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2023년 08월

석사학위논문

보스웰리아 (Boswellia serrata)

추출물의 향장소재 적용을

위한 항산화와 항균활성

조 선 대 학 교 산 업 기 술 창 업 대 학 원

미 용 향 장 학 과

바 스 마

보스웰리아 (Boswellia serrata)

추출물의 향장소재 적용을

위한 항산화와 항균활성

Antioxidant and Antimicrobial Activity of

Boswellia Serrata Extract for Cosmetic

Ingredient

2 0 2 3 년 0 8 월 2 5 일

조 선 대 학 교 산 업 기 술 창 업 대 학 원

미 용 향 장 학 과

바 스 마

보스웰리아 (Boswellia serrata)

추출물의 향장소재 적용을

위한 항산화와 항균활성

지 도 교 수 신 현 재

이 논문을 미용향장학 석사학위신청 논문으로 제출함

2 0 2 3 년 0 4 월

조 선 대 학 교 산 업 기 술 창 업 대 학 원

미 용 향 장 학 과

바 스 마

I

Contents

List of Tables ··································································· Ⅳ

List of Figures ·································································· Ⅴ

ABSTRACT ······································································· Ⅵ

Ⅰ. Introduction ··································································· 1

1.1. Background of the study ······················································ 1

1.1.1. Herbal cosmetics ····································································· 1

1.1.2.

Boswellia Serrata

······························································ 3

1.1.3. Scientific classification ··························································· 5

1.1.4. Economic importance ··························································· 12

1.2. Antioxidant ················································································· 18

1.2.1. Definition..................................................................................18

1.2.2. Free Radicals and Oxidative Stress ...................................19

1.2.3. Skin antioxidant ....................................................................19

1.3. Antimicrobial.................................................................................22

1.3.1. Defense systems of the skin .................................................22

II

1.3.2. Bacterial growth on skin .....................................................23

1.3.3. Protection by colonizing bacteria..........................................23

1.3.4. Microbes that cause skin disease ........................................24

1.3.5. Antibacterial .............................................................................27

1.4. Research Trends and Composition ···································· 28

1.4.1. Research Trends ···································································· 28

1.4.2. Structure of the study ·························································· 30

Ⅱ. Literally review ·························································· 31

2.1. Review of research on

Boswellia serrata

····················· 31

2.1.1. Ingredients and compounds of

Boswellia serrata

······· 31

2.1.2. Bioactive compounds in

Boswellia serrata

················· 36

2.1.3.

Boswellia serrata

in cosmetology ································ 39

2.2. A review of research on

Boswellia serrata

················· 47

Ⅲ. Materials and Methods ·············································· 50

3.1. Experimental Materials and Reagents ······························· 50

3.1.1. Materials ········································································ 50

3.1.2. Reagents ········································································ 50

3.2. Extraction and Isolation ····················································· 51

III

3.2.1. Sample collection ·································································· 51

3.2.2. Extraction ··············································································· 51

3.2.2.1 Soxhlet Extraction ······························································ 51

3.2.2.2 Immersion Extraction ························································· 53

3.2.3. floor separation ····································································· 55

3.3. Antibacterial activity ··························································· 56

3.3.1. Antibacterial agar disc diffusion assay ······························ 56

3.4. Antioxidant activity ......................................................................57

3.4.1. DPPH free radical scavenging assay..................................57

3.4.2. ABTS radical scavenging assay............................................59

3.5. Total polyphenol and flavonoid contents...............................61

3.5.1. Determination of total polyphenol content....................61

3.5.2. Determination of total flavonoid content.........................62

3.6. Analysis of polyphenol componds...................................................63

3.6.1 High performance liquid chromatography (HPLC) analysis63

3.6.2 HPLC-MS/MS Analysis Method.............................................64

Ⅳ. Results and Discussion ············································· 65

4.1. Yield of extract according to Immersion extraction method.......65

4.2. Antibacterial activity using well diffusion method ··················· 68

IV

4.3. Results of antioxidant activity................................................71

4.3.1. DPPH free radical scavenging............................................71

4.3.2. ABTS radical scavenging....................................................73

4.4. Results of total polyphenol and flavonoid contents............75

4.4.1 Total polyphenol contents (TPC).........................................75

4.4.2. Total flavonoid contents (TFC)...........................................76

4.5. Analysis results of polyphenol componds.....................................78

4.5.1 HPLC Analysis Results........................................................78

4.5.2 HPLC-MS/MS Analysis Results...........................................81

Ⅴ. Discussion.....................................................................86

Ⅵ. Conclusion ································································ 89

Ⅶ. Reference ·································································· 90

V

List of Tables

Table 1. Taxonomical Hierarchy of

Boswellia serrata

·················································· 7

Table 2. Vernacular names of

Boswellia serrata

··························································· 8

Table 3. products containing B. Serrata resin extracts in cosmetics..............................15

Table 4. The most common microbial infection...............................................................25

Table 5. Main ingredients and compounds in

Boswellia serrata

··························· 38

Table 6. Chemical composition of

Boswellia serrata

. resin and use as cosmetic

ingredients.............................................................................................................43

Table 7. The yield of each Fractions according to the extracted Boswellia Serrata...67

Table 8. Antibacterial activity of B. Serrata using the disk diffusion method ········· 69

Table 9. Radical scavenging activity and polyphenolic/Flavonoids content of different

Boswellia fractions................................................................................................77

Table 10. Polyphenol compounds identified in Boswellia serrata extracs quantified by

HPLC　(Unit :μg/mg).............................................................................................79

Table 11. Polyphenol compounds identified in Boswellia serrata extract quantified by

HPLC-MS/MS........................................................................................................82

VI

List of Figures

Figure 1. Tree parts of

Boswellia Serrata

................................................................9

Figure 2.

Boswellia Serrata

resin extracted method......................................................10

Figure 3. The four grades of

Boswellia Serrata

............................................................11

Figure 4. products containing

Boswellia Serrata

.resin extracts...................................17

Figure 5. Environmental causes causing the formation of free radicals.......................21

Figure 6. Chemical structures of boswellic acids............................................................35

Figure 7. The illustrated list of cosmetic applications of

Boswellia serrata

..........42

Figure 8. Soxhlet Extraction diagram of

Boswellia Serrata

....................................52

Figure 9. Immersion Extraction diagram of Boswellia serrata ······································ 54

Figure 10. Measurement of the activity of an antioxidant by the DPPH assay...........58

Figure 11. Measurement of the activity of an antioxidant by the ABTS assay...........60

Figure 12. Floor separation diagram of Boswellia Serrata..............................................66

Figure 13. Antibacterial activity of Boswellia Serrata of different concentrations

against M.pachydermatis, M.furfur, S.epidermidis, and C.acnes using the

disk diffusion method.......................................................................................70

VII

Figure 14. DPPH free radical scavenging activity results of Boswellia Serrata

extract.................................................................................................................72

Figure 15. ABTS radical scavenging activity results of Boswellia Serrata

extract.................................................................................................................74

Figure 16. HPLC profile of Boswellia serrata extracts and standard mixture using

diode array detection at 280 nm. (A) Boswellia serrata water extract; (B)

Boswellia serrata 70% EtOH extract; (C) Boswellia serrata EtOAc

extract; (D) standard mixture. Numbers indicate the following: (1) gallic

acid; (2) catechin; (3) (-)-epicatechin; (4) vanillic acid; (5)Narigin;

(6)Ethyl gallate; (7) p-coumaric; (8) ferulic acid; (9) benzoic acid; (10)

quercetin; (11) narigenin; (12) kaempferol; (13) 4-hydroxybenzoic

acid.....................................................................................................................80

Figure 17. Component analysis of Boswellia serrata water extract by HPLC

MS/MS...............................................................................................................83

Figure 18. Component analysis of Boswellia serrata 70% EtOH extract by HPLC

MS/MS...............................................................................................................84

Figure 19. Component analysis of Boswellia serrata EtOAc extract by HPLC

MS/MS...............................................................................................................85

VIII

ABSTRACT

Antioxidant and Antimicrobial Activity of Boswellia Serrata

Extract of Cosmetic Ingredient

Bssmah Ghazi Alraddadi

Advisor: Prof. Hyun-Jae Shin, Ph.D.

Department of Beauty and Cosmetology,

Graduate School of Industrial Technology and Entrepreneurship,

Chosun University

Boswellia serrata resin, which is an important source of gum oleoresin known as

Indian frankincense and is well documented for its pharmaceutical properties due to its

chemical structure, antibacterial and antioxidant properties, and the presence of several

compounds such as polyphenols, phenols, and terpenoids.

In the experimental study, it was observed that Boswellia Serrata resin extracts in-

hibited antibacterial activity in all strains for the relevant concentrations. The diameter

of the zone of inhibition for the B. serrata extract for S. epidermidis ranged from

13.3±0.58 to 10.3±0.58 mm, for M. furfur from10.6±0.58 to 9±0 mm, for M. pachy-

dermatis from13.25±0.35 to 9.75±1.06 mm, and for C. acnes from 11.83±0.29 to 9.5±0

mm in the anaerobic jar.

antioxidant activity was measured with DPPH It was confirmed that there was high-

er activity in the Water Fr. compared to the Other fractions; respectively, Water Fr.

IX

showed the highest scavenging activity 902.19 ± 35.53 µg/mL, Ethyl acetate Fr. 20436

± 652.19 µg/mL ,70% EtOH Fr. 8627.74± 369.22 µg/mL, and the Hexane Fr. Shows

No scavenging activity.

According to ABTS assay, the Water fraction demonstrated the maximum scaveng-

ing activity with a scavenging activity of 1845.08 ± 2265.74, followed by the ethyl

acetate fraction with a scavenging activity of 12167.16 ± 8152.82 µg/mL.

polyphenol and flavonoid content in the Water Fr. was the highest; it reached TPC

32.15 ± 0.75 ⅿg/mL and TFC 20.29 ± 1.47 ⅿg/mL.

This study shows that the Boswellia serrata resin have some biological activities, and

if they differ in terms of effectiveness and activity, they can be considered a good

component of cosmetic products.

1

Ⅰ. Introduction

1.1. Background of the study

1.1.1. Herbal cosmetics

Herbal cosmetics are products manufactured with phytochemicals from various bota-

nical sources that affect skin functions and provide nutrients for healthy skin or hair[1]

Cosmetics are materials that are intended to be rubbed, poured, sprinkled, sprayed, in-

jected into, or otherwise applied to the human body or any component of it for wash-

ing, beautifying, promoting attractiveness, or changing the appearance[2] Herbal cos-

metics are products made with one or more herbal substances legally used to provide

specific cosmetic benefits exclusively and a basis for various cosmetics[3] Active com-

pounds found in medicinal plants are substances produced by the plant's natural metabo-

lism and are crucial in treating many human illnesses, particularly infections brought on

by bacteria[4] Plant antimicrobials contribute significantly to the eradication of infections

caused by pathogenic microbes[5] These active components have multiple functions, in-

cluding improving skin elasticity, preventing collagen degradation, protecting against UV

radiation by antioxidant property and delaying the aging process of the skin by smooth-

ing out wrinkles[6] Individuals' skin and hair beauty are influenced by their health, rou-

tines, daily activities, environment, and upkeep[7] Utilizing a range of herbs and plants,

the science of Ayurveda enabled the development of efficient Ayurvedic cosmetics.

Ayurvedic cosmetics adorn the face and shield the body from external impacts[8] Many

herbal cosmetics are developed and extensively used in daily life. Herbal cosmetics like

facial washes, conditioners, soaps, shampoos, and others are popular with the general

public. Their best quality is that only herbs and shrubs make herbal cosmetics. Plant

cosmetics, often known as ayurvedic, still have the same beneficial properties.

