I N A L & AticP D Ic La E Nts Medicinal & Aromatic Plants
ntsPlaMedicinAromatic&alMedicinal & Aromatic PlantsISSN: 2167-0412Garg et al., Med Aromat Plants (Los Angels) 2017, 6:3DOI: 10.4172/2167-0412.1000290Open AccessResearch ArticlePhytochemical High Performance Thin Layer Chromatography basedEstimation of Lawsone in Lawsonia inermis (Henna) obtained from TwoNatural Habitats and Dye Products Collected from Local MarketGarg RK1, Tripathi R2, Batav N1 and Singh RK11Centreof Excellence in Biotechnology, MP Council of Science and Technology (MPCST), Vigyan Bhawan, Nehru Nagar, Bhopal, Madhya Pradesh, India2Departmentof Biochemistry, APS University, Rewa, Madhya Pradesh, India*Correspondingauthor: Garg RK, Centre of Excellence in Biotechnology, MP Council of Science and Technology (MPCST), Vigyan Bhawan, Nehru Nagar, Bhopal,Madhya Pradesh, India, Tel: 9826220316; E-mail: rkgargmpcst@gmail.comReceived date: March 14, 2017; Accepted date: May 08, 2017; Published date: May 14, 2017Copyright: 2017 Garg RK, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permitsunrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.AbstractThe present study elucidates the preliminary phytochemical analysis and High Performance Thin LayerChromatography (HPTLC) fingerprint analysis of lawsone extract of the leaves of Lawsonia inermis (henna) andevaluates their regions and products based quantitative estimations. The lawsone content has been evaluatedquantitatively in two plants samples collected from Rewa and Bhopal and one commercial henna powder procuredfrom open market of Bhopal city. Here we aimed to optimize high performance thin layer chromatography for thedetermination of lawsone. The chromatography development was carried out on the TLC silica gel 60 F254aluminum plate and good resolution was achieved with toluene: ethyl acetate: formic acid (22:16:2) as mobilephase. Lawsone detected was carried out densitometrically at 254 nm by absorption mode and a linear regressioncoefficient was obtained with Y 133.527 13.470 R 0.99092 6.43%. The methods were applied are validatedwith respect to specificity, linearity, accuracy precision and robustness, and also find out the linear calibration comewith respect to the concentration and regression coefficient. The developed method was validated as percentagerelative standard deviation of peak areas was found to be which indicates the suitability of system for the furthervalidation parameters. There was the presence of the peak at Rf 0.4 in the placebo, indicates the method is specificas none of the recipients interfered with the analytes of interest. The peak purity of Lawsone was achieved from560-1865 y; Aluminum plateinermis(henna);IntroductionThe name henna also refers to the dye prepared from the plant andthe art of temporary body art based on dyes it’s also know mehndi [1].Henna “Lawsonia inermis,” also known as henna, the henna tree, themignonette tree, and the Egyptian privet and the sole species of theLawsonia genus. The English name "henna" comes from Arabic ḥinnapronounced as “henna”. Henna is a traditional medicinal plant [2]. Theuse of henna as medicinal plant and also for body painting is describedin the Ebers papyrus [3].bark, seeds, or leaves. This versatility makes henna a very valuableelement in traditional medicines, particularly Ayurvedic practice. InIndia as a part of Hindu and Sikh weddings, henna is applied duringwedding ceremonies. It is a common practice among Indians,particularly elderly ones, to dye their hair using Henna. Our culturethough traditional henna many women may work together during alarge wedding, where in hundreds of guests have henna applied to theirbody parts. This particular event at a marriage is known as the MehndiCelebration or Mehndi Night, and is mainly held for the bride [4].Various physiological factors, such as skin type and temperature,hormone levels, and stress affect the appearance of mehendi on people.After the dried paste is scraped off the skin, air oxidation orperspiration can further darken the stain over the next 48 hours [5,6].Henna can be used in a wide variety of ways, including its dye form,as well as in aqueous extracts, tinctures, and salves, composed of theClassification of Genus:LawsoniaSpecies:L. inermisMed Aromat Plants (Los Angels), an open access journalISSN: 2167-0412Volume 6 Issue 3 1000290
