enrichment of the bioactive components from the residue of rosemary rosmarinus officinalis l leaf after distillation by using macroporous resin

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VIETNAM NATIONAL UNIVERSITY HO CHI MINH CITY HO CHI MINH CITY UNIVERSITY OF TECHNOLOGY

NGUYEN HUYNH HUONG THAO

ENRICHMENT OF THE BIOACTIVE COMPONENTS

FROM THE RESIDUE OF ROSEMARY (Rosmarinus

officinalis L.) LEAF AFTER DISTILLATION BY USING

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THIS THESIS IS COMPLETED AT

HO CHI MINH CITY UNIVERSITY OF TECHNOLOGY – VNU-HCM Supervisor: Dr. Le Xuan Tien

Examiner 1: Assoc. Prof. Mai Huynh Cang Examiner 2: Dr. Le Vu Ha

This master’s thesis is defended at HCM City University of Technology, VNU- HCM City on 26th January 2024.

Master’s Thesis Committee:

(Please write down full name and academic rank of each member of the Master’s Thesis Committee)

1. Assoc. Prof. Nguyen Thi Phuong Phong (Committee Chair) 2. Assoc. Prof. Mai Huynh Cang (Thesis Advisor 1)

3. Dr. Tong Thanh Danh (Committee Member) 4. Dr. Le Vu Ha (Thesis Advisor 2)

5. Dr. Nguyen Dang Khoa (Committee Secretary)

Approval of the Chair of Master’s Thesis Committee and Dean of Faculty of Chemical Engineering after the thesis being corrected (If any)

CHAIR OF THESIS COMMITTEE DEAN OF FACULTY OF CHEMICAL ENGINEERING

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VIETNAM NATIONAL UNIVERSITY - HO CHI MINH CITY

HO CHI MINH CITY UNIVERSITY OF TECHNOLOGY

SOCIALIST REPUBLIC OF VIETNAM Independence – Freedom - Happiness

THE TASK SHEET OF MASTER’S THESIS

Full name: Nguyen Huynh Huong Thao Student ID: 2270014Date of birth: 18th March 1999 Place of birth: Long AnMajor: Chemical engineering Major ID:

I. THESIS TITLE (In Vietnamese): Làm giàu hoạt chất kháng oxy hoá từ bã

chưng cất lá hương thảo (Rosmarinus officinalis L.) bằng nhựa hấp phụ.

II. THESIS TITLE (In English): Enrichment of the bioactive components from

the residue of rosemary (Rosmarinus officinalis L.) leaf after distillation by using

macroporous resin.

III. TASKS AND CONTENTS:

- Obtain the rosemary leaves’s residue after one hour of distillation, and evaluate the potential of reusing the residue extract.

- Investigate the conditions of adsorption and desorption process to enhance the content of actives in residue extract by using macroporous resin.

- Evaluate the antioxidant capacity of enriched extract in artificial sebum.

VI. SUPERVISOR: Dr. Le Xuan Tien

Ho Chi Minh City, 26th January 2024

DEAN OF FACULTY OF CHEMICAL ENGINEERING

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ACKNOWLEDGEMENTS

The process of scientific research over six years at the Ho Chi Minh University of Technology is marked by the distinguished pages of my master's thesis. Completing this thesis was not only my effort and dedication but also a journey filled with emotions alongside the collaborative atmosphere of Laboratory 401B2.

I greatly appreciate Dr. Le Xuan Tien - my dedicated mentor who guided me in the early days of research. He not only taught me diligently but also helped me build essential knowledge and develop comprehensive soft skills to break stereotypes about a master's degree.

Simultaneously, my family has always been a strong support, spiritual support whenever I felt weary. The encouragement from my family provided the strength for me to continue on the chosen path. A few lines of gratitude cannot describe the immense sacrifices of my parents, who always hoped to see their little daughter grow day by day and reach further in my career. Thanks to my grandmother and younger sister as well for taking care of every meal and sleep and always listening to me whenever I need.

Next, I extend my thanks to two partners, Luu Bao Chau and Nguyen Anh Khoa, who accompanied me during the research process. I appreciate their efforts and collaboration in resolving challenging issues. I wish them continued passion in their future endeavors.

Furthermore, the suggestions from Minh Chau significantly contributed to improving the quality of my thesis. I appreciate the valuable time she spent supporting me in the research process.

In addition to the direct support from teachers, family, and friends, I want to express my heartfelt thanks to Vo Minh Khai - a friend who was always by my side, providing moments of relaxation after the pressured research period. Thank you for overcoming the distance to join me for meals, I hope our future times together will be beautiful. A few words cannot encapsulate everything, and I want to express my final thanks to those friends and companions who stood by me throughout this journey.

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I hope to keep my passion for Chemistry and contribute to the research efforts for future generations.

Sincerely grateful.

Quá trình nghiên cứu khoa học hơn 6 năm tại ngôi trường Đại học Bách Khoa – ĐHQG TPHCM được đánh dấu bằng những trang luận văn thạc sĩ đầy danh giá. Để hồn thành bài luận văn này khơng chỉ là sự cố gắng và nỗ lực của riêng bản thân em, mà còn là cả một chặng đường đầy cảm xúc với tập thể phịng thí nghiệm 401B2. Em xin dành lời cảm ơn chân thành và sâu sắc nhất đến TS. Lê Xuân Tiến - người thầy tận tình chỉ dạy em trong những ngày đầu nghiên cứu. Đồng thời thầy là người giúp em xây dựng nền tảng kiến thức vững chắc, phát triển toàn diện các kĩ năng mềm để xoá tan định kiến về một thạc sĩ giấy từ lâu nay.

Song song đó, gia đình ln là điểm tựa vững chắc, là hậu phương mỗi khi em mệt mỏi, chính những sự động viên khơng ngừng từ gia đình đã tiếp thêm cho em dũng khí bước tiếp trên con đường mình đã chọn. Vài dịng tâm thư khơng thể sánh được với sự hi sinh lớn lao của ba mẹ, những người luôn mong đứa con gái bé nhỏ từng ngày trưởng thành, tiến xa hơn trên con đường sự nghiệp. Cảm ơn ngoại và em gái, người luôn chăm chút từng bữa cơm, giấc ngủ và là nơi lắng nghe những trải lòng của em sau tháng ngày dài nghiên cứu.

Tiếp đến là lời cảm ơn dành cho hai người em Lưu Bảo Châu và Nguyễn Anh Khoa đã đồng hành vùng em trong quá trình nghiên cứu. Cảm ơn các em đã không kể công sức, lăn xả cùng chị giải quyết những vấn đề khó khăn, chúc các em tương lai sau này mãi giữ lòng nhiệt huyết như ban đầu.