Additionally, instead of harming the body, the natural elements of the herbs nourish it

with nutrients and other advantageous minerals[8] Since the beginning of the practice of

medicine, natural materials with such a plant origin have been used in healthcare. The

evaluation of phytochemicals for pharmaceutical development has been widespread in re-

cent decades. However, only a tiny handful of these plant species have received a thor-

2

ough scientific inspection. Therefore, research into the bioactivities of these plants and

compounds is necessary. Even today, some of these historically used plants and prod-

ucts derived from plants are still important pharmacologically. One such healing herb is

Boswellia serrata

(Burseraceae)[9]

3

1.1.2.

Boswellia serrata

Natural resins have played an essential role since ancient times, as they were consid-

ered among the plants with primary resources for food, flavors, and aromas. They are

considered high-value ingredients for being an important component in the manufacture

of human medicines. These adhesives have also been utilized as coating materials, cos-

metic compounds, fragrances in religious ceremonies and daily rituals, and for their dif-

ferent medicinal properties[10]

Boswellia serrata

resin were used extensively in ancient times by the Hindus,

Babylonians, Persians, Romans, Chinese, Greeks and early Americans for incense and

embalming. They firmly believed that the smoke and aroma produced by these materials

when burned with fire not only helped to lift their spirits but also to appease their

gods. Their collective life revolves largely around the burning of these natural resins.

To prevent evil spirits from affecting their spirits or to commemorate the dead or the

living, they burned these resins during sacrifice ceremonies and as part of their daily

rituals[11] Since ancient times, these species have been utilized for their medicinal and

aromatic qualities. As a result, the ethnobotany of their immediate surroundings fre-

quently emphasizes the importance of their trees and shrubs. The resins of this species

have been used by indigenous communities for various purposes in traditional medicine,

including as an antiseptic and disinfectant, an external agent (cosmetics), and a wound

dressing[12]

Frankincense is a significant oleo-gum resin used in various industries, including the

pharmaceutical, culinary, perfumery, flavoring, liqueur, beverages, cosmetics businesses.

Since ancient times, people have valued frankincense for its ceremonial and sacred pur-

poses, even before Biblical times[13] is one of the most popularly used essential oils in

aromatherapy to treat breathing disorders. It facilitates respiration and benefits people

with asthma. Additionally, it relieves the symptoms of colds, asthma, bronchitis, and

laryngitis[14] used to treat various bacterial and fungal infections[15] utilized to cure

malignant disorders as well based on numerous studies have demonstrated its efficiency

4

in combating human leukemia[16]

Due to its ability to regulate the release of immune cytokines and the presence of

cortisone, which inhibits inflammation and does not have the adverse side effects asso-

ciated with synthetic cortisone, frankincense resin is used in the treatment of a variety

of illnesses as well as to strengthen the heart and brain, and treats forgetfulness, blood

diarrhea, treat arthritis, and other infections[17]

For its fresh, balsamic, dry, resinous, slightly green note, frankincense oil is em-

ployed in perfumery as a fixative and in oriental bases, ambers, flowers, colognes, and

manly scents[14] Due to its sweet scent, it is also used as incense[18]

5

1.1.3. Scientific classification

The family Burseraceae has between 560-600 species spread across 18 genera. This

family (Burseraceae) is where Boswellia serrata resin, also known as olibanum or

frankincense, The genus Boswellia is a little one with roughly 28 species [19] In honor

of Johann Boswell, who identified 25 different Boswellia species, the genus Boswellia

was named after his name[20] The Taxonomical Hierarchy and vernacular names of

Boswellia serrata are given in table1, table2

Humans have used botanical remedies like the Indian frankincense tree (Boswellia

serrata) as remedies since ancient times. It has significantly influenced the treatment of

several diseases. A medium-sized deciduous tree known as Boswellia serrata is mainly

found in Saudi Arabia, Oman, Southern Arabia, Yemen, India, Pakistan, Africa, Nigeria,

Somalia, and Asia.

A medium to a big tree, the

Boswellia serrata

can reach heights of 18 meters and

a width of 2.4 meters. The leaves have opposing leaflets and are imparipinnate and

packed together at the ends of the stems, and are frequently serrated. The tiny, white

flowers on this hermaphrodite plant develop in racemes at the axils. Its thin, green-

ish-gray bark turns yellow or crimson as the plant's petals ripen that are imbricated,

number from three to five, and ultimately take on the color of ash. The bark will

exude an exudate containing oleo gum resin after being hurt or having a natural crack.

The bark secretes tiny droplets of resin and sheds in brilliant crimson, papery, silky

flakes. Golden yellow, clear, aromatic oleo gum resin ultimately turns into crusts, tears,

or drips that are brownish-yellow [19] (Figure1) Frankincense resin is harvested from

the tree by creating an incision or wound in its bark that looks like milk or resin, then

dries outside to appear like olibanum[23] (Figure2) which is then kept in a bamboo

basket constructed especially for storage. The semi-solid gum resin is left in the basket

for about a month while the fluid inside flows out. The gum resin, the semi-solid to

the solid portion of the residue, progressively solidifies to form amorphous, tear-shaped

products with an aromatic scent. It is then manually cleaned of all impurities, such as

bark fragments, during the process of being broken into tiny bits with a wooden mallet

or chopper. After that, the gum-resin is rated based on its flavor, color, form, and

6

size[22] Color, scent, cluster size, tree age, and purity, harvest season, and geographic

position of the plant source are some of the variables that affect the quality and type

of luban (Frankincense resin) is offered commercially in four grades, under the Arabic

names Hoojri, Najdi, Shathari, and Shaabi (Figure3) There are four geographical loca-

tions in Oman, specifically the Dhofar region, where the resins is harvested. Hoojri

Grade I resins, is distinguished by its lighter color and larger mass size than the rest

of the species. This type it is gathered from trees that grow in the north of the

Samhan Mountains. It costs $83 per kilogram "It is the sample used in the present

work". While the Grade II, which is Najdi, is distinguished by its pale yellow color. It

is gathered from a plateau that is hidden by mountains of Dhofar. It costs 67$ per kg.

Shathari, Grade III resin it is darker color collected from northwest Dhofar, costs $31

per kilogram. Finally, Shaabi, Grade IV , which is also darker color and collected

from valleys, costs only $15 per kilogram[14]

7

Kingdom Plantae-Plants

Division Angiospermae

Class Dicotyledoneae

Order Geraniales

Family Burseraceae

Genus Boswellia

Species Serrata

Table 1. Taxonomical Hierarchy of Boswellia serrata [21]

8

English Indian olibanum or Indian frankincense

Arabic Luban or Luban dhakar or Luban mur or luban omani

Hindi Kundur, salai

Bangali Kundur, salai

Gujarati Dhup, Gugali

Kannada Chitta, Guguladhuph

Malayalam Parangi, Saambraani

Tamil Parangi, Saambraani

Telugu Phirangi, Saambraani

Sanskrit Ashvamutri, Kundara, Shallaki

Table 2. Vernacular names of Boswellia serrata [22]

9

Figure 1. Tree parts of

Boswellia Serrata

.

10

Figure 2.

Boswellia Serrata

resin extracted method.

11

Figure 3. The four grades of

Boswellia Serrata

.

12

1.1.4. Economic importance

Arabs, Middle Easterners, Indians and even Africans are interested in the Boswellia

serrata plant, because of its social, religious and economic importance in their

countries.

It is used on a daily basis, especially in the homes of Arabs, because they believe

that it helps to clean the house of bad energy and toxins. However, it helps to im-

prove mood and get rid of anxiety.

Also, based on its social standing, it is given as a very precious gift on special

occasions.

Oman is the country that produces the most Boswellia serrata plant, and frankincense

is called Omani because of the interest of the Arabs in frankincense.

There is a special tourism in the Dhofar region in Oman to buy the finest types of

Omani frankincense, which they call (incense and perfume tourism).

Subsequently, the world's attention became focused on Boswellia serrata, as

many pharmaceutical, cosmetic, and fragrance industries include the Boswellia serrata

plant.

a. medical uses

The resin is one of the most important herb medicines with remarkable efficacy in

many therapeutic fields[24] Asthma, Crohn's disease, osteoarthritis, rheumatoid arthritis

are the most prominent medical conditions that B. Serrata is used to treat. In addition,

it's employed to cure bronchitis and cough[19] Use for skin conditions, corneal ulcers,

osteoarthritis, dysentery, inflammatory conditions that last a long time, wound recovery,

and diarrhea[25] used as a weight-loss aid[26] and is advantageous for liver fibrosis[27]

and several cancers[19]Colitis and ulcerative colitis are both treated and prevented with

Boswellia resin. In the cerebrovascular system, B. Serrata exhibits adequate antioxidant

13

activity[28] used for antipyretic, anti-sclerotic, and analgesic[29] in pain, arthritis, in-

cluding osteoarthritis, allergies, and inflammatory bowel illness[30] It has additionally

been utilized in mouthwashes as an antimicrobial[31] also for the treatment of chronic

inflammatory illnesses[32] or the avoidance of skin and nail diseases[33] also benefits

of antiulcer, antiulcerogenic, antibacterial, and psychopharmacological effects[34]

b. Industrial uses

Cosmetology:

Cosmetic industries have become one of the leading industries in developing countries, as

these industries contribute to the country's economic growth.

International cosmetic companies depend on manufacturing skin, hair, and body care prod-

ucts from natural plant sources. On healthy skin, a look that is free from skin infections

that affect most people.

There are numerous applications for Boswellia serrata resin in cosmetics, including

anti-aging, soothing, and anti-inflammatory properties. It can also encourage quicker skin

regeneration and increase skin elasticity to lighten the skin. Some of the global cosmetic

products includes B. Serrata resin extracts are compiled in a table 3.

Perfumery:

Frankincense is of very high value in the manufacture of scented products due to its

beautiful aromatic scent, as it is an essential ingredient in the manufacture of incense,

perfumes, potpourri, creams, lotions, soaps, and detergents. It is frequently incorporated

into meditation combinations since it bolsters the soul and aids in achieving a deeper

level of meditation and relaxation[35] Because it holds its aroma for so long—some

say indefinitely—frankincense is a favorite ingredient in potpourris. These days, it is

still used for making incense, perfume, and medicines. Additionally, it is a component

of numerous contemporary perfumes[36] It is known to be helpful as a fixative in pot-

14

pourris and perfumes in addition to providing a unique fragrance to any blend. It is

utilized by perfumers in Oriental bases, ambers, powder perfumes, floral perfumes, cit-

rus colognes, spice blends, violet perfumes, manly fragrances, soaps, lotions, and

creams, among other things, as an absolute (by alcohol extraction), oil, or resinoid (by

hydrocarbon extraction)[35]

Food and beverages:

A few businesses that employ frankincense products include the beverages, con-

fectioneries, gelatins, nut goods, puddings, cans of vegetables, candies, chewing gum,

and most of food industry. In addition, they are frequently utilized in rubber products

as releasing agents, adhesive thickeners, stabilizers, flavor enhancers, fixing and emulsi-

fying substances in culinary products. 500 tonnes or so of olibanum are imported into

the Middle East, especially Saudi Arabia, to produce chewing gum[35]

15

Product Name Function

united states of America

Aveda—outer peace™ acne relief pads

Eliminate blackheads and stop fresh

breakouts from occurring.

I image—CLEAR CELL clarifying sal-

icylic tonic

Assist in calming sensitive skin and

refresh and clear congested pores.

Aveda—men pure-formance™ condi-

tioner

Refreshes scalp.

Dermadoctor—Ain’T Misbehavin’

Intensive 10% Sulfur Acne Mask &

Emergency Spot Treatment

Reduces the appearance of blackheads

and spots. Additionally, it aids in skin

healing.

