Luận văn thạc sĩ Kỹ thuật hóa học: Enrichment of the bioactive components from the residue of rosemary (Rosmarinus officinalis L.) leaf after distillation by using macroporous resin

LITERATURE REVIEW

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

2 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]

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

– 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]

5 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

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

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 Nutroxsun TM 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 Nutroxsun TM 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

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]

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 o C 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]

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]

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].

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 1,8- 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

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

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

10 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]

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 Cu 2+ - 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]

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.

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

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),

13 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

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

MIC MBC MIC MBC MIC MBC

X campestris pv campestris 1 60 NT NT NA -

NT: non-dectived NA: not applicable

Besides the antioxidant and antibacterial activities, the anti-inflammatory activity of rosemary leaves has also been extensively studied worldwide According to Scheckel and colleagues, rosmarinic acid can effectively inhibit the expression of the pro- inflammatory gene cyclooxygenase-2 (COX-2) - a factor that contributes to the risk of tumor formation in HT-29 colon and benign MCF10A breast tissue [40] In 2015, 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]

The polyphenolic compounds present in rosemary leaves are the main source of anti- 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]

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].

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]

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

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

Name Material Polarity Size (mm)

Acrylate Strong polar 0.50 – 0.56 500 500 a) HPD-100 b) DM-301 c) c) XAD-7HP Figure 1.6: The shape of three macroporous resins

The HPD-100 resin is commonly used for isolating compounds and enriching organic groups such as flavonoids [55], polyphenol [56], or tannins [66] This is a type of cycloaliphatic aromatic hydrocarbon resin that has been hydrogenated, is odorless, has good thermal stability, and is white and opaque [57]

The pore volume is 1.35 – 1.65 (mL/g) [58]

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]

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,

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

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]

In a study conducted in 2021 by Che Zain and colleagues, it was found that XAD- 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]

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

19 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].

AN OVERVIEW OF ACNE AND THE MECHANISM OF ACNE

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

20 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]

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

Types of fats The percentage in sebum (%)

The percentage on the surface of human skin

Triglycerides, diglycerides, and free fatty acids

Figure 1.7: The components of sebum in pore

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]

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 by- 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]

EXPERIMENTAL

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

- 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).

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

Methanol (HPLC) VWR Chemicals USA

Acetonitrile (HPLC) VWR Chemicals USA

Chengdu Pufeide Biotech China Carnosol 97,7%

MEASURING DEVICE

- Moisture analyzers Satorius (MA35, Sartorius, Gửttingen, Germany)

- Analytical balances (ED 224S, Sartorius, Gửttingen, Germany)

- UV-VIS Spectrophotometers (Thermo Genesys 10S UV-Vis, Waltham, MA, USA)

- Vacuum Evaporator IKA (RV8, IKA – Werke GmbH, Staufen, Germany)

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

RESEARCH CONTENT

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

26 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)

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 o C 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 o C to remove the solvent The resulting rosemary extract is measured and

Figure 2.3: The extraction process of rosemary leaves

27 the moisture content is recorded It is then stored in a dark vial and kept at -20 o C The process is illustrated in Figure 2.3 and the extraction yield is calculated by:

𝑚"*% -*)$"#*.: The mass of dried material before extraction (g)

2.4.4 Investigate the adsorption and desorption ratio of macroporous resins: a Pre-treatment of macroporous resin:

To eliminate monomers and contaminants still present in the pores of the resin during synthesis, pre-treatment of resin is essential Based on the method of Dong et al., the pre-treatment process is carried out with some modifications [81] The adsorbed resin is soaked in ethanol for 24 hours at the ratio of solid: liquid is 1:5 (g/mL), then washed three times with ethanol and subsequently rinsed twice with distilled water to remove any remaining alcohol The resin is then soaked in a 1.0 M NaOH solution for 5 hours at unchanged ratio of solid: liquid, rinsed twice with distilled water, followed by soaked in a 1.0 M HCl solution for 5 hours and thoroughly rinsed with distilled water Finally, the pre-treated resin is dried in a drying cabinet at 60°C until a constant weight is achieved before using b Selecting a suitable solvent to dissolve rosemary extract:

The study conducts an investigation into the solubility of the rosemary extract in 8 different solvents, including: water, ethanol 20 o , ethanol 40 o , ethanol 50 o , ethanol 65 o , ethanol 80 o and absolute ethanol A total of 0.25 g of the extract was dissolved in 50 mL (5000 ppm) of each solvent and then sonicated for 5 minutes at two different temperature points of 30 o C and 60 o C The observations are compared to select the appropriate solvent and dilution temperature

28 c Static adsorption of XAD-7HP, DM301, and HPD100 resins:

In order to determine the adsorption capacity of phenolic compounds of XAD-7HP, DM301, and HPD100 resins, the study aims to construct a dynamic adsorption kinetics curve for phenolic compounds in rosemary extract using the three types of macroporous resins mentioned above The Figure 2.4 summarized the procedure of performing the adsorption calculation The process was based on the study conducted by Lianzhu Lin and colleagues in 2012 with some modifications [16], as follows: rosemary extract was accurately weighed at 0.50 ± 0.01 g and completely dissolved in 100 mL of ethanol 50 o at the temperature 60 o C to obtain a homogeneous solution Then, precisely weigh 1.0 ± 0.1 g of the pre-treated adsorption resin into a 100 mL Erlenmeyer flask with a screw cap to limit alcohol evaporation during the adsorption process 100 mL of rosemary extract was added to the Erlenmeyer flask containing the adsorption resin The flask was continuously shaken at a speed of v = 175 rpm at

