Extraction, Isolation and Characterization of Rosmarinic Acid, a Major Polyphenol in Non-volatile Constituent of Mint Leaves

G. Jayanthy and S. Subramanian

Department of Biochemistry, University of Madras, Guindy Campus, Chennai -600 025. India.

*Corresponding Author E-mail: subbus2020@yahoo.co.in

ABSTRACT:

Mint (Mentha arvensis) is one of the most extensively cultivated aromatic plants worldwide for its essential oils. The non-volatile part of the plant serves as a rich source of medicinal agents. The present study was aimed to characterise the phytocomponents responsible for its wide spectrum of biological properties. The mint leaves were shadow dried, powdered and subjected to soxhlet extraction. The extract obtained was successively fractionated by column chromatography and the fractions were purified by preparative HPLC. The major component characterised by UV, IR, two dimensional 1H and 13C-NMR and Mass spectroscopy studies was found to be Rosmarinic acid (o-O-caffeoyl-3,4-dihydroxyphenyl-lactic acid). The presence of Rosmarinic acid in mint may be responsible for the various pharmacological properties.

KEYWORDS: Mint, Rosmarinic acid, HPLC, UV-Vis, IR, NMR.

INTRODUCTION:

Consumption of medicinal herbs protects and heals a number of ailments and they have been the principal treatment therapy in prehistoric times until the discovery of synthetic drugs in the nineteenth century¹. About 40% of prescription drugs are derived from herbs² and about half of the world’s best-selling pharmaceutical preparations are derivatives of natural products. Although plants possess a number of components such as proteins, vitamins, minerals which primarily contribute to growth and development, the secondary plant metabolites like flavonoids and polyphenols are found to be responsible for the efficient disease prevention. Recently there is an accelerating interest in identifying the novel phytochemicals in medicinal plants. Among the various herbal plants, culinary herbs are becoming increasingly important for their flavour enhancing and food preserving potency in addition to their medicinal properties^3,4. Plants belonging to Lamiacea family like mint, basil, rosemary, oregano, sage and lavender are among the widely used culinary herbs around the world.

Mint is considered as one of the important spice and rich source of traditional medicine. The genus Mentha (mint) comprises approximately of 30 species which possesses a great degree of morphological and phytochemical diversity. It is the largely cultivated aromatic plant. It is a perennial herbaceous plant with toothed leaves and grows to height of about one to two feet. The leaves are simple and arranged oppositely to each other at an angle of 90 degree. The stem is quadrangular and it possesses lilac coloured flowers⁵. Each mint plant can be harvested two to three times during growing season. Harvest is done by cutting the stems one inch from the ground. It can also be harvested just by picking the leaves when needed. They grow well in moist soil. Growing mint plants calls for less care. This makes the cultivation of mint easier. The essential oil isolated from mint yields great economic importance and has a long history of usage from pharmaceutical to domestic⁶ purpose.

The aerial part of the plant is used for herbal teas and condiments⁷, as spasmolytic, anti-bacterial, stimulant, carmative, anti-spasmodic, stomachich and diuretic and is used in treatment of gas pain, rheumatism, toothache and muscle pain. Mint also acts as an effective antioxidant and recent studies document that they are a rich source of phenolic acids^8,9. Various polyphenols like Caffeic acid, Rosmarinic acid and Ferulic acid at different concentrations have been determined in mint¹⁰.

The antioxidant property of mint is attributed to its phenolic acid content¹¹. Since the past researches focussed mainly on aroma and flavour of mint, the other non-volatile components of the plant drew less attention. Recently, the useful phytochemicals of the non-volatile constituent are being investigated.

Rosmarinic acid (RA) is a Caffeic ester with a wide array of biological properties¹². Recently, it is obtaining growing interest due to its plausible medicinal values. Rosmarinic acid was found to inhibit inflammation by interfering several cell signalling pathways^13-15. In the absence of systemic reports in the literature the present study was aimed at the isolation and characterisation of Rosmarinic acid from mint leaves using different qualitative and quantitative analysis.

MATERIALS AND METHODS:

Plant Material

The mint plants were procured and authentication of the plant was carried out by a Taxonomist in the Centre for Advanced Studies in Botany, University of Madras. The leaves were shadow dried and powdered using a pulverizer.

Preparation of ethanolic extract

The powdered leaf material was defatted with petroleum ether (60-80˚C) and then extracted with 95% ethanol in a Soxhlet apparatus. The solvent was selectively removed under reduced pressure, which yields a dark green sticky residue (26.5% w/w) with respect to the dried leaves. The dried leaf extract was stored in a desiccator till further investigation.

Analytical instruments

High Performance Liquid Chromatography for determination of the compound was carried out on water HPLC C 18 (Sun Fire) column 0.8 ml/min. The detecting wavelength was 330 nm, and the moving phase was methanol/water/glacial acetic acid (55:44.9:0.1). The UV-visible spectrum of the isolated compound was recorded on a Perkin-Elmer UV-Visible spectrophotometer with quartz cells of 1 cm path length, at 25 ˚C. IR spectra studies were carried out in the solid state as pressed KBr pellets using Perkin-Elmer FT-IR spectrophotometer in the range of 400-4000 cm^-1. The mass spectrum of the complex was obtained using JeolGcmate. The ¹H NMR and C¹³NMR were obtained using Burker AM-500 instrument at 500.13 and 125.758 MHz respectively. The spectra were recorded without any correction for instrumental characteristics.

