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Quantitative Determination of the Triterpenoids and Total Tannin in Korean Rubus species by HPLC
Quantitative Determination of the Triterpenoids and Total Tannin in Korean Rubus species by HPLC
Natural Product Sciences. 2014. Dec, 20(4): 290-295
Copyright © 2014, The Korean Society of Pharmacognosy
  • Received : August 24, 2014
  • Accepted : October 03, 2014
  • Published : December 31, 2014
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About the Authors
Min-Young Kim
Department of Pharmaceutical Engineering, Sangji University, Wonju 220-702, Republic of Korea
Leon Azefack Tapondjou
Department of Chemistry, Faculty of Science, University of Dschang, Box 183, Dschang, Cameroon
Hee-Juhn Park
Department of Pharmaceutical Engineering, Sangji University, Wonju 220-702, Republic of Korea
hjpark@sangji.ac.kr

Abstract
The triterpenoids contained in four Rubus species (Rosaceae) were quantitatively analyzed using HPLC to select plants with large quantities of niga-ichigoside F 1 . Unripe fruits, ripe fruits, and leaves were extracted to estimate the quantity of niga-ichigoside F 1 , together with Rubus -specific 19α-hydroxyursane-type triterpenoids, euscaphic acid, tormentic acid, and kaji-ichigoside F 1 , and a dimeric triterpenoid coreanoside F 1 . Niga-ichigoside F 1 was most abundant in the leaves of R. crataegifolius (23.4 mg/g dry weight). The amount of triterpenoid in the black, ripe fruits of R. coreanus was lower than the unripe fruits of the same plant. On the other hand, the ripe fruits of three plants, R. crataegifolius , R. parvifolius and R. pungens var. oldhami , which are reddish, contained higher or similar level of triterpenoids than their unripe fruits. In addition, the concentration of niga-ichigoside F 1 in the ripe fruit of R. crataegifolius was 20.5 mg/g, suggesting that the fruits could be used as a functional food. Methyl gallate and ellagic acid were used as quantitative indices of total tannin. Methyl gallate levels were higher in ripe fruits than unripe fruits in R. crataegifolius , R. pungens var. oldhami , and R. parvifolius . In R. crataegifolius , the quantity of methyl gallate was 30.5 mg/g in ripe fruit, but 1.19 mg/g in unripe fruit.
Keywords
Introduction
Rubi Fructus, a Chinese medicinal drug, is the dried unripe fruit of Rubus coreanus (Rosaceae). 1 R. Fructus is used to treat diarrhea, sexual disinclination, and diabetes mellitus in Chinese medicine. 2 We have previously reported the antinociceptive/anti-inflammatory, 3 anti-hyperlipidemic, 4 anti-rheumatic, and anti-gastropathic effects 5 of the constituents of niga-ichigoside F 1 and 23-hydroxytormentic acid as the active principles. We have also reported the anti-inflammatory activity of euscaphic acid, tormentic acid, kaji-ichigoside F 1 , and rosamultin as the 19α-hydroxyursane-type triterpenoids. 6
Although several Rubus species grow wild in Korea, only the fruits of R. coreanus are black when ripe. The fruits of other Korean Rubus species become reddish when ripe. Here, we quantified levels of the 19α-hydroxyursane-type triterpenoids, particularly niga-ichigoside F 1 , in the ripe and unripe fruits and leaves of four Korean Rubus species, R. coreanus , R. crataegifolius , R. pungens var. oldhami , and R. parvifolius . Six standard triterpenoid compounds were quantified by HPLC analysis. In addition, methyl gallate and ellagic acid were used as markers of total tannin in the same sources.
Experimental
Instruments and Reagents − HPLC chromatograms were measured using a Varian Prostar 210 solvent delivery module, a Prostar 325 UV-Vis detector, and a 20 μL sample loop. Separation was achieved on a Shiseido Capcell Pak C18 column (5 μL, 250 mm × 4.6 mm I.D.). All the solvents used for analysis were HPLC grade.
Plant material − The four plant species were collected on a mountain near Cheon-Eun Temple in Wonju city, Korea, during summer (July). The unripe fruits, ripe fruits, and the leaves were collected over June - July from Rubus coreanus (natchem # 45), R. crataegifolius (natchem # 46), R. pungens var. Oldhami (natchem # 47), and R. parvifolius (natchem # 48), dried and pulverized for HPLC analysis. The voucher specimens (natchem #46 - 48) were identified by Prof. Sang-Cheol Lim (Dept. of Horticulture and Landscape Artchtecture, Sangji University, Korea).
Extraction − One gram of the pulverized plant material was weighed on a chemical balance. Plant material (1 g) was extracted in methanol (40 ml) at 40 ℃ under untrasonication for 2 h, and then filtered and concentrated to dryness on a rotatory evaporator, a centrifuge evaporator, and finally a freeze dryer. The concentrated materials were weighed and diluted for the HPLC chromatogram.
