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LITHUANIAN UNIVERSITY OF HEALTH SCIENCES

FACULTY OF PHARMACY

DEPARTMENT OF DRUG TECHNOLOGY AND SOCIAL PHARMACY

MAHMOUD TABBIKHA

EXTRACTION OF PHENOLIC COMPOUNDS FROM GARDEN SAGE

(SALVIA OFFICINALISL) LEAVES USING ULTRASOUND

Master thesis

Supervisor

Doc. Dr. Giedrė Kasparavičienė

KAUNAS, 2017

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LITHUANIAN UNIVERSITY OF HEALTH SCIENCES

FACULTY OF PHARMACY

DEPARTMENT OF DRUG TECHNOLOGY AND SOCIAL PHARMACY

CONFIRM:

Dean of Pharmacy faculty Prof. Dr. Vitalis Briedis

Date

EXTRACTION OF PHENOLIC COMPOUNDS FROM GARDEN SAGE

(SALVIA OFFICINALIS L) LEAVES USING ULTRASOUND

Master thesis

Reviewer Supervisor

Doc. Dr. Giedrė Kasparavičienė

Student: Mahmoud Tabbikha

KAUNAS, 2017

3

CONTENT

Summary……………………………………………………………………………………………………..5

Santrauka……………………………………………………………………………………………………..6

Abbreviations and definitions………………………………………………………………………………..7

Introduction......................................................................................................................................................8

Literature review..............................................................................................................................................9

1.1 Salvia officinalis……………………………………………………………………………………...9

Classification of Sage………………………………………………………………………………...9

Bioactive compounds of Sage……………………………………………………………………….10

1.2 Quantitative analysis of Sage……………………………………………………………………….11

1.3 Pharmaceutical form………………………………………………………………………………..15

1.4 Method of actions…………………………………………………………………………………...16

Antioxidant activity………………………………………………………………………………....16

Antiseptic effects……………………………………………………………………………………18

Anti-inflammatory and antinociceptive properties………………………………………………....20

Cognitive- and memory-enhancing effects………………………………………………………....22

1.5 Methods of extraction……………………………………………………………………………....23

2. Methods………………………………………………………………………………………………….25

2.1 Materials……………………………………………………………………………………………..25

2.2 Equipment…………………………………………………………………………………………...25

2.3 Object..................................................................................................................................................25

2.4 Particle size analysis………………………………………………………………………………....25

2.5 Technology of sage extracts………………………………………………………………………....26

2.6 Determination of total phenolic content………………………………………………………….….26

2.7 Antioxidant activity DPPH method………………………………………………………………….26

2.8 Statistical analysis…………………………………………………………………………………...27

3. Results……………………………………………………………………………………………………28

4

3.1 Total phenolic content………………………………………………………………………………28

3.2 Antioxidant activity…………………………………………………………………………………31

3.3 Summarize of the results……………………………………………………………………………34

4. Conclusions…………………………………………………………………………………………..36

5. Practical recommendations……………………………………………………………………….......37

6. References…………………………………………………………………….………………………38

5

SUMMARY

M. Tabbikha, Master thesis, Master thesis supervisor Doc. Dr. G. Kasparavičienė, Lithuanian university of

health science , Faculty of pharmacy, department of drug technology and social pharmacy. Kaunas .

Extraction of phenolics from dried sage (Salvia officinalis L) using maceration and ultrasound.

Aim of the work: Evaluate quality of extracts from dried sage extracted using maceration and ultrasound.

Objects of the research: Water extracts of Garden Sage prepared by dynamic maceration using

ultrasound.

Tasks of the research: Extraction using maceration and ultrasound in different conditions.

Total phenolic content measurement by colorimetric oxidation/reduction reaction using the Folin-

Ciocalteau reagent.

Antioxidant activity determination by 2,2-diphenyl-1-picrylhydrazyl (DPPH•) free radical method.

Salvia officinalis has antioxidant properties due to high content of phenolic compounds and rosmarinic

acid.

The results of Total phenolic content of water extracts show that the total phenolic content (TPC) ranged

from 5.43±0.16 mg/gRAE till 12.12±0.48 mg/gRAE using 1.2 mm size of dry extracts. TPC was lower

and ranged from 7.053±0.44 mg/gRAE to 11.62±0.72 mg/gRAE using more than 1.2 mm size of dry

extracts. The highest extraction yield from big size particles was after 15 minutes of sonication using the

1:10 ratio of solid to solvent and 30 minutes, 45 minutes, 60 minutes using 1:20 and 1:30 rations. The

total phenolic content in water extract from smaller size particle was significantly higher than the big one

after 30 minutes of sonication using 1:30 ratio of solid solvent. The 30 and 45 minutes extraction showed

an increase in the phenolic amount.

The results of DPPH activity expressed by Trolox equivalent are presented in figures 6 and 7 Water

extracts after 15 min at 1:10, 1:20 and 1:30 ratios from the small size particles showed a significantly

higher activity while from the big particles one it was very high at the same conditions. The big size

particles demonstrated a higher result after 45and 60 min.

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SANTRAUKA

M. Tabbikha. Magistro baigiamasis darbas „Fenolinių junginių ekstrahavimas iš vaistinių šalavijų (Salvia

officinalis L.) žolės ultragarsu“/ mokslinė vadovė doc. dr. G. Kasparavičienė; Lietuvos sveikatos mokslų

universiteto, Medicinos akademijos, Farmacijos fakulteto, Vaistų technologijos ir socialinės farmacijos

katedra. – Kaunas.

Tyrimo tikslas – įvertinti vaistinių šalavijų ekstraktų pagamintų maceracijos būdu, kokybę.

Tyrimo objektas – vandeniniai vaistinių šalavijų ekstraktai, pagaminti dinaminės maceracijos būdu.

Tyrimo uždaviniai: įvertinti ekstrahavimo faktorių – ekstrahavimo laiko, žaliavos dalelių dydžio ir žaliavos

ir ekstrahento santykio įtaką vandeninių ekstraktų pagamintų dinaminės maceracijos būdu, kokybei.

Nustatyti bendrą fenolinių junginių kiekį ir antioksidacinį aktyvumą DPPH metodu.