30 o C in a shaker incubator, then the concentration of target compounds during the adsorption process was determined at various time intervals until reaching equilibrium (at intervals of 5, 10, 15, 20, 30, 40, 50, 60, 80, 100, and 120 minutes) The same procedure was repeated for the other three types of resins The adsorption capacity is calculated using the formula:

Figure 2.4: The procedure of calculating the adsorption

𝑞 ) : Adsorption capacity (mg/g dried resin) at time t (mins)

𝐶 2 : Initial adsorption concentration of target compounds (mg/mL)

𝐶 ) : Adsorption concentration of target compounds at time t (mg/mL)

𝑉 # : Volume of solution (mL) 𝑊: Mass of used resin (g) M: Moisture content of the adsorption resin (w/w %) The adsorption ratio is calculated using the formula:

𝐶 $ : The equilibrium concentration of target compounds in the solution (mg/mL) d Adsorption and desorption kinetics:

When dealing with adsorption, it is essential to consider both thermodynamic and kinetic aspects to understand its efficiency and underlying mechanisms In addition to the adsorption capacity, the kinetic performance of a particular adsorbent holds significant importance for practical applications Through kinetic analysis, one can determine the rate at which the solute is captured, which dictates the time required for the adsorption process to reach completion Moreover, kinetic information allows for the design of the adsorption equipment appropriately [16] e Adsorption isotherm and thermodynamics of the selected resin:

The adsorption isotherm depended on the equilibrium concentration of the adsorbed resin and the amount of adsorbent present in a solution at a particular temperature [82] Equilibrium data provides valuable insights into the interaction between solutes and adsorbents The Langmuir and Freundlich equations are commonly employed to assess linearity and to elucidate the nature of solute-adsorbent interactions [82] In detail, the Langmuir isotherm was initially formulated for gas-solid interactions but has found applications with various types of adsorbents It is an empirical model grounded in kinetic principles, where the adsorption and desorption ratios on the surface are assumed to be equal, resulting in zero accumulation at equilibrium conditions This model is based on several key assumptions, including:

1 Monolayer adsorption: It assumes that adsorption occurs as a single layer of solute molecules on the adsorbent surface

2 Homogeneous sites: The adsorption sites on the adsorbent surface are considered identical and uniform in their adsorption capacities

3 Constant adsorption energy: It assumes constant adsorption energy for all molecules being adsorbed

4 No lateral interaction between the adsorbed molecules: No significant interaction or interference exists between neighboring adsorbed molecules

These assumptions collectively provide a simplified framework for describing the adsorption behavior of solutes on surfaces, allowing for the derivation of the Langmuir isotherm equation The monolayer assumption necessitates uniform adsorption sites where only a single molecule can adhere to each site [83]

In terms of Freundlich, this empirical model applies to multilayer adsorption occurring on heterogeneous sites It considers nonuniform distributions of adsorption heat and affinities for the heterogeneous surface [83] The experiments were carried out following the subsequent procedure: the chosen resin (0.5 g) is added into flasks containing 50 mL of rosemary extract with varying concentrations Adsorption experiments are carried out at three distinct temperatures (30, 45, and 60 °C) using

100 mL flasks equipped with stoppers The adsorption isotherms for carnosol and carnosic acid are established by applying both the Langmuir and Freundlich equations: The Langmuir equation:

The Freundlich equation: ln 𝑞 7 = ln 𝑏 + 8 / ln 𝐶 7 (Equation 2.6)

𝑞 $ : Desorption capacity (mg/g dried resin)

𝐶 $ : Concentration of compounds in the desorption solution (mg/mL)

𝐾 5 : The Langmuir constant in L/mg

𝑞 2 : The maximum amount of adsorbed surfactant (mg/g)

𝐶 2 : Initial adsorption concentration of target compounds (mg/mL)

𝑅 5 : Equilibrium parameter b: An intercept of a plot of ln 𝑞 $ versus ln 𝐶 $ 1/𝑛: A slope of a plot of ln 𝑞 $ versus ln 𝐶 $ f The desorption process of XAD-7HP, DM301, and HPD100:

After the adsorption process reached equilibrium, the top layer of the solution was discarded The resin layer containing the adsorbed compounds was washed twice with deionized water, and then subjected to the desorption process using 50 mL of different solvents, including: absolute ethanol (ABS), acetonitrile (ACN), ispropyl alcohol (ISP) The Erlenmeyer flasks were shaken at 150 rpm for 120 minutes at

30 °C in a incubator [84] The concentration of carnosol and carnosic acid was determined by HPLC method

The desorption ratio was calculated using the formula:

The desorption capacity is calculated using the formula:

𝑞 ! : Desorption capacity (mg/g dried resin)

𝐶 ! : Concentration of carnosol and carnosic acid in the desorption solution (mg/mL)

𝑉 ! : Volume of the desorption solution (mL)

𝐶 2 : Initial adsorption concentration of target compounds (mg/mL)

