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Publication Ref No.: IJPRD/2010/PUB/ARTI/VOV-2/ISSUE-6/AUG/001 ISSN 0974 9446

International Journal of Pharma Research and Development Online

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ONLINE HPLC-DAD/UV-MS DETERMINATION OF MAJOR FLAVONOIDS RUTIN ANDISOQUERCITRIN IN TWO MORUSSPECIES.

Vijay Gokarn1,2*, Vidya Dighe1,Sasikumar Menon2, Bhalerao Khairnar1,2

1 Ramnarain Ruia College,Matunga (E), Mumbai- 400 019, India.

2 TDM Laboratory, Plot No. 194, Scheme No 6, Road No.15,Sion (E), Koliwada, Mumbai 400 022, India.

Email: [email protected]

ABSTRACT

Mulberry is one such plant that has tremendous therapeutic applications but not much exploitedfor its medicinal value. The taxonomy of Mulberry (Morus) is complex and disputed. There are noestablished methods to differentiate among existing species. The present paper demonstrates the workfocused on the quality aspects of medicinal plant research for standardizing Mulberry Species. Areversed-phase high performance liquid chromatographic method with simultaneous diode arraydetection and mass spectral analysis (HPLC-DAD/UV-MS) was developed to detect and quantify twoflavonoids, rutin and isoquercitrin as two major flavonoids from two Morusspecies, Morus albaLinn.and Morus australis Poir. collected from Maharashtra, India. The chromatographic separation wasperformed on a Cosmosil C8 column (150 x 4.6 mm, 5 m) with an isocratic mobile phase, comprising0.1 % formic acid in distilled water, acetonitrile and methanol (75:15:10 v/v/v) at a flow rate of 1.0mL/min. The wavelength selected for simultaneous quantitation of both analytes was 259 nm. Themethod developed with careful validation was successfully used for simultaneous quantification of twomajor flavonoids rutin and isoquercitrin from hydroalcoholic extracts of two Morus species. Thehydroalcoholic extraction of the finely powdered dried leaves of both the plants was carried out usingultrasonic extraction technique. The further confirmation of rutin and isoquercitrin in both Morus leaveswas done by developed online mass spectral analysis. An Online HPLC-DAD/UV-MS method thusestablished could be considered as a possible quality control tool for standardizing Morusspecies andeven in routine quality testing of various formulations having rutin and isoquercitrin in them.

Key Words: HPLC-DAD/UV-MS, Rutin, Isoquercitrin, Morus albaLinn., Morus australisPoir.

Vijay Gokarn

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INTRODUCTIONMorus or Mulberry is a genus of 1016

species of deciduous trees native to warm

temperate and subtropical regions of Asia,Africa, Europe, and America, with the majorityof the species native to Asia [1]. It has beencultivated in many Asian countries such asChina, India, Korea, Japan and Thailand wherethe leaves were used as food for silkworms [2].

There is an increasing interest on mulberryleaves because the leaves were found to havehypoglycemic, hypotensive, diuretic,bacteriostatic and antivirotic properties [3].

Mulberry leaves have traditionally been used forhealth promotion and the treatment of certaindiseases such as diabetic hyperglycemia. It hasbeen also reported in literature thatMorus plantalso possesses immunomodulating and active-oxygen scavenging properties[4].

The Morusplant is rich source of the isoprenoidsubstituted phenolic compounds, includingflavonoids [5], which may be the reason for itseffectiveness in the above mentioned diseases[6, 7],andflavonoids may be precursors of some

toxic substances in photosynthesis and cellularenergy transfer processes [8,9]. Flavonoids havebeen referred to as "nature's biologicalresponse modifiers" because of strongexperimental evidence of their inherent ability tomodify the body's reaction to allergens, viruses,and carcinogens. They show anti-allergic, anti-inflammatory [10], anti-microbial [11] and anti-cancer activity.Flavonoids are powerful antioxidants andscavengers of free radicals. Free radicals cause

cellular, and DNA damage in our body andconsequently induce age-related diseases suchas dementia and cancer [12].Considering these variable and contrastingproperties and keeping in view the biologicalimportance, the two major flavonoids inmulberry, rutin and isoquercitrin were selectedin our research work as quality control markers.