Citation:Garg RK, Tripathi R, Batav N, Singh RK (2017) Phytochemical High Performance Thin Layer Chromatography based Estimation ofLawsone in Lawsonia inermis (Henna) obtained from Two Natural Habitats and Dye Products Collected from Local Market. Med AromatPlants (Los Angels) 6: 290. doi:10.4172/2167-0412.1000290Page 2 of 72 [8]. Its molecule contains 10 carbon, 6 hydrogen’s and 3 oxygen’s(C10H6O3), giving a total molecular weight of 174.16 atomic units ofmass [10].Henna is known to be dangerous to people with glucose-6phosphate dehydrogenase deficiency (G6PD deficiency), which is morecommon in males than females. Infants and children of particularethnic groups, mainly from the Middle East and North Africa, areespecially vulnerable. Though user accounts cite few other negativeeffects of natural henna paste, save for occasional allergic reactions,pre-mixed henna body art pastes may have ingredients added todarken stain, or to alter stain color. The health risks involved in premixed paste can be significant [11]. The green leaves of Lawsoniainermis , a small tree that grows in warm, arid regions of the worldsuch as India, Pakistan, and Northern Africa and all other countries(Figures 1 and 2).Figure 1: Chemical Structure of lawsone compound.Henna contains natural ingredients which are vital for nourishmentof hair [12]. It has a bond with the hair structure as it serves topenetrate, cleanse and thicken the hair shafts thus improving itsquality. It also has great dandruff fighting ability. Henna is mainly usedas a coloring agent. Nature has been a rich source of therapeutic agentsfor thousands of years and an impressive number of modern drugshave been isolated from natural sources based on the uses of theseplants in traditional medicine. Henna is one such plant commonlyknown as Persian Henna or Lawsonia inermis, a bushy, flowering tree,commonly found in India. The most common forms for medicinalbenefits, and the high concentration of chemicals and nutrients in theplant gives it anti-inflammatory, hypotensive, antibacterial, astringent,and antiviral effects, among many others.The antioxidant capacity of henna has not been widely studied, theoil has been proven to be an astringent, which has led some people touse henna juice and oil on the skin to reduce the signs of aging andwrinkles, as well as unsightly appearance of scars and other blemishes.This is complemented by the antiviral and antibacterial effects that canprotect the body’s largest organ is the skin. Henna oil is used forrheumatic and arthritic pains. Ground leaves are applied to sore flintsto ease rheumatism. The juice of the medicinal plant can be applied tothe skin for headaches, and the henna oil is applied to hair to prevent itfrom graying [13,14].Figure 2: Photographic views of Lawsonia inermis.“Henna” is understood by people around the world in manydifferent ways. The majority of people probably associate henna withthe dark-red/brown dye for hair and skin that is traditionally used inEastern cultures, but the name also applies to the flowering plant fromwhich that dye is derived. Henna can be used in a wide variety of ways,including its dye form, as well as in aqueous extracts, tinctures, andsalves [7]. HPTLC was the methods used estimation of lawsonecompound which quantify through the HPTLC analysis.Henna, Lawsonia inermis contain a red orange pigment lawsone themolecules of which is also known as hennotannic acid. The name andmolecular structure of lawsone show its congeniality to naphthalene[8]. The phytochemistry of henna was largely studied and revealedinteresting information. Already in 1920, the dye principle was known.Lawsone, C10H6O3, the