Tyrimo metodai: bendras fenolinių junginių kiekis nustatytas spektrofotometriškai, naudojant Folin-

Ciocalteu reagentą; rezultatai išreikšti rozmarino rūgšties ekvivalentu ir perskaičiuoti sausam žaliavos

svoriui. Antioksidacinis aktyvumas nustatytas spektrofotometriškai, naudojant 2,2-difenil-1-pikrilhydrazyl

(DPPH•) laisvojo radikalo tirpalą; rezultatai išreikšti radikalo inaktyvinimo procentais.

Tyrimo rezultatai. Bendro fenolinių junginių (BFK) kiekio rezultatai svyravo nuo 5.43±0.16 mg/gRRE iki

12.12±0.48 mg/g RRE, ekstrahuojant mažesnes nei 1,2 mm dydžio žaliavos daleles. BFK buvo mažesnis ir

svyravo nuo 7.053±0.44 mg/g RRE iki 11.62±0.72 mg/g RRE ekstrahuojant didesnes nei 1,2 mm dydžio

žaliavos daleles. Didžiausia fenolinių junginių išeiga gauta po 15 min. ekstrahavimo 1:10 santykiu ir po 30,

45 ir 60 min. nauojant 1:20 ir 1:30 santykius iš didesnių žaliavos dalelių. Ekstrahuojant mažesnes žaliavos

daleles reikšmingai didesnis kiekis gautas po 30 min. santykiu 1:30. Ilgiau trunkanti ekstrakcija didino

fenolinių junginių kiekį.

Vandeniniai ekstraktai po 15 min. ekstrakcijos santykiu 1:10, 1:20 ir 1:30 iš mažesnių žaliavos dalelių

pasižymėjo reikšmingai didesniu aktyvumu negu iš didesnių dalelių, kurie pasižymėjo taip pat aukštu

aktyvumu tomis pačiomis sąlygomis. Ekstraktai iš didesnių dalelių parodė didesnį aktyvumą po 45 ir 60

min. ekstrahavimo.

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ABBREVIATIONS AND DEFINITIONS

ABTS - 3-ethylbenzthiazoline-6-sulfonic acid

ANOVA - Analysis of variance

ARP - Antiradical power

DNA - Deoxyribonucleic acid

DPPH - 2,2-diphenyl-1-picrylhydrazyl

FRAP - Ferric reducing antioxidant power

GBIF - Global biodiversity Information facility

HCl - Hydrochloric acid

HPLC - High-performance liquid chromatography

ICV - Intracerebroventrcular

IP - Intraperitoneally

LA - Luria agar

LB - Luria both

LDL - lower than detection limit

MHA - Muller Hinton agar

MHB - Muller Hinton broth

MIC - Minimum inhibitory concentrations

SWE - Subcritical water extractions

TROLOX - 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid

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INTRODUCTION

Relevance of the work:

Salvia officinalis is widely used in the traditional medicine. It has antioxidant properties due to high

content of phenolic compounds and rosmarinic acid. In various studies, rosemary and sage, both belonging

to the mint family (labiatae), have been shown to be the most potent natural antioxidants of the common

spices. There is increasing evidence to suggest that many degenerative diseases, such as brain dysfunction,

cancer, heart diseases and immune system decline, could be the result of cellular damage caused by free

radicals, and antioxidants present in human diet may play an important role in disease prevention. The

reported high antioxidant activity of sage and its traditional medicinal uses prompted us to investigate this

herb further to provide a better understanding of the chemistry involved. Phenolic compounds will be

extracted by well-known classical maceration method and innovative method ultrasound extraction.

Influence of extraction factors (particle size, temperature, contact time, solvent) on the extraction of the

active compounds dried sage and antioxidant activity of extracts will be compared.

The goal:

Evaluate quality of extracts from dried sage extracted using maceration and ultrasound.

The tasks:

1. Extraction using maceration and ultrasound in different conditions.

2. Total phenolic content measurement by colorimetric oxidation/reduction reaction using the Folin-

Ciocalteau reagent.

3. Antioxidant activity determination by 2,2-diphenyl-1-picrylhydrazyl (DPPH•) free radical method.

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1. LITERATURE REVIEW

1.1. Salvia officinalis

Natural products from a central pillar of the modern pharmaceutical industry as the use of new

chemical entities derived from natural sources has risen to values between 45% and 75%. In several

studies, herbs of the Lamiaceae family Natural products form a central pillar of the modern pharmaceutical

industry as the use of new chemical entities derived from natural sources has risen to values between 45%

and 75%. In several studies, herbs of the Lamiaceae family have been indicated significant potential

sources of secondary active compounds. So, among those most popular aromatic plants, the genus Sage

shows much medicinal and horticultural importance due to the high diversity in secondary metabolites

(essential oils and polyphenols)[1].

Plants of this genus grow all over the world and the specie of Salvia officinalis is native to Middle

East and Mediterranean areas [2].This Garden Sage (Salvia officinalis) is highly appreciated both for its

antioxidant and medicinal properties such as antihydrotic, spasmolytic, antiseptic, and anti-inflammatory

properties, antimicrobial, astringent, eupeptic, antimutagenic and hypotensive activity as well as beneficial

effects in the treatment of bronchitis, cancer, Alzheimer’s disease, mental and nervous conditions[3].

Figure 1 : Arial parts of Salvia officinalis[2]

.

Classification of Sage: According to GBIF Backbone taxonomy [4].

KINGDOM Plantae

PHYLUM Tracheophyta

CLASS Magnoliopsida

ORDER Lamiales

FAMILY Lamiaceae

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GENUS Salvia L.

Bioactive compounds of Salvia officinalis

The major phytochemicals in flowers, leaves, and stem of Salvia officinalis are well identified. A

wide range of constituents include alkaloids, carbohydrates, fatty acids, glycosidic derivatives(e.g., cardiac

glycosides, flavonoid glycosides, saponins), poly acetylenes, steroids, terpenes/terpenoids (e.g.

monoterpenoids, diterpenoids, triterpenoids, sesquiterpenoids), waxes,[2] phenolic compounds ( e.g.

rosomarinic acid, caffeic acid, flavonoids, tannis),[5] carbohydrates (e.g. arabinose, galactose, glucose,

mannose, xylose, uronic acids and rhamnose)[6] are found in Salvia officinalis. Among flavonoids, Salvia

officinalis infusion is rich in chlorogenic acid, ellagic acid, epicatecin, epigallocatechingallate, quercetin,

rosmarinic acid, rutin, and luteolin-7-glucoside. Rosmarinic acid and ellagic acid are the most abundant

flavonoids in Salvia officinalisinfusion extract, followed by rutin, chlorogenic acid, and quercetin[7].

