ISOLATION OF RUTIN FROM Ruta graveolens (RUTACEAE) CULTIVATED IN IRAQ BY PRECIPITATION AND FRACTIONAL SOLUBILIZATION

Headnote

ABSTRACT

Ruta graveolens L., a plant belongs to the family Rutaceae, is characterized by a particularly wide spectrum of different plant constituents. More than 100 compounds have been indentified, these belong to four major classes of substances namely alkaloids, coumarins, flavonoids, and essential oils. Rutin is the major flavonoid glycoside found in Ruta graveolens is the rhamnoglucoside of the flavonoid quercetin has been called vitamin P or the permeability factor. Several studies had been done for the isolation of rutin by different chromatographic method. In this study rutin was isolated from Ruta graveolens by precipitation and fractional solubilization without the use of any chromatographic technique. The isolated rutin was identified by measuring its melting point, UV absorption, FTIR spectra, HPLC and TLC.

Keywords: Rue; flavonoids; rutin; isolation.

INTRODUCTION

Ruta graveolens L., synonyms bitter herb or garden rue is a plant of the family Rutaceae. It is characterized by a particularly wide spectrum of different plant constituents. More than 100 compounds have been identified and belong to four major chemical classes which may be present in variable amounts in the aerial parts of the plant, namely alkaloids of quinoline and quinolinone (graveoline), furoquinoline (dictamine, skimmianine), pyrano-quinoline (rutalinium) and acridone (funacridone); coumarins, like dicoumarins, furacoumarins (bergaptene), pyranocoumarins; flavonoids (rutin) and essential oils.1 It is an evergreen shrub that grows up to one meter tall and well known for its offensive odor.2,3 Rue was used by the ancients as a medicinal plant. In Iraq, it was given to soldiers to instill courage.4 Flavonoids are among the most widely produced natural products in plants with over 2000 different compounds present both as free aglycons and glycosides.5 Recently, naturally occurring and synthetic flavonoids are used in modern medicine for various therapeutic effects, especially in USA, western and central Europe. They are used for treatment of various conditions related to capillary bleeding and increased capillary fragility and permeability.1,5 Rutin is the rhamnoglucoside of the flavonoid quercetin, and found in many plants1 and used for treatment of various diseases related to the vascular.6 It is quercetin-3-rutinoside or 3,3',4', 5,7-pentahydroxy flavones-3-rutinoside,7 and has a chemical formula C27H30O16. Major commercial sources of rutin include Saphora japonica, Eucalyptus spp., Fagopyrum esculatum and Ruta graveolens. As a constituent of Fagopyrum esculatum, the British and European pharmacopoeias requires a minimum content of 4.0% flavonoids determined by liquid chromatography with absorbance at 350 nm.7

Rutin (Figure 1) and quercetin were isolated from tobacco leaves by column chromatography and preperative TLC8 and from Ruta graveative and other plants using chromatographic techniques and ultrasonic methods.9,10 The present study was designed to isolate rutin from Iraqi Ruta graveolens based on its solubility in different solvents without any other separation techniques.

MATERIAL AND METHODS

HPLC (waters 1525), UV-visible spectrophotometer (Cary 100 conc), FT/IR (Jasco-4200). All chemicals and solvents are of analytical grade. Standard rutin was obtained from Sigma chemical company.

Plant material

The aerial parts of Ruta graveolens were collected from Al- Graiaat area in Baghdad and authenticated by the National Herbarium at Abu-Graib, they were dried in shade for several days at room temperature and then grinded as powder.

Extraction and isolation

Twenty grams of the powdered aerial part was extracted by Soxhlet apparatus with 250 ml of 80% ethanol till exhaustion. The extract was filtered and concentrated by evaporation under vacuum to about 10ml then mixed with 25ml distilled water, and extracted with petroleum ether (50ml x 3), then with chloroform (50 ml x 3).

After extraction, the aqueous layer was collected and leftto stand in a cold place for 72 hours; a yellow precipitate separated out of the solution. The precipitate was filtered and washed with a mixture of chloroform: ethylacetate: ethanol (50:25:25). The un-dissolved part of the precipitate was dissolved in hot methanol and filtered, the filtrate was evaporated to dryness to give 115 mg yellow powder (rutin), and its melting point was measured.

Identification of isolated rutin

The isolated rutin was identified by HPLC method and compared with standard rutin using hyperclone ODCC C18 V-25cm column and a mixture of methanol: water (70:30 ratio) as a mobile phase with a flow rate of 0.5ml/min, and detected at 280 nm.