HPLC is the method of choice for analysis ofthese phenolic compounds, because of itsextremely high versatility and precision [13- 15]. A

capillary electrophoretic method has beenreported in literature, which is used for theidentification of rutin and isoquercitrin presentas major antioxidant flavonoids in Solidagogiganteaand Morus nigra[16]. An HPLC methodis reported in literature for analysis of theflavonoids from Solidago Canadensis[17].However no methods are reported in literaturefor simultaneous quantitation of rutin andisoquercitrin from Morus alba Linn. and MorusaustralisPoir.

A rapid HPLC-UV-MS method has beenestablished for separation, identification andquantitation of two major flavonoids rutin andisoquercitrin present in the hydroalcoholicextracts of Morus albaLinn. and Morus australisPoir. The identities of the two major flavonoidswere confirmed by passing HPLC elutedcomponents through mass spectrometer andobtaining the parent masses (m/z values) of therespective peaks in the chromatogram whilecomparing with the reference standards. The

method has been developed, validatedaccording to the ICH requirements and appliedfor estimation of two major flavonoids, rutin andisoquercitrin from hydroalcoholic extracts ofMorus alba Linn. and Morus australis Poir. inthe present work. The hydro alcoholic extractsof both the plant leaf powders were prepared byusing ultrasonic extraction technique.

Objective:

To establish quality control method with thehelp of modern analytical technique forstandardizing Mulberry (Morus) species.

EXPERIMENTAL AND METHOD :

Standard, Reagents

The reference standards rutin hydrate (95%)and isoquercitrin (90%) were purchased from

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Sigma-Aldrich Chemie GmbH (Aldrich Division,Steinbeim, Germany).

Acetonitrile (purity- 100 %, UV cutoff- 188 nm)

and methanol (purity- 100 %, UV cutoff- 205nm) used in the present work were of HPLCgrade (J. T. Baker) and were procured fromOrochem India Pvt. Ltd. Water used in thepresent research work, was purified with a Milli-Q water purifying system (Millipore, USA). Thesolvents were filtered through 0.5m membrane(Millipore) and degassed in an ultrasonic bath.Formic acid (100 %, GR grade) was procuredfrom Merck Specialties Pvt. Ltd.

Plant MaterialThe leaves of Morus alba Linn. were collectedfrom wild plants found in Keshav Srushti,Mumbai, India and the leaves of Morus australisPoir. were collected from Mahabaleshwar,Satara, India.Herbaria of both the plant species wereprepared and authenticated from BotanicalSurvey of India (BSI), Pune, India and NationalBotanical Research Institute (NBRI), Lucknow,India respectively.The leaves of Morus alba Linn. and MorusaustralisPoir. were shade dried, powdered andthen sieved through BSS mesh size 85 andstored at 25oC, in light resistant and airtightcontainers.

Preparation of standard stock solutions ofrutin and isoquercitrin

Individual standard stock solutions of rutin(1000 g/mL) and isoquercitrin (1000 g/mL)were prepared in methanol. Weights equivalentto 10 mg of rutin and isoquercitrin were weighedaccurately and dissolved separately in 5.0 mL ofmethanol, followed by sonication for 1 min andfinally making up the volume of solutions to 10.0mL, with methanol.

Preparation of mixed working standardsolutions

Desired aliquots of stock solutions of rutin (1000g/mL) and isoquercitrin (1000 g/mL) weretransferred to a 10 mL standard volumetric

flask. Solution in the flask was then diluted uptothe mark with the mobile phase to obtain mixedworking standard solutions of rutin andisoquercitrin in concentration range of 1g/mLto 150 g/mL for rutin and 3.0 g/mL to 250.0g/mL for isoquercitrin.

Preparation of sample solutions

Accurately weighed about 100 mg of finelypowdered leaf powder of Morus albaLinn. and

500 mg of finely powdered leaf powder of Morusaustralis Poir. were taken in two separatetarsons tubes of capacity 15 mL and to eachtube, 10 mL of methanol: Milli-Q water (1:1) wasadded and the tubes were vortex mixed for 30sec and kept in ultrasonic bath (Frequency- 50Hz) for extraction of phytoconstituents for anoptimized time period of 15 mins. The extractswere then high speed centrifuged at 15,000 rpmusing a microcentrifuge and the clearsupernatant sample solutions were finally

filtered using 0.45 m nylon filters (Millipore).The hydro alcoholic extracts of both the plantleaf powders were thus prepared and used forfurther chromatographic analysis.

