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
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
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
3.1 Total phenolic content………28
3.2 Antioxidant activity………31
3.3 Summarize of the results………34
4. Conclusions………..36
5. Practical recommendations………...37
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.
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.
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
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.
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
PlantaePHYLUM
TracheophytaCLASS
MagnoliopsidaORDER
LamialesGENUS
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 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
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
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
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
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
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
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 ± 39.3tetramethychroman -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,
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 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
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
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
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.
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].
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.
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
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.
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
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.
Total phenolic content of water extracts of different size particles of Salvia Officinalis using 1:20 (solid and solvent) ratio are presented in figure 5.
*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.
Total phenolic content of water extracts of different size particles of Salvia Officinalis using 1:30 (solid and solvent) ratio are presented in figure 6.
*Small particles: less than 1.2 mm, Big particles: from 1.2 mm.
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.
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 (solid and solvent) ratio are presented in figure 7.
*Small particles: less than 1.2 mm, Big particles: from 1.2 mm.
Figure 7. Antiradical activity of water extracts of different size particles of Salvia Officinalis using 1:10 (solid and solvent) ratio.
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
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.
*Small particles: less than 1.2 mm, Big particles: from 1.2 mm.
Figure 8. Antiradical activity of water extracts of small particles of different size Salvia Officinalis using 1:20 (solid and solvent) ratio.
According to our results shown in figure 8, antiradical activity of water extracts varied from 57 till 74 %. 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).
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.
*Small particles: less than 1.2 mm, Big particles: from 1.2 mm.
Figure 9. Antiradical activity of water extracts of small particles of different size Salvia Officinalis using 1:30 (solid and solvent) ratio.
According to our results shown in figure 9, antiradical activity of water extracts varied from 24 till 74 %. 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.
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.
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].
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.
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.
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