𝐶 $ : The equilibrium concentration of target compounds in the solution (mg/mL)

𝑊: Mass of used resin (g) M: Moisture content of the adsorption resin (w/w %) g Investigate the volume of elute solvent:

After selecting a suitable solvent for the best dissolution ability, the influence of the solvent volume on the dissolution rate is investigated The experiment is conducted with unchanged parameters, but during the solvent dissolution step, the solvent volume is increased gradually to 30 mL, 50 mL, 70 mL, and 100 mL The increasing solvent volumes correspond to an increase in the proportion of solvent used for dissolution relative to the volume of the extracted solution After 120 minutes, the polyphenol content is determined for each sample The experiment is conducted twice, and the average results for each solvent volume are calculated Finally, the dissolution

33 rate is determined using Equation 2.8 to select the optimal solvent volume for the dissolution rate h Investigate the desorption time:

After 60, 120, 180, 240, 300, 360, and 400 minutes, 0.5 mL samples are taken, and the polyphenol content of each sample is determined by UV-VIS equipment The experiments are performed twice, and the average results are calculated for each desorption time Finally, the desorption percentage is determined using Equation 2.8, and the desorption time for the highest desorption ratio is selected

Dynamic desorption experiments for carnosol and carnosic acid are carried out on glass columns (50 cm x 6 cm) wet-packed with 8.0 g resin of selected hydrated resin The bed volume (BV) of resin is 30 mL After packing the column, a thin layer of cotton is covered on the resin surface to prevent cracking of the column Firstly, the column is washed with 4BV of H2O and then eluted with an ethanol-water solution

2.4.6 The quantification method of target compounds in extract after adsorption in macroporous resin: a Methodology:

RESULT & DISCUSSION

Distillation efficiency of rosemary essential oil by steam distillation method

After being processed and dried, rosemary leaves were distilled using steam under conditions similar to Dr Le Xuan Tien and his colleagues [102] distillation time was calculated from collecting the first drop of essential oil until 1 hour later The efficiency of essential oil distillation after 1 hour was obtained in two distillations, reaching 1.83 ± 0.22 % (n=2) – based on the amount of dried leaves Therefore, with the same distillation conditions, this study yielded a higher efficiency than that of Boutekedjiret and his colleagues - only 1.2 % [92]

Compared with the results obtained by direct steam distillation in other studies, the steam distillation method was much more effective Specifically, the study of Boutekedjiret and colleagues showed that with the same amount of initial dry material, direct steam distillation only achieved an efficiency of 0.44 % [92] However, Carvalho and his colleagues have shown that the yield of rosemary essential oil can reach 5 % if supercritical CO2 distillation is applied [93] In addition, the supercritical

CO2 distillation method is still limited by its expensive investment costs and high technical requirements Therefore, the steam distillation method is still the most suitable for this thesis

Besides, the yield of essential oil can be affected by the processing of raw materials, such as drying and crushing rosemary leaves before distillation This contributes to breaking down the structure of the leaves, breaking the essential oil-containing sacs on the leaves, thereby increasing the effectiveness of attracting critical oils Thanks to that, the components in the essential oil will evaporate quickly, thereby shortening the distillation time and limiting the decomposition of active ingredients in the residue

The rosemary residue after distillation for 1 hour had high moisture due to direct contact with steam during the distillation process After the rosemary leaves finish the distillation process, the residue is dried at room temperature until the humidity reaches a constant value below 11 % and stored in a sealed zip bag with silica gel desiccant bags to limit moisture

Most aromatic medicinal species are extracted from essential oils for commercial purposes However, the content of essential oil only takes a tiny part of rosemary leaves, the majority consists of non-volatile compounds such as carnosol, carnosic

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

43 acid, rosmanol, rosmarinic acid, and flavonoids - valuable in anti-oxidation, antioxidant antibacterial, anti-cancer, anti-inflammatory [94] Therefore, rosemary distillate residue is predicted to have considerable potential for the recovery and enrichment of antioxidant-active ingredients

After drying, rosemary distillation residue (RDR) was extracted by 65 o ethanol solvent with a solid: liquid ratio of 1:7.5 (g/mL) The extraction efficiency results were shown in Figure 3.2 and compared with the initial dried rosemary leaf sample

Based on the results in Figure 3.2, it can be seen that the yield of a 65 o ethanol extract from the initial dried rosemary reached 30.42 ± 0.59 % Under the same extraction conditions, the efficiency for RDR extract occupied at 25.45 ± 0.21 %, which was lower than the extraction from initial dried rosemary leaves This phenomenon can be attributed to the post-distillation process, where the rosemary residue undergoes drying and exposure, leading to a significant reduction in the surface area of the leaves Additionally, some active compounds may be lost or degraded during this stage [54]

Ex tr ac tio n yi el d (% )

