Anti-HIV Activity of Phytosterol Isolated from Aerva lanata Roots BY Rajendra Prasad Gujjeti and Estari Mamidala

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Background: AIDS in humans, which is caused by the HIV remains among the leading causes of death worldwide. Objective: The study was undertaken to investigate the HIV-RT inhibitory activity of Phytotesrols extracted from roots of Aerva lanata. Methodology: Sequential maceration method was performed for preparation of extracts using hexane, chloroform, ethyl acetate, acetone and methanol solvents. RetroSys HIV-1 RT (Innovagen, Sweden) kit was used to determine the HIV-RT inhibitory activity of all solvents extracts. Results & Discussion: All extracts showed signifcant inhibitory activity. Chloroform extracts shows highest inhibition of recombinant HIV-RT (89.0%) at 2 and mg/ml concentration. Phytosterols isolated from chloroform extracts. These results were remarkable and can be used to develop a new drug for HIV treatment or other infectious diseases caused by the pathogenic micro-organisms, for which plant sterols may play a distinctive role. Conclusion: Further work is in progress to evaluate the advanced spectroscopic studies are required for the structural elucidation, identifcation and characterization of the active compound from this plant. Key words: Aerva lanata, HIV-1, Phytosterols, Reverse transcriptase, Characterization, Structural elucidation
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  Pharmacogn J. 2017; 9(1):112-116 A multifaceted peer reviewed journal in the field of Pharmacywww.jyoungpharm.org | www.phcog.net Pharmacognosy Journal, Vol 9, Issue 1, Jan-Feb, 2017   112 Original Article INTRODUCTION Human immunodeficiency virus type 1 (HIV-1) in-ection continues to spread throughout the world despite a downward trend o HIV new inections, an estimated 35.3 million people are currently living with this virus. 1  Now a days, a large variety o natu-ral products rom medicinal plants, such as alkaloids, phytosterols, flavonoids, lignans, polysaccharides and so on, have been ound to inhibit unique en-zymes and proteins crucial to the lie cycle o HIV, including the reverse transcription process,  virus entry, the integrase or protease. 2-4  Herbal medi-cine is now globally accepted as a legal, alternative system o therapy or treatment and cure o various diseases and physiological conditions in traditional treatments in the orm o pharmaceuticals. 5  Previ-ous investigations established that different medicinal plant extracts inhibit HIV reverse transcriptase in non-specific manner. 6,7 Phytochemical analysis con-ducted on the plant extracts revealed the presence o constituents which are known to exhibit medicinal as well as physiological activities. 8  Te medicinal value o plants lies in some chemical substances that have a definite physiological action on the human body. Di-erent phytochemicals have been ound to possess a wide range o activities. 9  Tereore, screening o po-tential anti-HIV agents rom medicinal plants may be a rapid and effective way or drug discovery. However, drug toxicity, drug resistance, adverse drug–drug interactions, and accompanying poor patient adher-ence are still the major actors leading to treatment ailure. 10  Tere is still an acute need or less toxic and more potent HIV drugs and continues be the concern. Te aim o research in HIV drug development is to find new drugs that are specific to HIV, or to develop a method that alters the nature o the drug adminis-tered such that it acts only on the target cells and not the regular normal unctioning cells, thereby reduc-ing the side effects. Te present study was to evaluate the HIV reverse transcriptase inhibitory activity o phytosterol o  Aerva lanata roots. MATERIAL AND METHODS Plant collection Plant was selected or this study is based on its tradi-tional medicinal use. 11    Aerva lanata roots were   col-lected rom the Chintur mandal, Khammam district o elangana, India, in the month o September 2012. Te plant voucher specimen identification was done with the help o taxonomist Pro. Vastsavaya. S. Raju Department