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Phytochemical Analyses and Evaluation of Antioxidant Efficacy of in vitro Callus Extract of East Indian Sandalwood Tree (Santalum album L.)

Phytochemical Analyses and Evaluation of Antioxidant Efficacy of in vitro Callus Extract of East Indian Sandalwood Tree (Santalum album L.)

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Phytochemical Analyses and Evaluation of Antioxidant Efficacy of in vitro Callus Extract of East Indian Sandalwood Tree (Santalum album L.)
Phytochemical Analyses and Evaluation of Antioxidant Efficacy of in vitro Callus Extract of East Indian Sandalwood Tree (Santalum album L.)

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Published by: Biswapriya Biswavas Misra on Sep 27, 2012
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ISSN 2278- 4136ZDB-Number: 2668735-5IC Journal No: 8192
Volume 1 Issue 3Online Available atwww.phytojournal.com
Journal of Pharmacognosy and Phytochemistry
 
Vol. 1 No. 3 2012 www.phytojournal.com Page | 8 
Phytochemical Analyses and Evaluation of Antioxidant Efficacyof 
in vitro
Callus Extract of East Indian Sandalwood Tree(
Santalum album L.)
 
Biswapriya B. Misra
1*
, Satyahari Dey
2
 1.
 
Post-Doctoral Fellow, Center for Chemical Biology, Universiti Sains Malaysia [CCB@USM], 1st Floor Block B, No 10, Persiaran Bukit Jambul, 11900 Bayan Lepas, Penang, Pulau Pinang, Malaysia,Phone: +6-01037002012.
 
Professor, Plant Biotechnology Laboratory, Department of Biotechnology, Indian Institute of TechnologyKharagpur, Kharagpur-721302, West Bengal, India
 
The phytochemical constitution and antioxidant activity of in vitro grown callus cultures of EastIndian Sandalwood tree (Santalum album L.) were investigated. The extractive yield for adichloromethane-methanolic (1:1) solvent mixture was 4.3 %. The phytochemical screeningrevealed the extract’s richness in phenolics (18.2 µg), terpenoids (16.4 µg), saponins (9.4 µg)and flavan-3-ols (7.4 µg) per milligram of extract, as major constituents. This extract showedantioxidant activity in ferric reducing assay power (FRAP), total antioxidant capacity (TAC),metal ion chelation, inhibition of lipid peroxidation and in scavenging of hydroxyl radical (OH.),2, 2
-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid (ABTS.), di (phenyl) (2, 4, 6-trinitrophenyl) iminoazanium (DPPH.) and nitric oxide (NO.) free radical scavenging andreducing power assays that was comparable to sandalwood oil and reference antioxidant such as
quercetin, gallic acid and α
-tocopherol. We conclude that in vitro propagated callus showsimmense potential as a renewable resource of antioxidant constituents.
 Keyword:
Antioxidant, Callus, In Vitro, Phenolics, Sandalwood, TerpenoidCorresponding Author’s Contact information:Biswapriya B. Misra*Plant Biotechnology Laboratory, Department of Biotechnology, Indian Institute of Technology Kharagpur,Kharagpur-721302, West Bengal, IndiaE-mail: bbmisraccb@gmail.com
 
INTRODUCTION: 
Santalum album
L., the EastIndian Sandalwood tree is an important medicinaltree. This woody and tropical member of Santalaceae is the major source of sandalwoodessential oil, a mixture of sesquiterpenoidalcohols, i.e., 90 % santalols. Deposited in thecore of heartwood of the tree, a 50 year oldmatured tree may yield 2.5- 6 % of essential oil,and is influenced by several factors. The oil finds
 
Biswapriya B. Misra*, Satyahari Dey
 Vol. 1 No. 3 2012 www.phytojournal.com Page | 9 
use in traditional medicine system Ayurveda asan antiseptic, antipyretic, antiscabietic, diuretic,expectorant, stimulant and for the treatment of  bronchitis, dysuria, urinary infection andgonorrhea as it seems to contain antibacterial andantifungal properties [1]. The hydrolyzedexhausted sandalwood powder demonstrates anti-remorogenic, anti-inflammatory, anti-mitotic,anti-cancer, anti-hypertensive, anti-pyretic andsedative properties
 