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

Figure 3.3 The rosemary extract after 1-hour distillation

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

Although the same extraction conditions were applied, the total polyphenol content in the extract from dried rosemary leaves reached 223.738 ± 4.185 (mg GAE/g dry leaves) - significantly higher than a previous study, which only achieved 202.7 ± 5.73 mg GAE/g dry leaves [95] This difference can be due to various environmental factors, such as UV radiation, soil conditions, temperature, precipitation, and humidity affecting the biosynthesis [104] Furthermore, the temperature and harvest season are also two most important factors affecting polyphenol content in rosemary leaves According to Wahid’s research in 2006, the synthesis of polyphenols is highly temperature-sensitive within rosemary leaf cells [104] Additionally, a study by Bolling and colleagues indicated that variations in the content of polyphenols, flavonoids, and tannins in plants can be season-dependent [97] In this thesis, rosemary leaves were harvested in late spring to early summer, during cool and suitable weather conditions conducive to plant growth This partly explains why the total polyphenol content in dried rosemary leaves studied in this thesis is higher than in previous studies

Figure 3.4 illustrated the polyphenol content in two examined extracts, where the polyphenol content in the one-hour distillation extract was slightly lower than that in the initial dried rosemary leaves

Figure 3.4 The total polyphenol content in the initial dried rosemary leaves and the

The polyphenol content in the 65 o ethanol extract from the RDR accounted for 198.643 ± 4.949 (mg GAE/g dry weight), which was lower slightly than the value obtained from the initial dried leaves (223.738 ± 4.185 mg GAE/g dry weight) This decrease can be explained by the fact that during distillation, water vapor entrainment may have led to the loss of some polyphenolic compounds [98] However, overall, the polyphenol content in the distillation residue was not significantly lower than in the initial dried rosemary leaves, indicating that the distillation residue still possesses the potential for reuse Therefore, it can be concluded that reusing the distillation residue after one hour to enrich polyphenols is feasible, as the residue contains many valuable non-volatile compounds Furthermore, reusing the residue helps address the issue of plant waste from the essential oil distillation industry and conserves natural resources Next, the study compared the content of two active compounds, carnosic

Th e to ta l p ol yp he no l c on te nt (m g G A E /g d ry w eig ht)

46 acid, and carnosol, in dried rosemary leaves and essential oil The results were presented in Figure 3.5

It can be observed that after a 1-hour steam distillation process, the carnosol content in RDR extract reached 52.57 ± 0.55 mg/g dry weight, which was approximately similar to the carnosol content in dried rosemary leaves (51.43 ± 0.40 mg/g dry weight) On the other hand, the carnosic acid content in RDR extract was 96.26 ± 1.43 mg/g dry weight, reaching only 91% of the content found in dried leaves samples (105.82 ± 0.71 mg/g dry weight) This is because of the carnosic acid in the rosemary leaves is transformed into carnosol when exposed to high temperatures during the 1- hour steam distillation process [108] However, considering the potential for the residue, it can still be seen as a valuable source of raw material since it still contains a relatively high amount of carnosol, known for its potent antioxidant properties Furthermore, reusing the residue in commercial applications also helps reduce the cost of raw materials, thus increasing the value of the final product

Th e co nt en t o f ac tiv es in e xt ra ct (m g/g e xtra ct)

Figure 3.5 The content of carnosic acid and carnosol in initial rosemary dried leaves and

Investigation of factors affecting the adsorption capacity of

According to Wang and colleagues (2020), the adsorption process of phenolic compounds on different types of macroporous resins occurs based on the physical mechanisms between the resin material and the combinations to be adsorbed, involving Van der Waals forces or hydrophobic interactions [82] Therefore, the adsorption capacity of macroporous resins depends on the interaction between the compounds to be adsorbed and the resin's structure [109] Consequently, this study compares the adsorption capacity of three different macroporous resins with varying forms and polarities, namely HPD100, DM301, and XAD–7HP, to select the best resin type Additionally, the study investigates two factors affecting the resin's adsorption capacity: adsorption time and temperature

3.2.1 Selection of the suitable solvents for dissolving rosemary extract:

The rosemary extract was tested for solubility using the following solvents: water, EtOH 40 o , EtOH 50 o , EtOH 65 o , EtOH 80 o , and EtOH 99.5 o (v/v) The tests were conducted with the same amount of extract (0.25 g) in the same solvent volume (50 mL) Table 3.1 showed significant differences in the solubility of the extract in these six solvents at two different temperature levels

Table 3.1 The solubility of rosemary extract in various solvents

Poorly soluble, and contain residue

Partly soluble, and contain residue

Sparingly soluble, and contain residue

Observing Table 3.1, it was easy to notice that, with the same amount of rosemary extract and solvent volume, rosemary extract dissolved well in two solvents, EtOH

50 o (v/v) and EtOH 65 o (v/v) This can be explained by the fact that these are two solvents with moderate polarity, making it easier to dissolve compounds with moderate polarity, such as carnosol and carnosic acid, as well as the sticky substances,

49 proteins, and starch present in the extract [110] However, as the alcohol content increases, indicating less polarity in the solvent, the solubility of the rosemary extract gradually decreases, and it seems to be utterly insoluble in absolute ethanol (EtOH 99.5 o ) This is because the sticky substances, proteins, and starch in the extract do not dissolve in less polar solvents like ethanol [92] Furthermore, when rosemary extract is dissolved in water, it forms a turbid solution and does not dissolve completely This phenomenon is explained by the fact that most polyphenolic compounds and chlorophyll in the extract have poor solubility in highly polar solvents like water, resulting in an insoluble solution [100] Therefore, the two solvent systems EtOH – H2O with alcohol concentrations of 50 o and 65 o are highly suitable for dissolving the extract