Bên cạnh đó, chính những lời góp ý chân thành từ chị Minh Châu đã giúp em hoàn thiện bản thảo luận văn tốt hơn, cảm ơn chị đã dành thời gian quí báu của mình hỗ trợ em trong quá trình nghiên cứu.

Bên cạnh những sự hỗ trợ trực tiếp từ thầy cô, người thân và bạn bè, em muốn gửi lời cảm ơn chân thành đến bạn Võ Minh Khải - người bạn luôn bên cạnh em, mang đến những phút giây thư giãn sau khoảng thời gian nghiên cứu đầy áp lực. Cảm ơn bạn đã không ngại khoảng cách xa xơi sang đón mình đi ăn, mong rằng khoảng thời gian sau này của hai chúng ta mãi mãi tốt đẹp.

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Đôi lời muốn nói khơng thể gói gọn trong vài trang giấy, em xin dành lời cảm ơn gửi đến những người bạn, người em đã luôn bên cạnh em trong suốt chặng đường qua. Bản thân em hi vọng chính mình ln giữ lửa đam mê ngành Hố, góp phần đóng góp vào cơng cuộc nghiên cứu cho thế hệ mai sau.

Trân trọng cảm ơn.

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ABSTRACT

Rosemary (Rosmarinus officinalis L.) is known as a plant with a pleasant fragrance,

and its essential oil is often extracted by steam distillation. Additionally, the portion of polyphenols that are less volatile in rosemary residue is considered a rich source of antioxidant compounds. Therefore, the study aims to utilize rosemary residue to increase the content of carnosol and carnosic acid in the extract by using macroporous resin, serving the application of antioxidant properties in artificial sebum. The results showed that DM301 resin was suitable for the adsorption process, achieving an adsorption ratio of 82.51 ± 0.52% for carnosol and 93.21 ± 0.27% for carnosic acid. Regarding desorption, the thesis determined that 99.5% (v/v) ethanol was an effective type of solvent for removing target compounds from the DM301 resin surface, with a desorption time of 60 minutes and a resin: solvent ratio of 1:70 g/mL. Under these conditions, desorption ratios were 88.02 ± 0.49% for carnosol and 83.95 ± 0.01% for carnosic acid.

The research was examined the purity of carnosol in static desorption extract (32.99%) and gradient concentration extract (23.24%). For carnosic acid, the purity in the gradient concentration extract (70.69%) was 4.4 times higher than that in static desorption (15.99%).

Lastly, the concentration of the 400 ppm enriched extract (gradient concentration extract) exhibited a 3.5 times higher antioxidant capacity for artificial sebum compared to the RDR extract at the same concentration, with values of 45.66 ± 0.47 mg MDA/kg sample and 154.90 ± 0.22 mg MDA/kg sample, respectively.

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THE COMMITMENT OF AUTHOR

The thesis was carried out at Ho Chi Minh University of Technology and I declare that this is an original work. Under the guidance of Dr. Le Xuan Tien, the research results and conclusions in this thesis are honest and not copied from any source in any form. This work has yet to be submitted for any other award at any other university.

Nguyen Huynh Huong Thao

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LIST OF CONTENTS

ABSTRACT ... vi

LIST OF FIGURES ... xi

LIST OF TABLES ... xiii

CHAPTER 1 : LITERATURE REVIEW ... 1

1.5. MACROPOROUS ADSORPTION RESIN: ... 15

1.5.1. Characteristics of macroporous resin: ... 16

1.5.2. The HPD – 100 resin: ... 17

1.5.3. The DM – 301 resin: ... 17

1.5.4. The XAD – 7HP resin: ... 18

1.5.5. The potential applications of macroporous resins in the future: ... 18

1.6. AN OVERVIEW OF ACNE AND THE MECHANISM OF ACNE FORMATION ON THE SKIN: ... 19

1.7. SUMMARIZE THE FOREIGN AND DOMESTIC RESEARCH: ... 21

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2.4.2. Method for measuring moisture content: ... 25

2.4.3. The extraction process of rosemary leaves: ... 26

2.4.4. Investigate the adsorption and desorption ratio of macroporous resins: 27 2.4.5. Dynamic desorption: ... 33

2.4.6. The quantification method of target compounds in extract after adsorption in macroporous resin: ... 33

CHAPTER 3 : RESULT & DISCUSSION ... 41

3.1. Distillation efficiency of rosemary essential oil by steam distillation method: ... 41

3.1.1. The extraction efficiency: ... 43

3.1.2. The polyphenol content in dried rosemary leaves and RDR extract: ... 44

3.2. Investigation of factors affecting the adsorption capacity of macroporous resin: ... 47

3.2.1. Selection of the suitable solvents for dissolving rosemary extract: ... 47

3.2.2. Investigation of the adsorption capacity of three types of resins: ... 50

3.2.3. Selecting the macroporous resin based on the content of carnosol and carnosic acid: ... 52

3.2.4. Adsorption isotherm: ... 56

3.3. Investigation of factors affecting the desorption capability of the resin:59 3.3.1. Selection of desorption solvent: ... 59

3.3.2. Selecting the desorption time: ... 61

3.3.3. Selection of desorption solvent volume: ... 62

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3.4. Comparison of the carnosol and carnosic acid content in the initial

extract and purified extract: ... 65

3.4.1. Enrichment of carnosol and carnosic acid by resin column: ... 65

3.4.2. Qualitative analysis by thin-layer chromatography (TLC): ... 66

3.4.3. Quantification of the content of target compounds by HPLC: ... 67

3.5. Determine the TBARS value in artificial sebum: ... 70

CHAPTER 4 : CONCLUSION AND DISCUSSION ... 72

CONCLUSION: ... 72

DISCUSSION: ... 73

REFERENCES ... 74

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LIST OF FIGURES

Figure 1.1: Map of rosemary distribution in the world [98] ... 2

Figure 1.2: The shape of rosemary species ... 5

Figure 1.3: The structure of carnosic acid ... 9

Figure 1.4: Meat sample before and after metmyoglobin formation ... 10

Figure 1.5: The structure of carnosol ... 11

Figure 1.6: The shape of three macroporous resins ... 17

Figure 1.7: The components of sebum in pore ... 21

Figure 2.1: Rosemary dried leaves ... 25

Figure 2.2: Rosemary leaves after grinding ... 25

Figure 2.3: The extraction process of rosemary leaves ... 26

Figure 2.4: The procedure of calculating the adsorption ... 28

Figure 2.5: Carnosic acid standard calibration ... 35

Figure 2.6: Carnosol standard calibration ... 35

Figure 2.7: Gallic acid standard calibration ... 37

Figure 2.8: The reaction between MDA and TBA ... 39

Figure 2.9: The MDA calibration in artificial sebum ... 40

Figure 3.1: The main (a) and the secondary products (b) of steam distillation method from dried rosemary leaves ... 42

Figure 3.2: The comparison between the extraction efficiency of initial dried rosemary leaves and RDR extract ... 43

