Journal of Chromatographic Science Advance Access published June 17, 2015 Journal of Chromatographic Science 2015;1– 5 doi:10.1093/chromsci/bmv071
Article
Determination of Rutin and Isoquercetin Contents in Hibisci mutabilis Folium in Different Collection Periods by HPLC Diangang Liu1, Qing Mei2,†, Xiangluan Wan3, Hongling Que1, Luyang Li2 and Dingrong Wan2* 1 Foreign Languages School, Hubei University of Traditional Chinese Medicine, Wuhan 430065, China, 2Pharmaceutical School, SouthCentral University for Nationalities, Wuhan 430074, China, and 3Luojia College of Wuhan University, Wuhan 430065, China
*Author to whom correspondence should be addressed. Email: [email protected] †
Co-first author.
Received 6 January 2015; revised 1 May 2015
Hibisci mutabilis Folium (HMF), the dried leaf of Hibiscus mutabilis (Malvaceae), is commonly used in traditional Chinese medicine. This article aimed to establish a high-performance liquid chromatography method for the determination of rutin and isoquercitrin contents in the HMF, and to compare the content variation in all the samples, including nearly withered yellow leaf, in different collection periods, so as to provide research basis for the quality evaluation and determination of the optimal collection period. A reversed-phase HPLC separation method was employed, with a BDS Hypersil C18 column (4.6 m 3 250 mm, 5 mm), under the following conditions: acetonitrile – 0.3% phosphoric acid (15:85, v/v) solution as the mobile phase, flow rate 1.0 mL/min at 3088 C and detection wavelength 254 nm. The calibration curves for rutin and isoquercitrin were linear over the range of 1.5–48 and 0.25 – 8 mg/mL, and the average recoveries were 99.92 and 100.45% (RSD: 2.39% and 2.11%, respectively)]. Based on the analysis results, it was found that contents of rutin and isoquercitrin in HMF (mature green leaf) harvested in different periods had significant difference, and reached the highest in mid-December. It was also found that the contents of the two components in the mature green leaf were much higher than those in the nearly withered leaf from the same collection period. In conclusion, the results indicated that the HPLC method was easy-to-operate and precise, and could be applied for the determination of rutin and isoquercitrin contents in the HMF. The experimental data also showed that early winter should be the most suitable collection period for HMF.
Introduction Hibisci mutabilis Folium (HMF), the dried leaf of Hibiscus mutabilis (Malvaceae), is commonly used in traditional Chinese medicine. It has the function of cooling the blood, relieving toxin, reducing swelling and alleviating pain, and has long been used in the treatment of ulcer, swelling, herpes zoster, scald, bruises, etc. Besides, HMF is also widely used in ethnic minority areas for migraine and otitis media (by Dong people), pulmonary hemoptysis (by She people), menorrhagia (by She, Jingpo, Lisu people), appendicitis (by Yao people), epidemic parotitis (by Lisu, Yao people) and so on (1 – 3). This crude drug contains chemical components such as flavonoid glycosides, phenols, tannins, phenylalanine and volatile oil, etc. (4). Its ethanol extract has antibacterial effect (5) and aqueous extract has effects on anti-inflammation and abirritation (6). Flavonoid glycosides are main active constituents of HMF, among which rutin, quercitrin, hyperoside and isoquercitrin
have already been reported (7). They have significant effects on anti-inflammation and liver protection, and have the ability to prevent and treat ischemia reperfusion injury of the kidney (8, 9). Determination of the rutin content in HMF was reported in the other studies (10, 11), so was that of isoquercitrin. However, there was no research being done to simultaneously determine the contents of rutin and isoquercitrin in HMF. It is known that rutin and isoquercitrin have significant pharmacological activities of anti-inflammatory, analgesic, antibacterial, antioxidation and antitumor (12, 13). Moreover, rutin also has protective action on heart, brain and kidney injuries after ischemic reperfusion and gastric mucosa injury (12). Therefore, the determination of rutin and isoquercitrin contents in this crude drug has certain significance for its quality control. It is known that several factors, such as habitat, harvest time and so on, significantly affect the quality of herbal medicines. However, there was no report about the optimal harvest season of HMF in the literatures apart from the Chinese Pharmacopoeia (2010) and so on, which reported it should be collected in summer or fall (1, 3). Usually, determination of harvest season of raw medicinal materials is based on conventional folk experience without any scientific evidence; therefore, it is necessary to determine the optimal harvest season of this medicine by the related study. In our prophase research, the best harvest time was preliminarily determined after studying the total flavonoids content of HMF in different collection periods (14). But for the more effective quality evaluation, we developed another analytical method by HPLC to simultaneously determine the contents of rutin and isoquercitrin in HMF in different harvest period. Therefore, the best harvest season was further confirmed and the quality of the crude drug could be well evaluated. Experimental Apparatus and reagents The HPLC analysis was performed using Dionex Ultimate 3000 Series (Dionex, USA) Chameleon data processing system equipped with a binary pump, an autosampler, a column thermostat, temperature-controlled sample trays, an online degasser and a diode array detector. The reference substances of rutin and isoquercitrin were purchased from the National Institute for the Control of Pharmaceutical and Biological Products in hina (batch number: 111809-201102, 100080-200707). Chromatographic grade
# The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: [email protected]
acetonitrile was purchased from Tedia, Fairfield, USA (batch number: AS1122-001). Water for HPLC was purified by an Aquapro Hi-End Water Treatment Solution Provider (ASWO-0005-U, Ever Yong Enterprises). Ethanol and phosphoric acid were of analytical grade. Plant materials Seven batches of HMF were collected from South-Central University for Nationalities (SCUEC, Wuhan, Hubei province) in different seasons by ourselves and one batch of sample mixed with a few yellow leaves was purchased from Bozhou (Anhui), as presented in Table I. These samples were identified by Professor Dingrong Wan (Pharmaceutical College, SCUEC) and were deposited after removing impurities and drying in shade.