Standard triterpenoids − Six triterpenoids, euscaphic acid, tormentic acid, 23-hydroxytormentic acid, kajiichigoside F 1 , niga-ichigoside F 1 , and coraenoside F 1 , were used after isolation from R. crataegifolius or R. coreanus as in our previous reports). 4 , 5 The compounds were identified by comparison of physicochemical data (mp, [α] D , 1 H-NMR and 13 C-NMR) with the data previously reported. 7 - 10 The structures are shown in Fig. 1 . These six compounds were used as internal or external standards for HPLC analysis.
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Structure of triterpenoids, ellagic acid, and methyl gallate for HPLC analysis. 1, Niga-ichigoside F1; 2, kaji-ichigoside F1; 3, coreanoside F1; 4, 23-hydroxytormentic acid; 5, euscaphic acid; 6, tormentic acid; 7, methyl gallate; 8, ellagic acid.
HPLC conditions and quantitative analysis for triterpenoids − The samples and standard compounds were dissolved in 80% aqueous MeOH and the mixture was filtered through a 0.50 μm syringe for injection. The UV detector was fixed at 206 nm. The mobile phase was the mixed solvent of deionized water with 1.25% phosphoric acid (solvent A) -MeOH (solvent B) (solvent A : solvent B = 30 : 70). The HPLC chromatogram was run for 30 min and the flow rate was 0.70 ml/min. Triterpenoid quantification in the eleven samples was performed after making a calibration curve by running HPLC chromatograms at 100, 250, 500 and 1000 μg/mL. The equations are shown in Table 1 .
Calibration curve equations of 6 standard triterpenoids, ellagic acid, and methyl gallate
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y (peak area), x (mg/mL)
HPLC analysis for total tannin − Zhentian et al . 11 developed a new method for quantitative analysis of tannin using the indicators, ellagic and gallic acid, after treatment with anhydrous methanolic HCl. Using this method, HPLC was performed for analysis of total tannin. Ten mg of ground samples was placed in a microtube (2 ml) and anhydrous methanolic HCl (2 ml) was added. Tubes were sealed tightly and placed in a centrifugal evaporator at 90 ℃ for 90 min. The mixtures were then cooled to room temperature and filtered (0.50 μm). The filtrates were evaporated to dryness and the residues were dissolved again in 2 ml of 80% aqueous methanol. The filtrate was injected onto the same HPLC column used for measuring triterpenoids. Ellagic acid and methyl gallate levels were detected at the wavelength of 252 nm and 280 nm, respectively.
The mobile phase was MeOH (solvent A) and 0.2% aqueous trifluoroacetic acid (solvent B). The gradient elution was as follows: 0 min, 0% A : 100% B; 0 - 40 min, 100% A : 0% B; 40 - 41 min, 0% A : 100% B; 41 - 50 min, 0% A : 100% B. Run time was 40 min and the flow rate was 0.75 mL/min. The two standard compounds dissolved in 80% aqueous methanol were used to make the calibration curve.
Results and Discussion
Quantity of triterpenoids − The MeOH extraction method of triterpenoid from Rubus plants was reported by Ono et al . 12 Their work made it possible to separate nigaichigoside F 1 and rubusside A using HPLC analysis from the blackberry. Chen et al . 13 used a detection wavelength of 206 nm because triterpenoids had the largest absorption and sensitivity in this wavelength, so we also used this parameter.
Structures and HPLC chromatograms of the six triterpenoids (euscaphic acid, tormentic acid, kaji-ichigoside F 1 , 23-hydroxytormentic acid, and niga-ichigoside F 1 , and coreanoside F 1 as the 19α-hydroxyursane-type triterpenoids) in the leaves of R. crataegifolius are shown in Fig. 1 and 2 . Analysis of R. crataegifolius , R. pungens var. oldhami , and R. parvifolius extracts were performed by HPLC, while the unripe fruits (green), semi-ripened fruits (reddish) and ripened fruits (black) in R. coreanus were tested. Retention times of the peaks are shown in Table 1 . The R 2 values of calibration curve equations were more than 0.999. As shown in Fig. 2 and Table 2 , niga-ichigoside F 1 was the most abundant triterpenoid, and 23-hydroxytormentic acid, which could be transformed by glycosidation to niga-ichigoside F 1 , 14 was the most abundant genin (mean 2.29 mg/g).
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HPLC chromatogram of triterpenoids in R. crataegifolius leaves. 1, Niga-ichigoside F1; 2, kaji-ichigoside F1; 3, coreanoside F1; 4, 23-hydroxytormentic acid; 5, euscaphic acid; 6, tormentic acid.
Triterpenoid contents of the fourRubussamplesa
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Abbreviation: NigaF1 (niga-ichigoside F1), KajiF1 (kaji-ichigoside F1), CorF1 (coreanoside F1), 23-HTA (23-hydroxytormentic acid), EA (euscaphic acid), TA (tormentic acid), tr (trace), nd (not detected). a Average of duplicate analyses. b Reported in mg/g.