Moreover, the essential oil of Salvia officinalis revealed the presence of 40 components that made up

99.58% of the total composition. Cineole, borneol, α-thujone, ledene, β-pinene, α-humulene and trans-

caryophyllene were the major components of the oil[8].

Figure 2: Structure of main flavonoids isolated from Salvia officinalis.

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Figure 3: Structure of main terpenes and terpenoids isolated from Salvia officinalis.

1.2. Quantitative analysis of Salvia officinalis

Total phenolics can be estimated by the Folin–Ciocalteu assay and results are expressed as

micrograms of Gallic acid equivalents per mL of infusion (µg GAE/mL) So Sage contains 43.1 ± 3.7 µg

GAE/mL.

Polyphenols are identified and quantified using a Waters 600 HPLC–UV/VIS system, equipped

with an auto sampler and an UV/Vis detector.

Table 1: Polyphenolic profile of Salvia officinalis infusion by HPLC–UV/VIS [7].

Phenolic compound ng/mL

Chlorogenic acid 1.8

Epicathechin 1.1

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Hesperidin LDL

Gallic acid 0.8

Coumaric acid 0.5

Gallogacatechingallate 1 ± 0.1

Caffeic acid 1.1

Epigallocatechingallate LDL

Rutin 2.2 ± 0.1

Sinapic acid 1

Vanillin 1.2 ± 0.1

Ellagic acid 4 ± 0.1

Rosmarinic acid 6.27 ± 0.3

Naringenin 1.1 ± 0.1

Quercetin 1.6 ± 0.01

Resveratrol 0.5

Also there is a study demonstrating that the amount of total phenolics varies according to the type

of solvent used in the extraction. So, Results revealed that methanol and ethanol are better solvents than

diethyl ether and hexane in extracting phenolic compounds from the extracts due to their polarity and good

solubility for phenolic components from plant materials. Results in Table 3 showed that methanol was the

best solvent for extracting phenolic compounds, followed by ethanol then diethyl ether and hexane where

5.95, 5.80, 4.70, and 4.25 mg GAE/g DW for Sage.Total phenolic content is expressed as mg Gallic acid/g

dry extract (mg GAE/g DM) [9].

Table 3: Total phenolic contents of the yield extracted Salvia officinalis[9].

Solvent Extract yield (%) Total phenols (mg

GAE/g DM)

Methanol 23.41 ± 2.65 5.95 ± 2.65

Ethanol 18.24 ± 1.73 5.80 ± 1.00

Diethyl ether 5.38 ± 1.98 4.70 ± 2.00

Hexane 4.63 ± 1.73 4.25 ± 1.00

Moreover, to determine the composition of the essential oil of Salvia officinalis, it can be analyzed

by gas chromatography-FID followed by mass spectrophotometry and then the constituents will be

13

identified by comparison of their mass spectra fragmentation, retention indices, and standard materials

with authentic compounds or with data from the literature.

Table 4: Chemical composition of the essential oil of Salvia officinalis aerial parts [8].

No Compound Percent of

compound

Retention

indices

1 Borneol 13.77 13.896

2 Cineole 13.69 8.717

3 Alpha.-Thujone 12.46 11.487

4 Ledene 11.05 31.239

5 Beta-Pinene 7.00 6.966

6 Alpha.-Humulene 6.92 25.803

7 Trans-Caryophyllene 5.28 24.413

8 Beta-Thujone 4.56 11.864

9 Alpha -Pinene 3.89 5.771

10 Camphor 3.58 12.946

11 Naphthalene 3.27 46.877

12 Camphene 2.86 6.165

13 Bicyclo 1.75 18.839

14 Limonene 0.94 8.586

15 Caryophyllene oxide 0.84 30.821

16 Beta.-Myrcene 0.69 7.310

17 Alpha Terpineol 0.64 14.823

18 Gamma.-Terpinene 0.63 9.616

19 Oxabicyclo 0.47 31.817

14

20 Cyclohexen 0.37 14.279

21 Alpha-Thujene 0.33 5.570

22 Dimethy 0.33 32.807

23 Alpha.-Terpinene 0.32 8.157

24 Alpha-Terpinolene 0.32 10.714

25 Linalool 0.32 11.224

26 Delta.-Cadinene 0.27 28.538

27 Sabinene 0.25 6.835

28 Bicyclo 0.25 9.919

29 Cyclohexadiene 0.24 12.751

30 Aromadendrene 0.22 25.162

31 H-Cycloprop 0.22 26.043

32 H-Cycloprop 0.22 30.615

33 cis-Ocimene 0.21 8.849

34 Benzene 0.19 8.425

35 Isoaromadendrene

epoxide

0.18 32.664

36 Naphthalenemethanol 0.18 33.310

37 Bicyclo 0.17 11.126

38 Alpha.-Amorphene 0.17 26.690

39 Phenanthrene 0.15 42.288

40 Isoaromadendrene

epoxide

0.14 34.077

15

1.3. Pharmaceutical form [10]:

• Comminuted herbal substance as herbal tea for oral use.

• Comminuted herbal substance for infusion for or mucosal and cutaneous use.

• Herbal preparations in solid or liquid dosage forms for oral use.

• Herbal preparations in liquid or semi-solid dosage forms for cutaneous use or for or mucosal

use.

• The pharmaceutical form should be described by the European Pharmacopoeia full standard

term.

Clinical particulars [10]:

Therapeutic indications:

• Indication 1: Traditional herbal medicinal product for relief of mild dyspeptic complaints

such as heartburn and bloating.

• Indication 2: Traditional herbal medicinal product for relief of excessive sweating.

• Indication 3: Traditional herbal medicinal product for relief of inflammations in the mouth

or the throat.

• Indication 4: Traditional herbal medicinal product for relief of minor skin inflammations.

Contraindications:

Hypersensitivity to the active substance(s).

Special warnings and precautions for use:

• The use in children and adolescents under 18 years of age has not been established due to

lack of adequate data.

• If the symptoms worsen during the use of the medicinal product, a doctor or a qualified

health care practitioner should be consulted.