and isoquercitrin solutions were 1.5, 3, 6, 12, 24, 48 mg/mL and 0.25, 0.5, 1, 2, 4, 8 mg/mL, respectively. Calibration curves were generated based on the formula: Y ¼ aX þ b, among which the Y-axis was the value of peak area (mAU) and the X-axis was the concentration (mg/mL) injected into HPLC column of each standard. The regression equations of rutin and isoquercitrin calibration curves and the parameters for linearity are presented in Table II. The correlation coefficients were over 0.9999, which indicated good linearity under each range. Optimization of the sample extraction conditions Being aware that extraction of rutin and isoquercitrin from HMF is a method-dependent process, some important parameters determining the amount of extracted rutin and isoquercitrin were investigated.
Methods and results Preparation of reference solution 0.1 mg/mL reference solutions of rutin and isoquercitrin were prepared by dissolving accurately weighed rutin and isoquercitrin (Figure 1) in 50% ethanol, respectively. Sample preparation The powdered HMF (crushed and passed through a 60-mesh sieve) was extracted according to the description below. One gram of accurately weighed sample was placed in roundbottomed flask and then 50 mL of 50% ethanol solution was added and refluxed for 1 h. After cooling, 50% ethanol solution was added in order to make up the loss weight. Then the solution was shaken and filtered with a 0.45 mm syringe membrane. HPLC conditions The chromatographic separation was carried out with BDS Hypersil C18 column [4.6 250 mm, 5 mm, Thermo Scientific (San Jose, USA)]. Column temperature was set at 308C. Elution was conducted using acetonitrile and 0.3% phosphoric acid (15:85, V/V) as mobile phase. The flow rate was 1 mL/min. Ten microliters of each sample were injected into the column. UV spectra were acquired in the range of 200–380 nm and the quantitative wavelength was 254 nm. The chromatogram of standards and sample are shown in Figure 2. Preparation of standard calibration curves and analysis of linearity Six different concentration levels of mixed reference solutions were analyzed in this research. The six concentrations of rutin
Table I Samples and Their Sources No.
Habitat
Properties of samples
Collection date
1 2 3 4 5 6 7 8
Campus of SCUEC, Wuhan
Mature green leaves
10 June 2012 21 August 2012 18 November 2012 10 December 2012 18 November 2012 1 December 2012 16 December 2012 10 October 2012 (Production date)
Nearly withered yellow old leaves Bozhou, Anhui
2 Liu et al.
Mixed with a few yellow leaves
Optimization of the extraction methods 0.5 g accurately weighted sample (No. 7) was chosen for the comparative study of the two extraction methods. First, 25 mL of 50% (V/V) ethanol solution was added to reflux and then heated for 1 h. After cooling, the extract was filtered with a 0.45 mm syringe membrane to form the sample solution. This procedure was then repeated via ultrasonic method for comparative study. The HPLC analysis results showed that the content of rutin and isoquercitrin extracted via reflux was twice as much as that extracted via ultrasonic method, which demonstrated that refluxing was the optimal extraction method. Optimization of the extraction solvent Five accurately weighed samples (0.5 g) were refluxed by 25 mL of 20, 30, 40, 50 and 70% (V/V) ethanol solution for 1 h in parallel. The HPLC analysis results showed that extraction efficiency of the two compounds improved as the ethanol concentration increased, and increase of the content of the two compounds was fairly gradual when the ethanol concentration came to 30 –50%, then it began to decrease. Therefore, 50% (V/V) ethanol solution was chosen as the best extraction solvent. Optimization of the extraction time Four accurately weighted samples (No. 7), each one weighed 0.5 g, were refluxed with 50% (V/V) ethanol solution for 30, 60, 90, 120 min, respectively. The HPLC analysis results revealed that the two compounds had been extracted completely in 60 min. Hence, the optimal extraction time is 60 min. Optimization of ratio of the extraction solvent Five accurately weighted samples (No. 7), each one weighed 1.0 g, were refluxed with 50% (V/V) ethanol solution for 60 min. The solvent ratio was 20:1, 30:1, 40:1, 50:1, 60:1, respectively. The HPLC analysis results proved that when the solvent ratio transferred from 50:1 to 60:1, the extraction efficiency showed an upward tendency, but the change of it was smooth and gentle. Thus, 50:1 was chosen as the best extraction ratio (Figure 3). Optimization of chromatographic conditions Based on the results of the three-dimensional chromatograms of the two standard solutions by diode array detector (the wavelength range from 190 to 600 nm), the detection wavelength
Figure 1. Chemical structures of (A) rutin and (B) isoquercitrin.