HPLC- UV/DAD analysis

Separation for qualitative and quantitativeanalysis was performed by a Jasco HPLCsystem comprising an isocratic pump (JascoPU- 980, Intelligent HPLC Pump) with a

rheodyne injector valve having a fixed volumeloop of 20 L capacity and a photo diode arraydetector (Jasco MD- 910 Multiwavelengthdetector).Phytochemicals were separated on a reversed-phase Cosmosil C8 analytical column (4.6 mm x150 mm) 5 m particle size. The MS compatibleoptimized mobile phase comprised of 0.1%

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formic acid in Milli-Q water, acetonitrile andmethanol, in the ratio of 75: 15: 10 v/v/v. Theflow rate was maintained constant at 1.0 mL/

min. Before use, the mobile phase was filteredthrough 0.45 m nylon filters (Millipore) and de-aerated in an ultrasonic bath. Data wereintegrated by Jasco Borwin software, V 1.21.

Mass spectral analysisMass spectrometry was performed with anApplied Biosystems (MDS SCIEX) API 2000triple quadrupole mass spectrometer. A turboion-spray source was used in negative-ionmode with the settings: source temperature;

450

o

C; ion spray voltage; -4500 V; nebulizergas (N2), 60-40arbitrary units; curtain gas (N2),20 arbitrary units; entrance potential (EP), -10V; declustering potential (DP), -100 V; Full scanacquisition was performed between m/z 50 and800 U. Experiment was performed with thequadrupole (Q1) operated at unit resolution.All samples and solutions were filtered through0.45 m nylon filters (Millipore) before analysisby HPLC or Mass Spectrometry.

RESULTS

Method validation [18, 19]

The developed RP-HPLC method forsimultaneous determination of rutin andisoquercitrin was validated for linearity, limit ofdetection (LOD), limit of quantitation (LOQ),precision, accuracy and system suitability.

Linearity

To establish the linearity, calibration plots ofpeak area against concentration wereconstructed after triplicate analysis of mixedworking standard solutions of rutin andisoquercitrin, simultaneously, at seven differentconcentrations in the range of 1.00 150.00 g/mL for rutin and 3.0 250.00 g/ mL forisoquercitrin. The peak area values and

concentrations at seven different calibrant levelsof the respective working standard solutions ofrutin and isoquercitrin were subjected to

regression analysis to calculate the calibrationequation and correlation coefficient (r). Theresults are listed in Table 1.0.

Limit of Detection (LOD) and Limit ofQuantitation (LOQ)

LOD and LOQ were established at signal tonoise ratio of 3:1 and 10:1 respectively. Thevalues obtained are listed in Table 1.0.

Precision

The instrument precision was studied byinjecting mixed standard solution of rutin andisoquercitrin (10.0 g/mL each), in tenreplicates, in the chromatographic system underthe specified conditions. The results expressedas percent RSD of peak area, are listed in Table1.

Repeatability (Intra-day precision) of the methodwas assessed by triplicate analysis using themixed standard solutions at low (3 g/mL forrutin and, 9 g/mL for isoquercitrin), medium (75

g/mL for rutin and 100 g/mL for isoquercitrin)and high (130 g/mL for rutin and 180 g/mL forisoquercitrin) concentration levels covering theentire linearity range. The mean percent RSDwas found to be < 2 for all three levels.