TLC and paper chromatography

Isolated rutin was also compared with standard rutin using TLC method; a pre-coated aluminum sheet with silica gel GF254 with the following mobile phases: ethyl acetate: butanone: formic acid: water (50:30:10:10), ethyl acetate: formic acid: acetic acid: water (100: 11: 11:27). In paper chromatography, Watman No.1 filter paper was used as a stationary phase and mobile phases of: Acetic acid: water (15:85) and isopropyl alcohol: water (60:40).12

Spectrophotometric analysis

The isolated rutin was dissolved in methanol and its UV absorption peaks were determined and compared with standard rutin. Infrared spectrum of the isolated rutin was determined using KBr disk method.

RESULTS AND DISCUSSION

Isolated rutin showed a melting point at 194-196°C which is identical with that reported for rutin;7 the HPLC chromatograms of the aqueous fraction after extraction and isolation with chloroform and standard rutin are shown in figure 2, 3 and 4 respectively, while the Rf values of isolated and standard rutin in different mobile phases are shown in table 1.

The infrared spectrum is shown in figure 5, while the UV spectra of isolated and standard rutin are shown in figure 6. The spectrum of rutin showed two major absorption bands at 359 and 370nm which indicated the presence of flavonol structure. The first absorption maximum can be considered as originating from π-π* transitions in the ring A (aromatic system) and the second absorption maximum observed around 370nm, which may be assigned to transitions in ring B (cinnamayl system); this band appeared broad as a result of overlapping with LMCT band.13,14 The principle of this method depends on the differences in solubility between the glycoside and its aglycone.7

After concentrating the aqueous-alcoholic extract the solubility of the glycoside and the aglycones decrease and therefore they precipitate out of the solution and the mixture of precipitates is further fractionated by dissolving the aglycone in the chloroform: ethylacetate: ethanol mixture leaving the glycoside which was later on dissolved in hot methanol, and so this method of isolationdoes not need any chromatographic technique.

CONCLUSION

The flavonoid rutin can be isolated & purified from natural sources depending on the differences in solubility compared to its aglycone part particularly when rutin is the major glycoside found in the plant.

References

REFERENCES

1. The European Agency for the evaluation of Medicinal products, Committee for veterinary medicinal products "Ruta graveolens", EMEA-542, March, 1999.

2. Kostona I, Ivanova A, Mikhova B, Klaiber I; Alkaloids and comarins from Ruta graveolens. Monatshefte Chemie Fur 1999; 130(5):703-707.

3. Ekiert H, Czygan F ;. Accumulation of biologically active furacoumarins in agitated culture of Ruta graveolens L. and Ruta graveolens ssp. Divaricata. Gams Pharmazia 2005; 60(8):623-626.

4. Globol herbal supplies PTY Ltd, Crystal Brook, "Information about the herb Rue" 2009; 4800.

5. Robbers J E, Speedie M K, Tyler V E, (Eds.), Pharmacognosy and pharmaco-biotechnology, Williams and Wilkins Co., New York, 1996; 138.

6. Toker G, Turkoz S, Erdemogly N; High performance liquid chromatographic analysis of rutin in some Turkish plants II. J Chem Soc Pak. 1998; 20(4):240-243.

7. The Merck index, Merck & Co., Inc, USA, 14th edition. 2006; 8304.

8. Fathiazad F, Delazar A, Amiri R, Saker S D; Extraction of flavonoids and quantification of rutin from waste tobacco leaves. Iranian J Pharm Res. 2006; 3:222-227.

9. Alvarez C, Doroteo V H, Cobello I, Lock O; Analysis of rutin in Ruta graveolens or Ruta species using chromatographic techniques. Practica 2006; 6:5.

10. Doblas A M, Eisner A, Ventura K; Extraction of antioxidants from plants using ultrasonic methods and their antioxidants capacity. J Sep Sci. 2009; 32(2):288-294.

11. Stahl E, (Ed.), Thin layer chromatography, (2nd ed.), Oxford, London, 1969; 689.

12. Lederer E; Chromatography: A review of principles and applications, Elsevier publishing Co. (2nd edn.), 1957; 380-381.

13. Cvetkovic D, Markovic D, Cvetkovic D, Radovanovic B; Effects of continuous UV-irradiation on the antioxidant activities of quercetin and rutin in solution in the presence of lecithin as the protective target. J Serb Chem Soc. 2011; 81:1-16.

14. Uivarosi V, Barbuceanu S, Aldea V, Arama C; Synthesis, spectral and thermal studies of new rutin ranadyl complexes. Molecules. 2010; 15:1578-1589.