Table II Linear Equation, R 2 and Range of Rutin and Isoquercitrin for Quantitative Determination Compound
Regression equation
R2
Range (mg/mL)
Rutin Isoquercitrin
Y ¼ 234.42X 2 0.0228 Y ¼ 280.56X 2 0.0106
0.9999 0.9999
1.5 –48 0.25 –8
time, better resolution (R . 1.5) and higher theoretical plate numbers (n . 8000). Method validation
Figure 2. Chromatogram of reference solution and sample solution: (1) rutin and (2) isoquercitrin. This figure is available in black and white in print and in color at JCS online.
was set at 254 nm, where the two standard substances had the strongest absorption. A screening on the solutions of mobile phase was carried out in order to improve their chromatographic separation efficiency. The developed analytical method for the determination of the two components was based on the methods of Hibisci mutabilis Folium of Chinese Pharmacopoeia and the related literature. Three kinds of mobile phase (acetonitrile, tetrahydrofuran and methanol with 0.3% phosphoric acid, respectively) were studied and compared, and separating effect was detected by each chromatographic peak. As it had led to an increasing degree of separation, acetonitrile was preferred over methanol and tetrahydrofuran as the mobile phase. Then a series of solutions with different ratios of acetonitrile –0.3% phosphoric acid were studied and the ratio 15:85 was selected ultimately. In the end, the optimal chromatographic conditions were chosen, because of the better peak symmetry, shorter retention
Precision Ten-microliter reference substances’ solutions were injected into the HPLC instrument continuously under the optimized chromatographic conditions for five times. After determination, the results showed that relative standard deviations (RSDs) of the peak areas of the rutin and isoquercitrin were 0.11 and 0.17%, respectively, indicating that the instrument was highly precise. Repeatability Five samples (No. 8) were detected in parallel by using the above methods of sample preparation and chromatographic conditions. The RSDs of the peak area of the two components (rutin and isoquercitrin) were 1.52 and 2.89%, respectively, indicating nice repeatability. Stability One sample (No. 8) solution was prepared according to the sample preparation method discussed earlier, and then rutin and isoquercitrin peak areas were detected at 0, 2, 4, 8 and 12 h under the chromatographic conditions as described earlier. The RSDs of rutin and isoquercitrin peak area values were 2.07 and 3.24%, respectively. This showed that the two components in sample solutions were stable for at least 12 h. Recovery and accuracy The accuracy was determined by calculating the recovery after a standard substances addition procedure. The recoveries of Determination of Rutin and Isoquercetin Contents in HMF in Different Collection Periods 3
Table III Recovery of Rutin in Hibisci mutabilis Folium (No. 8) Sample amount (g)
Rutin in the sample (mg)
Added rutin amount (mg)
Determined rutin amount (mg)
Recovery (%)
0.5006 0.5005 0.5006 0.5003 0.5002
0.4808 0.4808 0.4808 0.4808 0.4808
0.504 0.504 0.504 0.504 0.504
0.9702 0.9763 0.9913 1.0006 0.9836
97.10 98.31 101.28 103.13 99.76
Mean recovery (%)
RSD (%)
99.92
2.39
Mean recovery (%)
RSD (%)
100.45
2.11
Table IV Recovery of Isoquercitrin in Hibisci mutabilis Folium (No. 8) Sample amount (g)
Isoquercitrin in the samples (mg)
Added isoquercitrin amount (mg)
Determined isoquercitrin amount (mg)
Recovery (%)
0.5006 0.5005 0.5006 0.5003 0.5002
0.0521 0.0521 0.0521 0.0522 0.0521
0.040 0.040 0.040 0.040 0.040
0.0919 0.0910 0.0925 0.0930 0.0931
99.50 97.25 101.00 102.00 102.50
Table V Amounts of Rutin and Isoquercitrin in Hibisci mutabilis Folium Harvested in Different Seasons Habitat
Sample/ harvested season
Content of Rutin (mg/g)
Content of isoquercitrin (mg/g)
Campus of SCUEC, Wuhan
Mature green leaf (June 10)
0.2979
0.0215
Mature green leaf (August 21) Mature green leaf (November 18) Mature green leaf (December 10) Nearly withered yellow old leaf (November 18) Nearly withered yellow old leaf (December 1) Nearly withered yellow old leaf (December 16) Mixed with a few yellow leave (October 10)
0.1364 0.1358 1.1305 0.2110
0.0199 0.0223 0.2491 0.0261
0.3840
0.0773
0.4250
0.0810
0.9772
0.1016
Bozhou, Anhui
Figure 3. Amount variation of the components with respect to different factors. (A) Ethanol concentration; (B) extraction time; (C) solid – liquid ratio. This figure is available in black and white in print and in color at JCS online.
quantification procedure of the standard substances were examined at five levels covering the linearity range of calibration curves. The five precisely weighed samples (No. 8) were extracted using the method described earlier and determined by HPLC after adding an appropriate amount of rutin and isoquercitrin (0.504 and 0.040 mg, respectively) to each sample. Recovery of the two component,and RSDs were calculated. The results are presented in Tables III and IV and showed that the method had a good accuracy. The content determination of rutin and isoquercitrin in different collection periods All samples were accurately weighed (each sample was prepared in triplicate), operated and determined according to the optimized extraction method and HPLC conditions. According to the detection results of the peak areas of rutin and isoquercitrin in HMF (including nearly withered yellow old 4 Liu et al.
