HPTLC FINGEPRINTING AND ANTIOXIDANT ACTIVITY OF Aloe vera

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HPTLC FINGERPRINTING AND ANTIOXIDANT ACTIVITY OF Aloe vera :

HPTLC FINGERPRINTING AND ANTIOXIDANT ACTIVITY OF Aloe vera Submitted by - Deepali R. Khangaonkar Project guide - Dr. Vinod S. Narayane

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What is Chromatography ? Chromatography is the technical separation of substance according to their Partition Coefficient in two immiscible phases. Basically, all chromatographic system consists of a stationary phase , which may be as solid or liquid that is immobilized and the mobile phase which may be liquid or gas, flows over or through stationary phase. The various components in a sample get separated essentially due to their differences in their partition or distribution behavior between the stationary phase and the mobile phase. This distribution is measured by Partition Coefficient which is defined as the ratio of concentration of compound in two phases at equilibrium.

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Types of Chromatography Planar Chromatography Column Chromatography

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What are Antioxidant? " anti" means against, in opposition to, or corrective in nature. An antioxidant is a molecule capable of inhibiting the oxidant of other molecules. Antioxidants are substances that may protect your cells against the effects of free radic als.

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Materials and Methods For phytochemical analysis of Aloe vera by TLC and HPTLC, sample was prepared by two different extraction methods. Maceration method Soxhlet method

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Different solvent systems used for TLC are : Ethyl acetate: Methanol: Distilled water(100:13.5:10) Ethyl acetate: Formic acid: Glacial acetic acid: DW(10: 1.1: 1.1: 1.1) Chloroform: Methanol(7:3) Different solvent systems used for HPTLC are : Ethyl acetate: Methanol: Distilled water(100:13.5:10) Ethyl acetate: Formic acid: Glacial acetic acid(10: 1.1: 1.1: 1.1) Derivatizing agents used for TLC/HPTLC For solvent system- Ethyl acetate: Methanol: Distilled water (100:13.5:10) derivatizing agents use is 5% Ethanolic KOH. (5gm KOH in 100ml ethanol). For solvent system- Formic acid: Glacial acetic acid(10: 1.1: 1.1: 1.1) derivatizing agents use is 5% AlCl3 in Ethanol.

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Antioxidant Activity of Aloe vera dried leaf powder can be determined by three assays Total Antioxidant Activity Nitric oxide Radical Scavenging Activity Ferric Reducing Activity

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Result and discussion HPTLC Fingerprinting of Aloe vera leaf powder extracted in different solvent by maceration method after dervitizing with 10% ethanolic KOH solution T 1 T 2 T 3 T 4 A. UV 366nm T 1 T 2 T 3 T 4 B. Visible Rf 0.5

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T 1: Ethylacetate extract T 2: Hydromethanolic extract T 3: Benzene extract T 4: Metahnolic extract

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Plate 2: HPTLC Fingerprinting of methanolic extract of Aloe vera leaf powder extracted by soxhlet method after dervitizing with 10% ethanolic KOH solution T 1 T 2 T 3 T 4 T 5 T 1 T 2 T 3 T 4 T 5 A. UV 366nm B. Visible

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T 1: Sample loaded at 10µl T 2: Sample loaded at 15µl T 3: Sample loaded at 20µl T 4: Sample loaded at 25µl T 5: Sample loaded at 30µl

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Table 1 : HPTLC Fingerprinting of Aloe vera leaf powder extracted in different solvent by maceration method after dervitizing with 10% ethanolic KOH solution. Extracts Peak No.s Ethyl acetate Hydromethanolic Benzene Methanol 1 0.29 0.08 0.09 0.07 2 0.35 0.15 0.39 0.26 3 0.71 0.25 0.67 0.40 4 0.78 0.53 0.72 0.50 5 0.85 0.61 0.77 0.61 6 - 0.62 0.83 0.63 7 - 0.68 0.91 0.68 8 - 0.82 - 0.83

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Plate 2: HPTLC Fingerprinting of methanolic extract of Aloe vera leaf powder extracted by soxhlet method after dervitizing with 10% ethanolic KOH solution Sample loaded Peak No. 10µl 15µl 20µl 25µl 1 0.07 0.07 0.07 0.06 2 0.09 0.14 0.09 0.11 3 0.11 0.17 0.19 0.17 4 0.20 0.35 0.38 0.20 5 0.24 0.41 0.44 0.36 6 0.39 0.46 0.56 0.76 7 0.45 0.48 0.74 0.78 8 0.50 0.66 0.78 0.91 9 0.57 0.75 0.91 - 10 0.68 0.79 - - 11 0.76 0.91 - - 12 0.80 - - - 13 0.93 - - -

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Total Antioxidant Activity of 1 mg of Aloe vera dried leaf powder is equivalent to 0.087mM of ascorbic acid

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NITRIC OXIDE RADICAL SCAVENGING ACTIVITY Aqueous extract significantly inhibited nitric oxide with the IC 50 value being 16.21µg/ml for standard and 17.76µg/ml for sample.

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FERRIC REDUCING ANTIOXIDANT ACTIVITY

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CONCLUSION Phytocemical analysis of Aloe vera dried leaf powder using four different solvents (Methanol, Methanol+ D/W, Ethyl acetate, Benzene) by three different solvent systems was performed. HPTLC analysis was performed by using two standardized solvent systems. Among four extract methanolic extract showed distinctive separation. For HPTLC fingerprinting two standardized solvent systems were used: Ethyl acetate: methanol: water (100: 13.5: 10). Ethyl acetate: Formic acid: Glacial acetic acid: Distilled water (10: 1.1: 1.1: 1.1)

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The distinctive separation of bands was observed by methanolic extract of Aloe vera dried leaf powder by using solvent system Ethyl acetate: methanol: water (100: 13.5: 10). Yellow bands of aloin and aloe emodin were observed at Rf value 0.50 Antioxidant activity of Aloe vera dried leaf powder was estimated by three methods total antioxidant activity, nitric oxide radical scavenging activity, ferric reducing activity and it was found to be significant.  

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