Figure 3.3: The rosemary extract after 1-hour distillation ... 44

Figure 3.4: The total polyphenol content in the initial dried rosemary leaves and the RDR extract ... 45

Figure 3.5: The content of carnosic acid and carnosol in initial rosemary dried leaves and RDR extracts ... 46

Figure 3.6: The adsorption capacity curves of the three types of resins over time . 50Figure 3.7: The adsorption ratio of the three types of resins based on the total polyphenol content ... 51

Figure 3.8: The adsorption capacity of carnosic acid ... 53

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Figure 3.9: The adsorption capacity of carnosol ... 53

Figure 3.10: The adsorption capacity of carnosol (a) and carnosic acid (b) by DM301 resin at three different temperature levels ... 56

Figure 3.11: The effect of extraction time on the desorption ratio of carnosol and carnosic acid ... 62

Figure 3.12: The effect of desorption solvent volume on the desorption ratio ... 64

Figure 3.13: The content of carnosic acid and carnosol were analyzed by HPLC method ... 65

Figure 3.14: Thin-layer chromatography (TLC) of solvent fractions ... 67

Figure 3.15: The desorption ratio of each fraction ... 68

Figure 3.16: The content of target compounds in extract ... 68

Figure 3.17: The MDA values of after enrichment rosemary extract at different concentrations and tocopherol at concentration 400ppm ... 70

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LIST OF TABLES

Table 1.1: The MIC and MBC values of rosemary extract in different solvents for various

bacteria. ... 13

Table 1.2: The specifications of the three types of macroporous resin. ... 16

Table 1.3: The percentage of different types of fats in sebum and on the surface of human skin ... 21

Table 2.1: The chemicals uesd in the thesis ... 24

Table 2.2: The process of preparing a working standard solution from an intermediate standard solution ... 34

Table 2.3: HPLC gradient program ... 35

Table 2.4: The content of artificial sebum ... 39

Table 3.1 The solubility of rosemary extract in various solvents ... 48

Table 3.2 The physical properties of three resin types [109], [103]. ... 52

Table 3.3 The adsorption capacity and adsorption ratio of DM301 and XAD-7HP resins. 55 Table 3.4 The Langmuir and Freundlich parameters ... 58

Table 3.5 The desorption ratio of DM301 resin in different solvents. ... 60

Table 3.6 The purity of extract based on the solvent volume ... 63

Table 3.7 The adsorption and desorption conditions ... 64

Table 3.8 The purity of each fraction ... 69

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CHAPTER 1 : LITERATURE REVIEW

In recent years, environmental pollution has become a highly prioritized topic of public concern. Among them, the skin is the most affected by factors such as UV rays, dust, and surrounding emissions. To contribute to preventing oxidative processes on the skin surface and protecting it from harmful agents, various skincare products with antioxidants have been developed. However, some products contained irritating or pore-clogging ingredients, leading to redness or acne. Therefore, the trend of applying natural and gentle skincare products is becoming increasingly popular among consumers.

Rosemary (Rosmarinus officinalis L.) known as a commonly used herb for essential

oil, is valued for its relaxing and stress-relieving properties, as well as its culinary use. Additionally, this plant has drawn significant attention from global researchers for its effective antioxidant, anti-inflammatory, and anti-bacterial capabilities. In the food industry, rosemary is utilized as a natural preservative, replacing synthetic preservatives like butylated hydroxyanisole (BHA) or butylated hydroxytoluene (BHT) [1].

Moreover, many researches have demonstrated the outstanding biological activities of rosemary, primarily stemming from the polyphenol group. Notable compounds include carnosic acid, carnosol, rosmarinic acid, ursolic acid, and oleanolic acid [1]. Despite this, the majority of rosemary’s value comes from extracting essential oil for commercial purposes, and the residue is oftern thrown away after the steam distillation process.

Therefore, the thesis “Enrichment of bioactive components from the residue of

rosemary (Rosmarinus officinalis L.) leaf after distillation by using macroporous resin” aims to utilize the rosemary residue after distillation to enrich the antioxidant

actives, coupled with investigating antioxidant activity on artificial sebum.

1.1. INTRODUCTION:

The scientific name of rosemary is Rosmarinus officinalis L. - belongs to the

Lamiaceae family [1]. This plant has a reputation for unique aroma and is grown all

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over the world, especially in the Mediterranean region. Archaeologists have evidence of rosemary's culinary uses in Egypt, Mesopotamia, mainland China, and India [2].

Several decades ago, rosemary leaves were distilled by Indian citizens to achieve essential oils. However, the different techniques can lead to different yields and quality of essential oils [3].

In 2013, K. Hcini et al. analyzed various types of rosemary grown in three regions in

Tunisia, including Beja, Sidi Bouzid, and Gabes. According to the result, the research concluded that soil, climate, and altitude are factors affecting the quality, composition, and content of compounds in rosemary essential oil [2]. In terms of distribution, this plant grows and develops strongly in semi-arid and tropical climates [3]. Moreover, rosemary adapts well in sandy, well-drained, slightly acidic soils with pH ranges from 6.0 to 7.0 [4].

Rosemary is a woody perennial shrub [5], also known as a plant containing similar aroma to lavender [3]. In general, rosemary leaf - the main part containing characteristic scent, which is dark green on the upper surface and slight green on the underside. Leaf has a tendency on growing opposite along the branches, and its length from 15 to 40 mm, without petioles [6].

In addition, rosemary leaves carry up to 2 % essential oil, and other substances such as tannin, ursolic acid, flavonol, vitamins, and other minerals occupy approximately

Figure 1.1: Map of rosemary distribution in the world [98]

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8 % [7]. Up to now, there are more than 20 types of rosemary that appear over the world [8] and are classified by the color, scent, or shape of the plant [5].

Trailing rosemary is called Prostrate, which height fluctuates from 5 to 30 cm, then spreads up to 2 m [9] (Figure 1.2 a). In contrast, upright rosemary can reach a peak of 180 cm, and its branches tend to grow up to 90 cm (Figure 1.2 b) [10].

In terms of color, rosemary is divided into three species, including: Tuscan Blue (Figure 1.2 c), Majorca Pink (Figure 1.2 d), and White Flower (Figure 1.2 e). Tuscan Blue has a dark gray-green flower, needle-shaped leaf, and can be taller than 90 cm when growing in hot climates [11]. About Majorca Pink, which can be called “Salem” – widely upright rosemary that has purple flower clustering on vertical branches. The maximum height is about 120 cm, and the leaf seems to be larger than those of other rosemary species [11].

“Albus rosemary” is a white-flower rosemary, hence it simply is called “White Flower”. Similar to the above species, white-flower rosemary belongs to upright group, which has a needle-shaped and dark green leaf. The plant often blooms and gives off its scent in the end of spring and summer [12].