leaves) harvested in different seasons, we calculated the amounts of the two components in the light of the calibration curves and listed in Table V and showed in Figure 4. Discussion and summary The scanning results suggested that rutin and isoquercitrin had the largest absorption peaks when the wavelength was 254 nm. Therefore, the detection wavelength was determined at 254 nm. As the determination results of the mature-green-leaf samples have shown, overall the contents of rutin and isoquercitrin in HMF collected in the period from mid-June to mid-November did not change a lot. While the contents of two components in the samples harvested in mid-December increased dramatically, they were even about 10 times as much as those contained in the samples of August. From these it can be inferred that the contents of rutin and isoquercitrin had very significant differences in different harvest times. Similarly, the contents of rutin and isoquercitrin were the highest in mid-December in the nearly withered yellow old leaf, but significantly lower than the mature
However, on the basis of the results of the total flavones content in HMF harvested in different periods, we suggested that the early winter should be recommended as the optimal collection period.
Acknowledgments This work was supported by Science Research Foundation of Health Department of Hubei Province, China (Grant No. 2013Z-Y02).
References
Figure 4. Amount variation of the two components in Hibisci mutabilis Folium collected in different periods. (A) Rutin and (B) isoquercitrin. This figure is available in black and white in print and in color at JCS online.
green leaf collected at the same harvest time. In addition, the HMF purchased from Bozhou had even higher levels of the amounts of rutin and isoquercitrin than most samples collected in SCUEC. So, the content difference of the samples of different habitats is also remarkable. Based on our prophase research of the total flavones contents in HMF harvested in different times by ultraviolet spectroscopy (UV), it had been found that the HMF purchased from Bozhou had a much lower level of the total flavones than each batch of samples collected in SCUEC (14), which conflicted with the results of the contents of rutin and isoquercitrin in this experiment. This suggests that sometimes the contents of total components in herbs have a negative correlation with single component among them. Therefore, it was inferred that there were some defects in using the content of single component to evaluate the quality of crude drugs. According to the regulations of Chinese Pharmacopoeia (2010), the HMF should be harvested in summer or autumn.
1. Chinese Pharmacopoeia Commission. Chinese pharmacopoeia 2010 edition, supplement 1 Chinese Medicine Science and Technology Press, Beijing, (2010), pp. 3. 2. Jia, M., Li, X.; Summary of Chinese ethical drugs . Chinese Medicine Science and Technology Press, Beijing, (2005), pp. 324. 3. Editorial Board of Chinese Materia Medica. Chinese materia medica (volume 5). Shanghai Science and Technology Press, Shanghai, (1999), pp. 349– 350. 4. Liyun, Y., Yang, L., Zenai, C.; Studies on chemical constituents of Hibiscus mutabilis; Chinese Traditional and Herbal Drugs, (2003); 34(3): 201–203. 5. Li, C.-L., Liu, S.-G., Wu, D., Hu, X., Feng, Z.-J.; Study on bacteriostasis of extracts of Hibiscus mutabili leaf; Science and Technology of Food Industry, (2009); 30(11): 97– 101. 6. Xu, Y., Zheng, Y.L., Lin, J.F.; Studies on anti-inflammatory and toxicity effect of Hibiscus mutabilis; Fujian Med J, (1989); 11(3): 24– 26. 7. Yuxing, L., Junren, S., Jie, Z.; Content determination of quercitrin in Hibisci mutabilis Folium by HPLC; Fujian Journal of Traditional Chinese Medicine, (2000); 31(6): 43 –44. 8. Zhang, L., Zhou, C.; Research trend in Hibisci mutabilis Folium; Guide of China Medicine, (2013); 11(1): 453–455. 9. Cushnie, T.P., Lamb, A.J.; Antimicrobial activity of flavonoids; International Journal of Antimicrobial Agents, (2005); 26(5): 343–356. 10. Hua, L., Zhijuan, S., Renqiang, M.; Determination of rurin content in Folium Hibisci mutabilis; Pharmacy Today, (2010); 20(1): 35. 11. Yu, Q., Su, X., Lin, C., Zheng, Q.; Determination of rutin in Hibiscus mutabilis at different harvest time; Chinese Archives of Traditional Chinese Medicine, (2013); 31(9): 2047–2049. 12. Long, Q., Yang, T.; Summary and prospect of rutin; Chinese Journal of Information on Traditional Chinese Medicine, (2002); 9(4): 39– 41. 13. Fernandez, J., Reyes, R., Ponce, H., Oropeza, M., Vancalsteren, M.R., Jankowski, C., et al.; Isoquercitrin from Argemone platyceras inhibits carbachol and leukotriene D4-induced contraction in guineapig airways; Journal of Pharmacology, (2005); 522(1/3): 108–115. 14. Wan, D., Mei, Q., Pan, S.; Determination of total flavonoids content in Hibisci mutabilis Folium from different harvest periods; Journal of South-Central University for Nationalities (Nat. Sci. Edition), (2013); 32(4): 46 –49.