• For tinctures and extracts containing ethanol, the appropriate labelling for ethanol, taken

from the ‘Guideline on excipients in the label and package leaflet of medicinal products for human use’,

must be included

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1.4. Method of actions:

Antioxidant activity

Evidence from several studies suggests that Salvia officinalis has potent antioxidant activities.

Enriching the drinking water of rats with Salvia officinalis extract increases resistance of rat hepatocytes

against oxidative stress. It protects hepatocytes against dimethoxynaphthoquinone and hydrogen peroxide-

induced DNA damage through elevation of glutathione peroxidase activity.

The most effective antioxidant constituents of Salvia officinalis are carnosol, rosmarinic acid, and

carnosic acid, followed by caffeic acid, rosmanol, rosmadial, genkwanin, and cirsimaritin. The radical

scavenging effect of carnosol is comparable to that of α-tocopherol.The superoxide scavenging activity of

the rosmarinic acid derivatives are 15–20 times more than trolox, a synthetic water-soluble vitamin E. In

streptozotocin-induced diabetic rats, rosmarinic acid increases activities of pancreatic catalase, glutathione

peroxidase, glutathione-S-transferase, and superoxide dismutase. In addition to rosmarinic acid, other

flavonoids of Salvia officinalis particularly quercetin and rutin have strong antioxidant activities [2].

Antioxidant activity of Sage can be evaluated by DPPH radical scavenging activity, ABTS

decolorization assay and ferric reducing antioxidant power (FRAP). Several studies have showed that the

antioxidant potential of Salvia officinalis is affected by the harvesting time so results have shown that the

best antioxidant activities were detected at the flowering season[11].

Table 5:Variations of the antioxidant capacity of Salvia officinalis methanolic extracts according to

the phenological stage[11].

Antioxidant test Vegetative

stage

Flowering

stage

Fruiting stage

DPPH(IC50, g/mL) 46.47 ± 4.26b 32.49 ± 2.26c 55.91± 2.88a

ABTS(M TE/mg) 262.12±16.57b 345.81±1.07a 181.84±9.94c

FRAP(mMFe(II)/mg) 112.87 ± 5.78c 171.82± 8.10a 130.73 ± 7.98b

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Moreover, there is a study showing that the antioxidant activity may be affected by the type of

solvent used for extraction. Two parameters have been used in this study to measure the antioxidant

activity. First, (EC50) that is defined as: [the moles of phenolic compounds divided by moles of DPPH•

necessary to decrease by 50% the absorbance of DPPH•]. The lower the EC50, the higher is the antioxidant

power. Another parameter is the antiradical power (ARP) which is calculated by dividing 1 by EC50. The

higher the value of antiradical power (ARP), the higher is the free radical scavenging activity. So results

showed that the highest antiradical power (ARP) noticed for Sage extracted by methanol being 833.

Table 6: Free radical scavenging activities expressed as EC50 and antiradical power (ARP), as

affected by solvent extraction.

ABTS decolorization assay:

Antioxidant activity of Salvia officinalis can be performed also by an improved ABTS [2, 2’-

azinobis-(3-ethylbenzothiazoline-6-sulfonic acid)] decolorization assay. This method is based on the

generation of ABTS radical cation (ABTS•+) by oxidation of ABTS with potassium persulfate and the

cation is reduced in the presence of such hydrogen-donating antioxidants. So, the influences of both the

concentration of antioxidant and duration of reaction on the inhibition of the radical cation absorption are

taken into account when determining the antioxidant activity[12].

Briefly, ABTS (7 mmol/L in water) is reacted with 2.45 mmol/L (final concentration) potassium

persulfate overnight in the dark. The radical solution is diluted in phosphate buffer (pH 7.0) to an

absorbance of 0.700 ± 0.020 at 734 nm on a spectrophotometer. Trolox (6-hydroxy-2,5,7,8-

Plant Solvent EC50 ARP

Salvia

Officinalis

Methanol 0.0012 ±0.0001 833 ± 87.0

Ethanol 0.0019±0.0003 526 ± 77.0

Diethyl ether 0.0017±0.0002 588 ± 75.0

Hexane 0.0027 ±0.0011 370 ± 39.3

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tetramethychroman -2-carboxylic acid) is as an antioxidant standard. Sage extracts are tested in

concentrations ranging from 0.5 to 5.0 mg/mL in phosphate buffer (pH 7.0), while phosphate buffer served

as the control. The assay was started by adding 1 mL of the diluted ABTS radical to a cuvette containing

10 μL standard, sample, or buffer, and measuring absorbance every 30 s for 5 min. The % inhibition was

calculated after 5 min compared to a control (phosphate buffer and ABTS radical)[13].

HPLC analysis of extract:

The analytical HPLC system consisted of a liquid chromatograph with a UV multi wavelength

detector. The separation is achieved by a column at ambient temperature. The mobile phase consists of

water with 1% glacial acetic acid (solvent A), water with 6% glacial acetic acid (solvent B), and

water/acetonitrile (65:30 v/v) with 5% glacial acetic acid (solvent C). The flow rate is 0.5 mL/min, and the

injection volume is 20 µL. The monitoring wavelength is 280 nm. The identification of each compound is

based on a combination of retention time and spectral matching[14].

Antiseptic effects

The essential oil and ethanolic extract of Salvia officinalis show strong bactericidal and

bacteriostatic effects against both Gram-positive and Gram-negative bacteria[15] as well antifungal,

antiviral and antimalarial effects[8]. Antimicrobial effects of Salvia officinalis are attributed to terpenes

and terpenoids compounds found in this plant. Camphor, thujone, and 1, 8-cineole have antibacterial

effects[16].

• To test antibacterial effect[15]:

Methods:

A. Bacteria and media:

The following bacterial strains are used:

Staphylococcus aureus ATCC25923,

Staphylococcus epidermidis ATCC12228,

19

Pseudomonas aeruginosa ATCC27853,

Escherichia coli ATCC25922,

Bacillus subtilis ATCC10707,

Streptococcus faecalis ATCC29212,

Escherichia coli K12 strains SY252 and IB112

Salmonella typhimurium TA100 and TA102

Bacteria were cultivated at 37ºC in Luria broth (LB) (yeast extract 5 g, bacto-tryptone 10 g, NaCl 5

g, distilled water 1 L) or Mueller Hinton broth (MHB) from Oxoid. Luria agar (LA, LB plus 15 g

agar) and Mueller Hinton agar (MHA) from Oxoid were used as solid media.