colouring matter contained in henna leaves, isfixed well by wool, silk and tenaciously by the skin [9]. In lawsone twooxygen are attached to the naphthalene carbon at position 1 and 4 toform 1,4 naphthoquninone and a hydroxyl group is present at positionMed Aromat Plants (Los Angels), an open access journalISSN: 2167-0412In the present investigation, we elucidated preliminaryphytochemical analysis and High Performance Thin LayerChromatography (HPTLC) based quantitative analysis of lawsone inLawsonia inermis and evaluates qualitative and quantitative status oflawsone in L. inermis.Materials and MethodologyThe study was design to make the comparative analysis of thequantity of the lawsone compound founds in henna (Mehndi). Hennapowdered dried leaves from samples of Lawsonia inermis L.,Lythraceae, were obtained from suppliers of plant materials fromdifferent places of Madhya Pradesh. Total three samples were collected.Two leaves samples of plant henna were collected from Bhopal andRewa respectively. And one packed powdered sample (Amena) iscollected from local market of Bhopal. Plant material referencestandard used Lawsonia inermis. The Samples were identified anddeposited into the botanical Herbal Garden of MPCST (Bhopal).Samples of henna were obtained from the market and confirmed bymorphological analysis (Table 1).Volume 6 Issue 3 1000290
Citation:Garg RK, Tripathi R, Batav N, Singh RK (2017) Phytochemical High Performance Thin Layer Chromatography based Estimation ofLawsone in Lawsonia inermis (Henna) obtained from Two Natural Habitats and Dye Products Collected from Local Market. Med AromatPlants (Los Angels) 6: 290. doi:10.4172/2167-0412.1000290Page 3 of 7SNLocationSample ID1Bhopal, Madhya PradeshSample-I (Bhopal)2Rewa, Madhya PradeshSample-I (Rewa)3Open Market of BhopalSample-I (Open Market)Table 1: Collected henna samples and there assigned ID.Sample preparationHenna powdered dried leaves of each sample taken (1 g) and wereseparately mixed with methanol (10 ml) and mixed in shaker forovernight. The mixture was centrifuge at 10,000 rpm for 10 minutes.The supernatant was collected in a fresh tube and was 10 times dilutedwith methanol before use again further dilutes the sample.Standard preparationStock solution of lawsone was prepared by accurately weighing 10mg of 2 hydroxy 1,4 naphthoquinone (lawsone), dissolving it inmethanol. The mixture was collected 1 ml and dissolved in 10 mlmaking up the volume with methanol.Quantification of lawsone in methanolic extract of Lawsoniainterims using high performance thin layer chromatographyThe HPTLC system (CAMAG, Muttenz, Switzerland) consisted of(I) a Linomat 5 sample applicator using 100 μl syringes connected to anitrogen tank (II) an Automatic Developing Chamber 2 containingtwin trough chamber of 20 10 cm (III) TLC Plate Heater III; (IV)TLC scanner 3 linked to winCATS software.Sample applicationThe standard was taken in syringe 50 µl loaded the syringe inlinomate-5 through which the sample is loaded in TLC plate (1-8 µl). 8Tracks of each standard applied according to the band with 8 mm, andapplication volume was 1-8 µl and gas flow 150 nl/s. Then after loadingthe standard in TLC plate again was the syringe 2 times wash bymethanol. Sample is taken in syringe 20 µl 2 tracks of each samplesapplied 1-2 µl. Total 14 tracks of samples and standards are applied inTLC plate (Table 2).Appl. positionAppl. VolumeVialSample IDActive15.0 mm1 μl1Std1Yes28.0 mm2 μl1Std2Yes41.0 mm3 μl1Std3Yes54.0 mm4 μl1Std4Yes67.0 mm5 μl1Std5Yes80.0 mm6 μl1Std6Yes93.0 mm7 μl1Std7Yes106.0 mm8 μl1Std8Yes119.0 mm1 μl2Sample-I (Bhopal)Yes132.0 mm2 μl2Sample-I (Bhopal)Yes145.0 mm1 μl3Sample-II (Rewa)Yes158.0 mm2 μl3Sample-II (Rewa)Yes171.0 mm1 μl4Sample-III (Open Market)Yes184.0 mm2 μl4Sample-III (Open Market)YesTable 2: Sample application.Development of optimum mobile phaseThe composition of the mobile phase for development ofchromatographic method was optimized by testing different solventmixtures of varying polarity. After sample loading plate weredeveloped to 10 10 cm Twin Through chamber before developingpre-saturated for 20 minute through filter paper. Chamber fill up 10 mlMed Aromat Plants (Los Angels), an open access journalISSN: 2167-0412of developing solvent toluene: ethyl acetate: formic acid (22:16:2).Plates were developed into 8 cm. The developed plate allowed dry 5Minutes by hair drier.Volume 6 Issue 3 1000290