Determination of Rutin and Isoquercetin Contents in HMF in Different Collection Periods 5
Article
Determination of Rutin and Isoquercetin Contents in Hibisci mutabilis Folium in Different Collection Periods by HPLC Diangang Liu1, Qing Mei2,†, Xiangluan Wan3, Hongling Que1, Luyang Li2 and Dingrong Wan2* 1 Foreign Languages School, Hubei University of Traditional Chinese Medicine, Wuhan 430065, China, 2Pharmaceutical School, SouthCentral University for Nationalities, Wuhan 430074, China, and 3Luojia College of Wuhan University, Wuhan 430065, China
*Author to whom correspondence should be addressed. Email: [email protected] †
Co-first author.
Received 6 January 2015; revised 1 May 2015
Hibisci mutabilis Folium (HMF), the dried leaf of Hibiscus mutabilis (Malvaceae), is commonly used in traditional Chinese medicine. This article aimed to establish a high-performance liquid chromatography method for the determination of rutin and isoquercitrin contents in the HMF, and to compare the content variation in all the samples, including nearly withered yellow leaf, in different collection periods, so as to provide research basis for the quality evaluation and determination of the optimal collection period. A reversed-phase HPLC separation method was employed, with a BDS Hypersil C18 column (4.6 m 3 250 mm, 5 mm), under the following conditions: acetonitrile – 0.3% phosphoric acid (15:85, v/v) solution as the mobile phase, flow rate 1.0 mL/min at 3088 C and detection wavelength 254 nm. The calibration curves for rutin and isoquercitrin were linear over the range of 1.5–48 and 0.25 – 8 mg/mL, and the average recoveries were 99.92 and 100.45% (RSD: 2.39% and 2.11%, respectively)]. Based on the analysis results, it was found that contents of rutin and isoquercitrin in HMF (mature green leaf) harvested in different periods had significant difference, and reached the highest in mid-December. It was also found that the contents of the two components in the mature green leaf were much higher than those in the nearly withered leaf from the same collection period. In conclusion, the results indicated that the HPLC method was easy-to-operate and precise, and could be applied for the determination of rutin and isoquercitrin contents in the HMF. The experimental data also showed that early winter should be the most suitable collection period for HMF.
Introduction Hibisci mutabilis Folium (HMF), the dried leaf of Hibiscus mutabilis (Malvaceae), is commonly used in traditional Chinese medicine. It has the function of cooling the blood, relieving toxin, reducing swelling and alleviating pain, and has long been used in the treatment of ulcer, swelling, herpes zoster, scald, bruises, etc. Besides, HMF is also widely used in ethnic minority areas for migraine and otitis media (by Dong people), pulmonary hemoptysis (by She people), menorrhagia (by She, Jingpo, Lisu people), appendicitis (by Yao people), epidemic parotitis (by Lisu, Yao people) and so on (1 – 3). This crude drug contains chemical components such as flavonoid glycosides, phenols, tannins, phenylalanine and volatile oil, etc. (4). Its ethanol extract has antibacterial effect (5) and aqueous extract has effects on anti-inflammation and abirritation (6). Flavonoid glycosides are main active constituents of HMF, among which rutin, quercitrin, hyperoside and isoquercitrin
have already been reported (7). They have significant effects on anti-inflammation and liver protection, and have the ability to prevent and treat ischemia reperfusion injury of the kidney (8, 9). Determination of the rutin content in HMF was reported in the other studies (10, 11), so was that of isoquercitrin. However, there was no research being done to simultaneously determine the contents of rutin and isoquercitrin in HMF. It is known that rutin and isoquercitrin have significant pharmacological activities of anti-inflammatory, analgesic, antibacterial, antioxidation and antitumor (12, 13). Moreover, rutin also has protective action on heart, brain and kidney injuries after ischemic reperfusion and gastric mucosa injury (12). Therefore, the determination of rutin and isoquercitrin contents in this crude drug has certain significance for its quality control. It is known that several factors, such as habitat, harvest time and so on, significantly affect the quality of herbal medicines. However, there was no report about the optimal harvest season of HMF in the literatures apart from the Chinese Pharmacopoeia (2010) and so on, which reported it should be collected in summer or fall (1, 3). Usually, determination of harvest season of raw medicinal materials is based on conventional folk experience without any scientific evidence; therefore, it is necessary to determine the optimal harvest season of this medicine by the related study. In our prophase research, the best harvest time was preliminarily determined after studying the total flavonoids content of HMF in different collection periods (14). But for the more effective quality evaluation, we developed another analytical method by HPLC to simultaneously determine the contents of rutin and isoquercitrin in HMF in different harvest period. Therefore, the best harvest season was further confirmed and the quality of the crude drug could be well evaluated. Experimental Apparatus and reagents The HPLC analysis was performed using Dionex Ultimate 3000 Series (Dionex, USA) Chameleon data processing system equipped with a binary pump, an autosampler, a column thermostat, temperature-controlled sample trays, an online degasser and a diode array detector. The reference substances of rutin and isoquercitrin were purchased from the National Institute for the Control of Pharmaceutical and Biological Products in hina (batch number: 111809-201102, 100080-200707). Chromatographic grade
# The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: [email protected]
acetonitrile was purchased from Tedia, Fairfield, USA (batch number: AS1122-001). Water for HPLC was purified by an Aquapro Hi-End Water Treatment Solution Provider (ASWO-0005-U, Ever Yong Enterprises). Ethanol and phosphoric acid were of analytical grade. Plant materials Seven batches of HMF were collected from South-Central University for Nationalities (SCUEC, Wuhan, Hubei province) in different seasons by ourselves and one batch of sample mixed with a few yellow leaves was purchased from Bozhou (Anhui), as presented in Table I. These samples were identified by Professor Dingrong Wan (Pharmaceutical College, SCUEC) and were deposited after removing impurities and drying in shade.