The essential oils were dissolved in ethanol (1/10) and applied in different concentrations. Ethanol

is used as a negative control and antibiotics (chloramphenicol, streptomycin, and gentamycin) as positive

controls.

B. Antibacterial activity:

The disk-diffusion assay is applied to determine the growth inhibition of bacteria by Sage extracts.

Overnight bacterial cultures (100 μL) are spread onto MHA. Sage extracts were applied to 10 mm disks

(Whatman paper No.1). After 24 h of incubation at 37ºC, the diameter of growth inhibition zones is

measured.

C. MIC determination:

The broth dilution test is performed in test tubes. In two-fold serial dilutions of EO or its fractions,

a standardized suspension (McFarland turbidity standard) of test bacteria (100 μL) is added to obtain a

final concentration of 5x105 CFU/mL. A growth control tube and sterility control tube were used in each

test. After overnight incubation at 37ºC, the MIC was determined visually as the lowest concentration that

inhibits growth, evidenced by the absence of turbidity.

D. Time kill assay:

Exponential cultures of test bacteria (100 μL) are inoculated into several tubes of LB containing

MIC concentration of Sage extract for S.aureus and double MIC for B.Subtilis. A growth control tube was

used in each test. Tubes were incubated at 37ºC for 24 h. At regular time intervals, samples are taken,

diluted to obtain a countable number of colonies, and plated onto LA plates.

Results:

20

Results have showed that Sage had an effect againt S.aureus, S.subtilis, S.faecalis and E.coli IB112.

Moreover, Gram-positive bacteria were more sensitive than Gram-negative bacteria to the killing effect of

EO.The largest zones of growth inhibition appeared with the highest tested concentration (30 μL/disk).

Streptococcus faecalis displayed lower sensitivity to EO than S. aureus and B. subtilis. Moreover, MIC

values reported that the concentration of EO required to inhibit bacterial growth 1.25µl/ml was higher for

S. aureus than for B. subtilis 0.3 µl/ml.

In addition, the kinetics of survival of S. aureus and B. subtilis in the presence of EO

showed that EO exhibited a strong bactericidal effect on S. aureus, and within 4 hours the bacterial

population was completely inactivated. In contrast, EO rapidly reduced the counts of B. subtilis, but

after the initial reduction a constant fraction of the bacterial population survived. The survival of B.

subtilis is probably due to the presence of endospores, which are resistant to conditions to which

vegetative cells are intolerant. So, the essential oil of Salvia Officinalis has an antibacterial activity.

Anti-inflammatory and antinociceptive properties

Salvia officinalis has anti-inflammatory and antinociceptive effects[17].This plant helps to

control neuropathic pain in chemotherapy induced peripheral neuropathy. Chloroform extract has an

important anti-inflammatory [18]. Flavonoids and terpenes contribute to the anti-inflammatory and

antinociceptive actions of the herb[17], [19], rosmarinic acid inhibits epidermal inflammation [20].

Salvia officinalis constituents are responsible for its antinociceptive effect in patient with pharyngitis

[21].

➢ Anti-inflammatory and Analgesic Properties of Salvigenin: Salvia officinalis Flavonoid Extracted

[17]:

Methods:

Salvigenin is one of the active flavonoids existing in this plant.

To confirm the activity of Salvigenin, several tests were done on 100 male albino mice (25–30

g) and 48 male wistar rats with 6-8 weeks old.

In a hot plate test: animals were divided randomly into 5 groups of 10. Group 1 was

considered as the control group which received 10 mg/kg normal saline intraperitoneally, groups 2, 3

21

and 4, received respectively 25, 50 and 100 mg/kg of Salvignin intraperitoneally. Group 5 received

morphine at a dose of 10 mg/kg. All medications after zero time (initial test hot plate) were performed.

Writhing test: In the writhing test, animals were divided randomly into 5 groups of 10. Group 1

was the control group which received 10 mg/kg normal saline intraperitoneally, groups 2, 3 and 4,

received respectively 25, 50 and 100 mg/kg of Salvigenin intraperitoneally. Group 5 received

indomethacin at a dose of 10 mg/kg. All injections were performed 15 minutes before the injection of

acid and acetic acid injected at a dose of 0.6 percent intraperitoneally and 5 minutes later, abdominal

contractions of the mice were counted for 30 minutes.

In the inflammatory test: animals were randomly divided into 6 groups of 8. Group 1 was

assigned as a control group which received 0.05 ml carrageenin in the left foot subcutaneously. Groups

2, 3 and 4, received Salvigenin, at doses of 25, 50 and 100 mg/kg intraperitoneally respectively. Group

5 received 10 mg/kg of indomethacin and then changes of the volume of all groups were measured.

Extract and drug were injected intraperitoneally 30 minutes before injection.

Inflammation induction method: Different doses of Salvigenin were dissolved in saline carrier.

Experimental groups received 25, 50 and 100 mg/kg Salvigenin intraperitoneally and the positive

control group received 10 mg/kg Indomethacin intraperitoneally and negative control received 5 ml/kg

normal saline intraperitoneally. After half an hour, 100 µl of carrageenin (1%) was injected into the

right paw of the rats subcutaneously. Right paw volume was measured once every hour for 4

consecutive hours using plethysmometer (model 7140, made in Italy). Immediately before the injection

of carrageenin, the volume of the paw was measured as zero time.

Results:

According to the results of the writhing test, salvigenin reduced the number of abdominal contractions

and increased the percentage of inhibition at doses of 50 and 100 mg/kg compared to the control group.

Increasing dose of Salvigenin, with reduction in abdominal cramps leaded to increasing of pain

inhibition, and the percentage of this inhibition was statistically significant. Also, indomethacin in

receiver group significantly reduced abdominal contractions compared to the control group and dose of

25 Salvigenin.

In addition, the results of the hot plate test, the duration of response to the pain between the groups in

the hot plate test at time zero was not significantly different before the injection. In contrast, 30

22

minutes after injection of 100 mg Salvigenin and morphine, significant difference was observed in

comparison with the control group. So, Salvigenin has dose-dependent analgesic effect.

Moreover, inflammation induction method has showed that salvigenin can be useful in controlling of

inflammations, acute and chronic pain in a dose of 100 mg/kg.