Similarly, the intermediate precision wasassessed by triplicate analysis of the above-mentioned low, medium and high concentrationlevels, on three different days. The results forthe precision experiment are listed in Table 2.1

and 2.2.System SuitabilitySystem suitability was carried out to verify thatthe resolution and reproducibility of the systemwere acceptable for the analysis.System suitability test was carried out byinjecting 20 l of mixed standard solution ofrutin and isoquercitrin each having

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concentration 10.0 g/mL, in six replicates, inthe chromatographic system under optimizedconditions. The chromatograms were recorded

after injecting 20 l of the mixed standardsolution of rutin and isoquercitrin (10 g/mL).The values of percent relative standarddeviation (% RSD) of peak area of rutin,retention time of rutin and resolution ofchromatographic peak of rutin and isoquercitrinwere taken as an indicator of system suitabilityand the results obtained are listed in Table 3.0.A representative chromatogram of mixture ofreference standard solution of rutin andisoquercitrin (10.00 g/mL each) is shown in

Fig. 1.0.Assay ExperimentThe developed and validated RP- HPLCmethod for the simultaneous detection of rutinand isoquercitrin was applied for quantitation oftwo major flavonoids rutin and isoquercitrinusing hydro alcoholic extracts of dried leafpowders of Morus alba Linn. and Morusaustralis Poir. The assay experiment wasperformed with seven determinations by

injecting 20l of each of the sample solutions,i.e., hydro alcoholic extracts obtained byultrasonic extraction technique, from the driedleaf powders of Morus alba Linn. and Morusaustralis Poir. Typical HPLC chromatographicfingerprint patterns of the hydroalcoholicextracts of both plant leaf powders are shown inFig. 2.0.The amounts of rutin and isoquercitrin werecalculated by use of appropriate calibrationcurves. Comparative evaluations of the resultsfor assay experiment are tabulated in Table 4.0.

Accuracy

Accuracy of the method was determined byperforming recovery experiments for both theplant leaf powders of Morus species, usingstandard addition method. The recovery of theadded standards rutin and isoquercitrin was

studied by spiking working standard stocksolutions at three different concentration levelsto the leaf powders of Morus alba and Morus

australis just before extraction of leaf powders.The accuracy was expressed as the percentageof rutin and isoquercitrin recovered by theassay. The results of accuracy experiment arelisted in Table 5.1 and Table 5.2. The resultsindicate good accuracy of the method for thesimultaneous quantitative determination of rutinand isoquercitrin from leaf powders of bothMorusspecies.

Mass Analysis

An MS compatible mobile phase was developedin the present research work facilitating anonline HPLC- DAD- MS analysis, that furtherconfirmed the presence of rutin andisoquercitrin in the hydroalcoholic extracts ofdried leaves of Morus alba Linn. and Morusaustralis Poir. The negative mode ESI-MS isreported to be effective for rutin [20] . Thus,infusion in negative ion mode was employed,which facilitated tuning of source gas andcompound parameters for achieving strong

signals of rutin and isoquercitrin that wereobserved at 609.8 and 463.5 [M - H]- as parentmolecular ions respectively in the negative Q1mode.Flavonol O- glycoside, rutin furnished thedeprotonated molecule at m/z 609 of theglycoside and an ion corresponding to thedeprotonated aglycone [A H]-. The latter ion atm/z 301 was formed due to the loss ofrhamnose and glucose from the glycosides.Similarly isoquercitrin furnished the

deprotonated molecule at m/z 463 of theglucoside and an ion corresponding to thedeprotonated aglycone [A H]-.The HPLC eluents that were first detected bythe UV detector were simultaneously subjectedto ESI-MS under the optimized conditions, in anegative mode to obtain individual massspectras.

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The MS spectra of rutin and isoquercitrinpresent in both the hydroalcoholic extracts ofdried leaf powders of Morus alba Linn. and

Morus australis Poir. are shown in Fig3.1 and3.2. that confirms the identity of both theflavonoids.

