Contents

● Introduction● Literature review ● Methodology of decantation followed by rotary

evaporator● Yield of extract ● Phytochemical screening methods ● Result● Discussion ● Conclusion

IntroductionSolvent Extraction● Method employed to separate substances based on

their solubility.● Used in the extraction of phytochemicals from plants.

Phytochemicals are:● Non-nutritive plants chemicals,● Not crucial to sustain human life but are

beneficial,● Some common examples are lycopene in tomatoes

and isoflavones in soy,● Have beneficial effects such as:

○ Antioxidant, Antimicrobial, Anticancer, etc● Can be obtained from a variety of plants like:

○ medicinal ones, herbs, fruits/nuts.

IntroductionSalvia officinalis ● Commonly known as sage,● Belongs to Lamiaceae (Labiatae) family,● Perennial herbaceous plant,● Can reach 50 cm in height,● Used in food as herbs and in beverages such as tea,● Used in the cosmetics industry.

Salvia leaves Salvia flower

Salvia tea

IntroductionSalvia officinalis ● Used in culinary and medicinal preparations,● Salvia species have many pharmaceutical properties

○ Antibacterial,○ Antiviral,○ Antioxidative,○ Anticancer and many others

● Contains large amounts of flavonoids● Suitable for further studies in Phytochemistry

Dried Salvia leaves

Literature review● According to Martins et al. (2014), S.officinalis has

antioxidant and antifungal properties.● Made use of decoctions, infusions and methanol/water

extracts● Testing for phenolic compounds● 21 compounds were detected by HPLC

○ 10 phenolic acids and 11 flavonoids● Some compounds were:

○ Luteolin digluceronide○ Sagecoumarin○ Caffeic acid → ○ Luteolin-7-O-glucoside

● More compounds detected in decoctions than water/ethanol and infusion

Literature review● Coisin et al. (2012) evaluated phytochemicals from 9

Salvia species.● Employed methanol extraction method and HPLC for

detection.● Flavonoids and Phenolic compounds were detected.● Some were rosmarinic acid, caffeic acid, luteolin-7-

glucoside and were in satisfying quantities● Salvia officinalis, Salvia verticillata, Salvia glutinosa

were found to be promising for further analyses.● On the overall, S.officinalis was found to dominate the

other in terms of phytochemical compounds.

Methodology - DecantationExtraction of non-volatile secondary metabolites by decantation:

● 28g of fresh Salvia leaves were measured

● 250 ml of methanol was measured into a conical flask

● The leaves were added to the flask, sealed with a cotton wool

and aluminium foil and wrapped in newspaper

● The flask was then put on a shaker for 48 hours

Shaker machine

Methodology● Afterwards, the laboratory vacuum

pump machine was used to remove unwanted solids from the solvent○ Tissue paper was put in the porous

funnel and the solution was passed through

○ To the filtrate obtained, MgSO4 powder was added to absorb any water present

○ The powder was continuously added until no more agglutination occurred

○ Filtration was again carried out using filter paper instead

Hooshna using the lab vacuum filter pump

Methodology● The solution was then concentrated using the rotary

evaporator machine.○ Mass of empty rotary round bottom flask was taken○ Water bath was set at 65oC, pressure was closed in

the condenser, pump machine was put on and water was allowed into the condenser.

Rotary evaporator machine

Methodology● The process was completed when all solvent got

evaporated into the collecting flask and when a concentrated extract was formed○ Mass of rotary round bottom flask + extract were

measured● The extract was removed using a pipette and 10 ml of

solvent was added again to dissolve any extract left. This was the diluted extract.

Calculation of yieldFrom the 5 groups:Total amount of methanol solvent = ~1050mlTotal amount of Salvia leaves = ~110g

Mass of empty rotary round bottom flask = 328.4gMass of flask + extract = 375gMass of extract = (375 - 328.4) = 46.6g

Yield of non volatile extract = Weight of extract x 100 % Weight of sample used

Yield = (46.6/110) x 100 = 42.37 %

Phytochemical screeningThere are 2 ways to do phytochemical analysis: by test-tube tests and by TLC methods.

1. Coumarins- Conc. ammonia solution added to crude extract- A smear of this sol. placed on a microscope slide- Viewed under UV light at 366nm

2. Steroid/Terpenes- Extract separated on TLC in solvent system (9:1 Chloroform/Methanol), (40:10)ml respectively- Plate developed by spraying of LB reagent and dried

Phytochemical Screening

3. Tannins- Extract washed with petroleum ether and filtered- Equal amount of freshly prepared ferric chloride and potassium hexacyanoferrate (dropwise) added to the filtrate

4. Phenols- Extract separated on TLC in solvent system (4:1 Chloroform/Methanol)- Plate developed by spraying of Folin reagent

Phytochemical Screening5. Alkaloids- Extract separated on TLC in solvent system (9:2:8:1 Ether/Methanol/Acetone/Ammonia)- Plate developed by spraying of Dragendorff reagent

6. Saponins- 0.5g of dried crushed leaves put in water bath (100oC) for 5 min- Cooled and shaken vigorously

Spraying Dragendorff on silica gel plate

Phytochemical Screening7. Anthraquinones- 10 drops extract dissolved in 10 drops water- Sol. filtered and extracted with benzene- Ammonia was then added and shaken

8. Leucoanthocyanins and flavonols- Extract washed with petroleum ether to extract all pigments- Ethanol added and filtered into 2 test tubes- Conc. HCl added in both test tubes- To one, Mg turnings added and allowed to stand for 10min- The other, placed in hot water bath and allowed to stand for 30 min

ResultsTable 1: Methanol extracts of leaves from Salvia officinalis

Compounds Salvia Plant

Coumarins No result

Steroids/ terpenes -

Tannins +

Phenols +

Alkaloids -

Saponins -

Anthraquinones +

Leucoanthocyanins and flavonols -

TLC result for Steroid/Terpenes

The positive result for presence of steroid/terpenes is a blue colour after spraying with LB reagent.