In traditional medicine, rosemary leaves are used as a type of antibacterial plant, and relieving muscle. Additionally, the essential oil of rosemary extracted from the flowers and leaves aids in treating headaches and alleviating spasms [13].

In 2014, the high-ethanol extract of rosemary revealed the presence of five new compounds, including officinoterpenoside A1 and A2 (diterpenoid glycoside), officinoterpenoside B and C (triterpenoid glycoside), and officinoterpenoside D (normonoterpenoid). Additionally, the pharmacological potential in rosemary is strongly manifested through the compounds carnosic acid and essential oil [13]. Rosemary is often distilled using steam distillation from the leaves to extract its essential oil – colorless to pale yellow, insoluble in water, and possessing the characteristic scent of thyme. The main components of rosemary essential oil include camphor (5.0 – 21.0%), 1,8-cineole (15 – 55%), borneol (1.5 – 5.0%), limonene (1.5

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– 5.0%), along with various other volatile compounds and its proportions vary depending on the growth stage and climatic conditions [8].

Regarding the bioactive content in the rosemary extract, the compounds in rosemary include phenolic, di- and triterpenes. Additionally, the most common polyphenols are apigenin, diosmin, luteolin, genkwanin, and phenolic acids (>3%), particularly rosmarinic acid, chlorogenic acid, and caffeic acid [14].

Rosemary extraction methods typically utilize the most potent parts in rosemary, such as leaves, roots, stems, or flowers, with suitable solvents. Key factors influencing the quality of the extraction process include the characteristics of plants, solvents, temperature, pressure, and extraction time [15]. Many traditional extraction methods are employed, such as Soxhlet extraction, maceration, decoction, and infusion, as well as modern methods like supercritical fluid extraction and solid-phase microextraction [16].

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a) Rosemary Prostrate b) Rosemary Barbeque

c) Rosemary Tuscan Blue d) Rosemary Majorca Pink

e) Rosemary White Flower

Figure 1.2: The shape of rosemary species

1.2. APPLICATION

In the study of anti-oxidant properties of more than 70 types of dried spices in 1956,

Lundberg et al. pointed out rosemary has various benefits, including anti-oxidant

capacity [13]. From that, rosemary attracts more and more attention and is applied widely in many fields: food, cosmetics, and pharmaceuticals.

1.2.1. Cosmetic industry

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Since ancient Egypt, humans had a demand to take care of the body, and the cosmetic field appeared to fulfill their demand [14]. Skin has a complex structure, which is sensitive to free radicals due to long-term exposure to oxygen and environmental irritants [15]. In modern life, customers become more and more strict in selecting skincare products and pay attention to vegan or natural cosmetics.

Since 2000, each year have approximately 120 articles are published on aspects of rosemary [14], including: massage oil, perfume, shampoo gel, make-up remover, or

sunscreen,… According to the research in the year of 1998, Isabelle C. Hay et al.

concluded that rosemary essential oil can stimulate hair growth, reduce sebum secretion, and have potential in hair-loss treatment. In addition, rosmarinic acid and other derivatives of caffeic acid in this herb are one of antioxidants protecting the hair and scalp from partial damage [16].

Today, the market appears with NutroxsunTM product – the combination of rosemary

(Rosmarinus officinalis L.) and grapefruit (Citrus paradisi) that has an anti-aging

capacity and can protect skin from damage from sunlight. In 2016, the Italian scientist Vincenzo Nobile and partners proved the effectiveness of NutroxsunTM product by measuring the skin’s redness, wrinkle depth and skin elasticity. The study evaluated 90 volunteers from February to April 2015; the results illustrated that rosemary extract could reduce redness at both concentrations of 100 and 250 mg when skin is exposed to 1 minimal erythemal dose (MED) of UVB. Regarding to the depth of wrinkles, the concentration of extract at 250 mg can reduce 9.1%, 12.6%, and 13.9% after 0.5; 1 and 2 months in respectively. The similar figure was true for the concentration of 100 mg. In terms of skin elasticity, two concentrations showed the ability to enhance the value of gross elasticity, and reached the highest point about 4.6% after 2 months [17].

Recently, Hadizadeh-Talasaz and colleagues initially investigated the pain-healing effect of cream containing rosemary extract. The clinical trial was conducted on 80 pregnant women in Shahid Motahari, Iran, from September 2019 to March 2020. The clinical trial was conducted on 80 pregnant women in Shahid Motahari, Iran from

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September 2019 to March 2020. A postoperative wound healing rate was calculated by the REEDA scale of Davidson [18], and a lower score indicates a higher potential for healing. After 4 days, the group using rosemary cream obtained 3.82 ± 0.93 score, compared to 4.25± 1.29 score of placebo group. After 10 days, however, the difference between two groups was detected in detail. The group using rosemary cream achieved at 0.75±0.74 score, higher nearly 4.5 times than that of the placebo group (3.32 ± 2.54 score). Through many evidences, rosemary extract is proven its bioactivities and becomes a promising herb for cosmetic application [19].

1.2.2. Food industries

Rosemary is a popular herb used to enhance the taste, especially in Mediterranean cuisine [6]. Besides that, the leaf is the most used part and can be stored in fresh or dried form. According to Veenstra et al., the polyphenol compounds present in rosemary can be used as preservatives. The research proved that rosemary can prevent microorganism development and slow down food oxidation at the same time [20]. The advantage of adding the natural anti-oxidants is the ability to combine different compounds to achieve “synergistic blend” – enhancing the effects of each other rather than a single substance. In closely related plant to rosemary as sage, the anti-oxidant compounds of sage form “chelating complexes” with acid citric in foods. Taking advantage of this interaction not only enhances the impact on the oxidant reaction at different time in the cycle, but also push the preservation efficiency rather than using a single substance [13]. Rosmarinic acid, in addition, has the ability to limit the effects from sunlight, and prolong expiry date of food through radical removal and prevent ultraviolet (UV) rays [13].

In recent research by Kamel et al. in 2022, the rosemary extract was added to yogurt

as a preservative within 16 days at 4 oC. The data showed that the content of extract

from 0,5 to 0,7% can resist Escherichia coli, Staphylococcus aureus, Salmonella

marcescens, as well as Aspergillus flavus, and Candida albicans. This exploration

opens a new direction to choosing preservatives that are both safe and effective in food grade [21].

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Yang et al. reinforced the perspective of using rosemary as a natural preservative by

using Rancimat method – measure induction period (IP) by detecting volatile acids formed during oxidation of cooking oil, including in soybean oil, cottonseed oil, and bran oil. The IP value of oil samples containing rosemary extract are higher than those containing synthetic preservative (BHT, BHA) [22].

Besides the advantages, the addition of rosemary as a natural preservative still has some difficulties due to the smell and taste degradation, as well as alter product appearance [13].