Flexpower—Soothe Lotion Soothes, anti-inflammatory.

Found—Marshmallow Calming Face

Serum

Skin conditioning.

Aveda—Outer Peace™ Foaming

Cleanser

Thorough pore cleaning without caus-

ing skin irritation or excessive drying.

Kate Ryan—Collagen Booster Intense

Repair Serum

Reduces lines and wrinkles.

LANCER—Soothe and Hydrate Serum

Balances skin tone while reducing skin

redness.

Skin Actives—Collagen Serum

Enhances the texture and tone of the

skin.

Kate Somerville—Liquid Exfolikate Soothes skin.

New Vitality Lumatone—Anti-Aging

Eye Cream

Antiaging.

Table 3. products containing B. Serrata resin extracts in cosmetics

16

Product Name Function

Asia countries (South Korea, Japan, Indonesia, Malaysia, Thailand)

Smooth-E—Acne Treatment Hydrogel

Reduce acne inflammation, treat

acne, and kindly calm the skin.

Dermedics—YOUTH EXPERT™ Instant

Relief Eye Serum

Anti-irritant.

Dermedics—MESO CALM Instant

Soothing Elixir

Relieves irritability.

Dermedics—YOUTH EXPERT™

Physiological Micellar Water

Anti-irritant.

NPURE—Day Cream Centella Antiacne.

Mitomo—Hyaluronic Acid +

Lithospermum Facial Essence Mask

Brightening, moisturizing, and

refining.

Yadah—Anti-T Mist Revitalize and smoothens skin.

Europe countries (UK, Italy, Spain, Netherlands)

Bioearth—Siero Idratante Lenitivo Soothes skin.

Cantabria Labs Biretix—Gel Ultra

Purifying

Hydrating and soothing activity.

Dermalex—Rosacea Treatment Relieve different skin conditions.

Biodermal—Couperose Crème Soothes skin.

ITreatSkin—Neem Cream

Soothes and reduces skin

Inflammation.

Table 3. (Continued)

17

Figure 4. products containing

Boswellia Serrata

.resin extracts

18

1.2. Antioxidants

It serves as a protection mechanism to shield the body's cells from harm. The body's

enzymes and some nutrients consumed in everyday food make up antioxidants. Free

radicals lose their capacity to oxidize after they form, become resistant, and change into

another form. A free radical is an atom or molecule with one electron in the outer

orbit. This forces it to seek out the lost electrons from other body compounds, damag-

ing the body's cells by rupturing the barrier surrounding them. It does this by interact-

ing with the phospholipids in cell membranes, which causes damage to everything from

DNA to the collagen layer of the skin.

Antioxidant proteins protect cells from potential damage caused by free radicals.

Accurate identification of proteins and understanding their role in antioxidant activity is

critical in contributing to delayed aging[37]

1.2.1. Definition

Oxidation is a chemical reaction, in an oxidation process hydrogen or a material

transfers its electrons to an oxidizing agent. Oxidation can generate free radicals.

processes. These radicals can spark subsequent chain reactions. A cell may sustain harm

or even perish when the chain reaction takes place inside of it. Additionally, oxidative

stress both causes and results in illness. Protein molecules known as antioxidants stop

these chain reactions by eradicating the free radical intermediates and prevent additional

oxidation processes. Antioxidants are frequently reducing substances like thiols, ascorbic

acid, or polyphenols because they are oxidized themselves in order to accomplish

this[38] The definition can be summarized as that an antioxidant is "Any chemical that

significantly slows down or prevents the oxidation of a substrate when present in con-

centrations below those of the substrate" Both compounds with an enzymatic and

non-enzymatic nature are covered by this classification. Naturally, the diversity of anti-

oxidants must correspond to that of oxidants[38]

19

1.2.2. Free Radicals and Oxidative Stress

Extrinsic damage to the skin arises from various causes: Ionizing radiation, high lev-

els of stress on physical and emotional, alcohol use, poor diet, overeating, environ-

mental pollution, and UV radiation exposure (UVR) (Figure4) when the formation of

ROS in the skin caused by UV exposure surpasses the target cell's antioxidant defense

capacity, oxidative stress results[39] Acute UVR exposure reduces the activity of the

catalase enzyme in the epidermis and elevates protein oxidation[40]

According to estimates, UVR contributes up to 80% of all environmental factors,

making it the primary environmental factor influencing the occurrence of skin cancer

and skin aging[41] UVR causes molecular reactions in the human epidermis primarily

through the photochemical production of ROS, particularly hydroxyl radical, singlet oxy-

gen, superoxide anion, and hydrogen peroxide (H2O2)[42] UVR passes through the

skin, enters the cells, and interacts with DNA to cause the creation of photoproducts

that render DNA inactive. There are two distinct ways that UVR might harm an organ-

ism: (a) the cellular components directly absorb the incident light, creating excited

states that trigger a chemical reaction; and (b) pathways for photosensitization, in which

light is absorbed by endogenous (or exogenous) sensitizers that have been excited to

their triplet states. Two methods exist by which the energized photosensitizers can harm

cells: (a) the production of free radicals through methods for hydrogen abstraction and

electron transport, or (b) the production of singlet oxygen through energy transfer with

oxygen[43]

1.2.3. Skin Antioxidants

Antioxidants serve as a network of defense for the epidermis. They include enzy-

matic antioxidants like glutathione peroxidase, superoxide dismutase, and catalase, as

well as nonenzymatic low-molecular-weight antioxidants such as different forms of vita-

min E, vitamin C, glutathione (GSH), uric acid, and ubiquinol.[44] The outer layer of

the skin, known as the epidermis, contains a higher concentration of antioxidants than

the dermis. Potent antioxidants include ascorbate, carotenoids, and sulphydrils, which are

20

also abundant in the epidermis. The water-soluble antioxidants glutathione, glucose, pyr-

uvate, uric acid, ascorbic acid, and bilirubin are all present in plasma. Ubiquinol-10, ly-

copene, -carotene, lutein, zeaxanthin, and alpha-carotene are lipid-soluble antioxidants

similar to alpha-tocopherol[45] The most noticeable antioxidant in the lipophilic phase is

α-tocophero, while the cytosol contains the greatest concentrations of vitamin C and

GSH. According to molar ratios, hydrophilic non-enzymatic antioxidants like GSH, uric

acid, and L-ascorbic acid appear to be the most abundant antioxidants in human epi-

dermis[46] It was discovered that the stratum corneum (SC) contains both hydrophilic

and lipophilic antioxidants. The SC was discovered to contain GSH, uric acid, and vita-

mins C and E (both αγ and α-tocopherol)[47]

21

Figure 5. Environmental causes causing the formation of free radicals.

22

1.3. Antimicrobial

Threats from bacterial diseases to human health have been on the rise. Numerous an-

ti-infectious agents have been developed to efficiently reduce bacterial contamination

due to advances in biological technology and general hygiene. Antibiotics have been

widely used to combat bacteria and have successfully treated many illnesses. However,

one of the biggest threats to public health continues to be the emergence of drug

resistance from inappropriate antibiotic use.

A substance known as an antibiotic prevents the growth of (bacteriostatic agent) or

kills microorganisms (microbicide)

Antimicrobial drugs can be categorized based on the microbes they are most effective

against. For example, antibiotics are used against bacteria, while antifungals are used

against fungi. They can also be grouped according to how they are put to use. The use

of antimicrobial drugs to treat and prevent infections is referred to as antimicrobial pro-

phylaxis and antimicrobial chemotherapy.

1.3.1. Defense systems of the skin

The epidermis is a barrier to stop the spread and invasion of dangerous germs.

Among the cutaneous antimicrobial defense mechanisms are mechanical rigidity of the

stratum corneum, low moisture content, lysozyme production, acidity (pH 5), and de-

fensin[48] Generally, the skin's surface areas are dry, preventing bacterial growth.

Colonizing bacteria are eliminated via the sloughing of dead keratinocytes. Skin is cool-

er than normal body temperature and slightly acidic; most bacteria survive best at a

neutral pH and temperature of 37°C. If organisms get beyond human cutaneous de-

fenses, the immune system or skin-associated lymphoid tissue (SALT) steps in as the

next line of defense[49]

23

1.3.2. Bacterial growth on skin

Numerous bacteria have a complex habitat on the epidermis. The human epidermis is

initially sterile, but it quickly becomes a host to residing bacteria after birth. The varie-

ty and density of bacteria are influenced by anatomical location, ambient humidity, the

quantity of sebum and sweat produced, the host's hormonal condition, and age[50] In

relation to the host, the bacterial epidermis flora is parasitic, symbiotic, or commensal.

Although changes the type of interaction created is frequently intrinsic to the bacteria,

even if the host's immunological condition is recognized to have a considerable

influence. The ability of germs to stick to skin epithelium and thrive in mostly dry en-

vironments, and acidic environment, and then quickly read here during the usual des-

quamation process leads to persistent colonization[51]

1.3.3. Protection by colonizing bacteria

Commensal bacteria can grow on the skin, which helps to both directly and in-

directly safeguard the host from pathogenic bacteria. The production of bacteriocins, the

creation of toxic byproducts, the formation of minimal potential for reduction-oxidation,

the depletion of vital nutrients, the prevention of the adhesion of rival bacteria, the

Toxins' breakdown and blockage of translocation are just a few examples of the direct

effects. competition between commensal microbes for resources, niches, and receptors.

For instance, Staphylococcus epidermidis binds to keratinocyte receptors and prevents

pathogenic S. aureus from adhering to the skin[52] Commensals have the ability to pro-

duce bacteriocins, which are species-specific antibiotics. As an illustration, the S. aureus

strain 502A produces bacteriocins that prevent the growth of other virulent staph-

ylococcal pathogens[53] Indirectly, microbes can cause the host to produce more inter-

feron, cytokines, and phagocytosis, as well as more antibodies, phagocytosis, and clear-

ance mechanisms. For instance, Propionibacterium acnes releases fatty acids from lipid

decomposition, which acidifies the environment and stops Streptococcus pyogenes from

growing[54]

24

1.3.4. Microbes that cause skin diseases

Skin acts as a protection barrier and is home to numerous colonizing microorganisms.

Generations of dermatologists have claimed that microbes affect the natural course of

various skin conditions based on study and clinical observation. For instance,

Staphylococcus epidermidis is commonly cultivated from healthy skin and may shield

people from pathogenic bacteria[55] The most frequent bacterial skin and skin structure

infections are Scarlet fever, acute paronychia, staphylococcal scalded skin syndrome, cel-

lulitis, erysipelas, folliculitis, furunculosis, carbuncles, wound infections, abscesses, and

cellulitis[56] Bacteria are only one aspect of the relationship between microbes and hu-

man epidermis. Athlete's foot and chickenpox are just two examples of the well-known

human infections caused by fungi and viruses[57] a wide range of additional factors

may influence the microbial communities living on and in the epidermis. External ele-

ments including clothing types, use of lotions/creams, cleansers, deodorants, or anti-

perspirants, regularity of personal hygiene, seasonal temperature, ambient humidity, past

antibiotic use, and other environmental surfaces[58]

Microbes can cause skin infections. A wide variety of microorganisms can cause skin

infections. Most cases are brought on by streptococcus or staphylococcus aureus can be

treated with topical antibacterial. Table 3

25

Infections

Microorganisms Description Ref.

Nonbullous

Impetigo

staphylococcus

aureus

Lesions of nonbullous impetigo

typically begin on skin of the

face or extremities. a small vesicle

or pustule first appears, then

quickly transforms into a hon-

ey-colored, crusted plaque with a

diameter typically less than 2

centimeters. Lesions are typically

accompanied by little to no pain;

Pruritis occurs occasionally, and in

up to 90% of cases, regional ad-

enopathy is discovered.