The methanol extract (M) on the left, does not show any positive result.

Thus, steroid/terpenes were not present.

Figure 1: Thin Layer Chromatography of methanolic extracts of leaves from Salvia

officinalis.

TLC result for phenols

The positive result for presence of phenols is a colour of blue to grey after spraying of Folin reagent.

The methanol extract (M) on the left, shows positive result, being grey.

Thus, the presence of phenols is confirmed.

Figure 2: Thin Layer Chromatography of methanolic extracts of leaves from Salvia

officinalis.

TLC result for alkaloids

The positive result for presence of alkaloids when spraying with Dragendorff is orange-brown spots on a yellow background.

The methanol extract (M) on the left, does not show any positive result.

Thus, alkaloids were not present.

Figure 3: Thin Layer Chromatography of methanolic extracts of leaves from Salvia

officinalis.

Discussion

● Polarity is an ability of a molecule to form strong bonds with

other polar molecules (Barwick et al. 1997)

● The solvent methanol which was used is a polar organic

solvent having a hydroxyl group that attracts polar molecules.

● But, it can also dissolve the non-polar ones, although to a

lower conc.

● The phytochemical screening of Salvia gave positive results

for tannins, phenols and anthraquinones; implying the

presence of all 3.

Discussion● According to a study (Ramu et al., 2012), the presence of

terpenoids and steroids as well as tannins and flavonoids

yielded a positive result for the Salvia officinalis. However, it

is not the case in these experiments carried out.

● According to another study (Mattalib and Naqishbandi, 2012),

the presence of flavonoids, saponins and tannins were

positive.

● According to a study from Coisin et al. 2012,, flavonoids and

polyphenolic compounds were found present in all Salvia

species.

Discussion● According to a study by Muria et al. 2002, 11 abietane

diterpenoids, 3 apianane terpenoids, 1 anthraquinone, and 8 flavonoids were isolated from the Salvia officinalis plant.

➔ From the studies just mentioned, it can be concluded that the 3 compounds obtained (tannins, phenols and anthraquinones) from the decantation with methanol, are really present in the plant. However, other compounds such as terpenoids, steroids, flavonoids and saponins were not obtained.

➔ This would lead to speculate that decantation is not a suitable method and also that methanol might not have extracted some non-polar compounds

ConclusionThe Salvia officinalis has a lot of potential in terms of research

for phytochemicals.

It has been shown that a large variety of flavonoids and phenolic

compounds are available in this plant. However, decantation has

not shown as being a good method for extraction.

For much detailed or in depth research, much precise

equipments must be used such as HPLC or mass spectrometry.

References● British Herbal Medicine Association. (1971). British herbal pharmacopoeia. Nr.

Keighley: British Herbal Medicine Association.● COISIN, M., NECULA, R., GRIGORAŞ, V., GILLE, E., ROSENHECH, E., and

ZAMFIRACHE, M. M. (2012). PHYTOCHEMICAL EVALUATION OF SOME SALVIA SPECIES FROM ROMANIAN FLORA. Analele Stiintifice Ale Universitatii Alexandru Ioan Cuza Din Iasi. Sectiunea II A, Biologie Vegetala 58(1). Retrieved from http://www.bio.uaic.ro/publicatii/anale_vegetala/issue/2012F1/05-2012F1.pdf

● Kontogianni, V. G., Tomic, G., Nikolic, I., Nerantzaki, A. A., Sayyad, N., Stosic-Grujicic, S. and Tzakos, A. G. (2013). Phytochemical profile of Rosmarinus officinalis and Salvia officinalis extracts and correlation to their antioxidant and anti-proliferative activity. Food Chemistry 136(1), 120–129.

● Martins, N., Barros, L., Santos-Buelga, C., Henriques, M., Silva, S., and Ferreira, I. C. F. R. (2015). Evaluation of bioactive properties and phenolic compounds in different extracts prepared from Salvia officinalis L. Food Chemistry 170, 378–385.

● Muira, K., Kikuzaki, H. and Nakatani N. (2002). Antioxidant activity of chemical components from sage (Salvia officinalis L.) and thyme (Thymus vulgaris L.) measured by the oil stability index method. Journal of Agricultural Food Chemistry, 50(7), pp 1845-1851.

● Muttalib, L.Y. and Naqishbandi, A.M. (2012). Antibacterial and phytochemical study of Iraqi of Salvia officinalis leaves extracts. Iraqi Journal of Pharmaceutical Science, 21(1).