Citation:Garg RK, Tripathi R, Batav N, Singh RK (2017) Phytochemical High Performance Thin Layer Chromatography based Estimation ofLawsone in Lawsonia inermis (Henna) obtained from Two Natural Habitats and Dye Products Collected from Local Market. Med AromatPlants (Los Angels) 6: 290. doi:10.4172/2167-0412.1000290Page 4 of 7EvaluationThe plate was dried then inspected under a D2 white lamp at 254nm web scanned at 254 nm with using D2 white lamp. Spectra scanweb absorbed performed on plate and common spectra scanned webobtained by CAMAG Scanner 3 and analyzed by winCATS software(Table 3).RfSubstanceMaximum wavelength0.04Lowsone461 nm0.04Lowsone288 nm0.04Lowsone286 nm0.04Lowsone285 nm0.04Lowsone284 nm0.04Lowsone283 nm0.04Lowsone280 nm0.04Lowsone283 nm0.04Lowsone292 nm0.04Lowsone291 nm0.03Lowsone294 nm0.03Lowsone292 nm0.04Lowsone289 nm0.04Lowsone289 nmTable 3: Web scanned at 254 nm with using D2 white lamp.Results and DiscussionThe qualitative and quantitative estimation of henna samples werecarried out using HPTLC method. Various different concentration ofsample was applied to the TLC plate were used to quantification onimpact regional variations of lawsone. The chromatographicconditions in particular the developing solvents were carefullyoptimized and then formulation samples were analyzed scanned at 254nm using D2 white lamp the result observed good separation for allcompounds. A reference marker compound of lawsone was separatedon the plate for the authentication of each sample.Sample I Bhopal, Sample II Rewa, Sample III Open market Bhopal.The specificity of the method was ascertained by analyzing. Variousdifferent concentration of standard was made and also scanned forcomparisons. Bands were scanned at D2 white lamp. Different peaksand bands can be observed in the graph (Figure 3) and concentrationcalculated can be observed.Figure 3: Standard plot of standard of lawsone (pink in colour) andsample of Lawsonia inermis.The spots for lawsone in samples were confirmed by comparing theRf and spectra of the spot with that of standard. The peak purity oflawsone was assessed by comparing the spectra at three different levels,i.e., peak start, peak apex, and peak end positions of the spot. WinCATsand integration software 1.4 was used to calculate the amount oflawsone. Under the chromatographic conditions described inprocedure the Rf for lawsone was found to be 0.4. For positiveidentification, the sample must exhibit bands with chromatographiccharacteristics, including colors and Rf values similar to those ofreference marker compound. It was observed that lawsone appeared atRf 0.4 in samples which is similar to the Standard Lawsone.Developed and validated HPTLC method for estimation of Lawsonein henna was found to be simple, accurate, precise and robust.Different mobile phase was tried and good resolution was achievedMed Aromat Plants (Los Angels), an open access journalISSN: 2167-0412Volume 6 Issue 3 1000290
Citation:Garg RK, Tripathi R, Batav N, Singh RK (2017) Phytochemical High Performance Thin Layer Chromatography based Estimation ofLawsone in Lawsonia inermis (Henna) obtained from Two Natural Habitats and Dye Products Collected from Local Market. Med AromatPlants (Los Angels) 6: 290. doi:10.4172/2167-0412.1000290Page 5 of 7with toluene: Ethyl acetate: formic acid (22:16:2 v/v/v). Samplepreparation was optimized by using methanol as solvents. Lawsonewas detected at 0.04 Rf value at 254 nm by absorbance mode [15]. Thedeveloped method was validated as percentage relative standarddeviation of peak areas was found to be which indicates the suitabilityof system for the further