1.2.3. Pharmaceutical:

Rosemary has a wide application in the medical industry, especially in traditional medicine. The rosemary extract is used as medicine to treat many kinds of sickness like rheumatism, gout, neurosis, eczemas, and other health problems [7]. In the other hand, rosemary is considered to help cholesterol in the blood lower, and reduce the risk of diabetes as well as obesity [23]. In 2014, Labban investigated rosemary plant grown in Fayom region, Egypt to support the evidence about its bioactivity. With three doses of 2, 5, and 10 g rosemary leaf powder per day, the glucose levels in the blood decreased by 11.2%, 15.74%, and 18.25%, respectively. After 8 weeks, the dose of 2 g powder per day seemed to be useless, meanwhile the dose of 10 g showed the significant reduction of glucose in blood [24]. Furthermore, rosemary essential

oil can resist Escherichia coli and 𝛽-lactamase. In the range of MIC from 18.0 to 20.0 𝜇L/mL, Escherichia coli was inhibited by rosemary essential oil at 18.5 𝜇L/mL [25].

Many in vivo studies indicate that rosemary extract or its essential oil have a positive effect on stress reduction or inflammation in gastro-intestinal tract [20].

1.3. AN OVERVIEW OF CARNOSIC ACID AND CARNOSOL:

Sienkiewicz et al. analyzed the components in rosemary essential oil distilling by

steam distillation. The results indicated that more than 40% of the content was cineole, followed by camphor (11.4%) and 𝛼 -pinen (11%) [25]. Furthermore, rosemary extract also contains many natural components such as polyphenols, flavonoids, diterpenoids, and other caffeic acid derivatives.

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1,8-The highlighted bioactivities as anti-inflammatory, anti-diabetic, hepatoprotective, and antibacterial are mostly related to the polyphenol group (mainly rosmarinic acid, carnosol, and carnosic acid) [26]. Therefore, this thesis concentrates on researching and evaluating the content of polyphenols in rosemary plants, including carnosic acid, carnosol, and rosmarinic acid.

1.3.1. Carnosic acid

Figure 1.3: The structure of carnosic acid

Carnosic acid is ortho-diphenolic diterpene – which was found in rosemary plant and was proven to have cytoprotective effects in mammals [27]. Due to the appearance of the phenol group, carnosic acid is often classified as a polyphenol. However, the properties and biosynthesis of this compound are similar to terpenoids [27]. In 1964,

this compound was discovered firstly in Salvia officinalis L. by Linde, and then Wenkert et al. was found that the content of carnosic acid in Rosmarinus officinalis

L. leaf was higher than that of sage plant [28]. Carnosic acid can be considered to be a specific compound in Lamiaceae family, and was explored in the Gymnospermatophyta group in the year 2002 [29]. Carnosic acid is distributed in plant parts randomly, for example, this component is found mainly in photosynthetic tissues of rosemary, such as leaf, sepal, and petal [27]. The remarkable point of its structure is two hydroxyl (-OH) at C11 and C12 [27] that contribute to enhancing the antioxidant activity significantly [1].

Satoh et al. showed that carnosic acid and its derivatives could protect HT22 neurons

from glutamate toxicity. The study also pointed out that the toxicity of carnosic acid

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to the nervous system was less than that of carnosol – one of the typical components present in rosemary leaf [30].

Figure 1.4: Meat sample before and after metmyoglobin formation

According to the research of Naveena and colleagues, using a concentration of carnosic acid higher than 130 ppm contributed to controlling the lipid oxidation and metmyoglobin oxidation in red meat – which is a reason for making the dark brown color of red meat (Figure 1.4). This compound can be used as an additive to maintain the storage time [31]. On the other hand, carnosic acid is also a multi-functional compound as an antibacterial, anti-cancer, and HIV-1 protease inhibitor [32].

Although the synthesis of carnosic acid from rosemary leaves has not been

investigated, Brückner et al. (2014) proposed an intermediate compound, abietatriene,

with an aromatized C-ring and a molecular weight of 270. Subsequently, the dual hydroxylation process of abietatriene on the 20 C-framework of diterpenoid will yield the carboxyl group in carnosic acid [33].

Similar to other antioxidants, the radical scavenging activity mechanism of carnosic acid is attributed to the presence of two O-phenolic hydroxyl groups at positions C11 and C12 (catechol moiety). At 60 °C, carnosic acid exhibits a higher antioxidant capacity in lipid systems compared to 𝛼-tocopherol. However, at higher temperatures, the consumption rate of carnosic acid becomes faster than that of alpha-tocopherol, indicating that the oxidation products of carnosic acid significantly contribute to the antioxidant reaction [33]. Notably, the recovery efficiency of this active compound varies from 68.1% to 96.2% depending on the sample matrix [34].

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1.3.2. Carnosol

Figure 1.5: The structure of carnosol

Carnosol was isolated from sage (Salvia officinalis L.) in 1942 firstly, and was discovered its structure by Brieskorn et al. in 1964 [33]. This substance is a main

product in carnosic acid oxidation, and is one of the potential antioxidants in rosemary [34]. Although carnosol and carnosic acid only account for 5% of the total weight of dried rosemary leaves, however, the antioxidant capacity of these substances occupies more than 90% [35]. Similar to carnosic acid, carnosol is an ortho- diphenolic acid that has an abietane framework linked to hydroxyl (-OH) groups at C11, C12 and a part of lactone ring on the B ring (Figure 1.5).

In the year of 2010, a Japanese team developed a semi-synthetic process of carnosic acid and carnosol by using pisiferic acid from Sawara leaf – a cypress plant origins in Japan [36]. Notably, carnosic acid exposes to methanol for 7 days at room temperature can be oxidized to carnosol [33].

Aruoma and colleagues have demonstrated that carnosol can scavenge peroxyl radicals and inhibit the Cu2+- induced oxidation process caused by low-density lipoproteins, leading to the generation of free lipid radicals in rat liver [37]. Another mechanism for the effective inhibition of lipid peroxidation by carnosol is its capability to alter the order of phospholipid membranes. The antioxidant activity of the compound is observed to be significantly enhanced, up to 4 to 6 times, when analyzing phospholipid membranes compared to those without phospholipid membranes [38].

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However, carnosic acid is converted into carnosol through the oxidation process, which has physical, thermal, and light instability properties. Therefore, to limit this transformation, the method of supercritical fluid extraction can be employed (at low temperatures) [13]. Additionally, carnosol has been shown to activate various types of antioxidant enzymes, collectively referred to as cell-protective proteins.

1.4. BIOACTIVITY:

Since the discovery of rosemary, this plant has attracted much attention not only because of its characteristic aroma, but also because of its diverse biological activities. Studies around the world have shown that rosemary is effective in antioxidant, antibacterial, anti-tumor formation, as well as reducing stress and depression.

