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ONLINE HPLC-DAD/UV-MS DETERMINATION OF MAJOR FLAVONOIDS RUTIN AND ISOQUERCITRIN IN TWO MORUS SPECIES. Vijay Gokarn1,2*, Vidya Dighe1, Sasikumar Menon2, Bhalerao Khairnar1,2 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: vijay.g12@rediffmail.com
1

Vijay Gokarn

ABSTRACT Mulberry is one such plant that has tremendous therapeutic applications but not much exploited for its medicinal value. The taxonomy of Mulberry (Morus) is complex and disputed. There are no established methods to differentiate among existing species. The present paper demonstrates the work focused on the quality aspects of medicinal plant research for standardizing Mulberry Species. A reversed-phase high performance liquid chromatographic method with simultaneous diode array detection and mass spectral analysis (HPLC-DAD/UV-MS) was developed to detect and quantify two flavonoids, rutin and isoquercitrin as two major flavonoids from two Morus species, Morus alba Linn. and Morus australis Poir. collected from Maharashtra, India. The chromatographic separation was performed on a Cosmosil C8 column (150 x 4.6 mm, 5 m) with an isocratic mobile phase, comprising 0.1 % formic acid in distilled water, acetonitrile and methanol (75:15:10 v/v/v) at a flow rate of 1.0 mL/min. The wavelength selected for simultaneous quantitation of both analytes was 259 nm. The method developed with careful validation was successfully used for simultaneous quantification of two major flavonoids rutin and isoquercitrin from hydroalcoholic extracts of two Morus species. The hydroalcoholic extraction of the finely powdered dried leaves of both the plants was carried out using ultrasonic extraction technique. The further confirmation of rutin and isoquercitrin in both Morus leaves was done by developed online mass spectral analysis. An Online HPLC-DAD/UV-MS method thus established could be considered as a possible quality control tool for standardizing Morus species and even in routine quality testing of various formulations having rutin and isoquercitrin in them. Key Words: HPLC-DAD/UV-MS, Rutin, Isoquercitrin, Morus alba Linn., Morus australis Poir.

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INTRODUCTION Morus 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 majority of the species native to Asia [1]. It has been cultivated in many Asian countries such as China, India, Korea, Japan and Thailand where the leaves were used as food for silkworms [2]. There is an increasing interest on mulberry leaves because the leaves were found to have hypoglycemic, hypotensive, diuretic, bacteriostatic and antivirotic properties [3]. Mulberry leaves have traditionally been used for health promotion and the treatment of certain diseases such as diabetic hyperglycemia. It has been also reported in literature that Morus plant also possesses immunomodulating and activeoxygen scavenging properties [4]. The Morus plant is rich source of the isoprenoid substituted phenolic compounds, including flavonoids [5], which may be the reason for its effectiveness in the above mentioned diseases [6, 7] , and flavonoids may be precursors of some toxic substances in photosynthesis and cellular energy transfer processes [8,9]. Flavonoids have been referred to as "nature's biological response modifiers" because of strong experimental evidence of their inherent ability to modify the body's reaction to allergens, viruses, and carcinogens. They show anti-allergic, antiinflammatory [10], anti-microbial [11] and anticancer activity. Flavonoids are powerful antioxidants and scavengers of free radicals. Free radicals cause cellular, and DNA damage in our body and consequently induce age-related diseases such as dementia and cancer [12]. Considering these variable and contrasting properties and keeping in view the biological importance, the two major flavonoids in mulberry, rutin and isoquercitrin were selected in our research work as quality control markers.