Increasing the dissolution temperature did not significantly affect the solubility of rosemary extract in the solvents, except for EtOH 50 o , which showed a vastly improved solubility When comparing polarity, EtOH 65 o is less polar than EtOH 50 o On the other hand, fewer polar solvents can easily attach to the surface of fewer polar adsorption resins, reducing their ability to adsorb other compounds [110] Although both can completely dissolve the rosemary extract, EtOH 50 o was preferred over EtOH 65 o in this study due to its better polarity, and the temperature was 60 o C

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

This study examined all three types of resins, HPD100, XAD–7HP, and DM301, under the same adsorption conditions However, their adsorption capacities showed significant differences Based on the graph in Figure 3.6, it can be seen that after 120 minutes, the adsorption capacities of polyphenols by the three resin types reached equilibrium They achieved values of 0.0105 ± 0.0009 mg polyphenol/g resin for HPD100, 0.0205 ± 0.0001 mg polyphenol/g resin for DM301, and 0.0212 ± 0.0006 mg polyphenol/g resin for XAD–7HP HPD100 exhibited less than half the adsorption capacity compared to DM301 and XAD–7HP

Th e ad so rp tio n ca pa ci ty b as ed o n th e co nt en t o f to to tal p ol yp hen ol ( m g/ g resi n)

Figure 3.6 The adsorption capacity curves of the three types of resins over time

On the other hand, the adsorption capacity of the resin was also represented based on their adsorption ratio in Figure 3.7 below

Figure 3.7 The adsorption ratio of the three types of resins based on the total polyphenol content

Among the three surveyed resin types, XAD-7HP and DM301 exhibited the best adsorption capabilities for polyphenolic compounds, with adsorption ratios of 41.08 ± 0.42 % and 39.95 ± 1.12 %, respectively In contrast, HPD100 showed relatively poor adsorption ability, with an adsorption ratio of only 17.77 ± 0.90 % - roughly half that of the other two resin types According to the studies by Li and colleagues [77], and Yang et al [90] the adsorption capacity of resin depends on the chemical and physical properties of the resin, with essential factors including intermolecular forces, surface polarity, particle size, and surface area The properties of the three resin types examined in this study are presented in Table 3.2 below

Th e ad so rp tio n ra tio b as ed o n th e co nt en t o f to tal p ol yp hen ol ( % )

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

Particle size (𝝁𝒎) Polarity Pore size

Surface area (m 2 /g) Material HPD100 300 – 1200 Non-polar 85 – 90 650 – 700 Polystyrene

XAD–7HP 500 – 560 Strong polar 500 500 Acrylate

As shown in Table 3.2, XAD–7HP was the most polar resin among all three types, and it also exhibited the highest adsorption capacity for polyphenols compared to the other resins (Figure 3.6) According to Wang and partners, the macroporous resin was chosen depending on the polarity of the components Specifically, strong polar compounds with benzene rings and hydrogen groups necessitate the use of moderately polar resin, while weakly polar compounds were treated with non-polar resins [54] This result was attributed to the similarity between the resin's surface polarity and the polarity of the compounds to be adsorbed [109] n addition, Yan et al pointed out that the polarity of polystyrene resin could be affected by the crosslinking of the divinylbenzene group [104]

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

According to a study by K Brückner and colleagues (2016), the levels of the target antioxidant compounds, carnosol, and carnosic acid, only take a small portion of the total polyphenol content in rosemary extract [37] Therefore, evaluating the adsorption capacity of the resin solely based on the total polyphenol content is insufficient for concluding Hence, the research continued by assessing the adsorption ratio of DM301 and XAD-7HP resins by quantifying carnosol and carnosic acid using the HPLC method

The graphs represented the adsorption capacity of the two compounds, carnosol and carnosic acid, by DM301 and XAD-7HP resins over time, as depicted in Figure 3.8 The adsorption capacity of DM301 resin gradually increased from the initial 10 minutes to 30 minutes, then steadily increased until 60 minutes, reaching 16.32 ± 0.04 mg/g resin for carnosic acid and 5.97 ± 0.02 mg/g resin for carnosol XAD-7HP resin also exhibited a similar trend in adsorption over time to DM301 resin, reaching equilibrium at 60 minutes with values of 14.20 ± 0.08 mg/g resin for carnosic acid and 2.93 ± 0.05 mg/g resin for carnosol After 60 minutes, the adsorption capacity of both resin types appeared to remain stable until 90 minutes This could be explained

Th e ad so rp tio n ca pa ci ty Q t (m g/g d rie d re sin )

Figure 3.9 The adsorption capacity of carnosol

Th e ad so rp tio n ca pa ci ty Q t (m g/g d rie d re sin )

Figure 3.8 The adsorption capacity of carnosic acid

54 by the fact that during the initial 30 minutes, polyphenol compounds such as carnosic acid and carnosol were rapidly adsorbed onto the resin surface before diffusing into the porous structure [105] After 30 minutes, the adsorption process became slower and tended to approach equilibrium by 90 minutes This result was achieved because the complex structure of the resin material hinders the movement of large molecules towards the resin surface [105] Although the equilibrium adsorption time for the total polyphenol content was 120 minutes when considering only the two antioxidant compounds, carnosic acid and carnosol, the adsorption time reached equilibrium after only 60 minutes Therefore, the optimal adsorption time for the two antioxidant compounds, carnosic acid and carnosol, using XAD-7HP and DM301 resins was 60 minutes, and this was also the fixed adsorption time for subsequent experiments Selecting a shorter equilibrium adsorption time (60 minutes) helped save experimental operation time and reduced energy consumption

