, China)

- C2H9NaO5.3H2O (Sodium acetate trihydrate, Xilong Scientific Co., Ltd, Shantou City, China)

- CH3COOH (Glacial acetic acid, Merck KgaA, Germany)

- TPTZ (2,4,6-Tris(2-pyridyl)-s-triazine, Sigma-Aldrich Pty Ltd, Castle Hill, NSW, Australia)

- FeCl3 (Ferric chloride, Sigma-Aldrich Pty Ltd, Castle Hill, NSW, Australia)

- HCl (Hydrochloric acid, Xilong Scientific Co., Ltd, Shantou City, China)

- Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid, Sigma-Aldrich Pty

Ltd, Castle Hill, NSW, Australia)

3.1.3 Equipments

The dryers are used in this project: including convective drying, vacuum drying, infrared drying, heat pump drying (cold drying system) and other equipment are shown in the figures below

Figure 3.1 Convective dryer (Memmert, Germany)

Figure 3.2 Vacuum dryer (Memmert, Germany)

Figure 3.3 Infrared drying (DS.IR – 03, HCMC University of Technology and Education,

Vietnam)

Figure 3.4 Heat pump drying (cold drying system) (DSL – P – L – T – 02, HCMC University of

Technology and Education, Vietnam)

Figure 3.5 Analytical balance 10mg (LT

2200C, Precisa, Switzerland)

Figure 3.6 Analytical balance 1mg

(LS3200C and LS320, Precisa, Switzerland)

Figure 3.7 Powder grinder machine (China)

Figure 3.8 Spectrophotometers UV-Vis (UH 5300, Hitachi, Japan)

Tubers were put into sealed, clean plastic bags and kept in a freezer at 4oC before the commencement of the tests

Determination of drying time and moisture content

Infrared drying

Vacuum drying

Heat pump drying

Slice 2mm Slice 5mm

Figure 3.10 Curculigo orchioides after cutting hairy roots

Figure 3.11 Curculigo orchioides in size 2mm and size 5mm

b/ The process of drying [60] :

- For drying methods (convective, infrared, heat pump dryers):

+ Slice samples were weighed about 30 g and were put in the drying tray and then placed it at the dryer The drying experiments were carried out similarly to the samples dried over time and were weighed to unchanged mass

+ The drying process was observed and sample slices were weighed every 1 hour + The tables of moisture variation were made to build the drying curve

+ The drying experiments were finished until the weight was unchanged and each one was done in at least three replicates

 Convective dryer: set temperatures at 60oC, 80 oC, 100 oC

 Vacuum dryer: set temperatures at 40 oC, 50 oC, 60 oC and vacuum pressure

at 100 mbar

 Infrared dryer: set temperature at 80 oC and air velocity at 6m/s

 Heat pump dryer: set temperater at 40 oC and air velocity at 8m/s

- For sun drying method:

+ Slice samples were spread on a tray under daylight Until the evening, put these samples in a place out of the wind

+ Slices were weighed in the afternoon and evening of the day

- The variations of the moisture content over time determined by the following formula: G o (100 – W o ) = G j (100 – W j )

After each one hour:  = j , the sample has a weight of Gj (g)

𝐆𝐣(𝟏𝟎𝟎 − 𝐖𝐨)

Where:

W o: the first moisture value of the drying sample (%)

W j: the moisture value of the drying sample after time at j (%)

G o: the first weight of the drying sample (gram)

G j: the weight of the drying sample after time at j (gram)

3.3 Extraction of samples

Step 1, the herbal samples were ground with a coarse grinder, then 0.2 g of the crushed sample was put into the beaker, dissolved in 10 mL of Methanol, sealed the beaker with foil and stored in a place protected from light

Step 2, the sample was decanted after soaking for 1 hour, the liquid is poured into a new container, sealed with foil, the sediment in the beaker will be added 5 mL of Methanol and left to soak for 1 hour

Step 3, the process carried out the same as step 2 and left immersion for 30 minutes After 30 minutes, the solution was filtered with a filter paper 102 with a filter in size of 0.11cm to retain the extract, for the residue it would be sealed and kept in the freezer The extract of the third extraction will be examined for antioxidant activity first If the

absorbance of the sample is less than 0.2, it means that the concentration of the sample is still high, continue as in the 3rd time with the residue that has been collected after three times of extraction If the absorbance is greater than or equal to 1, the concentration of the sample is acceptable and the extraction completed; after that, transfer this extract into the previous total extract together

Figure 3.12 Diagram of extraction of samples

Decanting

Soaking in Methanol (2 nd time)

Decanting

Soaking in Methanol (3 rd time)

Filtrating

The extracted

30 minutes

3.4 Analytical methods

3.4.1 Determination of moisture content

 Principles

The experiment was depended on Vietnamese Standardization (TCVN 9741:2013) and

some documentaries [60], [61] to evaluate the moisture content of Curculigo orchioides

samples

 Equipments and supplies

Dryer, which maintained at temperature 105oC (at the laboratory area B, Ho Chi Minh City University of Technology and Education)

Analytical balance

Petri and lid

Tray

Desiccator

 The process of drying

- The lid of the petri dish was opened and both were dried for about 1 hour in an oven at

105oC Then are cooled in a desiccator After cooling to room temperature, the lid was closed and was weighed accurately to 0.001 g

- Sliced samples were weighed approximately 10 g immediately in a prepared petri dish with a lid on the side

- The petri dish with the measured sample and the lid opened was dried directly in the oven

at 105oC to constant weight for 4 hours The oven door should not be opened during the drying period

- Once the oven was opened and the petri dish was covered right after and transfered to the desiccator, the lid was opened (still leave the lid in the desiccator) then cooled it in a sealed desiccator for at least 30 min

- The petri were removed from the desiccator, covered and weighed to calculate the first moisture value This process was continued until the difference between the two weighings was not exceed 0.005 g.The experiment was performed in triplicates (n = 3)

- Determine the percentage of moisture according to the formula below:

𝐖 = 𝐆𝐨− 𝐆𝟏

𝐆𝐨 × 𝟏𝟎𝟎 Where:

G o: the weight of the sample before drying (gram)

G 1: the weight of the sample after drying (gram)

W: the moisture percentage of dried samples (%)

3.4.2 Analysis total phenolic content

 Principles

The total phenolic content was determined by UV-VIS measurement with Ciocalteu’s phenol reagent and mentioned in TCVN 9745-1-2013 [62] This method is based on the ability to react with phenolic compounds of Folin-Ciocalteu’s phenol reagent The molybdostungtate complex salt mixture is very sensitive to reducing agents, so the presence of polyphenolic compounds in a slightly alkaline medium, it will be reduced to a blue compound with the strongest absorbance at 756 nm of wavelength

Folin-Figure 3.13 Reaction of evaluation of total phenolic content

 Chemicals and equipments

Folin-Ciocalteu’s phenol reagent

Gallic acid (3,4,5-trihydroxybenzoic acid)

Na2CO3 (Sodium carbonate)

Distilled water

Spectrophotometer (UH5300)