Cognitive- and memory-enhancing effects:

Ethanoic extract of Salvia officinalis increases memory retention[22]. Hydroalcoholic extract

and its main flavonoid rosmarinic acid improve cognition and prevent learning and memory deficits

induced by diabetes[23].Salvia officinalis enhances cognitive performance both in healthy participants

and patients with cognitive impairment or dementia[24]. Moreover, its essential oil enhances

prospective memory performance in healthy adults[25]. Also, hydroalcoholic extract of Salvia

officinalis improves cognitive functions in patients with mild to moderate Alzheimer's disease[26].

Salvia officinalis inhibits acetylcholinesterase activity. Inhibitors of acetylcholinesterase are the leading

therapeutics of Alzheimer's disease and Salvia officinalis might be a promising source for developing

therapeutic agents for this disease [27].

➢ To confirm that ethanolic extract of Salvia officinalis increases memory

retention, the following study was done[22]:

The experiment was done on one hundred ninety male Wistar rats that weighed 200 to 250 g

and were allowed to habituate to the laboratory environment 1 h before each training or testing session.

The ethanolic extract of plant leaves was obtained in a soxhlet apparatus.

Methods:

Retention test: Twenty-four hours after training, a retention test was performed to determine

long-term memory. Each animal was placed in the light compartment for 5 s, the door was opened, and

the step through latency was measured for entering into the dark compartment; the test session ended

when the animal entered the dark compartment or remained in the light compartment for 300 s

(criterion for retention). During these sessions, no electric shock was applied.

Injection of drugs: The drugs used were pilocarpine, the muscarinic cholinoceptor agonist;

scopolamine N-butylbromide, the muscarinic receptor antagonist; nicotine, the nicotinic cholinoceptor

agonist ; and mecamylamine, the nicotinic receptor antagonist. Drugs were dissolved in saline and

23

administered by intracerebroventricular (ICV) injection and in a volume of 2 µL/rat immediately after

the training session. The ethanolic extract was diluted by distilled water and administered

intraperitoneally (IP) in a volume of 1 mL/rat.

To analyse data, analysis of variance (ANOVA) followed by Newman Keuls test were used.

Results:

It is concluded that the ethanolic extract (50mg/kg) of salvia officinalis potentiated memory

retention and also it has an interaction with muscarinic and nicotinic cholinergic systems that is involved in

the memory retention process.

1.5. Methods of extraction

Subcritical water extractions (SWE)

SWE is performed in batch-type high-pressure extractor with internal volume 450 mL and

maximum operating pressure of 200 bars and temperature 350 ˚C, connected with temperature controller.

Temperature (120 – 220 ˚C), extraction time (10 – 30 min) and HCl concentration (0 – 1.5%) : those

parameters are independent variables while all others are constant. 20.0 g of plant material is mixed with

200 mL of solvent and extraction is performed on isobaric conditions (30 bars). Stirring by magnetic stirrer

is employed in order to increase mass and heat transfer, and prevent local overheat on the inner walls of

extractor. Extracts were immediately filtered through filter paper under vacuum. Extracts are then collected

into glass flasks and stored at 4 °C until the analysis[28].

Maceration and percolation

Maceration is a traditional extraction procedure was performed by maceration of Sage herbal dust

with water at room temperature (25ºC) for 7 days. Maceration procedure follows: dried material placed in

the closed vessel, poured with extraction solvent (in various solid to solvent ratios); allowed to stand for

some days by shaking occasionally; decantation of liquid part then filtered through a paper filter. Extracts

are then collected into glass flasks [29].

Other classical extraction method is percolation. The percolation is the most common procedure for

the preparation of tinctures and fluid extracts. The percolator is a conical vessel with a top opening in

which is placed a circular drilled lid allowing the pass of liquid and subjecting the materials placed on it to

a slight pressure. The bottom has an adjustable closure to allow passage of the fluid at a convenient rate.

24

The plant material is moistened prior to their placement in the percolator with a proper amount of

menstruum, it´s placed in a sealed container and leave stand for approximately four hours. After that time

the plant material must be conveniently placed in the percolator so as to allow the even passage of fluid

and the complete contact with the plant material. The percolator must be filled with liquid and covered up.

The bottom outlet is opened until get a regular dripping and then closes. More menstruum is added to

cover all the material and must stand to soak in the percolator closed for 24 hours. After this time leave it

to drip slowly and added enough menstruum to a proportional volume of 3/4 of the total volume required

for the final product. The wet mass is pressed to extract the maximum residual fluid retained and

supplemented with sufficient menstruum to get the proper proportion, it´s filtered or clarified by

decantation. For example, percolation procedure: dried rosemary leaves were moistened with 50% ethanol

for 1.5 h and 4 h (1:10) → all material was placed into the percolator and allowed to stand for 3 days →

the first part (85% of final volume) of extract was percolated at a control rate (3 drops per minute) → the

second part (15% of final volume) of extract was percolated in the same rate with continuous addition of

fresh solvent (till all needed volume for extraction was used) → the second part was concentrated by

evaporation and filtered → the first and second parts were mixed and stored at 8 °C for 72 h → filtration

through a paper filter → extract

Ultrasound extraction

The milled plant material was added to a tube and extracted with water using different mass/volume

of plant material and solvent for different timing using the ultrasound bath. Ultrasound extraction is

performed in an ultrasound cleaning bath) by the mode of the indirect sonication, at fixed-frequency. After

extraction, extracts are filtered through filter paper. Extracts are then collected into glass flasks[30].

Extraction of essential oil from arial parts of Sage

After harvesting and cleaning of leaves and flowers of Sage in a shaded, well-aired place for 15

days; Fifty grams of the dried plant material is cut into small pieces and placed in 500 ml distilled water for

2.5 hours at 100°C after boiling at 290°C with a clevenger apparatus. Then, the oils are obtained with n-

pentane as a collecting solvent, and are dried over anhydrous sodium sulfate [8].

25

2. METHODS

2.1 Materials

1. Drygarden sage leaves (Salvia officinalis L), (UAB "AcorusCalamus", Lithuania).

2. 2,2-diphenyl-1-picrylhydrazyl (DPPH•) radical, > 99 %, ("Sigma-Aldrich," Germany).