Table 3.3 showed that DM301 resin exhibited a significantly higher adsorption ratio for both target compounds than XAD-7HP resin For carnosic acid, DM301 resin achieved an adsorption ratio of 93.21 ± 0.27 % after 90 minutes of adsorption, while XAD-7HP resin only reached 81.02 ± 0.51 % Regarding carnosol, this compound was very effectively adsorbed by DM301 resin, with an adsorption ratio of 82.51 ± 0.52 %, nearly double the adsorption ratio observed with XAD-7HP resin (41.51 ± 0.62 %) - the adsorption capacity and adsorption ratio of DM301 and XAD-7HP resins

Table 3.3 The adsorption capacity and adsorption ratio of DM301 and XAD-7HP resins

Carnosic acid Carnosol Carnosic acid Carnosol

This could be explained by the fact that even though both resin types achieve an approximate 40 % adsorption ratio based on the polyphenol content (Figure 3.7), there was a significant difference in the adsorption ratio of the two compounds, carnosol and carnosic acid, with DM301 resin being markedly superior This can be attributed to the pore size of DM301 resin, which falls within the range of 140 – 170 Å [103], making it a moderately sized pore, whereas XAD-7HP resin has a significantly larger pore size (550 Å) [54], allowing other polyphenol molecules to be adsorbed and attached to its surface (Table 3.2) In addition to pore size, the interaction between the polarity of the resin's structural material and the polarity of the compounds to be adsorbed also affects the adsorption ratio of the resin [54] Studies related to resin adsorption for separating moderately polar compounds containing benzene rings and hydrogen groups tend to choose resins with moderate to high polarity This study investigated the adsorption capacity of phenolic compounds, mainly two compounds, carnosol and carnosic acid, which were moderately polar due to carboxylic and catechol groups [54] DM301 resin exhibits significantly higher adsorption capacity because it is moderately polar, making it easier for carnosol and carnosic acid molecules to attach to the resin's surface due to their similarity in polarity

Furthermore, both resin types exhibited better adsorption capacity for carnosic acid than carnosol The explanation is the π-π interactions between the benzene ring and the components in polystyrene adsorbents [54] The carnosic acid has the π-π bonds

(carboxyl group), leading to a higher adsorption ratio, while the carnosol only has a lactone ring Moreover, the carnosic acid content in the initial rosemary extract reached 96.26 ± 1.43 mg/g of the extract (Figure 3.5), which was much higher than the carnosol content in the same extract (52.57 ± 0.55 mg/g of the extract)

Due to its superior adsorption properties, DM301 resin has been identified as a potential resin for enriching polyphenolic compounds, especially carnosol and carnosic acid, from rosemary extract

Temperature is an essential factor in the adsorption process of resins It can influence the adsorption capacity of the resin by altering the molecular kinetic energy and the

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

57 collision rate between active compounds and the resin surface [105] Therefore, it is crucial to identify an optimal temperature to achieve the best adsorption performance

Investigation of factors affecting the desorption capability of the resin:59 1 Selection of desorption solvent

Exploring the factors influencing the desorption capability of the resin is a key step when desorbing compounds from the resin surface Typical factors that can affect the resin's desorption capability include the desorption solvent's polarity, desorption time, and solvent volume [54] In this thesis, these factors were investigated and optimized based on the desorption ratio to ensure the efficiency of the process for separating carnosol and carnosic acid from rosemary extract using adsorption resin

The choice of desorption solvent is a critical factor in determining the desorption capability of the target compounds Additionally, the polarity of the desorption solvent significantly affects the experimental results [54] Therefore, this study selected different solvents with varying polarities and investigated the desorption ratio of DM301 resin in these solvents The desorption ratio was compared and presented in Table 3.5

Table 3.5 The desorption ratio of DM301 resin in different solvents

Desorption ratio (%) Purity (%) Desorption ratio (%) Purity (%)

The results in Table 3.5 showed that the desorption ratio of carnosol and carnosic acid increases as the polarity of the solvent decreases Specifically, 70 o ethanol, the most polar solvent, had the lowest desorption ratio for both carnosol and carnosic acid, achieving only 11.71 ± 0.12% for carnosic acid and 21.58 ± 0.08% for carnosol In contrast, 99.5 o ethanol exhibited significantly higher desorption capabilities for both compounds, with a desorption ratio of 79.68 ± 0.60% for carnosol and double that for carnosic acid compared to 70 o ethanol

In terms of purity, this value was determined based on the mass of the target compounds desorbed relative to the total mass of extract in each solvent Purity indicated the content of the desired compounds in the final desorbed extract, and it was an essential parameter for assessing the desorption efficiency of the solvent The data in Table 3.5 showed that 99.5 o ethanol was a potential solvent for the desorption process, with purities of 11.61% and 29.72% for carnosol and carnosic acid, respectively Isopropyl alcohol and acetonitrile followed closely, with little difference compared to absolute ethanol, and the lowest purity was observed with 70% ethanol for both compounds, below 11% This can be explained by the tendency of most impurities, oils, or starch to be less soluble in non-polar solvents like acetonitrile,