HPLC is the method of choice for analysis of these phenolic compounds, because of its extremely high versatility and precision [13- 15]. A capillary electrophoretic method has been reported in literature, which is used for the identification of rutin and isoquercitrin present as major antioxidant flavonoids in Solidago gigantea and Morus nigra [16]. An HPLC method is reported in literature for analysis of the flavonoids from Solidago Canadensis [17]. However no methods are reported in literature for simultaneous quantitation of rutin and isoquercitrin from Morus alba Linn. and Morus australis Poir. A rapid HPLC-UV-MS method has been established for separation, identification and quantitation of two major flavonoids rutin and isoquercitrin present in the hydroalcoholic extracts of Morus alba Linn. and Morus australis Poir. The identities of the two major flavonoids were confirmed by passing HPLC eluted components through mass spectrometer and obtaining the parent masses (m/z values) of the respective peaks in the chromatogram while comparing with the reference standards. The method has been developed, validated according to the ICH requirements and applied for estimation of two major flavonoids, rutin and isoquercitrin from hydroalcoholic extracts of Morus alba Linn. and Morus australis Poir. in the present work. The hydro alcoholic extracts of both the plant leaf powders were prepared by using ultrasonic extraction technique. Objective:

To establish quality control method with the help of modern analytical technique for standardizing 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- 205 nm) used in the present work were of HPLC grade (J. T. Baker) and were procured from Orochem India Pvt. Ltd. Water used in the present research work, was purified with a MilliQ water purifying system (Millipore, USA). The solvents were filtered through 0.5m membrane (Millipore) and degassed in an ultrasonic bath. Formic acid (100 %, GR grade) was procured from Merck Specialties Pvt. Ltd. Plant Material The leaves of Morus alba Linn. were collected from wild plants found in Keshav Srushti, Mumbai, India and the leaves of Morus australis Poir. were collected from Mahabaleshwar, Satara, India. Herbaria of both the plant species were prepared and authenticated from Botanical Survey of India (BSI), Pune, India and National Botanical Research Institute (NBRI), Lucknow, India respectively. The leaves of Morus alba Linn. and Morus australis Poir. were shade dried, powdered and then sieved through BSS mesh size 85 and stored at 25oC, in light resistant and airtight containers. Preparation of standard stock solutions of rutin and isoquercitrin Individual standard stock solutions of rutin (1000 g/mL) and isoquercitrin (1000 g/mL) were prepared in methanol. Weights equivalent to 10 mg of rutin and isoquercitrin were weighed accurately and dissolved separately in 5.0 mL of methanol, followed by sonication for 1 min and finally making up the volume of solutions to 10.0 mL, with methanol. Preparation of mixed working standard solutions

Desired aliquots of stock solutions of rutin (1000 g/mL) and isoquercitrin (1000 g/mL) were transferred to a 10 mL standard volumetric flask. Solution in the flask was then diluted upto the mark with the mobile phase to obtain mixed working standard solutions of rutin and isoquercitrin in concentration range of 1g/mL to 150 g/mL for rutin and 3.0 g/mL to 250.0 g/mL for isoquercitrin. Preparation of sample solutions Accurately weighed about 100 mg of finely powdered leaf powder of Morus alba Linn. and 500 mg of finely powdered leaf powder of Morus australis Poir. were taken in two separate tarsons tubes of capacity 15 mL and to each tube, 10 mL of methanol: Milli-Q water (1:1) was added and the tubes were vortex mixed for 30 sec and kept in ultrasonic bath (Frequency- 50 Hz) for extraction of phytoconstituents for an optimized time period of 15 mins. The extracts were then high speed centrifuged at 15,000 rpm using a microcentrifuge and the clear supernatant sample solutions were finally filtered using 0.45 m nylon filters (Millipore). The hydro alcoholic extracts of both the plant leaf powders were thus prepared and used for further chromatographic analysis. HPLC- UV/DAD analysis Separation for qualitative and quantitative analysis was performed by a Jasco HPLC system comprising an isocratic pump (Jasco PU- 980, Intelligent HPLC Pump) with a rheodyne injector valve having a fixed volume loop of 20 L capacity and a photo diode array detector (Jasco MD- 910 Multiwavelength detector). Phytochemicals were separated on a reversedphase Cosmosil C8 analytical column (4.6 mm x 150 mm) 5 m particle size. The MS compatible optimized mobile phase comprised of 0.1%