validation parameters. There was the absenceof the peak at Rf 0.04 in the placebo, indicates the method is specific asnone of the excipients interfered with the analytes of interest. The peakpurity of Lawsone was achieved 1000.Total 3 plants sample were applied during quantification of lawsone.Each sample were applied in duplicate of them track 1, having sample1 µl, however they were in another track is 2 µl. sample 1 track 1occupied 560.14 Au, sample 1 track 2 occupied 1004.50 Au, sample2nd track 1st occupied 517.47 Au, Sample 2nd track 2nd occupied theareas of 996.31 Au, similarly samples 3 track 1st occupied 1100.48 Ausample 3rd track 2nd occupied area of 1865.16 Au. Above absorptiondepicted that all sample are within the range of standard accept sample3 track 2, having the out of range of the standard (Tables 4-6, Figure 4).Sample IDSample application Volume (µl)Maximum position (Rf)Maximum height (AU)Area (AU)Standard1 (µl)0.0432.6292.182 (µl)0.0439.3412.923 (µl)0.0447.8553.624 (µl)0.0449.4617.475 (µl)0.0463.9749.666 (µl)0.0482.9963.257 (µl)0.0499.11117.138 (µl)0.0486.8890.191 (µl)0.0457.7560.142(µl)0.0488.81004.501 (µl)0.0351.3517.472 (µl)0.0379.0996.311 (µl)0.04103.71100.482 (µl)0.04165.01865.16Sample ISample IISample IIITable 4: TLC plate scanned at 254 nm using D2 white lamp.SubstanceRfX(Average)CV[%]nRegression remarkStd. lawsone0.0456.17 ng0.0001LinearS.B. lawsone0.0448.17 ng48.4302LinearS.R. lawsone0.0346.28 ng54.3162LinearS.M. lawsone0.0471.79 ng0.0001LinearTable 5: Calibration results per analysis.Med Aromat Plants (Los Angels), an open access journalISSN: 2167-0412Volume 6 Issue 3 1000290
Citation:Garg RK, Tripathi R, Batav N, Singh RK (2017) Phytochemical High Performance Thin Layer Chromatography based Estimation ofLawsone in Lawsonia inermis (Henna) obtained from Two Natural Habitats and Dye Products Collected from Local Market. Med AromatPlants (Los Angels) 6: 290. doi:10.4172/2167-0412.1000290Page 6 of 7well as methods applied are validated with respect to specificity,linearity, accuracy precision and robustness. We also find out the linearcalibration come with respect to the concentration and regressioncoefficient.Figure 4: Spectra scan for all tracks for lawsone scanned 254 nmwavelength.All standard and all sample were detected at 0.04 Rf value andwavelength 254 nm by absorption mode (Tables 6 and 7) and a linearregression coefficient was obtained Figure 5 with Y 133.527 13.470 R 0.99092 6.43%. After comparison of the all unknown sampleswith standards it has found that the average concentration of thestandard solution is 0.999 µg/µl. plants samples collected from HumanHerbal Heath Care Garden (HHHCG) achieved the concentration 1.91µg/µl in the crude extract of the plants as 1 µg/ µl. The plants sampleswhich were collected from Rewa showed the concentration 1.77 µg/µlin the crude extract of the plants as 1 µg/µl. However, the 3rd samplewas collected from open market make Amina Herbal Hair Powder(coded as sample 3rd). Quantitative the lawsone is 3.76 µg/µl. Thesimilar type of research work has been carried out by many scientist[15,16] for the quanti ication of Lawsonia inermis is compared ourresult with this research studied it has concluded that our results asFigure 5: 3D graph for specifity for 1-Lawsone standard 1 µg/µl, 2Lawsone standard 2 µg/µl, 3-Lawsone standard 3 µg/µl, 4-lawsonestandard 4 µg/µl, 5-Lawsone standard 5 µg/µl, 6-Lawsone standard6 µg/µl, 7-lawsone standard 7 µg/µl, 8-Lawsone standard 8 µg/µl, 9Sample Bhopal 1 µg/µl, 10-Sample Bhopal 2 µg/µl, 11 Sample-Rewa1 µg/µl, 12-Sample Rewa 2 µg/µl, 13-Sample Market 1 µg/µl, 14Sample Market 2 µg/µl.VialRfAmountAreraX (cal)10.0410.00
2 Rewa, Madhya Pradesh Sample-I (Rewa) 3 Open Market of Bhopal Sample-I (Open Market) Table 1: Collected henna samples and there assigned ID. Sample preparation Henna powdered dried leaves of each sample taken (1 g) and were separately mixed with methanol (10 ml) and mixed in shaker for overn
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