[2]. The oil also possessantiviral activity against herpes simplex virus
 
[3]and anti-
 Helicobacter pylori
properties [4], thecausative organism for gastric cancer and pepticulcer.Epidemic phytoplasmal ‘spike’ disease leading tosevere destruction of natural population, illegal poaching and over exploitation owing toincreased global demand are the reasons of thetree being inducted into IUCN, Red List of Threatened Species [5] as vulnerable.Unsurprisingly, the first
in vitro
 micropropagation study on any woody forest treewas reported in sandalwood (callusing fromembryos) followed by further advances in biotechnological routes of micropropagation i.e.,somatic embryogenesis, regeneration, suspensioncultures, somatic embryo production andmaturation in air lift bioreactors
 
[6]. Furthermore,
in vitro
callus is known to yield sandalwood oilconstituents [7].However, till date there are no reports availablethat investigated the antioxidant potential of sandalwood oil or the
in vitro
callus of sandalwood. This comparative study wasundertaken to probe the antioxidant properties of a dichloromethane: methanol extract from
in vitro
 callus, with sandalwood oil and referenceantioxidants. Moreover, it is important toestablish appropriate means to evaluate andquantify effective antioxidant principles of economically viable resources for plant-basedtherapeutics. To our knowledge, this is the firsttime effort towards evaluation of biologicalactivities of any
in vitro
material fromsandalwood tree.
Materials and methodsReagents
The chemical reagents were obtained as follows,i.e., 2,2
-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS
.
), linalool and butylatedhydroxy toluene (BHT) were obtained fromFluka, Switzerland; 2-deoxy-2-ribose,trichloroacetic acid (TCA), sodium nitroprusside(SNP), sulfanilamide, naphthylethylenediaminedihydrochloride (NED), rutin, catechin, gallicacid, quercetin, ferrozine, Bradford reagent andPhytagel were obtained from Sigma, St. Louis;Folin-Ciocalteau reagent, dimethylaminocinnamaldehyde (DMACA), aluminum chloride(AlCl3), ferric ammonium sulfate[NH
4
Fe(SO4)
2
.12H
2
O], sodium carbonate[Na
2
CO
3
.10H
2
O], ammonium molybdate[(NH
4
)
6
Mo
7
O
24
.4H
2
O], sodium nitrite [NaNO
2
],ferric chloride [FeCl
3
], ferrous chloride [FeCl
2
],dichloromethane (HPLC grade), methanol (HPLCgrade) and n-butanol (Spectroscopy grade) were procured from Merck, India; sapogenin, bovineserum albumin (BSA), polyvinyl polypyrrolidone(PVPP), thiobarbituric acid (TBA), di (phenyl)-(2, 4, 6- trinitrophenyl) iminoazanium (DPPH),casein, vanillin, potassium persulfate, potassiumferricyanide [K 
3
Fe (CN)
6
], ethylene diaminetetraacetic acid (EDTA), ascorbic acid, WoodyPlant Medium (WPM) and 2,4-dichlorophenoxyacetic acid [2, 4-D], 2, 4, 6-tripyridyl-s-triazine (TPTZ), were purchasedfrom HiMedia, India while authentic sandalwoodoil samples were procured from Cauvery
TM
,Bangalore, India.
Plant materials
 In vitro
callus was from a highly proliferating cellline (IITKGP/ 91), grown aseptically on solidmedia i.e. Woody Plant Medium [8]supplemented with 2, 4-D (1 mg/ L), 3 % sucrose,and 0.35 % Phytagel, pH 5.8 ± 0.5 in culturetubes, in dark 25 ± 2 °C and were maintained bysub culturing at intervals of 3 weeks, in thelaboratory in the plant tissue culture facility of Department of Biotechnology.
 