and isoquercitrin solutions were 1.5, 3, 6, 12, 24, 48 mg/mL and 0.25, 0.5, 1, 2, 4, 8 mg/mL, respectively. Calibration curves were generated based on the formula: Y ¼ aX þ b, among which the Y-axis was the value of peak area (mAU) and the X-axis was the concentration (mg/mL) injected into HPLC column of each standard. The regression equations of rutin and isoquercitrin calibration curves and the parameters for linearity are presented in Table II. The correlation coefficients were over 0.9999, which indicated good linearity under each range. Optimization of the sample extraction conditions Being aware that extraction of rutin and isoquercitrin from HMF is a method-dependent process, some important parameters determining the amount of extracted rutin and isoquercitrin were investigated.
Methods and results Preparation of reference solution 0.1 mg/mL reference solutions of rutin and isoquercitrin were prepared by dissolving accurately weighed rutin and isoquercitrin (Figure 1) in 50% ethanol, respectively. Sample preparation The powdered HMF (crushed and passed through a 60-mesh sieve) was extracted according to the description below. One gram of accurately weighed sample was placed in roundbottomed flask and then 50 mL of 50% ethanol solution was added and refluxed for 1 h. After cooling, 50% ethanol solution was added in order to make up the loss weight. Then the solution was shaken and filtered with a 0.45 mm syringe membrane. HPLC conditions The chromatographic separation was carried out with BDS Hypersil C18 column [4.6 250 mm, 5 mm, Thermo Scientific (San Jose, USA)]. Column temperature was set at 308C. Elution was conducted using acetonitrile and 0.3% phosphoric acid (15:85, V/V) as mobile phase. The flow rate was 1 mL/min. Ten microliters of each sample were injected into the column. UV spectra were acquired in the range of 200–380 nm and the quantitative wavelength was 254 nm. The chromatogram of standards and sample are shown in Figure 2. Preparation of standard calibration curves and analysis of linearity Six different concentration levels of mixed reference solutions were analyzed in this research. The six concentrations of rutin
Table I Samples and Their Sources No.
Habitat
Properties of samples
Collection date
1 2 3 4 5 6 7 8
Campus of SCUEC, Wuhan
Mature green leaves
10 June 2012 21 August 2012 18 November 2012 10 December 2012 18 November 2012 1 December 2012 16 December 2012 10 October 2012 (Production date)
Nearly withered yellow old leaves Bozhou, Anhui
2 Liu et al.
Mixed with a few yellow leaves
Optimization of the extraction methods 0.5 g accurately weighted sample (No. 7) was chosen for the comparative study of the two extraction methods. First, 25 mL of 50% (V/V) ethanol solution was added to reflux and then heated for 1 h. After cooling, the extract was filtered with a 0.45 mm syringe membrane to form the sample solution. This procedure was then repeated via ultrasonic method for comparative study. The HPLC analysis results showed that the content of rutin and isoquercitrin extracted via reflux was twice as much as that extracted via ultrasonic method, which demonstrated that refluxing was the optimal extraction method. Optimization of the extraction solvent Five accurately weighed samples (0.5 g) were refluxed by 25 mL of 20, 30, 40, 50 and 70% (V/V) ethanol solution for 1 h in parallel. The HPLC analysis results showed that extraction efficiency of the two compounds improved as the ethanol concentration increased, and increase of the content of the two compounds was fairly gradual when the ethanol concentration came to 30 –50%, then it began to decrease. Therefore, 50% (V/V) ethanol solution was chosen as the best extraction solvent. Optimization of the extraction time Four accurately weighted samples (No. 7), each one weighed 0.5 g, were refluxed with 50% (V/V) ethanol solution for 30, 60, 90, 120 min, respectively. The HPLC analysis results revealed that the two compounds had been extracted completely in 60 min. Hence, the optimal extraction time is 60 min. Optimization of ratio of the extraction solvent Five accurately weighted samples (No. 7), each one weighed 1.0 g, were refluxed with 50% (V/V) ethanol solution for 60 min. The solvent ratio was 20:1, 30:1, 40:1, 50:1, 60:1, respectively. The HPLC analysis results proved that when the solvent ratio transferred from 50:1 to 60:1, the extraction efficiency showed an upward tendency, but the change of it was smooth and gentle. Thus, 50:1 was chosen as the best extraction ratio (Figure 3). Optimization of chromatographic conditions Based on the results of the three-dimensional chromatograms of the two standard solutions by diode array detector (the wavelength range from 190 to 600 nm), the detection wavelength
Figure 1. Chemical structures of (A) rutin and (B) isoquercitrin.