o Botany Kakatiya University, Warangal and the same was deposited at Inectious Diseases & Metabolic Disorders Research Lab, Department o Zoology, Kakatiya University, Warangal. Preparation of plant extract Afer collection o selected medicinal plant mate-rial sample was dried at room temperature until they were ree rom moisture. Te selected part o plant subjected to size reduction to get coarse powder was then stored in a clean dry air tight container. Te air dried powder was subjected to sequential maceration method used by different solvents (hexane, chloro-orm, ethyl acetate, acetone, and methanol etc ;) or seven days. Te extract was filtered mass was ob-tained and it was finally dried at low room tempera-ture under pressure in a rotary vacuum evaporator (Termotech, buchi type model th-012). HIV-1 Reverse Transcriptase Inhibition Assay Te HIV reverse transcriptase enzyme in-hibition due to each extract was deter- Anti-HIV Activity of Phytosterol Isolated from  Aerva lanata Roots Rajendra Prasad Gujjeti and Estari Mamidala * ABSTRACT Background:  AIDS in humans, which is caused by the HIV remains among the leading causes of death world-wide. Objective:  The study was undertaken to investigate the HIV-RT inhibitory activity of Phytotesrols ex-tracted from roots of Aerva lanata  . Methodology : Sequential maceration method was performed for prepara-tion of extracts using hexane, chloroform, ethyl acetate, acetone and methanol solvents. RetroSys HIV-1 RT (Innovagen, Sweden) kit was used to determine the HIV-RT inhibitory activity of all solvents extracts. Results & Discussion:  All extracts showed significant inhibitory activity. Chloroform extracts shows highest inhibition of recombinant HIV-RT (89.0%) at 2 and mg/ml concentration. Phytosterols isolated from chloroform extracts. These results were remarkable and can be used to develop a new drug for HIV treatment or other infectious diseases caused by the pathogenic micro-organisms, for which plant sterols may play a distinctive role. Conclu-sion:  Further work is in progress to evaluate the advanced spectroscopic studies are required for the structural elucidation, identification and characterization of the active compound from this plant. Key words : Aerva lanata, HIV-1, Phytosterols, Reverse transcriptase, Characterization, Structural elucidation. Cite this Article: Gujjeti RP, Mamidala E. Anti-HIV Activity of Phytosterol Isolated from Aerva lanata   Roots. Pharma-cogn J. 2017;9(1):112-6. Rajendra Prasad Gujjeti and Estari Mamidala * Department of Zoology, Infectious Diseases and Metabolic Disorders Research Lab, Kakatiya University, Warangal-506009, INDIA. CorrespondenceEstari Mamidala, Department of Zoology, Infectious Diseas-es & Metabolic Disorders Research Lab, Kakatiya University, Warangal-506009, Telangana, INDIA.Phone no: 9848309231 E-mail:  estari08@gmail.comHistoryã Submission Date: 04-07-2016; ã Review completed: 22-08-2016; ã Accepted Date: 10-09-2016. DOI :   10.5530/pj.2017.1.19Article Available online http://www.phcogj.com/v9/i1 Copyright © 2016 Phcog.Net. This is an open- access article distributed under the terms of the Creative Commons Attribution 4.0 International license.  Estari : Anti-HIV Activity of Phytosterol Pharmacognosy Journal, Vol 9, Issue 1, Jan-Feb, 2017   113 mined using HIV R inhibition assay by using o Retro Sys HIV-1 R activity kit (Innovagen, Sweden). o determining R activity on inhibiting substances that are to be analysed are serially diluted. Te diluted substances are then added to a plate with reaction mixture. Afer 30 minutes o pre-incubation at 33°C, the reaction is started by the addition o a standardised amount o R. Te R will now incorporate BrdUMP depending on the level o inhibition. Te product is quantified by the addition o the R Product racer which binds to the incorpo-rated BrdUMP. Afer removing excess tracer the amount o bound tracer is determined by an alkaline phosphatase/pNPP colour reaction. 