3. Ethanol 96 % V/V (AB "Vilniaus degtinė", Lithuania),

4. Folin-Ciocalteureagent (Darmstadt, Germany),

5. Sodium carbonate, > 99,5 proc. (Sigma-Aldrich, Germany),

6. Distilled water (LUHS laboratory).

2.2 Equipment

1. Analitical weight "Axis AD510"Poland.

2. Ultra sound bath "Ultrasonic bandelin electronic DT 156 BH" Germany.

3. Sieves "RetschAs 200 basic,"Germany.

4. Centrifuge " Sigma 3-18 KS,"Germany.

5. UV Spectrofotometer " Model UV-1800 240V IVDD, Shimadzu", Germany.

6. Automatic pipetes "Pipetmangilson,"France.

2.3 Object

Water extracts of Garden Sage prepared by dynamic maceration using ultrasound.

2.4 Particle size analysis

Sieve test was used for the analysis. 50,0 g of dry material was sieved through 2,5 mm, 2 mm, 1,6 mm, 710

µm, 450 µm, 125 µm sieves. Time 10 min., amplitude – 60 mov/min.

26

2.5 Technology of sage extracts

1. Separation of Sage into two sizes using sieve nest machine. Big size (from 1.2 mm), small size (less

than 1.2 mm).

2. For both sizes, we were doing manual maceration and dynamic maceration (ultrasound)

3. For dynamic maceration Sage particles were added to a tube and extracted with water using

different mass/volume of plant material and solvent (1:10, 1:20, 1:30) for different timing (15, 30, 45, 60

minutes) using the ultrasound bath. Ultrasound extraction is performed in an ultrasound cleaning bath) by

the mode of the indirect sonication, at fixed-frequency.

4. After extraction, tubes were transferred to the centrifuge machine for 10 minutes and 4700 rpm,

extracts are filtered through filter paper. Extracts are then collected into glass flasks.

5. For manual maceration extraction procedure was performed by maceration of Sage herbal dust with

water at room temperature (25ºC) for 7 days. We couldn't test the extract because it was musty; also maybe

the water was a bad solvent for this maceration or due to herbal materiel.

2.6 Determination of total phenolic content

The method is based on a colorimetric oxidation/reduction reaction using the Folin-Ciocalteau reagent with

modifications [31].The standard calibration (0.0625–1.0 mg/ml) curve is plotted (R2 = 0.9989) using

rosmarinic acid dissolved in distilled water. 0,5 ml of the Salvia officinalis extract, 2,5 ml of Folin–

Ciocalteu reagent (diluted 10 times with water) and 2 ml of sodium carbonate (75 g/L) are added. The

sample is left for 30 min and the absorbance at 765 nm is measured. Results will be expressed as

milligrams of rosmarinic acid equivalent per milliliter of tested extract (RAE mg/ml) [32]

2.7 Antioxidant activity DPPH method

Antioxidant activity was determined by 2,2-diphenyl-1-picrylhydrazyl (DPPH•) free radical inactivation

method. DPPH is characterized as a stable free radical by virtue of the delocalization of the spare electron

over the molecule as a whole, so that the molecules do not dimerize, as would be the case with most other

free radicals. The delocalization also gives rise to the deep violet color, characterized by an absorption

band in ethanol solution centered at about 515 nm [33]. Analyzed extracts of Salvia officinalis(0.1 mL) are

27

mixed with 0.1 m Methanolic (96 %) DPPH• solution (2.90 mL) in 1 cm path length disposable

cuvette[34].

The decrease in absorption at 515 nm was recorded after incubation period 20 min at room

temperature. The percent inhibition of the 2, 2-diphenyl-1-picrylhydrazyl (DPPH•) free radical was

calculated according to the formula:

DPPH• (% inhibition) = [(Ablank – Asample)/Ablank] ×100.

Where Ablank is the absorbance of the blank solution and Asample is the absorbance sample and 2, 2-

diphenyl-1-picrylhydrazyl (DPPH•) free radical solution after 20 min.

2.8 Statistical analysis

Statistical analysis of results was performed using Microsoft Office Excel 2010 program.

Average, standard daviation and correlation were calculated.

28

3. RESULTS

3.1 Total phenolic content

The main goal of this research was to evaluate the influence of extraction factors to the quality of

extracts. Determination of total phenolic content was one of quality parameters for the water extracts of

sage. The results are presented by the different ratios of herbal material and solvent.

Total phenolic content of water extracts of different size particles of Salvia Officinalis using 1:10

(solid and solvent) ratio are presented in figure 4.

*Small particles: less than 1.2 mm, Big particles: from1.2 mm.

Figure 4. Total phenolic content of water extracts of different size particles of Salvia Officinalis using

1:10 (solid and solvent) ratio.

According to our results shown in figure 4, the highest concentration of phenolic content was in small

particles after 60 minutes of sonication using the 1:10 ratio of solid to solvent. Also, we can see that the

29

concentration is increasing when the time of sonication increases from 108.90±6.65 (30 min) till

200.53±9.22 mg/gRAE (60 min) using less than 1.2 mm size particles of dry extracts. As well as for big

particles, the concentration increases by increasing sonication time. The smallest amount of total phenolics

was after 15 min extraction from both sizes of dry particles of sage. After 30 min of extraction the amount

of phenolics was similar, just after 45 min we can see difference – higher amount from smaller particles of

dry particles of leaves.



*Small particles: less than 1.2 mm, Big particles: from 1.2 mm.

Figure 5. Total phenolic content of water extracts of different size particles of Salvia Officinalis

using 1:20 (solid and solvent) ratio.

According to our results shown in figure 5, also the highest concentration of phenolic content was in small

particles after 45 minutes of sonication using the 1:20 ratio of solid to solvent as well as big particles but

with less concentration. The smallest amount of total phenolics was after 15 min extraction from both sizes

of dry particles of sage. At 1:20 ratio after 30 min of extraction we can see difference – higher amount

from smaller particles of dry particles of leaves.

30




According to our results shown in figure 6, the highest concentration of phenolic content was is small

particles after 60 minutes of sonication using the 1:30 ratio of solid to solvent. The smallest amount of total

phenolics was after 15 min extraction from both sizes of dry particles of sage. At 1:30 like 1:20 ratio after

30 min of extraction there was higher amount from smaller particles of dry particles of leaves in water

extracts of sage.

Between all the ratios, the best concentration was after 60 minutes of sonication using 1:30 ratio of solid to

solvent as well as for big particles the highest concentration was at 45 minutes of sonication using 1:30

ratio of solid to solvent.