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formic acid in Milli-Q water, acetonitrile and methanol, in the ratio of 75: 15: 10 v/v/v. The flow rate was maintained constant at 1.0 mL/ min. Before use, the mobile phase was filtered through 0.45 m nylon filters (Millipore) and deaerated in an ultrasonic bath. Data were integrated by Jasco Borwin software, V 1.21. Mass spectral analysis Mass spectrometry was performed with an Applied Biosystems (MDS SCIEX) API 2000 triple quadrupole mass spectrometer. A turbo ion-spray source was used in negative-ion mode with the settings: source temperature; 450o C; ion spray voltage; -4500 V; nebulizer gas (N2), 60-40 arbitrary units; curtain gas (N2), 20 arbitrary units; entrance potential (EP), -10 V; declustering potential (DP), -100 V; Full scan acquisition was performed between m/z 50 and 800 U. Experiment was performed with the quadrupole (Q1) operated at unit resolution. All samples and solutions were filtered through 0.45 m nylon filters (Millipore) before analysis by HPLC or Mass Spectrometry. RESULTS Method validation [18, 19] The developed RP-HPLC method for simultaneous determination of rutin and isoquercitrin was validated for linearity, limit of detection (LOD), limit of quantitation (LOQ), precision, accuracy and system suitability. Linearity To establish the linearity, calibration plots of peak area against concentration were constructed after triplicate analysis of mixed working standard solutions of rutin and isoquercitrin, simultaneously, at seven different concentrations in the range of 1.00 150.00 g/ mL for rutin and 3.0 250.00 g/ mL for isoquercitrin. The peak area values and

concentrations at seven different calibrant levels of the respective working standard solutions of rutin and isoquercitrin were subjected to regression analysis to calculate the calibration equation and correlation coefficient (r). The results are listed in Table 1.0. Limit of Detection (LOD) and Limit of Quantitation (LOQ) LOD and LOQ were established at signal to noise ratio of 3:1 and 10:1 respectively. The values obtained are listed in Table 1.0. Precision The instrument precision was studied by injecting mixed standard solution of rutin and isoquercitrin (10.0 g/mL each), in ten replicates, in the chromatographic system under the specified conditions. The results expressed as percent RSD of peak area, are listed in Table 1. Repeatability (Intra-day precision) of the method was assessed by triplicate analysis using the mixed standard solutions at low (3 g/mL for rutin 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 for isoquercitrin) concentration levels covering the entire linearity range. The mean percent RSD was found to be < 2 for all three levels. Similarly, the intermediate precision was assessed by triplicate analysis of the abovementioned low, medium and high concentration levels, on three different days. The results for the precision experiment are listed in Table 2.1 and 2.2. System Suitability System suitability was carried out to verify that the resolution and reproducibility of the system were acceptable for the analysis. System suitability test was carried out by injecting 20 l of mixed standard solution of rutin and isoquercitrin each having

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concentration 10.0 g/mL, in six replicates, in the chromatographic system under optimized conditions. The chromatograms were recorded after injecting 20 l of the mixed standard solution of rutin and isoquercitrin (10 g/mL). The values of percent relative standard deviation (% RSD) of peak area of rutin, retention time of rutin and resolution of chromatographic peak of rutin and isoquercitrin were taken as an indicator of system suitability and the results obtained are listed in Table 3.0. A representative chromatogram of mixture of reference standard solution of rutin and isoquercitrin (10.00 g/mL each) is shown in Fig. 1.0. Assay Experiment The developed and validated RP- HPLC method for the simultaneous detection of rutin and isoquercitrin was applied for quantitation of two major flavonoids rutin and isoquercitrin using hydro alcoholic extracts of dried leaf powders of Morus alba Linn. and Morus australis Poir. The assay experiment was performed with seven determinations by injecting 20l of each of the sample solutions, i.e., hydro alcoholic extracts obtained by ultrasonic extraction technique, from the dried leaf powders of Morus alba Linn. and Morus australis Poir. Typical HPLC chromatographic fingerprint patterns of the hydroalcoholic extracts of both plant leaf powders are shown in Fig. 2.0. The amounts of rutin and isoquercitrin were calculated by use of appropriate calibration curves. Comparative evaluations of the results for assay experiment are tabulated in Table 4.0. Accuracy Accuracy of the method was determined by performing recovery experiments for both the plant leaf powders of Morus species, using standard addition method. The recovery of the added standards rutin and isoquercitrin was