DISCUSSION

The capillary electrophoretic method reported inliterature [16] serves to be a viable alternative toanalysis by HPLC method with respect toQualitative analysis but the Quantitation of theflavonoids in the reported method is performedby HPLC. The methods reported in literaturemainly use gradient mode of elution whichusually poses a great challenge for repeatabilityof results in case of the complex herbalmedicines and the column needs to beequilibrated after each sample run. Theproposed method uses an isocratic mode ofelution due to its simplicity, providing stablebaseline and unvarying response factor; alsothe isocratic elution facilitated best possibleseparation for the specified pair of peaks andalso for the other phytoconstituents separated inthe respective chromatograms Fig.2.0.Almost all reported methods use C18 analyticalcolumn of 250 mm length, whereas the presentmethod uses a shorter C8 analytical column of150 mm length which provides a goodseparation of the phytoconstituents with valuesof Rs 1.5 which is acceptable resolutionaccording to the Snyder method [21] . Thepresent method uses a mobile phasecomprising 0.1% formic acid, acetonitrile andmethanol in the ratio 75:15:10v/v/v. Formic acidhelps in the ionization of molecules and mixtureof acetonitrile and methanol facilitates favorableUV transmittance and low viscosity.The mobile phase pH- 2.72 was selected withfollowing considerations; a low pH protonatescolumn silanols and reduces theirchromatographic activity, is generally preferredand a low pH (< 3) is usually quite different from

the pKa values of common acidic and basicfunctional groups. Therefore, at low pH theretention of the compounds are not affected by

small changes in pH and the reversed phasechromatographic method will be more rugged[18].For qualitative purpose the method wasevaluated by taking into account retention timeprecision, peak asymmetry and resolution of thetwo peaks of rutin and isoquercitrin. Theretention time was highly repeatable, with RSDvalues below 2% and even for peak asymmetryand peak resolution (Table 3.0). Linearity, LODand LOQ (Table 1.0), precision (Table 2.1 and

Table 2.2) and accuracy (Table 5.1 and Table5.2) were evaluated for quantitative purposes.High recovery (ranging from 99.16- 100.10 %)and repeatability of both the flavonoids fromboth the plant powders in presence of the otherphytoconstituents was indicative of satisfactoryaccuracy of the method.The wavelength for detection and quantitation ofthe phytoconstituents was selected by use ofthe diode array detector. The HPLC- DADscans providing UV spectra obtained along with

the respective chromatogram for mixedstandard solution of rutin and isoquercitrin(10.00 g/mL each) prepared in mobile phaseare shown in Fig. 4.0The isocratic elution using HPLC-DAD allowedseparation and quantitation of the twoflavonoids rutin and isoquercitrin found as themajor polyphenols in hydroalcoholic extracts ofboth the Morusspecies and m/z values for rutinand isoquercitrin Fig. 3.1 and Fig. 3.2 obtainedby online mass spectrometry facilitated furtherconfirmation of both the flavonoids incomparison with the individual referencestandards.Rutin as well as isoquercitrin which were foundto be the major flavonoid phytoconstituents inline with the literature can serve as possiblequality control indicators in Morus species forfuture prospects since changes in the ratio ofrutin to isoquercitrin (and/or any other

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phytoconstituents) would indicate chemicalchange/degradation in the hydroalcoholicextract of the raw material or the finished

formulation made from mulberry.The average marker contents in both theselected plants Morus alba Linn. and Morusaustralis Poir. varied remarkably (Table 4.0).The average rutin and isoquercitrin contents indried leaf powder of Morus australisPoir. were38.34 and 55.33 times higher than that in thedried leaf powder Morus albaLinn.

CONCLUSION

A simple, precise and accurate HPLC- DAD-

MS method has been developed and validatedfor simultaneous detection and quantitation ofrutin and isoquercitrin with successfulapplication to hydro-alcoholic extracts of leafpowders of Morus alba Linn. and Morusaustralis Poir. Sample preparation was simpleand no tedious cleanup was necessary. Rutinand Isoquercitrin present as the two majorflavonoids in both the Morus species can beselected as the phytochemical markers forchromatographic fingerprints and for routine

quality control methods, thus for standardizationof Morus species. The method can also beapplied as a quality control tool for variousformulations having rutin and isoquercitrin inthem.

REFERENCES

1. Morus (plant). Wikipedia, the freeencyclopedia.

URL

[http://en.wikipedia.org/wiki/Morus(plant)];accessed February 2010.2. Nuengchamnong N, Ingkaninan K,

Kaewruang W, Wongareonwanakij S,Hongthongdaeng

B. Quantitative determination ofdeoxynojirimycin in mulberry leaves usingliquid

chromatography- tandem massspectrometry. J. Pharm. Biomed. Anal. 44(4),2007, 853-858.