1.4.1. Antioxidant activity:

Antioxidant activity is one of the activities that has received extensive research and has demonstrated the effectiveness of this activity in rosemary. This is explained by the presence of many typical antioxidants such as rosmarinic acid, sagenoic acid, carnosic acid, and carnosol in rosemary [37]. In addition, the combination of ursolic acid and oleanolic acid in rosemary extract also has a certain antioxidant effect [37].

According to Hu et al., an antioxidant is a substance that can interrupt lipid oxidation

by breaking the oxidation mechanism chain or protecting the underlying oxidants to resist the formation of free radicals in the initial stage. The characteristic of lipid oxidation is oxygen consumption, in which peroxyl radicals can be directly measured by electrochemistry [39].

In a study in 2005, Neura Bragagnolo et al. found a significant formation rate of free

radicals when frying chicken breast at 95 °C. Adding rosemary to this dish helped slow down the rate of oxygen consumption and the trend of free radical formation was slower than the sample of chicken breast without this plant [38].

More than 90% of antioxidant properties come from carnosol and carnosic acid, which serve as inhibitors for lipid peroxidation in both liposomal and microsomal systems. Carnosol and carnosic acid effectively eliminate CCl3O2 (peroxyl radicals),

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decrease cytochrome c, and remove hydroxyl radicals. In particular, carnosic acid scavenges H2O2 and may also function as a substrate for the peroxidase system [1]. Munné-Bosch and colleagues pointed out that the crucial aspect of rosemary’s antioxidant effectiveness lies in the connection between diterpenes and their ability to scavenge radicals [39]. The essential components in rosemary's structure include the aromatic ring (C11–C12) within the catechol group, coupled with the conjugation of the three fundamental rings.

1.4.2. Antibacterial activity:

In 2006, Moreno et al. tested the antibacterial activity of rosemary extract in different

solvents using the disk diffusion method. Bacteria were cultured at 37 °C for 24 hours in Muller Hinton Broth, while yeast was cultured at 30 °C in Sabouraud Dextrose Agar (SDA). The results showed that after 24 hours of incubation, rosemary extract at a concentration of 250 μL/mL showed 100% inhibition efficiency. However, after 48 hours, bacteria were observed to develop again [39].

Table 1.1 shows the MIC (minimal inhibitory concentration) and MBC (minimal bactericide concentration) values of rosemary extract in different solvents for various bacteria.

Table 1.1: The MIC and MBC values of rosemary extract in different solvents for

various bacteria.

Bacteria

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Rocha et al. demonstrated that the concentration of rosmarinic acid at 25 mg/kg

helped effectively reduce foot swelling after 6 hours and reduce signs of organ dysfunction by regulating NF-κB and metalloproteinase-9 in a heat injury model [41].

In the study conducted by Poeckel et al., it showed that diterpene phenolics - typified

by carnosic acid and carnosol - have the ability to regulate genes, activate the gamma receptor to increase peroxisome proliferation (PPARγ) [42]. In addition, these components also contribute to preventing the formation of leukotrienes - inflammatory mediators in white blood cells - and inhibiting the activity of the enzyme 5-lipoxygenase [43]. However, these studies are only evaluating the anti-inflammatory potential of each individual compound. In reality, extract enriched with carnosic acid and carnosol from rosemary leaves may have stronger effects than each individual component. In particular, rosemary extract has the ability to inhibit pro-inflammatory cytokines, reducing the release of TNF-α (a tumor necrosis factor), IL-1β (an inflammatory response mediator), and IL-6 (an inflammatory response activating agent for body protection) in the THP-1 white blood cell model [44].

1.4.4. Anti-cancer activity:

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The polyphenolic compounds present in rosemary leaves are the main source of cancer activity and have been widely studied in recent years. The anti-cancer process of rosemary extract is described through three stages of cancer development, including tumor initiation - preventing the formation of cancer cells, tumor promotion - anti-proliferation activity, and progression - anti-metastasis activity [42]. The anti-cancer activity may be closely related to the antioxidant capacity of this plant, especially the removal of free radicals in the body, thereby preventing damage caused by ROS reactions to lipids, proteins, and DNA [42].

anti-A study on HepG2 liver cancer cells has shown that carnosic acid is capable of limiting the proliferation process, reducing the instability of the mitochondrial membrane, thereby releasing proapoptotic proteins into the cell. Then, these proteins activate other proteins, typically caspase-3, promoting the process of programmed cell death (apoptosis) [45].

In 2015, Petiwala and Johnson demonstrated that rosemary extract can promote the process of androgen receptor (AR) degradation by creating stress-inducing proteins in the endoplasmic reticulum (ER), binding immunoglobulin protein (BiP), and C/EBP homologous protein (CHOP) [46].

Furthermore, many reports have used in vivo models to investigate the anticancer activity in diseases such as leukemia, breast, lung, pancreas, prostate, colon, cervix, and ovarian cancer cells [42].

1.5. MACROPOROUS ADSORPTION RESIN:

Macroporous adsorption resin is an organic adsorption material formed from polymers that has been used since the 1960s. This material has a three-dimensional porous structure and a large surface area that helps increase its ability to adsorb substances, especially organic compounds [47]. In addition, resin adsorbent can be used flexibly to refine natural compounds according to their polarity, molecular weight, and solubility in solvents [47]. Using resin adsorbent has been demonstrated to be one of the most effective techniques for enriching and recovering polyphenol groups to date [48].

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Compared to commonly used adsorbents such as silica gel, alumina, or activated carbon, resin adsorbent is considered a more reasonable substitute due to its outstanding physicochemical properties as mentioned above [49]. Furthermore, the relatively low cost of use, coupled with the ease of recovery and reuse, is also one of the reasons why resin adsorbent is increasingly being applied in scientific research [50].

The thesis conducts to investigate the adsorption capacity and desorption capacity of polyphenol compound in three types of macroporous resin, including: HPD100, DM301, and XAD-7HP. The final target is:

- Find out the appropriate resin for adsorbing two main biologically active compounds in rosemary extract: carnosic acid, and carnosol.

- Choose the safe and friendly environmentally solvent for desorption.

- Investigate the factors affecting to adsorption and desorption process throughout high-performance liquid chromatography (HPLC) quantification method.

- Test the antioxidant activity of the treated extract in black-head reduction, and then apply extract to cream formulation.

1.5.1. Characteristics of macroporous resin:

Unlike other adsorbent materials, macroporous resin do not contain exchange groups but operate through van der Waals to separate molecules in the extract and then collect the separated molecules by using an appropriate eluted solvent [51]. The physical properties and characteristics of the three types of macroporous resin are described in Table 1.2.

Table 1.2: The specifications of the three types of macroporous resin.