Fig 2: UV Visible spectrum of Rosmarinic acid

Isolation process

The ethanolic extract (7.6 g) was successively subjected to Reverse Phase (RP) silica gel coloumn chromatography with ethanol:water (4:6, 7:3, 1:0) as an eluent to give five fractions (A-E).

Fraction A (3.3 g) was fractionated by a silica gel CC with AcEt: EtOH (6:4) to yield five parts (A₁-A₅). 1.1g of fraction A₂was further purified using sephadex CC with EtOH.¹⁶

Characterisation of the isolated compound

The isolated compound was investigated using HPLC and various analytical spectral studies such as UV-VIS, IR, Mass and NMR.

RESULTS AND DISCUSSION:

In the column chromatography analysis, the further purification of fraction A₂ yielded about 129mg of Rosmarinic Acid. This was further confirmed by HPLC and its structural characteristics were elucidated by UV, IR, NMR and Mass spectra. HPLC analysis of the purified fraction showed that the isolated crystal component had similar retention times (approximately 8.9 min) to the Rosmarinic standard¹⁷(Fig 1). This observation provided further validation that the extracted compound is Rosmarinic acid. Similar to the previous reports, the maximum absorbance of UV spectra was obtained at 330 nm and a shoulder at 290 nm (Fig 2). The IR spectrum of the compound is shown in fig 3. The presence of OH groups is confirmed by the appearance of broad band around 3400 cm^-1to 3700 cm^-1. The band around 2920 cm^-1 depicts the presence of aliphatic stretching. The bands appearing around 1720 cm^-1 and 1640 cm^-1 is for the C=O and C=C bonds respectively. Aromatic rings are assigned to the bands appearing around 1550 cm^-1 and 1450 cm^-1. The C-O, C-C and C-H groups are depicted by the bands appearing around 1350, 1230-1080 cm^-1 and 940-670 cm^-1. The mass spectrum of Rosmarinic acid is illustrated in fig 4. The mass fragment profile at 45eV showed a very small molecular radical ion (M⁺) at m/z of 360.2 (intensity = 0.1%) which did not appear in 70eV as it was decomposed. At 45eV m/z 198 had an intensity of 10% and 8% at 70eV which belonged to C₉H₁₀O₅⁺. The base peak of m/z 123 was at 45eV and it had 75% intensity at 70eV. This corresponds to C₇H₇O₂^-.(Fig 5)

The NMR data of the compound depicting the structure of Rosmarinic Acid^18,19 (Fig 6) were as follows

Fig 6: Rosmarinic Acid

₁H-NMR (500 MHz in d6- DMSO ): δ12.90 (₁H, s, OH-18), 9.68 (₁H, s, OH-4), 9.20 (₁H,s, OH-15), 8.81 (₁H, s, OH-3), 8.75 (₁H, s, OH-14), 7.47 (₁H, d, J=16_(7,8)Hz, H-7), 7.06 (₁H, s, H-2), 7.01 (₁H, d, J=8_(6,5)Hz, H-6), 6.77 (₁H, d,J=8_(5,6)Hz, H-5), 6.69 (₁H, s, H-13), 6.64 (₁H, d,J=7.6_(14,15)Hz, H-16), 6.53 (₁H, d, J=7.6_(15,14)Hz,H-17), 6.24 (₁H, d, J=16_(8,7)Hz, H-8), 5.03 (₁H,dd, J=8.6_(17,16)Hz, J=4_(17,16)Hz, H-10), 2.99 (₁H,dd, J=14_(16,16)Hz, J=4_(16,17)Hz, H-11), 2.91(₁H,dd, J=14_(16,16)Hz, J=8.6_(16,17)Hz, H-11). (Fig 7)

₁₃C-NMR and DEPT 135˚ (125 MHz in d6- DMSO): δ171.84 (s, C-18), 166.81 (s, C 9),149.49 (s, C-15), 146.73 (d, C-7), 146.47 (s, C-14), 145.79 (s, C4), 144.85 (s, C-3), 128.28 (s,C12), 126.20 (s, C-1), 122.45 (d, C-6), 120.90(d, C-17), 117.55 (d, C-13), 116.64 (d, C-5),116.25 (d, C-1 6), 115.74 (d, C-2), 114.18 (d, C- 8), 73.84 (d, C-10), 37.01(t, C-11). (Fig 8)

As one of the major polyphenolic compound, Rosmarinic Acid is a proven potent therapeutic agent for the treatment of various ailments. RA contribute the plants of mint species to receive considerable attention for medicinal usage as they possess high content of polyphenols. Recent reports revealed the astringent^20,21, anti-mutagen,anti-tumor²², anticholinesterase²³, hepatoprotective²⁴effects of RA in addition to other important medicinal properties.

CONCLUSION:

The results of the present study establish the presence of significant amounts of Rosmarinic acid in the mint leaves. The occurrence of Rosmarinic acid in appreciable amounts in the mint leaves may account for the various pharmacological properties of the plant.

ACKNOWLEDGEMENT:

The research fellowship (UGC-BSR) of the University Grants Commission (UGC), New Delhi, India, to Mrs. G. Jayanthy is gratefully acknowledged.

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Received on 16.10.2013 Modified on 26.10.2013

Asian J. Research Chem. 6(12): December 2013; Page 1160-1165