Total triterpenoid contents of the unripe, semi-ripened and ripe fruits of R. coreanus were 13.89 mg/g, 8.14 mg/g and 2.85 mg/g, respectively ( Table 2 and Fig. 4 ). Nigaichigoside F 1 contents of the conditional fruits were 3.80 mg/g, 2.87 mg/g and 2.85 mg/g, respectively ( Table 2 ). These results indicate that the ripening process accelerated triterpenoid distribution throughout the fruit and also reduced triterpenoid content. The triterpenoid content was particularly low in the black, ripe fruits. The triterpenoid content of the leaves in R. coreanus was 14.79 mg/g, higher than its fruit, suggesting that triterpenoids produced in the fruits may be transported to leaves.
The triterpenoid contents in R. crataegifolius are shown in Fig. 4 and Table 2 . Unlike R. coreanus , the ripe fruits had higher triterpenoid content than unripe ones. The quantity of niga-ichigoside F 1 in the ripe fruits was 20.49 mg/g and the quantity in the unripe one was 9.07 mg/g. The pattern of triterpenoids abundance was usually more similar to R. crataegifolius than R. coreanus ; i.e., triterpenoid quantities in ripe fruit were similar or higher than in unripe fruit, suggesting that triterpenoids can accumulate during the ripening process. Species with red, ripe fruits contained a large amount of triterpenoids, with only R. coreanus having more at the unripe stage. Ohtani et al . 10 reported that 19α-hydroxyursane-type triterpenoid compounds are present in R. coreanus , and that these compounds have bioactivity. So, R. coreanus may be different than other Rubus species in systematic botany. Niga-ichigoside F 1 contents in the leaves of R. parvifolius and R. pungens var. oldhami were 21.38 mg/g and 4.80 mg/g, respectively, providing information on the best plant resource for producing large amounts of nigaichigoside F 1 with anti-inflammatory, 3 anti-hyperlipidemic 4 and antirheumatic 5 effects.
The niga-ichigoside F 1 content in R. pungens var. oldhami was as follows: leaves > ripe fruits > unripe fruits. The leaves exhibited the highest amount of coreanoside F 1 of any other part of this plant or other Rubus species. Although Wang et al . 15 have previously reported the isolation of several triterpenoids from R. pungens var. oldhami , our result is the first quantitative analysis.
Using HPLC analysis, this research confirmed the validity that Rubi Fructus, which is a Chinese traditional medicine, should be the unripe fruit of R. coreanus . In contrast, red, ripe fruits of other three Rubus species had higher triterpenoid levels than unripe fruits. The ripe fruits could be used as a functional food, with high quantities of 19α-hydroxyursane-type triterpenoids, especially nigaichigoside F 1 .
Quantity of total tannin − Hydrolysable tannins have been reported in Rubus species. 16 In general, gallic acid or hexahydroxydiphenic acid are esterified to monosaccharides to form tannins. On methanolysis, tannins produce methyl gallate or ellagic acid ( Fig. 3 ). Ellagitannin has been isolated from R. sanctus . 16 In this experiment, the quantities of methyl gallate and ellagic acid were evaluated as indicative of tannins ( Fig. 1 and 3 ). The leaves of the four Rubus species exhibited the highest amount of methyl gallate and ellagic acid, suggesting that they contain a large quantity of tannins ( Table 3 and Fig. 4 ). In R. crataegifolius , R. pungens var. oldhami and R. parvifolius , methyl gallate levels were higher in ripe fruit than unripe fruit. In R. crataegifolius , the quantity of methyl gallate was 30.50 mg/g in ripe fruit but 1.19 mg/g in unripe fruit. However, in R. coreanus , there was no considerable difference between the ripe and unripe fruits. Thus, differences in levels of triterpenoids lead to different total tannin levels in the unripe fruits; therefore, R. coreanus has been used in Chinese medicines.
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HPLC chromatogram of methyl gallate (A) and ellagic acid (B) in R. crataegifolius leaves. 7, Methyl gallate; 8, Ellagic acid.
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Comparison of the total triterpenoid and total ellagic and methyl gallate in four Rubus plants. ● , Total triterpenoid; ○ , Total ellagic and gallic acid.
Ellagic and methyl gallate contents of fourRubussamplesa
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a Average of duplicate analyses. b Reported in mg/g.
In conclusion, we can measure the peak products of ellagic acid and methyl gallate at two different wavelengths (252 and 280 nm). Total triterpenoids and tannins content were highest in leaves of the four Rubus species. The results of this study can lead to useful products using other secondary metabolites from leaves of the Rubus species.
Acknowledgements
The authors report no conflicts of interest. We gratefully acknowledge the financial support provided by the Rural Development Administration of the Korean Ministry of Agriculture and Forests.
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