[59-61]

B u l l o u s

Impetigo

staphylococcus

aureus

Most frequently, skin on the

cheeks, buttocks, trunk, perineum,

and extremities develops flaccid,

transparent bumps. Bullae readily

rupture, leaving a thin scale rim

at the edge of a moist, shallow

erosion.

[60, 62]

Table 4. The most common microbial infection

26

Infections

Microorganisms Description Ref.

Erysipelas

Group A be-

t a - h e m o l y t i c

S t r e p t o c o cc u s

p y o g e n e s

(GABHS)

Small erythematous patch with

well-defined, slightly raised bor-

ders that quickly turn brilliant red,

edematous, indurated, and shiny. It

is most commonly seen on the

central face and legs. The in-

fection spreads quickly, unevenly,

and laterally over a short period,

and it has the potential to worsen

into an infection with the develop-

ment of bullae and severe

necrosis.

[63]

cellulitis

s t r e p t o c o c c i

and

S

.

aureus

Hemorrhagic cellulitis may re-

sult from petechiae and ecchy-

moses, with numerous bullae de-

veloping on inflamed skin. are

rapidly spreading skin diseases

that are more severe than er-

ysipelas and affect the subcuta-

neous tissues. Regional lympha-

denopathy and lymphatic streaking

associated with the condition are

intermittent, and local con-

sequences like necrosis and ab-

scesses are more common than in

erysipelas.

[64]

27

1.3.5. Antibacterial

Antibacterial activity is linked to substances that kill or retard the development of

bacteria locally while not generally being toxic to nearby tissue. The majority of mod-

ern antibiotics are natural substances that have been chemically altered[65] The agents

can commonly fall into two categories: bacteriostatic, which inhibits bacterial develop-

ment, and bactericidal, which kills bacteria. Antibacterial medications are crucial in the

battle against infectious diseases. However, due to their widespread use and abuse, bac-

terial resistance to antibacterial drugs is now a regular occurrence, which is a sig-

nificant issue. Resistance is typically founded on evolutionary processes that happen

during, for example, antibiotic therapy and can be passed down inheritably.

Additionally, tolerance may develop through horizontal gene transfer via conjugation,

transduction, or transformation. [66]

28

1.4. Research Trends and Composition

1.4.1. Research Trends

Due to the tight relationship between human life and plant life as food, bot-

any research and identification are crucial. In addition to using plants as food, people

have linked the diseases they suffer from to the plants that cover the earth's surface

and have used these plants or parts of them as medicines.

Since the discovery of the advantages of plant extracts until now, many developed

nations have turned their attention to the significance of plant extracts, and the pharma-

ceutical and biological industries are flourishing as a result[67] With the discovery of

pure and efficient plant compounds in the nineteenth century due to the development of

chemical sciences and laboratories, the use of natural extracts continued and became a

significant part of molecular sciences. This led to the identification and extraction of

many compounds, such as polyphenols, and the importance of plant compounds that

have medicinal properties.

In America, 25% of medicines are herbal, an environmentally friendly product

free of synthetic chemicals is now widely accepted by people[68]

Additionally, the majority of the plant includes one or more chemicals, either nat-

urally or through active chemicals extracted from it, in small or large concentrations

that can treat one or more specific diseases or lessen their symptoms.

The beauty field is seeing the emergence of a new business in addition to the

pharmaceutical one: "green cosmetics." Since 2007, the market for natural and organic

skin and body care items has increased by an average of 6% annually, reaching over

$50 billion[69]

Since the beginning of time, people have been preoccupied with beauty and out-

ward looks. As a result of numerous bothersome external factors, this leads to the de-

velopment of melasma, wrinkles, hair loss or unwelcome hair growth, and skin diseases.

Male or female, people were eager to find solutions to these issues and to present

themselves in the best and most attractive way possible, so they took care of their

29

skin, its freshness, the length of their hair, its luster, and everything else related to

beauty[70]

Different kinds of cosmetics emerged over time and became a necessity in the

lives of the majority of people as human nature and living circumstances improved.

The types and uses of cosmetics differ, and among the best cosmetics that are

significantly superior to other preparations are those made from only naturally occurring

materials. It has a negative impact on the face, and many allergic individuals have dis-

covered that natural products, whether in the form of oils, creams, or other for-

mulations, work best for these issues.

Natural products play a part in all aspects of the body's health and beauty, not

just in medical treatments. Natural extracts and essential oils can whiten and color-coor-

dinate skin, remove excess hair, lengthen hair and smooth feet, and provide a variety

of other health advantages.

In light of this, natural and organic cosmetics are still in high demand from con-

sumers who have been influenced by recent media and social media posts providing

facts about green cosmetics, who produces them, how they are better for your skin

than conventional cosmetics, and the health risks of traditional colorants and poses.

Health-conscious consumers purchase these products to limit their exposure to poten-

tially harmful chemicals like parabens, and they demand that businesses be transparent

about the ingredients used in the products they use on a daily basis. They also discuss

what is required to create a product that falls into the category of eco-friendly prod-

ucts[71]

Manufacturers and business owners were under much pressure due to all these

customer expectations and demands, so they began spending much money to encourage

scientists and researchers to find natural-source compounds for use in developing new,

marketable products[72]

30

1.4.2. Structure of the study

Boswellia Serrata

resin are one of the primary resin used in this field because their

chemical compounds have beneficial effects on the body, such as their anti-in-

flammatory, anti-bacterial and antioxidant effects. Therefore, this study aimed to find the

best way to extract antioxidants and antimicrobial from

Boswellia Serrata

resin.

Boswellia Serrata

is one of the considered one of the most important frankincense

found in the homes of Arabs, Middle Easterners. Families trust this plant for its safe

and practical uses. Recent studies demonstrated that boswellic acids and

B

.

serrata

ex-

tract have a significant safety margin According to many studies, it has also been re-

ported for antioxidant, antimicrobial activity, which is attributed to compounds with free

radical scavenging activity due to its phenolic content. The

Boswellia Serrata

resin

extract generally contains boswellic acids, Fatty acids, and bioactive components.

Therefore, the experiment was designed to antioxidant and antimicrobial properties

Boswellia Serrata

resin extract and assessment of the ability to root scavenging using

DPPH and ABTS assay and measurement of phenolic components TPC and TFC. be-

side the experiment of HPLC analysis to confirm the polyphenols present in the

extract.

In addition to containing effective antimicrobial materials, a test was carried out

Antibacterial agar disc diffusion assay.

The purpose of this study is to investigate the possibility of using Boswellia Serrata

resin extract as functional ingredients of cosmetics by studying the results of root

scavenging and antibacterial tests according to the extraction method.

31

Ⅱ. Literally review

2.1. Review of research on

Boswellia serrata

2.1.1. Ingredients and compounds of

Boswellia serrata

The polysaccharides in oleo gum-resins (65% arabinose, galactose, and xylose) are

soluble in water. The resins (30–60%) and essential oils (5%–10%) are soluble in or-

ganic solvents. The resinous part of Boswellia serrata possesses monoterpenes, di-

terpenes, triterpenes, tetracyclic triterpenic acids and four major pentacyclic triterpenic

acids i.e. β-boswellic acid, acetyl-β-boswellic acid, 11-keto-β-boswellic acid and ace-

tyl-11-keto-β-boswellic acid, responsible for inhibition of pro-inflammatory enzymes.

One of these four boswellic acids, acetyl-11-keto-boswellic acid, is the most potent in-

flammatory enzyme inhibitor. 5-lipoxygenase[22]

According to a phytochemical analysis using According to thin-layer chromatography,

terpenoids, phenolic compounds, flavonoids, and phenylpropanoids were the major con-

stituents of B. serrata's gum resin[34]

a. Resin \ Gum

Oleo-gum resin analysis revealed: Moisture 10-11%, volatile oils 8-9%, resins

55-57%, gums 20-23%, and insoluble materials 4% - 5%.

The techniques for separating oleo gum resin into its various components and gum

enzymes like diastase and oxidase have also been studied. The gum has 0.16% nitrogen

in it by[73] The gum was hydrolyzed by heating it with 3% H2SO4 recognized the

sugars as arabinose, xylose, and galactose after 8 hours by [74] Comparable to those

made with 5% Acacia mucilage were tablets made with 9% B. serrata mucilage. identi-

fied water soluble protein of gum resin, 4-O-methyl-glucuronoarabinogalactan[25]

Boswellia serrata gum resin extracts are effective at treating several inflammatory dis-

eases, include asthma, rheumatoid arthritis, osteoarthritis, inflammatory bowel disease de-

pending on animal research and preliminary clinical trials[75]

32

b. Oil

A-thujene, a-pinenes, a significant ingredient, and b-phellandrene were discovered in

minute quantities in the low boiling oil fractions. Terpenol, methyl chavicol, and sesqui-

terpenes were the three main constituents that worked out the high boiling fractions in

detail. It has also been claimed that acetyl-b-boswellic acid separation was accomplished

using spectral data and interconversion. The qualities and applications of essential oils,

as well as the methods for separating them from gum and resin.

Due to its diverse sources, oil has a wide range of physicochemical properties.

A-pinene dipentene, phellendrene, cadinene, camphene, p-cymene, d-borneol, verbenone,

and verbenol are some of the components of the oil. According to numerous research,

the principal components of the essential oil include a-thujene (50%) a-pinene (6.2%),

dlimonene (4.5%), p-cymene (14%), cadinene (4%), geraniol (0.8%), and elemol (1.3%).

The main components are the a and b-pinenes and d-emonene. Terpinyl acetate 3.5%,

methyl chavical 2%, linalool 1.5%, and terpinol 1% are present.[25]

c. Boswellic acids

boswellic acids in particular have been found to be active constituents; their chemical

structures are shown in (Figure5)

The content of six boswellic acids [keto boswellic acid (1), 3-O-Acetyl 11-keto β

-boswellic acid (2), α-Boswellic acid (3), β-Boswellic acid (4), 3-O-Acetyl-ɑ-boswellic

acid (5) and 3-O-Acetyl-β-boswellic acid (6)] had been used as the standard index to

appraise the quality of boswellia gum resin and its products[76]

In 11-keto BAs (KBA) and 3-O-acetyl-BAs (AKBA), respectively, a carbonyl and

acetyl groups are present, providing structural variation[77] The main anti-inflammatory

properties of boswellic acids are attributed to suppression of leukotriene formation via

inhibition of 5-lipoxygenase (5-LO) by 11-keto BAs (KBA) and 3-O-acetyl-BAs

(AKBA)[75]

In addition to inhibiting human leukocyte elastase, which is produced in inflammatory

33

and hypersensitive conditions, boswellic acids also have antiphlogistic effects[78]

d. Terpenoids

On the basis of b-boswellic acid, which makes up more than 30% of the total tri-

terpene acids, the estimation of the total triterpene acids present in the various forms of

B. serrata was made. B-boswellic, 11 ketoboswellic, and acetyl 11-keto b-boswellic

acids are all types of triterpene acids. Utilizing functional groups analysis, triterpene

acids were estimated individually or in conjunction with one another. Acetyl and hy-

droxyl groups at position 3 and keto groups at position 11 were the functional groups

examined[25]

e. Fatty acids

The Boswellia serrata tree's bark contains Fatty acids like Myristate, Palmatic, Oleic,

Linolic, Arachidate, Arachidatenate, and Lignocerate. It was discovered using a

GLC-MAS tool, which has proven to be effective against bacteria[79, 80] The GLC

technique was used to identify the fatty acids in the extract of frankincense resin. The

equipment produced GLC-chromatograms and extractor retention times that were com-

pared to standard chemicals. Each fatty acid's concentration in the extract was also

known. The following fatty acids were identified: (Myristate, Palmatic, Oleic, Linolic,

Arachidate, Arachidatenate, and Lignocerate). From highest concentration to lowest con-

centration, the fatty acids identified with the GLC device are as follows: (Palmatic

0.129, Myristate 0.119, Lignocerate 0.114, Lenolic 0.026, Arachidatenate 0.008, Oleic

0.007). Additionally, it displayed each fatty acid's concentration and retention period af-

ter being separated from olibanum. According to the study's by [80] Olic acid was

found to be present in the lowest percentage 0.007 and palmatic acid in the greatest

percentage 0.129 followed by the remaining separated fatty acids. Numerous studies

have established that the Boswellia serrata plant includes a variety of fatty acids, in-

cluding Oleic, Linoleic, Arachidic, Arachidonic, Palmitic, and Lauric acids[17]

34

f. Phenols

The Boswellia serrata tree resin demonstrated that includes phenolic chemicals that

have demonstrated their effectiveness as antibacterials, including thujene, camphene,

b-pinene, myrcene, limonene, and cis-verbenol.by [34]

Previous studies have proven that Due to its anti-inflammatory properties, the frank-

incense resin of the Boswellia serrata plant, which contains free phenols, is used in the

treatment of many human illnesses, particularly reducing the symptoms of arthritis.