1  Afer correction or background signal, the measured residual R activity or each substance dilution is calculated as a percentage o the measured R activity in absence o inhibiting substances. Plot the percentage o residual R activity against the concentrations o the substance dilutions or each o the tested substances. AZ (Azidothymidine) was used as control. Te inhibitory effect o each substance is expressed by R activity and is determined with the aid o the obtained graph. All ractions were selected or anti-HIV activity. Te percentage inhibition o HIV-1 R was calculates as,Inhibition (%) = [(A control-A sample)/A control] × 100Where, A is Optical Density (OD). Isolation of active compound from  Aerva lanata  crude extract Thin layer chromatographic studies LC as per conventional one dimensional ascending method using silica gel 60F254, 7×6 cm (Merck) were cut with ordinary household scissors. Plate markings were made with sof pencil. Glass capillaries were used to spot the sample on LC volume 1 µl by using capillary at distance o 1 cm at 1 track. o find which solvent system is suitable or separation o compounds, the five solvent systems were selected. Tese are solvent system I (hexane: acetic acid 9:1), solvent system II (hexane: ethyl acetate: acetic acid 5:4:1), solvent system III (hexane: ethyl acetate: acetic acid 4:4:2), solvent system IV (hexane: ethyl acetate: acetic acid 3:6:1) and solvent system V (hexane: ethyl acetate: acetic acid 2:7:1). Te chloroorm extract was applied on pre-coated LC plates and developed in LC chamber. Te developed plates were air dried and iodine vapors were used to detect the bands on the LC plates. In this study, the LC chromatograms were developed in five eluent systems o different polarity. Finally solvent system V (hexane: ethyl acetate: acetic acid 2:7:1) was selected or column chromatography. Te movement o the active compound was expressed by its retention actor (R    ), values were calculated or different samples by ollowing equation: R   = Distance travelled by the solute/Distance travelled by the solvent ront LC plates Column chromatographic studies Te column can be prepared using a column chromatography flask. Glass wool was inserted at the bottom o the flask to prevent the silica rom escaping the column. Te mixture o hexane: ethyl acetate: acetic acid in the volume ratio (2:7:1) used in LC was used as the mobile phase. 4 gm o plant active crude extract was added to 8 gm o silica gel. Tis was then added to a packed column (silica gel packed with hexane which is the least polar in the solvent mixture). Te selected mobile phase (hexane: ethyl acetate: acetic acid 2:7:1) was continuously poured to the top with the aid o a dropper. Te bottom outlet o the column was opened, allowing the eluent to flow through the column. As the eluent passed down the column, the compound raction moved down the column. Te separated raction flowed out o the column where the different eluates were collected in separate test tubes. Tis was repeated until all the dissolved extract was adsorbed on to the silica gel.Te total o 24 ractions was collected within 6 hr 15 min each raction containing 5 ml. All ractions were concentrated under rotary reduced pressure evaporation rotating at 100 rpm and with water bath at 40°C (Termotech, buchi type model th-012). All ractions were spotted on LC plate and on visualized using iodine vapors. Te R     values o 24 ractions were recorded and same R     value ractions were pooled. 24 ractions were reduced in to three categories afer pooling. Anti-HIV activity o these three ractions was done by Retro Sys HIV-1 R activity kit (Innovagen, Sweden). Active ractions were tested or LC to detect the purity. For urther purification this active raction processed to column chromatography by using solvent system chloro-orm and ethyl acetate in the ratio o 9:1. Six ractions were collected and pooled based on similar R     values. Six ractions were reduced in to three ractions. Tree ractions tested or anti-HIV activity. Active raction is selected or urther purification process. Column chromatography was done on above mentioned active raction by using solvent system chlo-roorm and ethyl acetate in the ratio 8:2. Finally single spot was obtained in LC o active raction. Phytochemical screening of active compound Qualitative phytochemical analysis to detect the presence o alkaloids, carbohydrates, glycosides, saponinis, phytosterols, phenols, tannins, flavonoids, proteins, aminoacids and diterpens was carried out by using the standard methods and the intensity o the coloration determines the abundance o the compound present. 