studied by spiking working standard stock solutions at three different concentration levels to the leaf powders of Morus alba and Morus australis just before extraction of leaf powders. The accuracy was expressed as the percentage of rutin and isoquercitrin recovered by the assay. The results of accuracy experiment are listed in Table 5.1 and Table 5.2. The results indicate good accuracy of the method for the simultaneous quantitative determination of rutin and isoquercitrin from leaf powders of both Morus species. Mass Analysis An MS compatible mobile phase was developed in the present research work facilitating an online HPLC- DAD- MS analysis, that further confirmed the presence of rutin and isoquercitrin in the hydroalcoholic extracts of dried leaves of Morus alba Linn. and Morus australis Poir. The negative mode ESI-MS is reported to be effective for rutin [20] . Thus, infusion in negative ion mode was employed, which facilitated tuning of source gas and compound parameters for achieving strong signals of rutin and isoquercitrin that were observed at 609.8 and 463.5 [M - H]- as parent molecular ions respectively in the negative Q1 mode. Flavonol O- glycoside, rutin furnished the deprotonated molecule at m/z 609 of the glycoside and an ion corresponding to the deprotonated aglycone [A H]-. The latter ion at m/z 301 was formed due to the loss of rhamnose and glucose from the glycosides. Similarly isoquercitrin furnished the deprotonated molecule at m/z 463 of the glucoside and an ion corresponding to the deprotonated aglycone [A H]-. The HPLC eluents that were first detected by the UV detector were simultaneously subjected to ESI-MS under the optimized conditions, in a negative mode to obtain individual mass spectras.

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The MS spectra of rutin and isoquercitrin present in both the hydroalcoholic extracts of dried leaf powders of Morus alba Linn. and Morus australis Poir. are shown in Fig3.1 and 3.2. that confirms the identity of both the flavonoids. DISCUSSION The capillary electrophoretic method reported in literature [16] serves to be a viable alternative to analysis by HPLC method with respect to Qualitative analysis but the Quantitation of the flavonoids in the reported method is performed by HPLC. The methods reported in literature mainly use gradient mode of elution which usually poses a great challenge for repeatability of results in case of the complex herbal medicines and the column needs to be equilibrated after each sample run. The proposed method uses an isocratic mode of elution due to its simplicity, providing stable baseline and unvarying response factor; also the isocratic elution facilitated best possible separation for the specified pair of peaks and also for the other phytoconstituents separated in the respective chromatograms Fig.2.0. Almost all reported methods use C18 analytical column of 250 mm length, whereas the present method uses a shorter C8 analytical column of 150 mm length which provides a good separation of the phytoconstituents with values of Rs 1.5 which is acceptable resolution according to the Snyder method [21] . The present method uses a mobile phase comprising 0.1% formic acid, acetonitrile and methanol in the ratio 75:15:10v/v/v. Formic acid helps in the ionization of molecules and mixture of acetonitrile and methanol facilitates favorable UV transmittance and low viscosity. The mobile phase pH- 2.72 was selected with following considerations; a low pH protonates column silanols and reduces their chromatographic activity, is generally preferred and a low pH (< 3) is usually quite different from

the pKa values of common acidic and basic functional groups. Therefore, at low pH the retention of the compounds are not affected by small changes in pH and the reversed phase chromatographic method will be more rugged [18] . For qualitative purpose the method was evaluated by taking into account retention time precision, peak asymmetry and resolution of the two peaks of rutin and isoquercitrin. The retention time was highly repeatable, with RSD values below 2% and even for peak asymmetry and peak resolution (Table 3.0). Linearity, LOD and LOQ (Table 1.0), precision (Table 2.1 and Table 2.2) and accuracy (Table 5.1 and Table 5.2) were evaluated for quantitative purposes. High recovery (ranging from 99.16- 100.10 %) and repeatability of both the flavonoids from both the plant powders in presence of the other phytoconstituents was indicative of satisfactory accuracy of the method. The wavelength for detection and quantitation of the phytoconstituents was selected by use of the diode array detector. The HPLC- DAD scans providing UV spectra obtained along with the respective chromatogram for mixed standard solution of rutin and isoquercitrin (10.00 g/mL each) prepared in mobile phase are shown in Fig. 4.0 The isocratic elution using HPLC-DAD allowed separation and quantitation of the two flavonoids rutin and isoquercitrin found as the major polyphenols in hydroalcoholic extracts of both the Morus species and m/z values for rutin and isoquercitrin Fig. 3.1 and Fig. 3.2 obtained by online mass spectrometry facilitated further confirmation of both the flavonoids in comparison with the individual reference standards. Rutin as well as isoquercitrin which were found to be the major flavonoid phytoconstituents in line with the literature can serve as possible quality control indicators in Morus species for future prospects since changes in the ratio of rutin to isoquercitrin (and/or any other 6