Additionally, the Boswellia serrata extract included free phenols in a variety of concen-

trations, and these had a somewhat stronger impact than the fatty acid extract on patho-

genic microbes[17]

35

Figure 6. Chemical structures of boswellic acids.

36

2.1.2. Bioactive compounds in Boswellia serrata

The Boswellia serrata plant contains gum and essential oil. Monoterpenes, diterpenes,

and sesquiterpenes are all present in their essential oil. Essential oils also contain phe-

nolic substances and diterpene alcohol(serratol). The drug's gum component comprises

Pentose and Hexose sugars and certain oxidizing and digesting enzymes. The major

component of the resin is pentacyclic triterpene acid, whose active moiety is boswellic

acid.[81] NMR and mass spectroscopy were used to corroborate the structure of the

fraction produced 3-hydroxy-lup 20(29) ene-24-oic acid following further purification

with EtoAC- Hexane (1:1)[82] B. serrata gum-resin samples from India and Africa that

underwent HPLC analysis produced 12 different pentacyclic triterpene acids[21]

Pinene and cymene were discovered through thin layer chromatography (TLC) exami-

nation of the essential oil from B. serrata leaves using silica gel and spraying reagents

with vanillin-sulphuric acid. Their Rf values were 85 and 33, respectively. While GLC

tests using OV-17 and SE-30 at 69-200°C produced thirteen components, including

d--thujene (32%), p-cymene, and d-limonene as minor constituents in the lower boiling

fraction, and -terpineol, methyl chavicol, and four unidentified compounds in the high

boiling fraction[83] Using a highly sensitive reverse phase HPLC technology, boswellic

acids in Boswellia serrata were found and analyzed was used at 210 and 254 nm with

an acidic mobile phase at 60oC[84]

Boswellia serrata n-hexane extract was steam distilled, and GC-MS analysis of the

essential oil fraction revealed 33 components[21] comprising monoterpenes (9.9%) and

diterpenes (7.1%), esters (62.1%), and alcohol (15.4%). This essential oil was dis-

covered to contain the following compounds: α-thujene, α-pinene, camphene, sabinene,

β-pinene, myrcene, o-methylanisole, αterpinene, hexyl acetate, p-cymene, 1-8-cineole, li-

monene, cis-β-ocimene, trans-β-ocimene, γ-terpinene, 1-octanol, terpinolene, linalool,

1-decanol, terpinen-4-ol, α-terpineol, 1- octylacetate, bornyl acetate, citronellyl acetate,

neryl acetate, geranyl acetate, hexyl hexanoate, 1-decyl acetate, hexyl octanoate, iso-

cembrene, cembrene, iso-incensole and incensole[85] By employing column chromatog-

raphy with silica gel-G with n-hexane and ethyl acetat, the tetracyclic triterpene acids

E, F, G, and H from resin of Boswellia serrata were obtained[21]

37

Boswellia serrata oleoresin was successfully adapted for determine using solid phase

microextraction and gas chromatography/mass spectrometry the volatility and polarity of

terpenoids. As a consequence, cembrane and incensole were captured as unique

diterpenes. Additionally, 50 monoterpenes were obtained by gas chromatography at 40°C

using poly-dimethylsiloxane/divinylbenzene fiber, with roughly 15 of them having a

yield of more than 1%. after trimethylation yielded 15 triterpenes i.e. α-boswellic acid,

β-boswellic acid, 3-acetyl-αboswellic acid, 3-acetyl-β-boswellic acid, α-amyrin, β-amyrin,

3-epi-αamyrin, 3-epi-β-amyrin, lupeol, 3-epi-lupeol, αamyrenone, β amyrenone, lupenone,

3α-hydroxy lup20(29)en-24 oic acid and 3-O-acetyl hydroxy lup-20(29) en-24-oic acid

on GC-MS studies. In addition, three distinctive degradation products were discovered:

24-norroleana-3,12-diene (a), 24-norursa-3,12-diene (b), and 24-norlupa-3,20(29)-diene

(c)[21] The key characteristics and chemical composition of B.serrata extracts are out-

lined in the table 3.

38

ingredients and compounds in

Boswellia serrata

Ref.

Galacturonic acid, glucose, galactose, fructose, sitosterol, phe-

nol-o-cresol, m-cresol, p-cresol, thymol, and carvacrol are all

sugars.The acid campholenic Campholytic acid and 2,2,4-trimethylcy-

clopent-3-en-1-yl acetic acid

[86]

percentage (97.3%) rich in limonene and e-ocimene. Sesquiterpene

content in E-caryophyllene is (2.7%).

[87]

Resin acids comprise between 60% to 70% of the mixture, together

with water-soluble gum (20%) and monoterpene essential oil

(3%–10%).

[88]

oil(45%), α-thujene (12%), α-pinene (8%), sabinene (2.2%),

β-pinene (0.7%), myrcene (3.8%), α-phellandrene (1%), pcymene

(1%), limonene (1.9%), linalool (0.9%), perillene (0.5%),

methylchavicol (11.6%), methyleugenol (2.1%), germacrene D (2.0%),

kessane (0.9%), cembrene A (0.5%) and cembrenol (1.9%), a

monoterpene 5,5-dimethyl-1-vinylbicyclo- hexane (2%) and

m-camphorene (0.7%) and p-camphorene (0.3%)

[89]

Diterpenes, incensole, incensole acetate, and cembrenol (serratol)

[90, 91]

Lupeolic acids, oleanane(α-boswellic acids), ursane-(β-boswellic

acids) , and lupane-type lipophilic pentacyclic triterpene acids, and an

ether-insoluble fraction polysaccharides (arabinose, galactose, and xy-

lose) soluble in water

[19, 92]

Table 5. Main ingredients and compounds in

Boswellia serrata

.

39

2.1.3.

Boswellia serrata

in cosmetology

Plants have been used in cosmetics since antiquity, and modern scientific research

continues to center on this topic. It is now possible to draw even more intricate images

because of advances in our knowledge of how plants and skin change over time. Plants

are intelligent beings that respond to their environment by creating different metabolites.

Applying phytomolecules to the skin impacts the skin's health and appearance through

interacting with skin cells. Numerous plants with the potential to enhance modern cos-

metic products have been identified through both physico-chemical research and ethno-

botanical studies[93]

Plants offer precious active substances for both therapeutic and aesthetic uses.

Humans have utilized products meant to improve skin issues and appearance for hun-

dreds of years, and these products have evolved into modern cosmetics. Our skin is a

physical barrier between ourselves and the outer world and protects us from danger.

Plants have been proven to create compounds that help soothe and protect the skin. In

addition, Modern cosmetics can adjust the skin's elastic qualities along with hydrating

the skin and minimizing redness[94]

The major fibers that make up the skin's extracellular matrix are collagen and elastin;

the former is in charge of tensile strength, and the latter is in charge of elasticity. The

aging process is accompanied by a decline in collagen and elastin production and

strength, which results in wrinkles[95] Additionally, the creation of the enzymes colla-

genase and elastase, which break down collagen and elastin is additionally responsible

for the intrinsic aging of the skin[96]

Several things can cause damage to the outer skin, including intense UV radiation

exposure, a poor diet, and physical and mental stress. Oxidative stress, which produces

free radicals, is brought on by the production of reactive oxygen species by the impact

of UV rays on the skin[41], And research has effectively demonstrated a clear link be-

tween free radicals and the onset of premature aging[97]

According to estimates, UV rays account for up to 80% of all environmental influen-

ces, making them the most significant environmental component in the development of

40

skin cancer and skin aging[41]

Some of the applied antioxidants have the ability to preventively block the negative

effects of free radicals, resulting in normal generation of the structural proteins of the

skin[97] Vitamins and antioxidants applied topically in cosmetics are thought to enhance

protection and maybe even undo harm by neutralizing free radicals[98]

Since the skin serves as the body's external barrier against the environment, it is at

the forefront of the fight against external influences to destroy free radicals. Free radi-

cals are unpaired electron compounds that are very reactive that cause damage to the

surrounding molecules and tissues. It is known that ultraviolet light and environmental

pollutants are among the initiators to cause free radicals[99]

Several studies conducted on boswellia serrata material reported the presence of

many compounds such as polyphenols and terpenoids, These terpenoids, which are

thought to be the most potent components of Boswellia resin, are mostly represented by

boswellic acids[100] The researchers discussed polyphenols' antioxidant properties.

Terpenoids have been shown to have antioxidant capacity, and research has shown that

they are highly active molecules that can help slow skin aging[101]

Since ancient times, the Arabs and Indians have used Boswellia serrata and consid-

ered it an essential plant in their culture for its cosmetic properties and countless bene-

fits for skin and hair health.

Due to its antioxidant, antibacterial and anti-inflammatory properties, it helps get rid

of the most common skin and hair problems.

It works on common skin problems like acne, blemish-prone skin, aging skin, dry

skin, etc., and is suitable for all skin types. Frankincense essential oil's anti-in-

flammatory and antibacterial properties make it beneficial for acne-prone skin. It offers

sebum for aged skin and soothes greasy, acne-prone skin. Additionally, it serves as a

natural tone, enhances skin tone, and hides pores. It is a strong astringent and works

wonders on the skin's wrinkles, fine lines, scars, and stretch marks. Additionally, frank-

incense essential oil encourages the production of new skin cells, keeps the skin supple,

and calms dry, chapped skin[102]

41

It helps to even out the skin tone by preventing or reducing the occurrence of age

spots, sun spots, and other spots. Additionally, it lessens skin inflammation and redness

and helps obtain a homogeneous skin tone. It is also used as a treatment for bruises

and sore sores. It is a powerful anti-wrinkle and anti-aging agent that treats various

skin conditions, including psoriasis and eczema, and helps reduce hair loss, also for

suppression of skin and nail infections[19] Moreover, Boswellia serrata extracts have

been shown to reduce redness and irritation of the skin, act as a soothing agent for

sensitive skin and prevent the appearance of red dots after hair removal, and help even

skin tone[103](Figure6)

42

Figure 7. The illustrated list of cosmetic applications of

Boswellia serrata

.

43

Compound name Chemical structure

Proof of efficacy as an ingredient

in cosmetics

Ref.

Gallic acid

Gallic acid's ability to func-

tion as a cosmetic ingredient is

complicated by its heat

instability. Gallic acid was cou-

pled with a peptide to combat

this problem. It was possible to

produce galloyl-RGD, a promis-

ing candidate for the cosmetic

ingredient.

[104

, 105]

Vanillic acid

Vanillic acid helps the skin

of humans become lighter and

less pigmented. Since its skin

penetration has been established

and the toxicity test has been

successful, its use on the skin

has expanded.