Table II Linear Equation, R 2 and Range of Rutin and Isoquercitrin for Quantitative Determination Compound
Regression equation
R2
Range (mg/mL)
Rutin Isoquercitrin
Y ¼ 234.42X 2 0.0228 Y ¼ 280.56X 2 0.0106
0.9999 0.9999
1.5 –48 0.25 –8
time, better resolution (R . 1.5) and higher theoretical plate numbers (n . 8000). Method validation
Figure 2. Chromatogram of reference solution and sample solution: (1) rutin and (2) isoquercitrin. This figure is available in black and white in print and in color at JCS online.
was set at 254 nm, where the two standard substances had the strongest absorption. A screening on the solutions of mobile phase was carried out in order to improve their chromatographic separation efficiency. The developed analytical method for the determination of the two components was based on the methods of Hibisci mutabilis Folium of Chinese Pharmacopoeia and the related literature. Three kinds of mobile phase (acetonitrile, tetrahydrofuran and methanol with 0.3% phosphoric acid, respectively) were studied and compared, and separating effect was detected by each chromatographic peak. As it had led to an increasing degree of separation, acetonitrile was preferred over methanol and tetrahydrofuran as the mobile phase. Then a series of solutions with different ratios of acetonitrile –0.3% phosphoric acid were studied and the ratio 15:85 was selected ultimately. In the end, the optimal chromatographic conditions were chosen, because of the better peak symmetry, shorter retention
Precision Ten-microliter reference substances’ solutions were injected into the HPLC instrument continuously under the optimized chromatographic conditions for five times. After determination, the results showed that relative standard deviations (RSDs) of the peak areas of the rutin and isoquercitrin were 0.11 and 0.17%, respectively, indicating that the instrument was highly precise. Repeatability Five samples (No. 8) were detected in parallel by using the above methods of sample preparation and chromatographic conditions. The RSDs of the peak area of the two components (rutin and isoquercitrin) were 1.52 and 2.89%, respectively, indicating nice repeatability. Stability One sample (No. 8) solution was prepared according to the sample preparation method discussed earlier, and then rutin and isoquercitrin peak areas were detected at 0, 2, 4, 8 and 12 h under the chromatographic conditions as described earlier. The RSDs of rutin and isoquercitrin peak area values were 2.07 and 3.24%, respectively. This showed that the two components in sample solutions were stable for at least 12 h. Recovery and accuracy The accuracy was determined by calculating the recovery after a standard substances addition procedure. The recoveries of Determination of Rutin and Isoquercetin Contents in HMF in Different Collection Periods 3
Table III Recovery of Rutin in Hibisci mutabilis Folium (No. 8) Sample amount (g)
Rutin in the sample (mg)
Added rutin amount (mg)
Determined rutin amount (mg)
Recovery (%)
0.5006 0.5005 0.5006 0.5003 0.5002
0.4808 0.4808 0.4808 0.4808 0.4808
0.504 0.504 0.504 0.504 0.504
0.9702 0.9763 0.9913 1.0006 0.9836
97.10 98.31 101.28 103.13 99.76
Mean recovery (%)
RSD (%)
99.92
2.39
Mean recovery (%)
RSD (%)
100.45
2.11
Table IV Recovery of Isoquercitrin in Hibisci mutabilis Folium (No. 8) Sample amount (g)
Isoquercitrin in the samples (mg)
Added isoquercitrin amount (mg)
Determined isoquercitrin amount (mg)
Recovery (%)
0.5006 0.5005 0.5006 0.5003 0.5002
0.0521 0.0521 0.0521 0.0522 0.0521
0.040 0.040 0.040 0.040 0.040
0.0919 0.0910 0.0925 0.0930 0.0931
99.50 97.25 101.00 102.00 102.50
Table V Amounts of Rutin and Isoquercitrin in Hibisci mutabilis Folium Harvested in Different Seasons Habitat
Sample/ harvested season
Content of Rutin (mg/g)
Content of isoquercitrin (mg/g)
Campus of SCUEC, Wuhan
Mature green leaf (June 10)
0.2979
0.0215
Mature green leaf (August 21) Mature green leaf (November 18) Mature green leaf (December 10) Nearly withered yellow old leaf (November 18) Nearly withered yellow old leaf (December 1) Nearly withered yellow old leaf (December 16) Mixed with a few yellow leave (October 10)
0.1364 0.1358 1.1305 0.2110
0.0199 0.0223 0.2491 0.0261
0.3840
0.0773
0.4250
0.0810
0.9772
0.1016
Bozhou, Anhui
Figure 3. Amount variation of the components with respect to different factors. (A) Ethanol concentration; (B) extraction time; (C) solid – liquid ratio. This figure is available in black and white in print and in color at JCS online.
quantification procedure of the standard substances were examined at five levels covering the linearity range of calibration curves. The five precisely weighed samples (No. 8) were extracted using the method described earlier and determined by HPLC after adding an appropriate amount of rutin and isoquercitrin (0.504 and 0.040 mg, respectively) to each sample. Recovery of the two component,and RSDs were calculated. The results are presented in Tables III and IV and showed that the method had a good accuracy. The content determination of rutin and isoquercitrin in different collection periods All samples were accurately weighed (each sample was prepared in triplicate), operated and determined according to the optimized extraction method and HPLC conditions. According to the detection results of the peak areas of rutin and isoquercitrin in HMF (including nearly withered yellow old 4 Liu et al.