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phytoconstituents) would indicate chemical change/degradation in the hydroalcoholic extract of the raw material or the finished formulation made from mulberry. The average marker contents in both the selected plants Morus alba Linn. and Morus australis Poir. varied remarkably (Table 4.0). The average rutin and isoquercitrin contents in dried leaf powder of Morus australis Poir. were 38.34 and 55.33 times higher than that in the dried leaf powder Morus alba Linn. CONCLUSION A simple, precise and accurate HPLC- DADMS method has been developed and validated for simultaneous detection and quantitation of rutin and isoquercitrin with successful application to hydro-alcoholic extracts of leaf powders of Morus alba Linn. and Morus australis Poir. Sample preparation was simple and no tedious cleanup was necessary. Rutin and Isoquercitrin present as the two major flavonoids in both the Morus species can be selected as the phytochemical markers for chromatographic fingerprints and for routine quality control methods, thus for standardization of Morus species. The method can also be applied as a quality control tool for various formulations having rutin and isoquercitrin in them. REFERENCES 1. Morus (plant). Wikipedia, the free encyclopedia. URL [http://en.wikipedia.org/wiki/Morus(plant)]; accessed February 2010. 2. Nuengchamnong N, Ingkaninan K, Kaewruang W, Wongareonwanakij S, Hongthongdaeng B. Quantitative determination of deoxynojirimycin in mulberry leaves using liquid

chromatographytandem mass spectrometry. J. Pharm. Biomed. Anal. 44(4), 2007, 853-858. 3. Chu Q, Lin M, Tian X, Ye J. Study on capillary electrophoresis-amperometric detection profiles of different parts of Morus alba L. J. Chromatogr., A, 1116 (1-2), 2006, 286-290. 4. Tiamkao S., Sattayasai J., Puapairoj P. and Boonprakorb Y. Fens Forum Abstracts, 2004, 2. 5. Nomura T. and Hano Y. Nat. Prod. Rep 11(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. The Flavonoids. Advances in Research. Chapman and 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. Therapeutic potential of inhibition of the NF-B pathway in the treatment of inflammation and cancer". Journal of Clinical Investigation. 107 (2): 135. 11. Cushnie TPT, Lamb AJ. "Antimicrobial activity of flavonoids". International Journal of Antimicrobial Agents 26 (5): 2005, 343 356. 12. Flavonoids: Antioxidant Activity and Health Benefits. URL http://www.dietaryfiberfood.com/flavonoids2.p hp: 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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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 antioxidant flavonoids from asteraceae and moraceae plants 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 the Flavonoids 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), Tables and Figures

John Wiley and Sons, Inc., U.S.A. 1997. 19. Eksteen K R., Schoenmakers P. Chromatography Science Series, Handbook of HPLC, 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 of flavonoid glycosides utilizing high performance liquid chromatography/ mass spectrometric analysis. Phytochem. Anal., 8: 1997, 176- 180. 21. Snyder L.R., Kirkland J.J. Introduction to Modern Liquid Chromatography. John Wiley & Sons, London, 1974.

Table 1.0 Method validation parameters for the estimation of rutin and isoquercitrin by the proposed HPLC method. Results for Parameter rutin Linear range (n = 3) g/mL Correlation coefficient LOD g/mL LOQ g/mL Instrument precision (% RSD, n=10) 1.0- 150.0 0.99984 0.25 1.0 1.54

Results for isoquercitrin 3.0- 250.0 0.99931 0.63 3.0 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 Conc. of rutin g/mL 5.0 60.0 Mean* drug found g/mL 4.807 59.256 % RSD Day 1 0.0291 0.3658 0.61 0.62 4.807 59.256 Inter-day precision Mean* drug found g/mL Day 2 4.797 59.048 Day 3 4.814 59.470 Mean** drug found g/mL 4.806 59.258 % RSD 0.18 0.36 0.47

S.D.