[106

, 107]

Protocatechuic acid

Protocatechuic acid has the

capacity to treat skin aging in 8

weeks, according to research re-

sults on human skin.

108,

109]

Table 6. Chemical composition of

Boswellia serrata

. resin and use as cosmetic in-

gredients

44

Compound name

Chemical structure

Proof of efficacy as an

ingredient in cosmetics

Ref.

Syringic acid

Syringic acid slows down the

aging process of cells by

blocking ultraviolet B. Syringic

acid's effects as an antioxidant

and anti-aging agent con-

sequently increased the survival

rate of cells damaged by ultra-

violet B, suggesting that it can

be used as a natural phy-

tochemical in cosmetics.

[110,

111]

Cinnamic acid

Derivatives of cinnamic acid

are frequently utilized as UV

protection, antioxidant, and anti-

bacterial agents in cosmetic

goods.

[112,

113]

Caffeic Acid

Caffeic acids attracted much

attention because they are

promising and unaffected by

free radical toxicity. Due to its

antioxidant properties, it is

found in cosmetic goods.

[114,

115]

Table 6. (Continued)

45

Compound name

Chemical structure

Proof of efficacy as an

ingredient in cosmetics

Ref.

Ferulic acid

Ferulic acid is a protein in-

hibitor that catalyzes the gen-

eration of free radicals and en-

hances the action of scavenger

proteins. The primary skin

structures are protected by

ferulic acid.

In addition to enhancing an-

giogenesis and promoting

wound healing, it inhibits

melanogenesis. It is typically

used to fight photoaging as a

skin-brightening component in

skincare products.

[116,

117]

Sinapic acid

Sinapic acid protects the skin

cell from complete collagen

degradation by preventing UVB

activation, reducing the in vivo

effects of photoaging, and re-

ducing skin tissue inflammation.

[118,

119]

Table 6. (Continued)

46

Compound name

Chemical structure

Proof of efficacy as an ingredient

in cosmetics

Ref.

Pelargonic acid

A fatty acid called pelargonic

acid can serve as a surfac-

tant-cleansing agent as well as a

fragrance ingredient and emulsi-

fier in cosmetic products. This

chemical is safe to use in cos-

metic products to improve skin

penetration, according to the

Cosmetic chemical Review

(CIR) Expert Panel's earlier

ruling.

[120

, 121]

p-Hydroxybenzoic

acid

p-hydroxybenzoic acid is uti-

lized in cosmetics to prevent the

growth of microorganisms and

extend the shelf life of cosmetic

and personal care goods.

[122

, 123]

Catechin

By crosslinking, catechin can

improve collagen arrangement.

Studies show that catechin mol-

ecules attach to collagen fibers.

[112

, 124]

Table 6. (Continued)

47

2.2. A review of research on

Boswellia Serrata

is an excellent plant, serving as medicine and prevention. Due to

this, many researchers in the health sciences have focused their attention on figuring

out the most effective ways to use this plant to both prevent and treat a variety of

ailments.

Boswellia serrata

is a plant with significant medicinal and cosmetic value.

Numerous studies, trials, and applications have demonstrated promising therapeutic

effects.

a. Toxicity and safety

Boswellia serrata

is considered one of the most important frankincense found in the

homes of Arabs, Middle Easterners. Families trust this plant for its safe and practical

uses. Recent studies demonstrated that boswellic acids and B. serrata extract have a

significant safety margin.

Boswellia gum resin, which has been used as a medicine for thousands of years, is

thought to be secure because it hasn't caused any serious side effects[125] Boswellia

has remarkably low toxicity, and unlike many chemical anti-inflammatory drugs, its an-

ti-inflammatory effects have no negative impacts on blood pressure, pulse rate, respira-

tion, or other autonomic responses[126] Boswellia gum resin has been authorized for

use as a food additive by the US Food and Drug Administration (USFDA), and it is

included on its list of safe substances[127] The markets are sell over-the-counter an-

ti-inflammatory products that are oral preparations of B. serrata extract containing

AKBA[127] In previous studies, it was observed that no deaths were noted following a

single dosage of B. serrata extract administered to mice at a dose of up to 5 g/kg[128]

Mice were administered the extract orally for 28 days in a row without showing any

behavioral toxicity, and their hepatic and renal function biomarkers did not significantly

change as a result[129] Boswellia extract was used in animal toxicology tests, and the

findings showed that it was safe to use in herbal remedies[76, 130]

48

b. anti-inflammatory

Boswellia serrata has been shown to be a strong anti-inflammatory medication in

both clinical investigations and in-vivo animal models. Boswellic acid inhibits leuko-

triene production in a dose-dependent manner by acting as a selective, non-redox in-

hibitor of 5-lipoxygenase. Additionally, it has been shown to reduce levels of leuko-

triene B4 and 5-hydroxyeicosatetraenoic acid, two active chemotactic agents responsible

for increased vascular permeability and pro-inflammatory 5-lipoxygenase products. As a

result, less white blood cells are drawn to the area of inflammation, which dampens the

inflammatory response and promotes speedier healing when boswellic acid is used as a

treatment.

Boswellic acid reduces primary antibody production, polymorphonuclear leukocyte in-

filtration, and migration and nearly completely inhibits the classical complement system.

When boswellic acid was studied in vitro for its impact on the complement system,

both the traditional and alternative pathways were significantly inhibited[131]

c. Hypoglycemic

A herbal supplement by modulating hepatic gluconeogenesis, pyruvate carboxylase,

and phosphoenol pyruvate carboxykinase, it has been shown to have considerable an-

ti-diabetic activity on non-insulin dependent diabetes mellitus in streptozocin-induced di-

abetic rats[21]

d. Analgesic and Psychopharmacological activity

Boswellia serrata's non-phenolic fraction exhibits sedative and analgesic properties.

Additionally, the potentiated enhanced secobarbitone-induced hypnosis in rats, with no

discernible effects on the conditioned avoidance response[21]

49

e. Muscle Relaxant activity

It was revealed that the essential oil of B. serrata has stimulatory effects on skeletal

muscles and spasmogenic effects on the smooth muscle of the guinea pig ileum.

According to a prior study, the B. serrata essential oil operates directly on biological

tissues and is not activated by non-specific cell membrane action[21]

f. Anti-Alzheimer’s activity

Alzheimer's disease (AD) is a chronic neurological illness. A common and early

symptom of AD is increased oxidative stress. Antioxidant-active medicinal plants have

long been used to treat various human illnesses. Boswellia may be able to treat

AlCl3-induced Alzheimer's by increasing Ach levels and lowering AchE activity in

brain homogenates, according to the study. A time-dependent improvement in de-

mentia-type AD caused by i.c.v. Streptozotocin injection has also been found to be pos-

sible with frankincense[132]

g. Antidepressant activity

The plant's extract is used in a variety of tea blends and as aromatherapy. According

to reports, B. serrata is effective in treating acute depression. Boswellia exhibits strong

antidepressant efficacy in acute stress trials and decreases the immobility time in the

experimental forced swim model at a dosage of 100 mg/kg. It was discovered that B.

serrata, a traditionally significant medicinal plant, acts as a bacteriostatic agent[132]

50

Ⅲ. Materials and Methods

3.1. Experimental Materials and Reagents

3.1.1. Materials

The devices used are a sensitive scale, Autoclave, inoculated Petri dishes, LM2 mill.

filter paper (Whatman No. 1), Rotarapor, electromagnetic, vibrating heater, Elisa reader,

freeze dryer, different glassware (cups, conical flasks, standard flasks of 500ml ca-

pacity), funnels, graduated condensers, glass stem, condenser, thermometer) and distilled

water.

3.1.2. Reagents

Reagents used for extraction in this study were ethanol (DUKSAN), methanol

(DUKSAN), and ethyl acetate (DUKSAN). For various physiological activity tests,

1,1-Diphenyl-2-picrylhydrazyl (DPPH, SIGMA), L -ascorbic acid (SIGMA),

2,2'-Azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS, SIGMA), folin-ciocalteus

phenol regent (SIGMA), potassium persulfate (SIGMA), KCl (DUKSAN) , NaCl

(DUKSAN), NaCO3 (DUKSAN), Na2HPO4 (DUKSAN), KH2PO4 (DUKSAN), gllic

acid (SIGMA), aluminum chloride (SIGMA), potassium acetate (SIGMA), quercetin

(SIGMA), kaempferol (SIGMA), isorhamnetin (SIGMA), quercetin dihydrate (WAKO),

6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (trolox, SIGMA), methanol

(THERMO FISHER SCIENTIFIC), phosphoric acid (DUKSAN) and ethanol (THERMO

FISHER SCIENTIFIC) were used. All reagents used for HPLC analysis were HPLC

grade reagents.

51

3.2. Extraction and Isolation

3.2.1. Sample collection

The oleo-gum-resin of

B

.

serrata

used was purchased from the Murshed Market in

Dubai, United Arab of Emirates. 500g of resin were collected and shipment to

Gwangju Korea Chosun University.

3.2.2. Extractration

3.2.2.1 Soxhlet Extraction

The Soxhlet Extraction was measured by modifying the method of S. Mishra[132] A

thimble containing around 5mg of the material was filled before being placed on the

Soxhlet extractor. Methanol (100mL) was then added to a distillation flask. The ex-

traction process was done for 6 h. After completing the Soxhlet extraction process, the

extract has been set in the concentration device Rotary Evaporator (Eyela N-1100,

Shanghai Eyela, China). When the extract dries, should add 40ml of methanol and put

it in an Ultrasonic device to combine the extract. Then the extract was divided into

several tubes, provided that the content of one tube did not exceed 20ml, and dried by

a Centra-Vac (VS-802, Vision, Korea) For up to a day. For the last step, place the ex-

tract in Freeze Dryer for a whole day. Calculation of Yield Extract B. Serrata 2.72g =

54.4% yielded.

52

Figure 8. Soxhlet Extraction diagram of

Boswellia Serrata

.

53

3.2.2.2 Immersion Extraction (70% EtOH)

200g of the sample (powder) was placed in the Ethanol 1400ml / Water 600ml was

add to the immersion container. 1 week immersion in mix in room temperature. After

completing the immersion process, The extract is filter with paper (Whatman No.

2)After filtering the extract has been set in the vacuum device and a (rotary vacuum

concentrator) When the extract dries, should add 90ml of Ethanol \ 10ml of Water and

put it in an Ultrasonic device to combine the extract. Then the extract was divided into

several tubes, provided that the content of one tube did not exceed 20ml, and dried by

a Centra-Vac (VS-802, Vision, Korea) For up to a day. Calculation of Yield Extract

B. Serrata 59.717g = 29.86% yielded

54

Figure 9. Immersion Extraction diagram of

Boswellia Serrata

.

55

3.2.3. floor separation

Place 300 mL of the solution (

B

.

serrata

70% EtOH 29.36g solvent with 100%

MeOH) in a clean separatory funnel and extract with 600 mL of Hexane x3 times then

300 mL of Water with 600 mL of Ethyl acetate x3 times

The separatory funnel should be stopped, Gently shake the funnel and flip it over.

Open the stopcock while the funnel is in this position to relieve the internal pressure.

Releasing the internal pressure once more after closing the stopcock and giving the fun-

nel a brisk shake This process should be repeated four or five times. Remove the stop-

per, place the separatory funnel upright in the support ring, and allow it to stand still.

Collect the lower aqueous layer of the liquids after they have separated, being cautious

not to let any of the top layer leak through the tap. Run the top layer through the

separatory funnel's tap and into a different conical beaker.

56

3.3. Antibacterial activity

3.3.1. Antibacterial agar disc diffusion assay

The Antibacterial agar disc diffusion assay was measured by modifying the method

of B. Chand [133] The in vitro antibacterial activity of the

B

.