Surface area

Average pore radius

HPD100

[52]

Polystyrene Non-polar 0.30 – 1.20 650 - 700 85 - 90

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DM301

[53]

Polystyrene Moderate polar

0.30 – 1.25 330 – 380 90 - 110 XAD-7HP

The pore volume is 1.35 – 1.65 (mL/g) [58] The moisture content is 61.2 – 63.72% [55], [59]

In a study conducted on 15 different adsorbent resins, Tang and colleagues demonstrated that HPD-100 resin is the most effective for separating stilbene glycosides from Polygonum multiflorum. After treatment with HPD-100 resin, the amount of stilbene glycoside obtained was 819 mg/g with a recovery rate of over 74% [60].

1.5.3. The DM – 301 resin:

The DM-301 resin is spherical in shape, white and opaque in color, odorless, and can withstand maximum heat of 120°C. DM-301 is suitable for separating organic compounds from polar to weakly polar compounds such as flavonoids, stevioside,

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polyphenols [61], or saponins [62]. The moisture content of DM-301 resin ranges from 61.12% to 65.48% [59].

1.5.4. The XAD – 7HP resin:

The XAD-7HP resin is a non-ionic acrylic resin with good physical durability and thermal stability. Due to its large surface area and the nature of being composed of non-aromatic aliphatic compounds, XAD-7HP resin has the ability to adsorb non-polar compounds in polar solvents, as well as polar compounds in non-polar solvents [63].

Moisture retention ability: 61 – 69 % [66] Pore volume: 1.14 (mL/g) [65]

In a study conducted in 2021 by Che Zain and colleagues, it was found that 7HP resin has a higher ability to adsorb flavonoids in palm oil compared to DAX-8 and XAD-4 resins. This can be explained by the fact that this type of resin has an acrylic structure, moderate polarity, a relatively wide pore diameter, and a large surface area, making it suitable for adsorbing and desorbing flavonoid compounds, specifically C-glycosides [66].

XAD-1.5.5. The potential applications of macroporous resins in the future:

Currently, these resins are used for various purposes, such as adsorption, separation of compounds, purification, decolorization, odor removal, water softening, and in chromatographic analysis [67]. Furthermore, adsorbent resins play a crucial role in the refinement and recovery of waste materials in the food technology industry. These resins have excellent adsorption and desorption capabilities with polyphenols, sugars in extracts, and wastewater [68].

In the past, adsorbent resins were typically non-polar, had low selectivity, and relied mainly on π-π interactions and hydrophobic interactions for adsorption. To enhance the adsorption efficiency and selectivity, the structure of the adsorbent resin has been modified to achieve the desired outcome. However, directly substituting a benzene ring may reduce the ability to link other functional groups, which is not beneficial for the adsorption process. Therefore, the Friedel-Crafts catalytic reaction and the Blanc

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chloromethylation reaction are some of the main methods for adjusting the functional groups on the classic adsorbent resin. Successful modifications include amino-modified resin, phenolic hydroxyl-modified resin, and co-polystyrene resin [69]. In 2019, Xu and colleagues added an amino group to chloromethyl polystyrene resin using the Friedel-Crafts reaction to adsorb phenolic compounds. The result showed that the adsorption capacity of phenolic compounds was 412.9 mg/g, nearly 47% higher than that of the regular resin with the same physical structure [70].

To serve the research on natural compounds, the isolation and purification technology require high efficiency and have become an urgent issue that needs to be addressed. The adsorption efficiency of classic macroporous resins is usually not high, resulting in low levels of obtained active compounds, complex desorption processes, and difficult recycling. Therefore, creating a resin characteristic for each targeted adsorbate is a new and promising direction for the isolation and purification of active components in natural extracts. However, this approach needs to be economically and practically considered, as creating a new type of resin requires a lot of effort, and the success rate is uncertain [71].

1.6. AN OVERVIEW OF ACNE AND THE MECHANISM OF ACNE FORMATION ON THE SKIN:

On the human body, especially on the face, numerous sebaceous glands containing a mixture of fats are often found [70]. Table 1.3 shows that human sebum usually contains cholesterol, cholesteryl ester, squalene, fatty acids, and wax esters [70]. The skin typically produces oil to maintain moisture on the surface; however, some changes in the body can lead to endocrine disorders, causing an uncontrolled increase in oil production that can clog pores. Additionally, the formation of acne can be caused by changes in the keratinization process in hair follicles, folliculitis [72], or

the development of the gram-positive anaerobic bacterium Propionibacterium acnes

(P.acnes) [73].

The mechanism of acne formation mainly involves the production of sebum in the sebaceous glands, which becomes a source of nutrients for the growth and invasion

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of P.acnes bacteria into hair follicles [70]. This type of bacteria causes localized skin inflammation by producing neutrophil chemotactic factors (NCF), which continuously release inflammatory mediators such as reactive oxygen species (ROS) [74]. Acne can be visible to the naked eye and is classified into two types: closed comedone (whiteheads) and open comedone (blackheads) [75]. The difference between these two types of acne lies in the process of clogged hair follicles, creating different colors of acne. Specifically, whiteheads appear when hair follicles are completely clogged, and sebum cannot be released onto the skin's surface [75]. As for blackheads, hair follicles are partially clogged, allowing air to enter the follicles. Oxygen reacts with sebum, causing the acne core to turn dark brown or black, resembling dirt [75]. Although oxygen is an essential component for human existence, it also contributes to the creation of many oxidative reactions such as superoxide anion, hydrogen peroxide, and hydroxyl radicals [76]. In 1965, Allan L. Lorincz pointed out that oxidative breakdown of squalene and other lipids in sebum might be directly impacted on acnes formation [77].

Furthermore, ROS plays an important role in stimulating, breaking down tissues, and causing the development of inflammatory acne [78]. Normally, the process of generating free radicals occurs slowly and is naturally eliminated by antioxidant enzymes such as SOD (superoxide dismutase), CAT (catalase), and G6PD (glucose-6-phosphate dehydrogenase) present in cells [74]. However, when the immune system is compromised, the process of producing free radicals occurs more rapidly,

affecting the formation of acne on the skin. In the year of 2002, Alonso et al.

demonstrated that antioxidant compounds have the ability to scavenge free radicals, prevent ROS oxidative reactions, and reduce the appearance of acne [79].

Besides that, there is evidence that the levels of thiobarbituric acid reactive substances (TBARS) are higher in patients with acnes compared to those without acnes, which is related to an increase burden of oxidative stress [77].

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Table 1.3: The percentage of different types of fats in sebum and on the surface of

Figure 1.7: The components of sebum in pore

1.7. SUMMARIZE THE FOREIGN AND DOMESTIC RESEARCH:

Rosemary has been gaining attention from scientists worldwide due to its exceptional biological properties. A study conducted in 2006 found that rosemary extract in methanol is highly effective at killing gram-positive bacteria, gram-negative bacteria, and yeast with values of 2 – 15 mg/mL, 2 – 60 mg/mL và 4 mg/mL in, respectively.. The study also found that the antibacterial effectiveness of rosemary is directly related to its total polyphenol content [46].