3. Chu Q, Lin M, Tian X, Ye J. Study oncapillary electrophoresis-amperometricdetection

profiles of different parts of Morus albaL. J.Chromatogr., A, 1116 (1-2), 2006, 286-290.

4. Tiamkao S., Sattayasai J., Puapairoj P. andBoonprakorb Y. Fens Forum Abstracts, 2004,2.

5. Nomura T. and Hano Y. Nat. Prod. Rep11(2): 1994, 205-218.

6. Harborne J. B., Mabry J. J. The Flavonoids.

Chapman and Hall, London, 1975, 5-39.7. Robards K., Antolovich M. Analyst, 1997,122, 11R.

8. Harborne J. B., Mabry J. J. TheFlavonoids. Advances in Research. Chapmanand

Hall, New York, 1982.9. Meclure J. W. Plant Flavonoids in Biology

and Medicine, Biochemical,Pharmacological and Structure Activity

Relationships, Alan R. Liss, New York, 1986,

77.10. Yamamoto and Gaynor. Therapeuticpotential of inhibition of the NF-B pathway inthe

treatment of inflammation and cancer".Journal of Clinical Investigation. 107 (2): 135.

11. Cushnie TPT, Lamb AJ. "Antimicrobialactivity of flavonoids". International Journal

of Antimicrobial Agents 26 (5): 2005, 343356.

12. Flavonoids: Antioxidant Activity and Health

Benefits.URLhttp://www.dietaryfiberfood.com/flavonoids2.php: accessed October 2009.

13. Escarpa A. and Gonzalez M.C. J.Chromatogr., 897, 2000, 161.

14. Escarpa A. and Gonzlez M.C. Anal. Chim.Acta, 427, 2001, 119.

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www.ijprd.com8

15. Tsao R. and Yang R.J. J. Chromatogr.,1018, 2003, 29.

16. Krist Sz. T., Ganzler K., Apti P., Szke .

and Kry . Analysis of antioxidantflavonoids from asteraceae and moraceaeplants by capillary electrophoresis.

Chromatographia, 56: 2002, 121-126.17. Apati P., Szentmihalyi K., Balazs A.,

Baumann D., Hamburger M., Kristo Sz. T.,Szoke E.

and Kery A. HPLC Analysis of theFlavonoids in Pharmaceutical Preparations from

Canadian Goldenrod (Solidago canadensis).Chromatographia, 2002, 56: S-65-S-68.

18. Snyder L R., Kirkland J J., Glajch J L.Practical HPLC method development, (2nd Edn),

John Wiley and Sons, Inc., U.S.A. 1997.

19. Eksteen K R., Schoenmakers P.

Chromatography Science Series, Handbook ofHPLC,1998, 78.

20. Constant H.L., Slowing K., Graham J. G.,Pezzuto J.M., Cordell G.A., Beecher C.W.W.,

A general method for the dereplication offlavonoid glycosides utilizing high performance

liquid chromatography/ mass spectrometricanalysis. Phytochem. Anal., 8: 1997, 176- 180.21. Snyder L.R., Kirkland J.J. Introduction toModern Liquid Chromatography. John

Wiley & Sons, London, 1974.

Tables and Figures

Table 1.0 Method validation parameters for the estimation of rutin and isoquercitrin by the proposedHPLC method.

ParameterResults for

rutin

Results for

isoquercitrin

Linear range (n = 3) g/mL 1.0- 150.0 3.0- 250.0

Correlation coefficient 0.99984 0.99931

LOD g/mL 0.25 0.63

LOQ g/mL 1.0 3.0Instrument precision

(% RSD, n=10)1.54 0.30

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Table 2.1 Results of intra-day precision data (repeatability) and inter-day precision data for rutin,by the proposed RP-HPLC method.

Intra-day Precision Inter-day precisionMean*

drug foundg/mL

Conc.of ruting/mL

Mean*drugfoundg/mL

S.D. %RSDDay 1 Day 2 Day 3

Mean**drugfoundg/mL

S.D. %RSD

5.0 4.807 0.0291 0.61 4.807 4.797 4.814 4.806 0.0087 0.18

60.0 59.256 0.3658 0.62 59.256 59.048 59.470 59.258 0.2111 0.36

120.0 123.984 0.1825 0.15 123.984 125.099 124.242 124.441 0.5836 0.47*Mean value from n = 3 determinations.**Mean value of three days.