Serrata

total crude ex-

tract and fractions was evaluated via the disc diffusion method with

S

.

epidermidis

,

M

.

furfur

,

M

.

pachydermatis

and the Anaerobic jar

C

.

acnes

using Tyrptic Soy

Agar(TSA), Modified Leeming Notman Agar(MLNA), Sabouraud Dextrose Agar(SDA),

and Reinforced Clostridial Agar(RCA) with determination of inhibition zones diameter

measured in millimeter (mm). Sterile filter paper discs (8 mm) were impregnated with

Concentration 3mg, 4mg, 5mg of

B

.

Serrata

extract and then placed on inoculated

Petri dishes containing bacterial suspension. Ampicillin disc (10mg) were used as pos-

itive control whereas discs without samples (10% DMSO (dimethyl sulfoxide) acted as

negative control. The zones of inhibition including the diameter of the extract impreg-

nated dics were compared with those of the controls after incubation.

S

.

epidermidis

at 37°C for 24h,

M

.

furfu

r at 30~37°C for 3~days,

M

.

pachydermatis

at 30~37°C

for 2~3days and the Anaerobic jar

C

.

acnes

at 37°C for 3days The inhibition zone di-

ameter (IZD) was used as criteria for the definition of active or inactive sponge

extracts. The tests were carried out in triplicate for each extract.

57

3.4. Antioxidant activity

3.4.1. DPPH free radical scavenging assay

The DPPH radical scavenging ability was measured by modifying the method of HP.

Singh [134] Mix 500 μL of 0.123 mM DPPH reagent with 500 μL of each Fraction

and each concentration of Boswellia serrata resin extract solution (500:500 DPPH)

Similarly, each concentration of standard Gallic acid 200 μL : 800 μL was treated with

DPPH reagent 0.5 μL to use as positive control, and after reacting in the dark for 15

minutes, Biotek Absorbance, was measured at 517 nm wavelength using Synergy HT

multi-detection microplate reader equipment. A comparative experiment was conducted

using gallic acid as a positive control group. The radical scavenging ability was

calculated by the following equation and expressed as a percentage. Each reaction was

measured in triplicate

% of scavenging activity = (A control – A sample) / A control × 100

58

Figure 10. Measurement of the activity of an antioxidant by the DPPH assay.

59

3.4.2. ABTS radical scavenging assay

ABTS radical scavenging ability was measured by modifying the method of Gupta

[135] Prepare 2,2-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt

(ABTS, 14 mM) at a concentration of 7 mM and potassium persulfate (4.9 mM) at a

concentration of 2.45 mM to 1: After mixing in a ratio of 1. This solution was diluted

with phosphate-buffered saline (0.1 M, pH 7.4), value at 730 After mixing 1,000 μL of

ABTS solution with 200 μL of extracts of Boswellia Serrata resin extracts for each

Fraction and each concentration and reacting for 30 minutes, absorbance was measured

at 730 nm wavelength using Biotek Synergy HT multi-detection microplate reader

equipment. A comparative experiment was performed using quercetin as a positive

control. The radical scavenging ability was calculated by the following formula and ex-

pressed as a percentage. Each reaction was measured in triplicate.% of scavenging ac-

tivity = (A control – A sample) / A control × 100.

60

Figure 11. Measurement of the activity of an antioxidant by the ABTS assay.

61

3.5. Total polyphenol and flavonoid contents

3.5.1. Determination of total polyphenol content

Total polyphenol content was measured by modifying method of MA Ayub [34]

500 µL of 0.2 M Folin-Ciocalteu's phenol reagent and 500 µL of 2% sodium carbonate

aqueous solution (w/v) were mixed with 500 µL of extract solution of Boswellia

Serrata resin for each Fraction and each concentration and reacted for 40 minutes.

Absorbance was measured at 750 nm wavelength using Biotek Synergy HT

multi-detection microplate reader equipment. The final concentration of the extract was

10 ⅿg/mL, and the total polyphenol content was expressed as gallic acid (GAE) and

P-coumaric Acid mg/g equivalent based on the calibration curve.

62

3.5.2. Determination of total flavonoid content

Total flavonoid content was measured by modifying method of MA Ayub [34] After

adding methanol 750 µL, potassium acetate 50 µL, aluminum chloride 50 µL, and etha-

nol 1.4 mL to 250 µL of extract solution of Boswellia Serrata for each Fraction and

each concentration, react at room temperature for 40 minutes.

Then Biotek Synergy HT Absorbance was measured at a wavelength of 415 nm using

a multi-detection microplate reader. The final concentration of the extract was 500

ⅿg/mL, and the total flavonoid content was expressed as quercetin (QUE) and Rutin

mg/g equivalent based on a calibration curve.

63

3.6. Analysis of polyphenol componds

3.6.1 High performance liquid chromatography (HPLC) analysis

The polyphenols contained in Boswellia serrata extracts were quantitatively analyzed

by HPLC (SPD-20A, SHIMADZUCO., Japan). Gallic acid, catechin, (-)-epicatechin, val-

lic acid, narigin, ethyl gallate, p-coumaric acid, ferulic acid, benzoic acid, quercetin,

narigenin, kaempferol, and 4-hydroxybenzoic acid were used as polyphenol standards.

Standards were tested at a concentration of 100 μg/mL, and extracts were prepared at a

concentration of 1,000 μg/mL and filtered through a 0.45 μm syringe filter. The column

was analyzed using a Shimpack GIS-ODS (C18, 4.6 × 250 mm, 5.0 μm, Shimadzu

Co., Japan) with a flow rate of 1.0 mL/min and an injection volume of 10 μL. The

mobile phases were water (A, in 0.1% phosphoric acid) and acetonitrile (B). The gra-

dient conditions of the mobile phase were 0-10 min: B (8-35%), 10-15 min: B

(35-35%), 15-25 min: B (35-65%), 25-30 min: B (65-65%), 30-35 min: B (65-100%),

35-40 min: B (100-100%) based on the mobile phase (B), and the wavelength was

measured at 280 nm.

64

3.6.2 HPLC-MS/MS Analysis Method

In order to identify the active ingredients of goldenrod licorice extract, 24 poly-

phenols were analyzed by HPLC-MS/MS (AB SCIEX 4000 Q Trap LC/MS/MS System,

Shimadzu LC 20A System). A 10 μL aliquot of the sample was analyzed using a C18

column (Gemini 3 μm, C18 110A 50 mm*2.0 mm) at 40°C in a column oven and

15°C in an autosmpler. Anionic and cationic modes were analyzed using Turbo Ion

Spray, and water (0.1% formic acid) (A) and acetonitrile (0.1% formic acid) (B) were

used as mobile phases. The anionic mode was based on the mobile phase (B):

0-0.5min: B (20-20%), 0.5-2min: B (20-80%), 2-2.5min: B (80-80%), 2.5-2.6min: B

(80-20%), 2. 6-6min : B (20-20%) and 0-3min : B (30-30%), and the cationic mode

was analyzed as 0-0.2min : B (30-60%), 0.2-2min : B (60-60%), 2-2.1min : B

(60-30%), and 2.1-4min : B (30-30%).

65

Ⅲ. Results and Discussion

4.1. Yield of extract according to Immerion extraction method

The extraction of natural products also shows differences in the extraction yield

because the components differ according to the solvent Using Floor separation method.

Extraction yield was obtained by the formula









 





 

















    







× 

The Table summarizes the yield of each Fraction. according to the extracted

Boswellia Serrata, confirmed that the content of the Hexane Fr. was higher than other

Fractions.

66

Figure 12. floor separation diagram of

Boswellia Serrata

.

67

Table 7. The yield of each Fractions according to the extracted Boswellia Serrata

68

4.2. Antibacterial activity using well diffusion method

The results of the antibacterial investigations using the well diffusion method are giv-

en in Table 5. It indicates that different bacterial species demonstrated different levels

of sensitivities towards the tested samples of

B

.

Serrata

extract. The zone of inhibition

of the

B

.

Serrata

extract in all strains was Antibacterial activity for the corresponding

concentrations. The diameter for zone of inhibition for

B

.

Serrata

extract for

S

.

epi-

dermidis

ranged from 13.3±0.58 to 10.3±0.58 mm,

M

.

furfur

ranged from 10.6±0.58

to 9±0 mm,

M

.

pachydermatis

ranged from 13.25±0.35 to 9.75±1.06 mm, and the

Anaerobic jar

C

.

acnes

ranged from 11.83±0.29 to 9.5±0 mm.

In agar well method, the zone of inhibition was larger for all bacteria found to be

sensitive than the disk diffusion method.

69

Table 8. Antibacterial activity of B. Serrata using the disk diffusion method

70

Figure 13. Antibacterial activity of Boswellia Serrata of different concentrations against

M.pachydermatis, M.furfur, S.epidermidis, and C.acnes using the disk diffusion method.

71

4.3. Results of antioxidant activity

4.3.1. DPPH free radical scavenging

Measuring the radical scavenging activity of DPPH is a widely used experimental

method because the antioxidant activity of natural products can easily be measured with

DPPH, a relatively stable free radical, and is closely related to the actual antioxidant

activity. In this experiment, extracts of Boswellia Serrata were prepared at a concen-

tration for each Fraction 10 ⅿg/mL – 100 ⅿg/mL 70% EtOH Fr., 5 ⅿg/mL- 30 ⅿ

g/mL EtOAC Fr., 5 ⅿg/mL – 25 ⅿg/mL Water Fr. to confirm the radical scavenging

capacity of DPPH and measured IC50 values. The results are shown in Table 9, As a

result of the measurement for each Fractions, it was confirmed that there was higher

activity in the Water Fr. compared to the Other fractions, respectively, the Water Fr.

showed the highest scavenging activity 902.19 ± 35.53 µg/mL, Ethyl acetate Fr. 20436

± 652.19 µg/mL ,70% EtOH Fr. 8627.74± 369.22 µg/mL, and the Hexane Fr. Shows

No scavenging activity.

72

Figure 14. DPPH free radical scavenging activity results of Boswellia Serrata extract

73

4.3.2. ABTS radical scavenging

The root scavenging capacity of ABTS was confirmed by preparing Boswellia Serrata

extract at a concentration for each Fraction 5 ⅿg/mL – 30 ⅿg/mL 70% EtOH Fr., 5

ⅿg/mL- 30 ⅿg/mL EtOAC Fr., 10 ⅿg/mL – 75 ⅿg/mL Water Fr. and IC50 values

were measured. The results are shown in Table 9 As a result of the respectively,

which indicated the highest scavenging activity in the Water Fr. it showed a scavenging

activity 1845.08 ± 2265.74 µg/mL, Ethyl acetate Fr. 12167.16 ± 8152.82 µg/mL, 70%

ethanol Fr.13796.44 ± 660.7 µg/mL, the Hexane Fr. Shows No scavenging activity.

74

Figure 15. ABTS radical scavenging activity results of Boswellia Serrata extract.

75

4.4. Results of total polyphenol and flavonoid contents

4.4.1 Total polyphenol contents (TPC)

Polyphenol compounds are one of the secondary metabolites widely distributed in the

plant kingdom and have different structures and molecular weights. Moreover, they bind

easily to proteins and other large molecules due to the phenol hydroxyl group (OH)

and are usually used as antioxidants and anticancer agents. Therefore, in this study, the

total polyphenol content of Boswellia Serrata extract was measured, and the results are

shown in Table 9. As a result of the measurement for each Fractions, it confirmed that

the polyphenol content in the Water Fr. was the highest, it reached 32.15 ± 0.75 ⅿ

g/mL, Ethyl acetate Fr. 9.05 ± 0.91 ⅿg/mL, 70% ethanol Fr. 6.54 ± 1.04 ⅿg/mL, and

Hexane Fr. reached 5.36 ± 0.16 ⅿg/mL.

