Effect of Extraction Solvent on the Phytoconstituents of Aegle marmelos (L.) Correa

Ipomoea reniformis Chaos is claimed in Indian traditional medical practice to be useful in the treatment of epilepsy and neurological disorders. In the present study, pretreatment effect of methanolic extract of Ipomoea reniformis on epilepsy and psychosis was evaluated in rodents using standard procedures. Besides evaluating epileptic and behavioral parameters, neurotransmitters such as Gamma-Amino Butyric Acid (GABA) in epilepsy and in psychosis dopamine, noradrenaline and serotonin contents in the rodent brain were estimated. The extract pretreatment reduced maximal electro shock; Isoniazid (INH) and Pentylenetetrazole (PTZ) induced seizures and also significantly inhibited the attenuation of brain GABA levels by INH and PTZ in mice. These results suggested that the observed beneficial effect in epilepsy may be by enhancing the GABAergic system. The test drug also inhibited the apomorphine induced climbing and stereotyped behavior and showed significantly reduced levels of brain dopamine, noradrenaline and serotonin which may be due to blocking of central dopaminergic, noradrenergic and serotonergic pathways or by enhancing the GABAergic system. The results obtained in present study suggest that the title plant possesses antiepileptic and antipsychotic activities in rodents.


Introduction
Ipomoea reniformis (IR) also called as merremia emarginata (Burm. f.) is a procumbent herb belonging to the family convolvulaceae. In India, it is commonly known as Undirkana and Mushakparni. The plant is widely distributed in India, Sri Lanka, Philippines, Malaysia, Tropical Africa and mainly grows in rainy and winter season. In India, it is found in Southern part mainly counting Chennai, and some places of Andhra Pradesh [1]. Traditionally, IR has been used to treat diverse clinical conditions ranging from pain; fever to neurological disorders [2]. IR has been claimed to be useful for inflammation, headache, fever, cough, neuralgia, rheumatism and also in liver and kidney diseases [3]. The powder of leaves is used as a snuff during epileptic seizures. Juice acts as purgative and the root is having diuretic, laxative actions and applied in the disease of the eyes and gums [4].
The plant contains various neuroprotective chemical constituents such as caffeic, p-coumaric, ferulic and sinapic acid esters. Petroleum ether extract contains fats and fixed oil while aqueous extract contains amino acids, tannins (condensed and pseudo tannins) and starch [5]. IR has been reported to possess various pharmacological actions, mainly antidiabetic [6], antiinflammatory [7], nephroprotective [8], antibacterial [9], antioxidant and antimicrobial activity [10]. Further, the principle constituents of IR such as sinapic and ferulic acids have exhibited behavioural and pharmacological

Introduction
The demand for ayurvedic formulations is increasing both in the domestic market as well as internationally. The medicinal plant industry is posing great threat due to the unavailability of genuine raw drugs thereby resulting in the use of several substitutes / adulterants as the source plant. In the near future, many species may be totally unavailable for the use of industry due to over exploitation [1].
The tree Aegle marmelos (Rutaceae) commonly known as Bael is indigenous to India and found wild all over the Sub-Himalayan forests, in Central, and South India [2]. It is a rich source of coumarins, vitamin C, and riboflavin. The bark as well as fruit is reputed to be a valuable Ayurvedic medicine for dysentery and various intestinal complaints [3,4]. It possesses potentmicrofilarial [5], radio protective [6], analgesic [7], antihyperglycemic, antidyslipidemic [8], anticancer [9,10] and antidiabetic activity [11,12]. Solvent extraction is most frequently used technique for isolation of plant metabolites. However, the extract yields of the plant materials are strongly depend on the nature of extracting solvent, due to the different solubility of the chemical compounds present in it. Polar solvents are frequently employed for the recovery of polyphenols from a plant matrix. Identification of most effective extraction solvents to increase the yield of raw materials is of great importance as it helps reducing the quantity required for medicine manufacture. The possibilities of using organic solvents such as hydro alcohol instead of water for the preparation of Herbal drugs have been evaluated in the present investigation. For the present study, A. marmelos an important medicinal plant belonging to Dasamoola group, has been selected.

Collection of Plant Materials
The different parts such as root stem and fruit of A. marmelos were collected from Herb garden of Arya Vaidya Sala, Kottakkal, Kerala, India during April, 2013, and authenticated by Plant Systematics and genetic resources division of Centre for Medicinal Plants Research, Arya Vaidya Sala, Kottakkal, Kerala. The voucher specimens were deposited in CMPR Herbarium.

Chemicals
Folin-Ciocalteu reagent was procured from Sisco Research Laboratory (SRL), Mumbai, India, Gallic acid and quercetin were procured from Sigma Chemicals Co. (Bangalore, India). All other chemicals employed were of standard analytical grade from Merck India.

Extraction
The shade dried ground plant materials such as root, stem and fruit (5 g for each sample) were extracted with 250 ml each of the solvents -absolute ethanol, absolute methanol, aqueous ethanol (ethanol: water, 80:20 v/v), aqueous methanol (methanol: water, 80:20 v/v) hydro alcohol (ethanol: water 50:50) and water for 6 hours by reflux method. The extracts were concentrated into dryness in a rotary evaporator at 40 0 C under reduced pressure. The yields of extracts were calculated. 10 mg of each extract was dissolved in 10 ml of respective solvents and these extracts were used for total phenolic and flavonoid estimations.