2 RESULT AND DISCUSSION Percentage of yield extract Te yield o sequential extracts  Aerva lanata  (g) is shown in (able 1). Te amount obtained rom hexane, chloroorm, ethyl acetate, acetone and methanol extracts are 5.020 gm, 4.080 gm, 2.750 gm, 1.720 gm, and 3.750 gm respectively. Anti-HIV activity of  Aerva lanata  root extracts Inhibition o HIV-R by  Aerva lanata  root extracts were presented in Figure 1. Chloroorm and methanol extraction shows highest inhibition o recombinant HIV-R (91.0% and 89.0% respectively) at 2 mg/ml concentration. Hexane, ethyl acetate and acetone extractions showed highest inhibition o HIV-R at 2 mg/ml concentration (86.9, 85.2 and 77.5 respectively). While control drug (AZ) showing 91.7% at 2 mg/ml concentration.ALHE -  Aerva lanata  Hexane ExtractALAE -  Aerva lanata  Acetone ExtractALCE -  Aerva lanata  Chloroorm ExtractALME -  Aerva lanata  methanol ExtractALEAE-  Aerva lanata  Ethyl acetate Extract Table 1: Extractive values of different extracts of  Aerva lanata  roots SolventColor of extractYield of theextract (in gm)Percentage yield (%w/w) HexaneWhite5.0202.51%ChloroormLight brown4.0802.04%Ethyl acetateLight brown2.7501.37%AcetoneLight brown1.7200.86%MethanolDark brown3.7501.85%  Estari :Anti-HIV Activity of Phytosterol 114   Pharmacognosy Journal, Vol 9, Issue 1, Jan-Feb, 2017 AZ -  Azidothymin Thin layer chromatographic analysis Te LC chromatograms o the chloroorm extract o  A. lanata in different solvent systems were shown in solvent system I, 2 spots were visible with R     values 0.18 and 0.94. In solvent system II, 3 spots were detected with R     values 0.10, 0.82 and 0.90. In solvent system III, 2 spots were detected with R     values 0.05 and 0.90. In solvent system IV, 2 spots were visible with R     values 0.09 and 0.78. In solvent system V, 2 spots were obtained having R     o 0.24 and 0.35 (able 2). Solvent system V was selected or urther process o separation. Column chromatographic analysis   Te solvent system V used as the eluent while silica gel was used as the solid phase. A total o 24 ractions were eluted rom the roots o  Aerva lanata.  All ractions were spotted on LC plate and on vi-sualized using iodine vapors and the ractions (3-8, 9-20 and 21-24) were pooled into three main categories based on their similar retardation  values showed in able 3. F9-F20 pooled raction shows highest anti-HIV activity. Tereore this raction sample was processed urther purification step. Above active raction sample based on column chromatography by using solvent system chloroorm and ethyl acetate in the ratio o 9:1six sub ractions Figure 1:   In vitro  HIV-RT inhibitory activity of  Aerva lanata  root extractions Table 2: R f   values of TLC in different solvent systems Extract nameSolvent system ISolvent system IISolvent system IIISolvent system IVSolvent system V No. o spots detectedR    ValueNo. o spots detectedR    ValueNo. o spots detectedR    ValueNo. o spots detectedR    ValueNo. o spots detectedR    ValueChloroormextract20.180.9430.100.820.9020.050.9020.090.7320.240.35 Table 3: Eluted fractions and their masses, Number of spots detected and R f values Compound fractionMass(g)No. of spots detectedR f   valuesCompound fractionMass(g)No. of spots detectedR f   values Fraction 10.02--NilFraction 130.01020.32, 0.51Fraction 20.15--NilFraction 140.07020.32, .051Fraction 30.22020.35, 0.81Fraction 150.09020.32, 0.51Fraction 40.03020.35, 0.82Fraction 160.11020.32, 0.50Fraction 50.05020.35, 0.82Fraction 170.02020.32, 0.51Fraction 60.32020.35, .081Fraction 180.05020.31, 0.51Fraction 70.14020.31, 0.51Fraction 190.18020.32, 0.51Fraction 80.11020.32, 0.51Fraction 200.23020.32, 0.51Fraction 90.01020.32, 0.51Fraction 210.05020.55, 0.77Fraction 100.04020.32, 0.51Fraction 220.08020.55, 0.77Fraction 110.13020.32, 