76

4.4.2. Total flavonoid contents (TFC)

Flavonoids are polyphenol compounds, usually including catechin, quercetin, and rutin,

the most potent antioxidants. It is found in different plants such as stems, leaves, roots,

flowers, and fruits. In this study, the total flavonoid content of Boswellia Serrata ex-

tracts was measured, and the results are shown in Table 9. It also revealed vast differ-

ences in flavonoid contents, with the highest content of flavonoids in Water Fr. 20.29

± 1.47 ⅿg/mL, Ethyl acetate Fr. 8.81 ± 0.14 ⅿg/mL, 70% ethanol Fr. 6.72 ± 0.71 ⅿ

g/mL, and Hexane Fr. 3.12 ± 0.14 ⅿg/mL.

77

Table 9. Radical scavenging activity and polyphenolic/Flavonoids content of

different Boswellia fractions.

IC

50

= The concentration of compound that affords a 50% reduction in the assay; GAE = gallic

acid equivalent. QUE = Quercetine equivalent. - = not detectable.

78

4.5. Analysis results of polyphenol componds

4.5.1 HPLC Analysis Results

HPLC analysis of Boswellia serrata extracts showed that only some of the 13 stand-

ards were detected. The water extract was found to have the highest content of gallic

acid, while the 70% EtOH extract was found to have the highest content of quercetin.

The EtOAc extract was found to have the highest content of epicatechin.

79

No. Sample water 70% EtOH EtOAc

1 Gallic acid 0.19 0.00 0.00

2 Catechin 0.00 0.00 0.20

3 (-)epicatechin 0.00 0.70 1.01

4 vanillic acid 0.00 0.08 0.18

5 Narigin 0.00 0.14 0.23

6 Ethyl gallate 0.00 0.00 0.00

7 p-coumaric acid 0.10 0.07 0.16

8 Ferulic acid 0.00 0.00 0.00

9 Benzoic acid 0.08 0.24 0.64

10 Quercetin 0.01 2.24 0.15

11 Narigenin 0.01 0.04 0.14

12 Kaempferol 0.00 0.00 0.27

13

4-hydroxybenzoic

acid

0.00 0.11 0.17

Total 0.38 3.63 3.16

Table 10. Polyphenol compounds identified in Boswellia serrata extracs quantified by

HPLC　(Unit : μg/mg)

80

1

23

4

5

6

7

8

9

10

11

12

13

(D)

R.Time (min)

0 10 20 30 40

2

3

4

5

7

9

10

11

13

(C)

4

3

4

5

7

9

10

11

13

(B)

1

7

9

10

11

13

(A)

R.Time (min)

0 10 20 30 40

Figure 16. HPLC profile of Boswellia serrata extracts and standard mixture using diode

array detection at 280 nm. (A) Boswellia serrata water extract; (B) Boswellia serrata

70% EtOH extract; (C) Boswellia serrata EtOAc extract; (D) standard mixture. Numbers

indicate the following: (1) gallic acid; (2) catechin; (3) (-)-epicatechin; (4) vanillic acid;

(5)Narigin; (6)Ethyl gallate; (7) p-coumaric; (8) ferulic acid; (9) benzoic acid; (10)

quercetin; (11) narigenin; (12) kaempferol; (13) 4-hydroxybenzoic acid

81

4.5.2 HPLC-MS/MS Analysis Results

A total of 14 polyphenol components were identified by HPLC-MS/MS analysis. The

highest content of benzoic acid was found in the 70% EtOH and EtOAc extracts. The

highest content of 5-hydroxymethylfurfural was found in the water extract.

82

No. Compound

Calculated Concentration (μg/g)

Water 70% EtOH EtOAc

1

4-hydroxy benzoic

acid

1.87 0.77 0.64

2 Caffeic acid 0.11 - -

3 Coumaric acid 0.39 - 0.02

4 Naringein - 0.01 0.01

5 Benzoic acid 5.45 13.35 6.82

6 Nicotinic acid 0.07 0.06 0.07

7 Gallic acid - 1.87 1.86

8 Protocatechuic acid 0.26 0.42 0.39

9 Tannic acid - 0.01 0.01

10 Ethyl gallate - 0.02 0.02

11 L-Asparagine 0.18 - -

12

5-hydrox-

ymethylfurfural

6.51 3.62 1.92

13 Chlorogenic acid - 0.40 0.09

14 Quercetin - 0.06 0.05

Total 14.84 20.59 11.9

Table 11. Polyphenol compounds identified in Boswellia serrata extract quantified by

HPLC-MS/MS

83

Figure 17. Component analysis of Boswellia serrata water extract by HPLC MS/MS.

84

Figure 18. Component analysis of Boswellia serrata 70% EtOH extract by HPLC

MS/MS.

85

Figure 19. Component analysis of Boswellia serrata EtOAc extract by HPLC MS/MS.

86

Ⅴ. Discussion

This study highlights the chemical composition and biological activities of Boswellia

serrata and its influential role as a cosmetic ingredient. The results of previous studies

revealed its nutritional, pharmaceutical, and cosmetic benefits as it was used over time.

Boswellia Serrata is a natural source of medicines and cosmetics. It has anti-in-

flammatory, antibacterial, antioxidant, and anti-irritant properties. They also work on an-

ti-aging, anti-acne, brightening, and calming the skin. In addition, it reduces the con-

sequences of acne and spots. It also works well to prevent hair loss and maintain the

health of the scalp and hair[19]

Where the results of the study proved that Boswellia serrata possesses effective anti-

bacterial properties, it only shows us the results of Antibacterial activity using well dif-

fusion method

Based on the results shown in Table 5 provides the findings of the well diffusion

method-based antimicrobial research. It means that various bacterial species showed

varying degrees of sensitivity to the extract of B. Serrata samples that were analyzed.

The B. serrata extract inhibited antibacterial activity in all strains for the relevant

concentrations. The diameter of the zone of inhibition for the B. serrata extract for S.

epidermidis ranged from 13.3±0.58 to 10.3±0.58 mm, for M. furfur from10.6±0.58 to

9±0 mm, for M. pachydermatis from13.25±0.35 to 9.75±1.06 mm, and for C. acnes

from 11.83±0.29 to 9.5±0 mm in the anaerobic jar.

Boswellia serrata has also been shown to be effective to Radical scavenging activity

via diffrent antioxidant assays

Because the antioxidant activity of natural products can be easily measured with

DPPH, a relatively stable free radical, measuring the radical scavenging activity of

DPPH is a widely used experimental technique. In order to confirm the radical scav-

enging ability of DPPH and determine the measured IC50 values, extracts of Boswellia

serrata were produced at a concentration for each fraction of 10 ⅿg/mL – 100 ⅿ

g/mL 70% EtOH Fr., 5 ⅿg/mL- 30 ⅿg/mL EtOAC Fr., 5 ⅿg/mL – 25 ⅿg/mL

87

Water Fr.

As a consequence of the measurement for each fraction, the findings are displayed in

Table 9. It was confirmed that there was higher activity in the Water Fr. compared to

the Other fractions; respectively, the Water Fr. showed the highest scavenging activity

902.19 ± 35.53 µg/mL, Ethyl acetate Fr. 20436 ± 652.19 µg/mL ,70% EtOH Fr.

8627.74± 369.22 µg/mL, and the Hexane Fr. Shows No scavenging activity.

By preparing Boswellia Serrata extracts at a concentration for each Fraction, the root

scavenging ability of ABTS was verified. concentration for each Fraction 5 ⅿg/mL –

30 ⅿg/mL 70% EtOH Fr. , 5 ⅿg/mL- 30 ⅿg/mL EtOAC Fr., 10 ⅿg/mL – 75 ⅿ

g/mL Water Fr. IC50 values were measured. Table 9 presents the findings. Accordingly,

the Water fraction demonstrated the maximum scavenging activity with a scavenging ac-

tivity of 1845.08 ± 2265.74 , followed by the ethyl acetate fraction with a scavenging

activity of 12167.16 ± 8152.82 µg/mL and the 70% ethanol fraction with a scavenging

activity of 13796.44 ± 660.7 µg/mLHexane Fr. Shows No scavenging activity.

In the total polyphenol content of Boswellia Serrata extracts was measured, our result

of the measurement for each Fraction confirmed that the polyphenol content in the

Water Fr. was the highest; it reached 32.15 ± 0.75 ⅿg/mL, Ethyl acetate Fr. 9.05 ±

0.91 ⅿg/mL, 70% ethanol Fr. 6.54 ± 1.04 ⅿg/mL, and Hexane Fr. reached 5.36 ±

0.16 ⅿg/mL.

We found that the total flavonoid content of Boswellia Serrata extracts also revealed

vast differences in flavonoid contents, with the highest content of flavonoids in Water

Fr. 20.29 ± 1.47 ⅿg/mL, Ethyl acetate Fr. 8.81 ± 0.14 ⅿg/mL, 70% ethanol Fr. 6.72

± 0.71 ⅿg/mL, and Hexane Fr. 3.12 ± 0.14 ⅿg/mL.

HPLC analysis uses standards to perform qualitative analysis, while LC-MS/MS anal-

ysis performs both qualitative and quantitative analysis. Since HPLC analyzes by com-

paring the retention time with a standard, the peak at the same time may contain mul-

tiple substances. LC-MS/MS analysis separates the substances through HPLC, and then

each component is analyzed qualitatively and quantitatively through a mass spectrometer.

Since it is not a simple separation analysis, after HPLC analysis, LC-MS/MS analysis

88

is performed for accurate analysis. Therefore, the results of HPLC analysis and

LC-MS/MS analysis are bound to be different. In this study, HPLC analysis confirmed

that the highest content of gallic acid in the water extract, quercetin in the 70% EtOH

extract, and epicatechin in the EtOAc extract were found in the water extract.

LC-MSMS analysis confirmed the highest content of 5-hydroxymethylfurfural in the wa-

ter extract and benzoic acid in the 70% EtOH and EtOAc extracts. The reason for the

different analytical results is due to the difference in the analytical methods of the ana-

lyzing instruments. In addition, the unknown samples that were not identified in the

HPLC analysis require further study and substance identification using analytical instru-

ments other than HPLC-MS/MS.

89

Ⅵ. Conculusion

Boswellia serrata resin is an important oleo-gum resin used in many industries, in-

cluding the pharmaceutical, culinary, flavoring, alcoholic beverage, cosmetic, and per-

fume industries.

It is one of aromatherapy's most commonly used essential oils to treat breathing

disorders. It is also used to treat various bacterial and fungal infections.

From the current study, it is concluded that Boswellia serrata resin has significant

antibacterial and antioxidant activities. As it has proven effective in discouraging bacte-

rial strains that cause skin diseases and grains such a

S

.

epidermidis

,

M

.

furfur

,

M

.

pachydermatis

and

C

.

acnes

, and it also has the ability to resist free radicals. This

means it has many uses and applications in cosmetics, including anti-aging, soothing,

and anti-inflammatory properties. It can also encourage faster skin regeneration and in-

crease skin elasticity to lighten the complexion.

Global cosmetic companies have recently relied on manufacturing skin, hair and

body care products from natural plant sources. Which means that finding natural sour-

ces It is easier to get the most out of these plants when they have cosmetic features

and are used in the creation of cosmetics. This also helps to broaden the sources and

is likely to reduce environmental losses, such as burning plants and dumping environ-

mental trash. This increases income and contributes to the economic growth of the

country exporting these plants.

For future Works Expecting more research to analyze polyphenol componds that

were not identified in the HPLC analysis require further study and substance identi-

fication using analytical instruments other than HPLC-MS/MS.

90

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