Separation of Methanolic Leaf Extracts of Three Rutaceous Plants by Capillary Electrophoresis and High-Performance Liquid Chromatography Methods

Introduction: In the phytochemical analysis of various multi component mixtures, Capillary electrophoresis (CE) method was considered as the best alternative tool due to high efficiency resolution separations, low solvent consumption and less maintenance costs. Materials and Methods: This study uses CE and high-performance liquid chromatography (HPLC) methods to evaluate the major components of methanolic leaf extract of Rutaceous plants. And also, to assess the purity of the components in isolated fractions and most importantly for the qualitative identification of the individual components of extracts based upon the relative retention time and UV profile. Results: The separation of the compounds in HPLC was attained by using ACE-5-C 18 (250 x 4.6 mm) with a flow rate of 1.5 ml/min, with the UV detection at 254 nm and CE was equipped with a UV diode-array detector and bare, fused silica capillaries (56 cm 50 μm) with micro extended light path were used for the separations. Conclusion: Our study reveals that CE technique is a simple, economical and convenient method and shown high-resolution for multi-components found in plant extracts compared with HPLC profiles.


INTRODUCTION
Medicinal plants are the major sources for finding and identification of new compounds which may be having different biological activities.Based upon the traditional healers claim, some species of Rutaceae family demonstrated wide variety of medicinal properties in different parts of the world. [1]Clausena excavate is one of the important plant of Rutaceae family available in many continents [2] provides good source for isolation and identification of number of compounds and tested for various biological activities.It was observed that various parts of the plant namely roots, leaves, stems, barks, fruits, flowers and twigs are used for medicinal purpose, some of them are used as for food. [3]ytochemical survey reveals that wide range of secondary metabolites alkaloids, [4] Coumarins, [5] Pyrano-coumarins, [6] limnoids, [7] essential oils, [8] insecticides [9] were isolated from the different species of Rutaceous plants.Pharmacological survey reveals that researchers around the world identified anticancer, [10] antibacterial, [7] antifungal, [7] anti-HIV, [11] immunomodulatory, [12] antinociceptive, [13] antimalarial, [14] insecticidal [9] activities of extracts and isolated compounds.However, some of the species of Rutaceae family namely Tetractomia roxbhughiana and Glycosmis calcicola are not completely elucidated and this forms the basis of our study.
High Performance Liquid Chromatography (HPLC)is used for the separation, identification and quantification of different secondary metabolites in crude plant extracts, however method development and subsequent isolation procedures are found to be expensive. [15]In the phytochemical analysis of various multi component mixtures Capillary electrophoresis (CE) method was considered as the best alternative tool due to high efficiency resolution separations, low solvent consumption and less maintenance costs. [16]e present investigation was aimed at identification of major constituents of three Rutaceous plants using CE and HPLC methods and comparison of the resolution profiles of two methods and also weigh advantages and disadvantages of each method.
CE has been used for a long time as a highly efficient and alternative technique to HPLC in areas such as pharmaceutical analysis.It is widely used to separate a complex array of large and small molecules like protein, peptides, urine metabolites etc.According to literature reports [17] This technique is used for analysing extracts of leaves, bark, roots and marine organisms.Phytochemical survey shows that CE and Micellar electro kinetic chromatography (MEKC) techniques were used for the separation of wide range of diversified chemical compounds like saponins, [18] coumarins, [19] alkaloids, flavonoids, [20] isoflavonoids, steroids from different plant sources.Seven coumarin derivatives were resolved with MEKC using borate / phosphate buffer at pH 7. [21] Flavonoids are widely spread in most of the plant families, separation of a mixture of sulphated flavanoids and flavonoids, (-)-epicatechin sulphate (ECS), (+)-catechin sulphate (CS), quercetin sulphate (QS), Apigenin sulphate (AS), and 6, 2, 3-flavonoid sulphate (FS) was reported. [20]And there were many researchers reported the separation of saponins by using capillary zone electrophoresis [18,22,23] in different conditions.Using CE in both its capillary zone electrophoresis (CZE) and MEKC modes allows the analyst to resolve both ionic (by CZE) and neutral (by MEKC) compounds on the same capillary using buffers with or without an organic modifier, a micelle or cyclodextrin. [21,24]e present investigation was aimed to use the capillary electrophoretic technique to get high-resolution profiling of extracts and in particular to assess the purity of "semi-purified" or preparative LC-purified fractions of extracts.And compare the resolution profile of the HPLC and CE methods.

Plant material
The leaves of Rutaceous plants Clausena excavate, Tetractomia roxbhughiana and Glycosmis calcicola were collected from the northern regions of Malaysia and their identity and authentication was done by Prof. Bowen, Department of Pharmacognosy, University of Sunderland, and a specimen sample was deposited in the herbarium.The plant materials were dried well under shade and powdered using an electric blender.