leaves) harvested in different seasons, we calculated the amounts of the two components in the light of the calibration curves and listed in Table V and showed in Figure 4. Discussion and summary The scanning results suggested that rutin and isoquercitrin had the largest absorption peaks when the wavelength was 254 nm. Therefore, the detection wavelength was determined at 254 nm. As the determination results of the mature-green-leaf samples have shown, overall the contents of rutin and isoquercitrin in HMF collected in the period from mid-June to mid-November did not change a lot. While the contents of two components in the samples harvested in mid-December increased dramatically, they were even about 10 times as much as those contained in the samples of August. From these it can be inferred that the contents of rutin and isoquercitrin had very significant differences in different harvest times. Similarly, the contents of rutin and isoquercitrin were the highest in mid-December in the nearly withered yellow old leaf, but significantly lower than the mature
However, on the basis of the results of the total flavones content in HMF harvested in different periods, we suggested that the early winter should be recommended as the optimal collection period.
Acknowledgments This work was supported by Science Research Foundation of Health Department of Hubei Province, China (Grant No. 2013Z-Y02).
References
Figure 4. Amount variation of the two components in Hibisci mutabilis Folium collected in different periods. (A) Rutin and (B) isoquercitrin. This figure is available in black and white in print and in color at JCS online.
green leaf collected at the same harvest time. In addition, the HMF purchased from Bozhou had even higher levels of the amounts of rutin and isoquercitrin than most samples collected in SCUEC. So, the content difference of the samples of different habitats is also remarkable. Based on our prophase research of the total flavones contents in HMF harvested in different times by ultraviolet spectroscopy (UV), it had been found that the HMF purchased from Bozhou had a much lower level of the total flavones than each batch of samples collected in SCUEC (14), which conflicted with the results of the contents of rutin and isoquercitrin in this experiment. This suggests that sometimes the contents of total components in herbs have a negative correlation with single component among them. Therefore, it was inferred that there were some defects in using the content of single component to evaluate the quality of crude drugs. According to the regulations of Chinese Pharmacopoeia (2010), the HMF should be harvested in summer or autumn.
1. Chinese Pharmacopoeia Commission. Chinese pharmacopoeia 2010 edition, supplement 1 Chinese Medicine Science and Technology Press, Beijing, (2010), pp. 3. 2. Jia, M., Li, X.; Summary of Chinese ethical drugs . Chinese Medicine Science and Technology Press, Beijing, (2005), pp. 324. 3. Editorial Board of Chinese Materia Medica. Chinese materia medica (volume 5). Shanghai Science and Technology Press, Shanghai, (1999), pp. 349– 350. 4. Liyun, Y., Yang, L., Zenai, C.; Studies on chemical constituents of Hibiscus mutabilis; Chinese Traditional and Herbal Drugs, (2003); 34(3): 201–203. 5. Li, C.-L., Liu, S.-G., Wu, D., Hu, X., Feng, Z.-J.; Study on bacteriostasis of extracts of Hibiscus mutabili leaf; Science and Technology of Food Industry, (2009); 30(11): 97– 101. 6. Xu, Y., Zheng, Y.L., Lin, J.F.; Studies on anti-inflammatory and toxicity effect of Hibiscus mutabilis; Fujian Med J, (1989); 11(3): 24– 26. 7. Yuxing, L., Junren, S., Jie, Z.; Content determination of quercitrin in Hibisci mutabilis Folium by HPLC; Fujian Journal of Traditional Chinese Medicine, (2000); 31(6): 43 –44. 8. Zhang, L., Zhou, C.; Research trend in Hibisci mutabilis Folium; Guide of China Medicine, (2013); 11(1): 453–455. 9. Cushnie, T.P., Lamb, A.J.; Antimicrobial activity of flavonoids; International Journal of Antimicrobial Agents, (2005); 26(5): 343–356. 10. Hua, L., Zhijuan, S., Renqiang, M.; Determination of rurin content in Folium Hibisci mutabilis; Pharmacy Today, (2010); 20(1): 35. 11. Yu, Q., Su, X., Lin, C., Zheng, Q.; Determination of rutin in Hibiscus mutabilis at different harvest time; Chinese Archives of Traditional Chinese Medicine, (2013); 31(9): 2047–2049. 12. Long, Q., Yang, T.; Summary and prospect of rutin; Chinese Journal of Information on Traditional Chinese Medicine, (2002); 9(4): 39– 41. 13. Fernandez, J., Reyes, R., Ponce, H., Oropeza, M., Vancalsteren, M.R., Jankowski, C., et al.; Isoquercitrin from Argemone platyceras inhibits carbachol and leukotriene D4-induced contraction in guineapig airways; Journal of Pharmacology, (2005); 522(1/3): 108–115. 14. Wan, D., Mei, Q., Pan, S.; Determination of total flavonoids content in Hibisci mutabilis Folium from different harvest periods; Journal of South-Central University for Nationalities (Nat. Sci. Edition), (2013); 32(4): 46 –49.
Determination of Rutin and Isoquercetin Contents in HMF in Different Collection Periods 5