S.D.

0.0087 0.2111

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

Table 2.2 Results of intra-day precision (repeatability) and inter-day precision for isoquercitrin, by the proposed RP-HPLC method. Intra-day Precision Conc. of isoquercitrin g/mL 30 100 Mean* drug found g/mL 29.091 99.867 % RSD Day 1 0.0877 0.0999 0.30 0.10 29.091 99.867 Inter-day precision Mean* drug found g/mL Day 2 29.112 99.708 Day 3 29.151 99.942 Mean** drug found g/mL 29.118 99.839 % RSD 0.10 0.12 0.10

S.D.

S.D.

0.0300 0.1196

200 199.114 0.2420 0.12 199.114 199.372 199.001 199.162 0.1902 *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/mL each), by the proposed RP-HPLC method. Results for Parameter Rutin* Peak Area 306347.63 4668.190 % RSD for rutin 1.52

Results for isoquercitrin* 339587.78 1596.657

% RSD for isoquercitrin 0.47 9

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Retention time (Rt) in minutes Peak asymmetry Resolution*

6.772 0.1300 0.99 0.32

8.417 0.1434 1.31 0.43

1.21 0.61 0.21

1.30 0.83

3.72 0.09

*Mean S.D. for n = 6 determinations Table 4.0 Comparative evaluation of the average percent contents of rutin in aqueous methanolic extracts of dried leaf powders of Morus alba Linn. and Morus australis Poir., by the proposed RP-HPLC method. Average contents Average of % isoquerci content trin found of in isoquercit sample rin (g) n=7 67.670 2.0619

Sample

Wei ght of sam ple (g)

Average content of rutin found in sample (g) n=7 71.756 2.1502

Average % content of rutin

Dried leaf powder of Morus alba Linn.

0.50

0.01435

0.01353

Dried leaf powder of Morus australis Poir.

0.10

550.163 5.9696

0.55016

748.681 9.3953

0.74868

Table 5.1 Results of accuracy experiment for rutin, by the proposed RP-HPLC method. Average* % RSD of Amount amount the Average of rutin % Sample Level of rutin amount of % added Recovery recovered rutin Recovery g g recovered 0.500 g 7.176 99.76 0 Nil 3.00 Dried leaf 0.2150 10
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7.876 0.2028 8.544 2 1.40 0.1005 9.244 3 2.10 0.0608 55.016 0 Nil 0.5970 0.100 g 59.637 1 5.50 Dried leaf 0.7024 powder of 65.117 Morus australis 2 11.00 0.7836 Poir. 71.731 3 16.50 0.5405 * Amount of rutin recovered is expressed as average S.D. powder of Morus alba Linn. 1 0.70

2.57 1.18 0.66 1.09 1.18 1.20 0.75

100.01 99.63 99.66 98.55 99.16 98.64 100.30

Table 5.2 Results of accuracy experiment for isoquercitrin, by the proposed RP-HPLC method. Average* % RSD of amount Amount the Average of of rutin % amount of % Sample Level added isoquercitrin Recovery rutin Recovery recovered g recovered g 6.767 0.500 g 0 Nil 3.05 0.2062 Dried 7.488 leaf 1 0.70 2.74 100.28 0.2049 powder 99.99 of 8.165 2 1.40 2.01 99.98 Morus 0.1640 alba 8.840 3 2.10 1.43 99.70 Linn. 0.1263 74.868 0.100 g 0 Nil 1.25 0.9395 Dried leaf 82.060 1 7.50 0.94 99.63 powder 0.7706 100.10 of 90.142 2 15.00 0.94 100.30 Morus 0.8462 australis 97.738 3 22.50 0.61 100.38 Poir. 0.5953 * Amount of isoquercitrin recovered is expressed as average S.D.

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