31

3.2 Antiradical activity

DPPH has been widely used in the determination of the antioxidant activity of single compounds as

well as the different plant extracts through the ability of compounds to act as free radical scavengers or

hydrogen donors and thus to evaluate the antioxidant activity.

Antiradical activity of water extracts of different size particles of Salvia Officinalis using 1:10



Figure 7. Antiradical activity of water extracts of different size particles of Salvia Officinalis using


According to our results shown in figure 7, antiradical activity of water extracts varied from 30 till 75

%.Antiradical activity was increasing for big particles by increasing time of sonication from 64.44±1.13 %

(15 min) till 71.89±3.39 % (60 min). For small particles, the antiradical activity was less after 45 and 60

32

min than the one of big particles, but the highest after 15 min of extraction. The results of antiradical

activity do not correlate with the amount of phenolics.

Antiradical activity of water extracts of different size Salvia Officinalis using 1:20 (solid and

solvent) ratio are presented in figure 8.


Figure 8. Antiradical activity of water extracts of small particles of different size Salvia Officinalis using



%. Antiradical activity was increasing for big particles by increasing time of sonication from 59.95±1.34

% (15 min) till 73.72±1.9 % ( 45 and 60 min).

For small particles, the antiradical activity was less after 45 and 60 min than the one of big particles,

33

but the highest after 15 min of extraction. The results of antiradical activity do not correlate with the

amount of phenolics.

Antiradical activity of water extracts of small particles of different size Salvia Officinalis using 1:30

(solid and solvent) ratio.


Figure 9. Antiradical activity of water extracts of small particles of different size Salvia Officinalis using



%. Antiradical activity was increasing for big particles by increasing time of sonication from 23.86±5.2 %

(15 min) till 74.02±2.88 % (60 min). For small particles, the antiradical activity was almost the same and it

didn‘t vary threw time. The highest antiradical activity was after 45 and 60 min of extraction from bigger

particles, the smallest after 15 min of extraction from bigger particles. The results of antiradical activity do

not correlate with the amount of phenolics.

34

3.3 Summarize of results

The main antioxidative effect of sage has been reported to relate to the presence of phenolic triterpenes and

phenolic acids, and thus special attention in this study was paid to rosmarinic acid. These compounds were

characterized in terms of their retention times and UV and mass spectra.

The results of Total phenolic content of water extracts in figures 4, 5 and 6 show that the total phenolic

content (TPC) ranged from 5.43±0.16 mg/gRAE till 12.12±0.48 mg/gRAE using 1.2 mm size of dry

extracts after 15 minutes of sonication using 1:10 ratio solid to solvent. TPC was lower and ranged from

7.053±0.44 mg/gRAE to 11.62±0.72 mg/gRAE using more than 1.2 mm size of dry extracts. The highest

extraction yield from big size particles was after 15 minutes of sonication using the 1:10 ratio of solid to

solvent and 30 minutes, 45 minutes, 60 minutes using 1:20 and 1:30 rations. The total phenolic content in

water extract from smaller size particle (316.20±8.25 mg/gRAE) was significantly higher than the big one

(163.99±6.68) after 60 minutes of sonication using 1:30 ratio of solid solvent. The 30 and 45 minutes

extraction showed an increase in the phenolic amount. From those results of water extract we can conclude

that the highest amount was obtained after 45 to 60 minutes of sonication using small size of particles of

Salvia Officinalis .In addition, this highest amount of TPC (316.20±8.25 mg/gRAE) is significantly higher

than the concentration obtained by conventional extraction with water as a solvent in another research

(33.5±0.5 mg/gRAE)[35].

In addition, the results of DPPH activity expressed by Trolox equivalent are presented in figures 7, 8 and 9

Water extracts after 15 min at 1:10, 1:20 and 1:30 ratios from the small size particles showed a

significantly higher activity while from the big particles one it was very high at the same conditions. The

big size particles demonstrated a higher result after 45and 60 min. From those results of water extract we

can conclude that the highest amount (74.02±2.88 %) was obtained after 45 to 60 minutes of sonication

using big size of particles of Salvia Officinalis. This amount of antiradical activity is too close the one

obtained by extraction with sonication using aqueous methanol as a solvent (76.58±0.33 %) or ethanol as

solvent (72.97±0.09 %). This can be explained by the fact that more the solvent is polar more the

extraction of total phenolic content is efficient so the antioxidant activity will be greater because the

antioxidant activity is directly related to the amount of the total phenolic compounds [36].

During the extraction, it has been influenced by three major factors: size of particles, time of extraction and

solid to solvent ratio.

35

The main antioxidative effect of sage has been reported to relate to the presence of phenolic triterpenes and

phenolic acids, and thus special attention in this study was paid to rosmarinic acid. These compounds were

characterized in terms of their retention times and UV and mass spectra.

According to our results, there is not always a significant correlation between the total phenolic content

and the antiradical activity. In fact, the highest amount of total phenolic content ((316.20±8.25 mg/gRAE )

in small particles did not show a high correlation (0.544) with the antiradical activity. In contrast, a low

amount 5.438±0.16 after 15 min of sonication in the 1:10 ratio showed a significant correlation (0.998)

with the antioxidant activity. Also, other researches did not show a significant correlation between the total

phenolic content and the antiradical activity, so it maintains that a high yield of individual phenolic

compounds does not necessarily indicate the maximal antioxidant activity and presents a significant

correlation between TPC and DPPH results [32].

36

4. CONCLUSIONS

1. The influence of extraction factors – extraction time, herbal material and solvent ratio and particle

size of herbal material on the quality of sage extracts was evaluated.

2. The highest amounts of phenolics (316.20 ± 8.25 mg/g RAE) were determined at 1:30 solid to

solvent ratio after 60 min of sonication. 30-45 min was efficient time for the TPC extraction and the

decreasing particle size of dry particles increased the extraction yield.

3. Antiradical activity was comparable of all extracts produced at different extraction time and solid to

solvent ratios. The results present that the antioxidant capacity was higher in biggest size particles

and the highest amount (74.0225 ±2.88 %) was determined at 1:30 solid to solvent ratio after 60

min of sonication.

37

5. PRACTICAL RECCOMENDATIONS

Our results showed that amount of phenolics didn’t correlate with DPPH. This may be due the difference

in concentrations of individual phenolic compounds, so it would be interesting to identify the main

compounds and evaluate correlation with them.

38

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