0.50Fraction 230.02020.55, 0.77Fraction 120.03020.32, 0.51Fraction 240.06020.55, 0.77  Estari : Anti-HIV Activity of Phytosterol Pharmacognosy Journal, Vol 9, Issue 1, Jan-Feb, 2017   115 be an increasingly valuable reservoir o bioactive compounds o substantial medicinal merit. Te phytosterols produce a wide spectrum o biological activities in animals and humans and are particularly considered an efficacious cholesterol-lowering agent. Te anticancer activity o plant sterols has been reviewed by Awad and Fink. 12  Bouic 13  has reviewed the potential role o phytosterols both in the etiology as well as in the prevention o immunological diseases. Experiments in animals and cell cultures indi-cated that plant sterols may exert biological activities unrelated to the hypocholesterolemic effect. In experiments in vitro the substitution o cholesterol with phytosterols altered the physical properties o cell mem-branes, which could affect the activity o membrane-bound enzymes or signal transduction. 14,15 CONCLUSION Te study showed that  Aerva lanata  plant contained compounds o medicinal importance. Tis phytochemical screening and analysis revealed that the phytosterols are mainly present in the active compound. Te were collected (able 4) and tested or LC. Based on similar R    values SF1-SF2, SF3-SF4 and SF5-SF6 pooled and these pooled ractions were tested or anti-HIV activity. SF3-SF4 sub raction showed highest anti-HIV activity. Tereore the SF3-SF4 sample was processed urther purification step. On LC SF3-SF4 sub raction shown single spot. Tis determined the compound obtained was a single compound. Analysis of anti-HIV activity of active compound Te pooled 9-20 raction showing highest inhibition o recombi-nant HIV-R (88.2%) at 2 mg/ml concentration. Te other ractions 21-24   and 3-8 are showing highest inhibition o recombinant HIV-R (61.5 and 58.0% respectively) at 2 mg/ml concentration. Tereore concluded that the F9-F20 raction sample could exhibit potent activity. While control drug (AZ) showing 91.7% at 2 mg/ml concentra-tion. In the second purification step the obtained sub ractions SF3-SF4 showing highest inhibitions o recombinant HIV-R (89.0%) at 2 mg/ml concentration. Tereore the sub raction sample SF3-SF4 could exhibit potent activity against anti-HIV.Obtained raction sample in final purification step bring single compound and this sample shows anti-HIV activity nearly similar to used control drug (AZ) showed in Figure 2. Tis raction showed 88.1% HIV-R inhibition at 2 mg/ml, while AZ showed 91.7% at 2 mg/ml respectively. Phytochemical analysis of active compound In this study showed that  Aerva lanata  plant contained compounds o medicinal importance. Tis phytochemical screening and analysis revealed that the phytosterols are mainly present in the active compound. Te results obtained in this study thus suggest the identified phytochemical compounds may be the bioactive constituents and this plant proving to Table 4: Eluted six fractions from F9-F20 their masses, Number of spots detected and R f values Compound fractionMass (g)No. of spots detectedR f   values SF 10.10020.31, 077SF 20.16020.31, 0.77SF 30.14020.35, 0.65SF 40.09020.35, 0.65SF 50.22020.32, .055SF 60.18020.32, 0.55 Figure 2:   In vitro  HIV-RT inhibitory activity of single compound (from SF3-SF4) of  Aerva lanata  chloroform extract Table 5: Phytochemical analysis of isolated active compound of  Aerva lanata  root extract Sl. No.Phytochemicals testActive compound 1Test for Alkaloids aMayer’s est - bWagner’s est - cHager’s est - dDragendorff’s est -2Test for Carbohydrates aMolisch’s est - bBenedict’s est - cFehling’s est -3Test for Glycosides aBorntrager’s est - bLegal’s est -4Test for Saponin aFroth est - bFoam est -5Test for Phytosterols aSalkowski’s est + bLibermann Burchard’s test +6Detection of phenols aFerric Chloride est -7Detection of tannins aGelatin est -8Detection of flavonoids- aAlkaline Reagent est - bLead acetate est -9Detection of proteins and aminoacids aXanthoproteic est - bNinhydrin est -10Detection of diterpenes- aCopper acetate est -
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