Preparation of crude extract
The extraction of the powdered leaf material was successively carried out with petroleum ether, hexane, chloroform, dichloromethane, ethanol, and water.In each case 500 ml of the solvent used for extraction.After each successive extraction, and before the extraction with another solvent, the powdered material was air dried and weighed.Maximum extractive value is achieved with methanol.The methanolic leaf extract was used for further fractionation by HPLC and CE.Samples were prepared by dissolving 1 mg of the dried extract in 1 mL of HPLC grade methanol and passed through 0.45 m Acrodisc syringe filters (Pall Corporation, Ann Arbor, MI, USA).Sample injections were freshly prepared by dissolving a small amount of the stock solution with methanol before injection. [25]
Bare, fused silica capillaries (56 cm 50 μm) microextended light path were used for the separations.Sample and buffer solutions were passed through 0.45 m Acrodisc syringe filters (Pall Corporation, Ann Arbor, MI, USA) prior to use.In capillary electrophoresis, the medium consists of a solution of buffer salts adjusted to a desired pH.This buffer solution is contained within a fused silica capillary.When a current is applied, cations in the middle are migrated towards the cathode.These hydrated cations will drag aqueous buffer with them towards cathode.Then the uncharged species move at the same speed as the electro osmotic force, positively charged ions move faster and negatively charged ions move slower.CE system comprised of f two buffer reservoirs, a detector, a capillary with a transparent detection window, and a high voltage power supply.Commonly the sample is introduced into the capillary by hydrodynamic or electro kinetic means.The separation voltage applied was 14 kv and the temperature was maintained at 25°C.This involves either applying a small positive pressure in the inlet or a potential difference across the capillary.

RESULTS AND DISCUSSION
Generally, plant extracts contain complex multi-component mixtures.To resolve these, it was planned to use high-resolution separation techniques like CE as well as the LC approaches so as to better assess the number of phytoconstituents in isolated fractions, the purity of components and also ultimately qualitative identification of the individual constituents based upon the relative retention times and more importantly with UV profile.This technique is very convenient and has more resolving power, which is quite essential for analyzing multi-component mixtures usually found in plants. [1]Due to its high resolving power, it can overcome the problems of non-baseline resolution associated with HPLC as shown in Figure 1-3.The main disadvantage of using CE analysis was that it could not be used for preparative work but doing capillary electrophoresis preparative work had never remotely been a consideration.
The main problem encountered using HPLC of plant extracts and fractions was that with the higher loads, the resolution achieved with the micro injections could not be seen in the higher loads due to the complex nature of plant extracts and multi-component mixtures.It was clearly desirable to search for a high-resolution technique so that it could be used to resolve all the components of plant extracts and also to assess the purity of the components.With the selection of different MEKC buffers, the first aim was to know how the buffers performed when changes in experimental variables were made.It was observed that in the case of cholate buffer good resolution was obtained but the main disadvantage was the long migration times.However, the initial aim was to establish resolution so that, after achieving this, higher voltages would then be applied to shorten the migration times of the components (Figure 3).
Based on the investigation it was concluded that the cholate buffer as shown in Table 1 gave better resolution and peak sensitivity whereas, at the other end of the scale, SDS buffer blocked the capillaries.Monitoring of reaction purity was assessed by capillary electrophoresis and the electropherograms were shown in Figure 4-6.metabolites.Separation of standard mixtures of flavonoids namely rutin, 2,6-dihydroxyflavoneand quercetin as shown in Figure 7, quinolids, steroids and other mixtures were established and it was observed that, in general, alkaloids came first followed by flavonoids as shown in the (Table 1, illustrations were shown in Figure 8-13).CE separations, using the optimum cholate / SCD buffer, of methanolic leaf extracts of Rutaceous plants were carried out and compared with separation with that of the HPLC profile.CE had given higher resolution as expected, but still the non-polar components had not been resolved, particularly the standard steroidal compounds, with MEKC buffers.Nonetheless a suitable generic MEKC system, along with some possible alternatives, had been established and this was used to study the composition of other extracts, both semi-purified and purified.CE and MEKC provided high-resolution separations     for the methanolic leaf extracts of three Rutaceous plants when compared with their HPLC profiles.Although CE with UV photodiode array detection is a good confirmation method of known compounds.CE-MS is a good characterisation method of unknown compounds.At this point capillary electrophoresis method development for identification of plant metabolites is confined to the achievements of high-resolution separations, and establishment of migration times of common known typical plant metabolites have been achieved.In the case of plant metabolites no single buffer is capable of eluting all the classes of compounds in three buffer used (as shown Figure 1).This area needs further investigation.Alkaloids best resolved in sodium cholate system (B3 buffer), in B2 buffer alkaloids are not resolved properly.The migration times are quite high in three buffers run.

Initial work on plant extracts was performed by running standard mixtures of typical plant metabolites for the comparison of these buffers with respect to the migration of different classes of plant
One more problem encountered for the identification of plant metabolites was that UV profiles could not be established for all the eluted peaks; this area still needs to be investigated.However, the combined use of the profiles of HPLC and MEKC that gave anidea about the efficiency of preparative separation.With these CE experiments, it could be said that polar compounds had been easily eluted whereas, non-polarcomponents elution was needed to be further investigated.Further work could be carried out by altering the composition of sulphated beta cyclodextrin in MEKC method, or by looking at non-aqueous CE.HPLC (ACE-5-C18 (250 x 4.6 mm) with a flow rate of 1.5 ml/ min, with the UV detection at 254 nm and CE was equipped with a UV diode-array detector and bare, fused silica capillaries (56 cm x50 μm) with micro extended light path were used for the separations.In this study, the results were confirmed that the CE is a useful complimentary alternative to the LC approaches for profiling of plant constituents.This is the first ever approach to explore the separation ability of the technique for typical secondary metabolites found in plants.However further research is required to optimize various separation parameters.

Figure 11 :
Figure 11: Migration time of mixture of Alkaloids.

Figure 13 :
Figure 13: Migration time of other metabolites.