61 isopropyl alcohol, or absolute ethanol Therefore, 70% ethanol, containing water, resulted in the elution of both target compounds and impurities, reducing the purity of the final extract [110] In addition, the two target compounds: carnosol, and carnosic acid, readily dissolve in organic solvents such as ethanol, dimethyl sulfoxide (DMSO), or chloroform [61] Therefore, the content of these compounds mentioned above could be effectively extracted when using ethanol, isopropyl alcohol, and acetonitrile

Therefore, while absolute ethanol might not provide the highest desorption ratio compared to isopropyl alcohol and acetonitrile, it offers good selectivity for the two target compounds Furthermore, considering the safety and economic aspects of the three solvents, 99.5 o (v/v) ethanol was the least toxic, most benign, and cost-effective solvent [107] [108] [109] Thus, 99.5% (v/v) ethanol was chosen as the desorption solvent for the entire process

After selecting the adsorbent resin as DM301 and the eluent as 99.5 o (v/v) ethanol, the extraction time was investigated to determine its influence on the desorption ratio of the resin The desorption ratio of the resin at time intervals of 20, 40, 60, 120, 180,

240, and 300 minutes were compared in Figure 3.11

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

Figure 3.11 illustrated that the desorption ratio reached 43.69 ± 0.11% for carnosic acid and 31.69 ± 0.42% for carnosol within the first 20 minutes As the extraction time gradually increased from 20 to 40 minutes, the desorption ratio of DM301 resin in absolute ethanol increased progressively and reached equilibrium at 60 minutes This can be explained by the fact that the time intervals of 20 to 40 minutes were not long enough for carnosol and carnosic acid to be desorbed from the macroporous resin However, when the extraction time reached 60 minutes, the desorption ratio further increased to 72.30 ± 0.70% for carnosic acid and 79.59 ± 1.69% for carnosol, and it appeared to plateau as the time extended to 300 minutes Therefore, the minimum required extraction time for the desorption experiment is 60 minutes to achieve a good desorption ratio

3.3.3 Selection of desorption solvent volume:

In addition to extraction time, varying the volume of the desorption solvent also influences the content of active compounds desorbed from the resin Generally, as the volume of the desorption solvent increased, the desorption ratio of both carnosol and carnosic acid tended to increase (Figure 3.12) Specifically, at an absolute alcohol

Th e de so rp tio n ra tio (% )

Desorption time (mins)Carnosic acid Carnosol

63 solvent volume of 70 mL, both target compounds were desorbed with the highest ratio, reaching 88.02 ± 0.49 % for carnosol and 83.95 ± 0.01 % for carnosic acid However, these values gradually decreased beyond this point and fell below 80 % for both compounds when the desorption solvent volume was increased to 100 mL Additionally, the desorption ratio at 70 mL increased by nearly 10 % for both compounds compared to the data at a volume of 30 mL (carnosol 79.03 ± 1.14 % and carnosic acid 69.35 ± 0.13 %)

Regarding the purity of the eluate after desorption, data from Table 3.5 showed that the purity of the eluate increased gradually with the increase in desorption solvent volume, reaching the highest values at a volume of 70 mL (carnosol was 17.38 %, and carnosic acid was 30.45 %) However, when the desorption solvent volume was further increased to 100 mL, the purity significantly decreased, less than half of the value at 70 mL (7.29 % for carnosol and 13.63 % for carnosic acid) This may be because when insufficient desorption solvent is used, it is impossible to remove the target compounds from the resin's surface completely Still, when an excessive amount of desorption solvent is used, it elutes unwanted impurities, resulting in a lower desorption ratio [56] In conclusion, the optimal desorption solvent volume was

70 mL to achieve the best desorption ratio

Table 3.6 The purity of extract based on the solvent volume

(% mass of target compound/ mass of total extract)

Figure 3.12 The effect of desorption solvent volume on the desorption ratio

In summary, the DM301 resin was chosen and the suitable conditions for adsorption and desorption were presented in Table 3.7:

Table 3.7 The adsorption and desorption conditions

Th e de so rp tio n ra tio ( % )

Volume (mL)Carnosol Carnosic acid

3.4 Comparison of the carnosol and carnosic acid content in the initial extract and purified extract:

The quantification results using high-performance liquid chromatography (HPLC) in Figure 3.13 showed that the percentage of both antioxidant-active ingredients in the extract after the adsorption and desorption process was significantly higher than in dried leaves and RDR extract

The carnosic acid content in the sample after enrichment by resin adsorption accounted for 32.99 ± 0.20 % on the dry weight basis, which was almost 4 times higher than the RDR extract (8.42 ± 0.13 %) and 3.6 times higher than the dried rosemary leaf extract (9.26 ± 0.06 %) Additionally, the percentage of carnosol relative to the total mass of substances in the original rosemary leaves sample and the distilled sample was deficient, reaching only 4.55 ± 0.04 % and 4.65 ± 0.05 %, respectively, on the dry weight After enrichment, the content of the actives increased by nearly 3 times, reaching 15.90 ± 0.19 %