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Next, a research team from Northeast Agricultural University in China successfully demonstrated the antioxidant effectiveness of rosemary extract in cooking oil. Specifically, the induction periods of oil samples containing rosemary extract were significantly higher than blank oils and oil samples containing synthetic antioxidants [26].

Another study conducted in 2021 showed that creating a chitosan film from rosemary and sage extract enhances the film's antibacterial properties against Staphylococcus Aureus and Escherichia coli. The same study also discovered that rosmarinic acid is released from both rosemary and sage extracts during the creation of a biofilm [85].

In 2023, a study was conducted to encapsulate rosemary extract in liposomes to enhance its antioxidant potential. The results showed that liposome-encapsulated rosemary extract was more effective at inhibiting the oil oxidation process than conventional thyme extract and could be used as a substitute for synthetic preservatives like BHT [86].

In terms of domestic research, rosemary is primarily exploited for its essential oils and preservation in food. In 2020, Nguyen Tat Thanh University surveyed various methods of rosemary essential oil extraction on a laboratory scale, led by Master Nguyen Dinh Phuc and his colleagues. The efficiency of oil extraction through steam distillation reached 3.04%, with the vapor content in the essential oil being 23.63%, 15.35%, 5.56%, and 5.52%, respectively, for α-pinene, 1,8-cineole, borneol, and geraniol [87]. Subsequently, Can Tho

University published a paper titled 'Effects of rosemary (Rosmarinus officinalis L.) extract

on the quality changes of fish balls from knife fish (Chitala chitala) and striped catfish product during refrigerated storage.' The study demonstrated that blending 156 mg/kg of rosemary extract helps maintain the freshness of fish balls and preserves microbiological quality during a 2-week storage period [88].

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by-CHAPTER 2 : EXPERIMENTAL 2.1. OBJECTIVE AND RESEARCH CONTENT:

Rosemary plant is known as a herb rich in biological activity, particularly in antioxidant and anti-inflammatory activity. After the distillation process, however, only the essential oil of rosemary is commonly used for commercial purposes, and the remaining residue is often discarded. Therefore, this project aims to utilize the rosemary residue after steam distillation to evaluate the total polyphenol content (TPE). Combined with investigating the adsorption and desorption ability of different types of resins, a suitable resin is chosen for the enrichment process of the TPE value in the extracted rosemary residue. Based on the results obtained, the project continues to study the antioxidant activity of the extract after desorption for use in skincare products to help reduce black-heads on the skin. To achieve the objectives outlined above, the thesis includes the following content:

Content 1: Extraction of rosemary residue

- Dried rosemary leaves are steam distilled for 1 hour, and both the essential oil and the distilled residue are collected for further study.

Content 2: Evaluation of the potential reuse of rosemary distilled residue

- Extraction of the distilled residue for 1 hour under suitable conditions and determination of the total polyphenol content in the extracted material.

- Comparison of the total polyphenol content in the extracted material from the distilled residue with that of the dried rosemary leaves to evaluate the potential for reusing the residue.

Content 3: Use of adsorption resin to enrich the antioxidant compounds in the

rosemary distilled residue

- Investigation of the static adsorption model to evaluate the adsorption and desorption ability of the three types of resins based on the TPE.

- Investigation of the desorption ratio of the resin in different solvents with different polarities to select the best solvent for desorption ability.

Content 4: Evaluate the anti-oxidant capacity of the extract after desorption

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- Measure the TBARS values to investigate the anti-oxidant activities in artificial sebum.

- Comparison of antioxidant abilities on artificial sebum between rosemary after desorption and tocopherol (vitamin E).

2.2. CHEMICALS:

The chemicals used in the thesis are suitable for the analysis standards and are presented in detail in Table 2.1.

Table 2.1: The chemicals uesd in the thesis

2,2 – diphenyl – 1 - picrylhydrazyl

2-Thiobarbituric acid ≥98%

Carnosic acid 99,29%

Chengdu Pufeide Biotech China Carnosol 97,7%

Automation Technology China

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- Essential oil distillation equipment (made at Bao Chau essential oil production facility, Vietnam)

2.4. RESEARCH CONTENT: 2.4.1. Material processing:

All experiments have been investigated at Ho Chi Minh University of Technology since October 2022. Rosemary is harvested in Lam Ha, Lam Dong, in March 2022. As this is the time when rosemary plants thrive, and the essential oil content is at its highest, it is suitable for extracting active compounds from the plant [1]. After harvesting, the plants are cleaned and the leaves are collected to be dried in a greenhouse. The drying process should avoid direct exposure to UV rays from sunlight or high-heat sources to limit the degradation and modification of active compounds in the leaves. Then, rosemary leaves are finely ground using a specialized grinder until they reach a uniform size of 0.5 to 2.5 mm (Figures 2.1 and 2.2). The moisture content of the material must be below 12% for convenient storage and to prevent the growth of moisture-loving microorganisms. Finally, the processed material is placed in a zip-lock bag with a moisture-absorbing packet and stored in a dry place.

2.4.2. Method for measuring moisture content:

Spread about 0.1 g of the material evenly on an aluminum plate and put it into the Satorius MA35 moisture analyzer. The material is dried at high temperature until the

Figure 2.1: Rosemary dried leaves Figure 2.2: Rosemary leaves after grinding

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mass remains constant, and the device signals the end of the measurement process, displaying the results on the screen. Repeat the measurement process three times to obtain the average value, with a break between each measurement to allow the device to cool down and reduce measurement errors. The mass of the dry material is calculated using the following formula:

m

!"#$!

= m

%$#&'$!

ì (1 à)

(Equation 2.1)

Where:

𝑚!"#$!: Mass of the dry material (g) 𝑚%$#&'$!: Mass of the material weighed (g)

𝜇: Moisture content of the weighed material (%)

2.4.3. The extraction process of rosemary leaves:

The process of investigating the active ingredient extraction from rosemary leaves is carried out according to the conditions surveyed by Cuong T. Q., et al. [80].

Rosemary leaves, after processing, are extracted with a solid-liquid ratio of 1:7.5 (g/mL) in 65% (v/v) ethanol. The mixture is stirred continuously at 300 rpm for 15 minutes at 65 ± 5 oC. Then, the mixture is filtered using a vacuum filtration device, and two parts are obtained: the extract and the residue. The residue is used to perform a second extraction using the same procedure as before. The extract from the second extraction is combined with the extract from the first extraction, wrapped in a food wrap, and stored at room temperature.

The combined extract is then evaporated using a rotary evaporator at a temperature of 50 ± 5 oC to remove the solvent. The resulting rosemary extract is measured and

Figure 2.3: The extraction process of rosemary leaves

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