Estimation of Polyphenolic Compounds
The total phenolic content (TPC) was determined spectrophotometrically using Folin-Ciocalteu reagent [13]. Different Gallic acid standards were used for obtaining a standard curve. TPC was expressed as gallic acid equivalents (GAE) in mg / g of sample. Total flavonoid content (TFC) was measured by aluminium chloride colorimetric assay [14]. The flavonoid content was calculated from the calibration curve of standard quercetin. TFC was expressed as mg quercetin equivalents (mg EQ). The absorbance against the reagent blank was determined at 550 nm with UV-Visible spectrophotometer (Pharmaspec-1700, Shimadzu, Japan).

Thin Layer Chromatographic Analysis
Thin Layer Chromatography (TLC) Profiling of different extracts of Aegle marmelos was carried out on a precoated silica plate (F 254 Merck) using toluene, ethyl acetate and methanol as mobile phase in the ratio of 8:2:0.5. Two marker compounds, umbelliferone and scopoletin (Sigma Aldrich, Bangalore, India) were also spotted for their identification in the extracts. The developed plate was visualized and documented at UV 254 nm and 366 nm.

Percentage of Yield of Extracts
The efficiency of extraction is influenced by several parameters such as nature of phytochemicals, the method used for extraction, particle size, the solvent used, as well as the effect of nosy substances [15]. The percentage yield of extracts is given in Table 1. The yield of extract varied with extraction solvents. The variation was also observed with respect to parts used. Hydro alcohol showed maximum yield for all the extracts. The use of aqueous alcoholic solvent may facilitate the extraction of chemicals that are soluble in water and/or organic solvent. This may be the reason why yields of hydro alcohol extracts are higher [16]. Hydro alcoholic extract of fruit showed highest extractive value (32.06%) whereas absolute methanolic stem extract showed minimum yield (2.82%). The variation of yield of extracts compared with water extract is given in Fig. 1. When compared to aqueous extract, root showed an increase of 42.05% in hydro alcohol. This supports the possibilities of using hydro alcohol as extraction solvents in Ayurvedic medicine manufacturing industry and which may lead to the judicious use of raw materials especially where the roots are useful parts.

Total Phenolics (TPC) and Total Flavonoids (TFC)
Total polyphenolic content was estimated spectrophotometrically using gallic acid and Quercetin as standards for plotting calibration curve (Fig. 2 & 3). Efficiency of solvents and methods is strongly dependent on plant matrix used for extraction. Solvents, such as methanol, ethanol, acetone, propanol and ethyl acetate have been commonly used for the extraction of phenolics [17]. The properties of extracting solvents significantly affected the measured total phenolics content. The highest extract yields (up to 22.8%) were obtained with polar alcohol based solvents [18]. The present reports also showed that alcohol based extracts contain more phenolics.
The total phenolic and total flavonoid contents of various extracts of root of A. marmelos is given in The phenolics and flavonoids of various fruit extracts are presented in Table 4. The highest TPC was observed     Solvents, such as methanol, ethanol and their combinations with different proportions of water have been used for the extraction of phenolics from plant materials [19]. The present results were also showed that the aqueous based solvents are the most effective extraction solvent for polyphenols.

Comparative TLC Profiling
The chemical pattern of different extracts was compared using TLC profiling. Variations were observed in terms of number of bands and band intensity which indicate the qualitative and quantitative divergence in chemical constituents.
On visualizing under UV-254 (Fig. 4), a compound with R f 0.14 was observed in all the extracts of fruit and it was absent in stem and root. The band at 0.17 is specific for root extracts and the intensity is more for absolute ethanol and absolute methanol extracts. The compound with R f 0.27 is present only in stem extracts. A compound at R f 0.33 was observed in aqueous ethanolic and aqueous methanolic extracts of stem. A band with R f 0.38 was common for all the extracts of root and the same was also seen in absolute ethanol and absolute methanol extracts of stem. A compound at 0.61 was observed only in aqueous methanol and methanol extract of fruit.
Under UV 366 (Fig. 5) major compounds were observed at R f 0.17, 0.26, 0.33, 0.44, 0.48, 0.52, 0.70, 0.78 and 0.81. The differences in the intensity of bands indicate the quantitative variations of phytoconstituents in different extracts. Umbelliferone , the so far reported marker compound, is present in almost all the extracts with varying band intensities, but the same was found to be absent in aqueous extract of fruit. Scopoletin, a known coumarin was found to be present in all the extracts of root and stem.
1: aqueous ethanol extract of root, 2: aqueous methanol extract of root, 3: absolute ethanol extract of root, 4: absolute methanol extract of root, 5: hydro alcohol extract of root, 6: aqueous extract of root.

Conclusion
The results of the present studies revealed that hydro alcoholic extract exhibited better yield with comparable chemical constituents with that of aqueous extract. Possibility of reducing the quantity of raw drugs by 40 % can be explored if raw drugs are being extracted with hydro alcohol (ethanol: water 50:50) instead of water. In the case of root, the higher quantities of individual components are found in absolute alcoholic (both ethanol and methanol) extracts. But the banding pattern of aqueous extract is matching with that of hydro alcoholic extract. Two coumarins umbelliferone and scopoletin, so far reported markers in Aegle marmelos, were detected in all the extract except aqueous extract of fruit. Among the six solvents used for the extraction, hydro alcohol showed the maximum yield of extract. Contrary to extraction yield, the quantity of individual components was more in aqueous and absolute alcohols, both ethanol and methanol.