Biswapriya B. Misra*, Satyahari Dey
 Vol. 1 No. 3 2012 www.phytojournal.com Page | 10 
Preparation of extracts
 In vitro
grown callus (100 g) was collected,washed in ddH
2
O, freeze dried and pulverizedinto fine powder using a mortar and pestle, andextracted for 18 h in dichloromethane: methanol(1:1, v/ v) [9] at 40 ºC in a ratio of 1: 200 (w/ v)of plant material and solvent. Post- extraction,solid materials were excluded by filtration using aWhatman No. 1 filter paper and were centrifugedat 5, 000 g for 10 min. The supernatant wasconcentrated using an Eyela, N- N series, rotaryevaporator connected to an Eyela aspirator,Model: A 3S (Rikakikai Inc., Tokyo, Japan) at 40ºC under reduced pressure. The extract obtainedwas stored at - 20 ºC until further use.
Table 1.
Summary of methods followed for phytochemical characterization of sandalwood callus extract.
SerialNo.Assay ExtractamountsReagents Conditions MonitoringsystemStandardcurveCalculation Reference1
Total terpenoidcontent100 µg inmethanol500 µl of 2 %vanilin-H
2
SO
4
incold.60 ºC for 20min, cooled at25 ºC for 5 minand within 20minutes,absorbancemeasuredBlue-greencolor,absorbanceat 608 nmLinalool(20 – 100mg/ l)as µg/ mgextract[11]
2
Totalanthocyaninscontent100 µg inmethanola. 500 µl extract +6 ml of n-butanol:HCl (95:5,v/ v) b. 0.2 ml of 2 % (w/v) solution of  NH
4
Fe(SO
4
)
2
,12H
2
O in 2 M HClHeated tubes at92-95 °C for 40minDeep green/ brownsolution,absorbance at550 nmMultipliedOD
530
by
33.3 (ε=
cyanidinchloride of 30,000 )as mg of cyanidinchloride/ 100g extract[12]
3
Total oligomeric proanthocyanidinscontent1 mg/ ml inmethanola. 5 ml of 0.5%vanillin reagent. b. 5 ml volume of 4% methanolicHCl.Incubation for 20 min at roomtemperatureDark greenish-bluecolor,absorbanceat 500 nmCatechin(100-500µg)as mg or gcatechinequivalents per 100 gextract[13]
4
Total phenolicscontent1 mg/ ml in30 %methanola. 100 µl of extract+ 50 µl of FolinCiocalteau reagent(FCR), 10 min waitand up to 1 ml with Na
2
CO
3
.10H
2
O andwell shaken.Incubation for 2hours in dark atroomtemperature.Blue solution,absorbanceat 720 nmGallic acid(10-50 µg)as mg gallicacid/g extract[14]
5
Total polyphenolsand Casein/ BSA/PVPP-BoundTannins1 mg/ ml in30%methanol(total polyphenols)a. 200 µl of extract+ 800 µl of water. b. 100 mg of BSA/casein or PVPPadded.a. Shaken for 1h at roomtemperature andthen 1-2 hincubation at4°C b. Filtered or centrifuged at4000 rpm/ 15min. Filtrate (=Unbound polyphenolsBlue solution,absorbanceat 720 nmCatechin(100-500µg)Boundtannins= Total polyphenols-Unbound polyphenols;as mgcatechin/gextract[14]
6
Total flavonoidcontent100 µg/ mlin methanol1 ml of 2%methanolic AlCl
3
, 6H
2
O + 1 ml extractIncubation at10 min.Yellow color,absorbanceat 430 nmRutin(10-50 µg)as mg rutin/ gextract[15]
7
Total flavan-3-olscontent1 mg/ ml inmethanol200 µl sample + 1ml of p-dimethylaminocinnamaldehyde(DMACA) (0.1%in 1 N HCl inmethanol)Samplevortexed, 10min at roomtemperature.Blue color monitoredat 640 nmCatechin(25-150 µg)as mgcatechin/ gextract[16]
8
Total saponincontent1 mg / mlin methanol10 µl extract inmethanol+ 50 µl8% vanillin inethanol + 500 µl 72% H
2
SO
4
Heated to 60 °Cfor 20 min,followed by 4°C for 5 min.Yellow/ greencolor,absorbanceat 544 nmSapogenin(10-100 µg)as mgsaponin/ gextract[17]

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