Introduction Literature review Methodology of decantation followed by rotary evaporator Yield of extract Phytochemical screening methods Result Conclusion References

1. Solvent extraction of
Salvia leaves by
decantation using the
solvent MeOH
6th
November 2014
Presented by:
Bhagea Ritesh
Buctowar Rouksaar
Cécile Christabelle
Ghoorbin Keshavi
Nazeer Huda

2. Contents
● Introduction
● Literature review
● Methodology of decantation followed by rotary
evaporator
● Yield of extract
● Phytochemical screening methods
● Result
● Discussion
● Conclusion

3. Introduction
Solvent 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.

4. Introduction
Salvia 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

5. Introduction
Salvia 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

6. 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

7. 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.

8. Methodology - Decantation
Extraction 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

9. 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

10. 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 65o
C, pressure was closed in
the condenser, pump machine was put on and water
was allowed into the condenser.
Rotary evaporator machine

11. 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.

12. Calculation of yield
From the 5 groups:
Total amount of methanol solvent = ~1050ml
Total amount of Salvia leaves = ~110g
Mass of empty rotary round bottom flask = 328.4g
Mass of flask + extract = 375g
Mass 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 %

13. Phytochemical screening
There 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

14. 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

15. Phytochemical Screening
5. 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 (100o
C) for 5 min
- Cooled and shaken vigorously
Spraying Dragendorff
on silica gel plate

16. Phytochemical Screening
7. 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

17. Results
Table 1: Methanol extracts of leaves from Salvia officinalis
Compounds Salvia Plant
Coumarins No result
Steroids/ terpenes -
Tannins +
Phenols +
Alkaloids -
Saponins -
Anthraquinones +
Leucoanthocyanins and flavonols -

18. 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.

19. 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.

20. 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.

21. 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.

22. 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.

23. 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

24. Conclusion
The 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.

25. 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).