Table 2.2Results of intra-day precision (repeatability) and inter-day precision for isoquercitrin,by the proposed RP-HPLC method.

Intra-day Precision Inter-day precisionMean*

drug found

g/mL

Conc. ofisoquercitrin

g/mL

Mean*drug

foundg/mL

S.D. %

RSD Day 1 Day 2 Day 3

Mean**drug

foundg/mL

S.D. %

RSD

30 29.091 0.0877 0.30 29.091 29.112 29.151 29.118 0.0300 0.10

100 99.867 0.0999 0.10 99.867 99.708 99.942 99.839 0.1196 0.12

200 199.114 0.2420 0.12 199.114 199.372 199.001 199.162 0.1902 0.10*Mean value from n = 3 determinations.**Mean value of three days.

Table 3.0 System suitability data for simultaneous estimation of rutin and isoquercitrin (10 g/mLeach), by the proposed RP-HPLC method.

ParameterResults for

Rutin*

Results forisoquercitrin*

% RSDfor

rutin

% RSD forisoquercitrin

Peak Area 306347.63 4668.190

339587.78 1596.657

1.52 0.47

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Retention time(Rt) in minutes 6.772 0.1300 8.417 0.1434 1.21 1.30

Peak

asymmetry0.99 0.32 1.31 0.43 0.61 0.83

Resolution* 3.72 0.09 0.21

*Mean S.D. for n = 6 determinations

Table 4.0 Comparative evaluation of the average percent contents of rutin in aqueous methanolicextracts of dried leaf powders of MorusalbaLinn. and Morus australisPoir., by the proposed RP-HPLCmethod.

Sample

Weightof

sample(g)

Average

contentof rutinfound insample

(g)n=7

Average%contentof rutin

Averagecontents

ofisoquercitrin found

insample

(g)n=7

Average%

contentof

isoquercitrin

Dried leafpowder ofMorus alba

Linn.

0.50

71.756 2.1502

0.01435 67.670 2.0619

0.01353

Dried leafpowder of

Morus australisPoir.

0.10550.163

5.9696

0.55016748.681 9.3953

0.74868

Table 5.1 Results of accuracy experiment for rutin, by the proposed RP-HPLC method.

Sample Level

Amountof rutinadded

g

Average*amountof rutin

recoveredg

% RSD ofthe

amount ofrutin

recovered

%Recovery

Average%

Recovery

0.500 gDried leaf 0 Nil

7.176 0.2150

3.00 - 99.76

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1 0.70 7.876 0.2028

2.57 100.01

2 1.40 8.544

0.10051.18 99.63

powder ofMorus alba

Linn.

3 2.10 9.244 0.0608

0.66 99.66

0 Nil 55.016 0.5970

1.09 -

1 5.50 59.637 0.7024

1.18 98.55

2 11.00 65.117 0.7836

1.20 98.64

0.100 gDried leafpowder of

Morus australisPoir.

3 16.50 71.731

0.5405

0.75 100.30

99.16

* Amount of rutin recovered is expressed as average S.D.

Table 5.2 Results of accuracy experiment for isoquercitrin, by the proposed RP-HPLCmethod.

Sample Level

Amountof rutinadded

g

Average*amount

ofisoquercitrinrecovered

g

% RSD ofthe

amount ofrutin

recovered

%Recovery

Average%

Recovery

0 Nil 6.767 0.2062 3.05 -

1 0.70 7.488 0.2049

2.74 100.28

2 1.40 8.165 0.1640

2.01 99.98

0.500 gDriedleaf

powderof

MorusalbaLinn. 3 2.10

8.840 0.1263

1.43 99.70

99.99

0 Nil 74.868 0.9395

1.25 -

1 7.50 82.060 0.7706 0.94 99.63

2 15.00 90.142 0.8462

0.94 100.30

0.100 gDried

leafpowderof

Morusaustralis

Poir. 3 22.5097.738 0.5953

0.61 100.38

100.10

* Amount of isoquercitrin recovered is expressed as average S.D.

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