3.4.1 Enrichment of carnosol and carnosic acid by resin column:

According to the comparison results in section 3.4, the total content of the two target compounds, carnosol and carnosic acid, only reached approximately 50% of the total

Dried leaves extract After 1 hour - steam distillation extract After adsorption and desorption extract Th e c on te nt o f ac tiv es i n ex tr ac t (% g /g e xtra ct)

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

66 mass of the enriched extract (32.99 ± 0.20 % for carnosic acid, and 15.90 ± 0.19 % for carnosol) Therefore, the thesis aims to enhance the purity of product (the total content of the two target compounds) by using column chromatography Chromatography is a commonly used method in the purification, enrichment, and refinement of substances, especially in the pharmaceutical industry First, the thesis conducts a preliminary survey using thin-layer chromatography to determine the eluent concentration gradient, and then quantifies it using HPLC

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

Based on Figure 3.14 a) and b), it can be observed that throughout the elution process from 4BV of H2O fraction to 3BV of EtOH fraction, there was hardly any trace of the two compounds, carnosol and carnosic acid, compared to the standard compounds carnosol (label S in TLC plate) and carnosic acid (label A in TLC plate) This indicated that at this concentration range, only impurities were eluted The two target compounds did not interact with the solvent system and thus remained retained in the resin column This result aligned with the explanation in section 3.3.3 Selection of desorption solvent, as lower alcohol content leads to less drag on the target compounds As alcohol concentration increased, traces of carnosol and carnosic acid appeared

Determine the TBARS value in artificial sebum

Many studies are revealed that the formation of blackheads is due to the oxidation of sebum in the hair follicles [79] Therefore, determining the level of MDA produced in artificial sebum is the first step in assessing the process of inhibiting proliferation and the formation of blackheads on the skin Within the concentration range of 100 to 400 ppm, the antioxidant capacity of artificial sebum of enriched extract gradually increased with the increase in the concentration range and reached the highest value at a concentration of 400 ppm (45.66 ± 0.47 mg MDA/kg sample) In addition, the MDA value of RDR extract was lower nearly 3.5 times than that of enriched extract at the same concentration, only occupied 154.90 ± 0.22 mg MDA/kg sample

Compared to the tocopherol sample at the same concentration (400 ppm) in Figure 3.17, the enrichment rosemary extract had a lower antioxidant capacity for artificial sebum by about 2 times (tocopherol = 26.25 ± 1.49 mg MDA/kg sample)

The results were consistent with the study by Ortuủo et al [111] which demonstrated that the antioxidant capacity for lamb meat of rosemary extract was nearly 5 times lower than tocopherol In detail, Ortuủo and colleagues illustrated that the MDA levels produced when using tocopherol as a preservative were the lowest (1.0 mg

Figure 3.17 The MDA values of enriched rosemary extract at different concentrations and tocopherol at concentration 400ppm

MDA/kg sample) Meanwhile, at the same concentration, the combination of carnosol and carnosic acid in a 1:1 ratio showed an antioxidant capacity of approximately 5.5 mg MDA/kg sample

CONCLUSION AND DISCUSSION

The thesis “Enrichment of bioactive components from the residue of rosemary

( Rosmarinus officinalis L.) leaf after distillation by using macroporous resin ” aims to utilize macroporous resin to enrich the antioxidant compounds in rosemary residues after steam distillation The objective is to evaluate the antioxidant capacity of an artificial sebum to determine its effectiveness in black-head resistance The research has achieved some results:

• Demonstrated the potential of rosemary residues after steam distillation, with the content of carnosol and carnosic acid was 52.57 ± 0.55 and 96.26 ± 1,43 mg/g extract, respectively

• Selected DM301 resin as suitable for adsorption process, achieved adsorption ratio of carnosol and carnosic acid was 82.51 ± 0.52 % and 93.21 ± 0.27 % in respectively The equilibrium adsorption time was determined to be 60 minutes at 30 o C

• Identified the adsorption process of carnosol and carnosic acid by DM301 resin as a monolayer adsorption following the Langmuir equation

• In terms of desorption, the thesis was determined that Ethanol 99.5 % (v/v) was the most suitable solvent for removing target compounds from the surface of DM301 resin, with the desorption time being 60 minutes and the ratio of resin : solvent being 1:70 g/mL Under these conditions, the desorption ratios were 88.02 ± 0.49 % for carnosol, and 83.95 ± 0.01 % for carnosic acid

• The research was investigated the purity of carnosol in static desorption extract and gradient concentration extract, which was 32.99 % and 23.24 %, respectively In terms of the purity of carnosic acid, the figure for gradient concentration extract (70.69 %) was 4.4 times higher than that of static desorption (15.99 %)

• Finally, the concentration of 400 ppm enriched extract (gradient concentration extract) had a higher antioxidant capacity for artificial sebum by about 3.5

73 times than that of RDR extract (enriched extract = 45.66 ± 0.47 mg MDA/kg sample, and RDR extract = 154.90 ± 0.22 mg MDA/kg sample)

Besides the results achieved, a number of recommendations are proposed to improve the report, including:

• The study should investigate the concentration of adsorption solution to get the optimal adsorption capacity

• The research should conduct in vivo test on volunteers to obtain more specific results regarding the ability to inhibit black-head formation

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