Quantitative Determination of the Triterpenoids and Total Tannin in Korean Rubus species by HPLC

  • 투고 : 2014.08.24
  • 심사 : 2014.10.03
  • 발행 : 2014.12.31

초록

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 $19{\alpha}$-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.

키워드

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 effects5 of the constituents of niga-ichigoside F1 and 23-hydroxytormentic acid as the active principles. We have also reported the anti-inflammatory activity of euscaphic acid, tormentic acid, kaji-ichigoside F1, 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 F1, 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 F1, niga-ichigoside F1, and coraenoside F1, 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, 1H-NMR and 13C-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.

Fig. 1.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.

Table 1.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 F1 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 F1, 23-hydroxytormentic acid, and niga-ichigoside F1, and coreanoside F1 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 R2 values of calibration curve equations were more than 0.999. As shown in Fig. 2 and Table 2, niga-ichigoside F1 was the most abundant triterpenoid, and 23-hydroxytormentic acid, which could be transformed by glycosidation to niga-ichigoside F1,14 was the most abundant genin (mean 2.29 mg/g).

Fig. 2.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.

Table 2.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 F1 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 F1 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 F1 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 F1 with anti-inflammatory,3 anti-hyperlipidemic4 and antirheumatic5 effects.

The niga-ichigoside F1 content in R. pungens var. oldhami was as follows: leaves > ripe fruits > unripe fruits. The leaves exhibited the highest amount of coreanoside F1 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 F1.

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.

Fig. 3.HPLC chromatogram of methyl gallate (A) and ellagic acid (B) in R. crataegifolius leaves. 7, Methyl gallate; 8, Ellagic acid.

Fig. 4.Comparison of the total triterpenoid and total ellagic and methyl gallate in four Rubus plants. ● , Total triterpenoid; ○ , Total ellagic and gallic acid.

Table 3.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.

참고문헌

  1. Moon, G. S. Constituents and uses of medicinal herbs; Iweolseogak: Republic of Korea, 1991; pp 310-311.
  2. Kim, T. J. Botanical Resources in Korea; Publishing Center of Seoul National University: Seoul, 1996; pp 142-145.
  3. Choi, J.; Lee, K. T.; Ha, J.; Yun, S. Y; Ko, C. D.; Jung, H. J; Park, H. J. Biol. Pharm. Bull. 2003, 26, 1436-1441. https://doi.org/10.1248/bpb.26.1436
  4. Nam, J. H.; Jung, H. J.; Tapondjou, L. A.; Lee, K. T.; Choi, J. W.; Kim, W. B.; Park, H. J. Nat. Prod. Sci. 2007, 13, 152-159.
  5. Nam, J. H.; Jung, H. J.; Choi, J.; Lee, K. T.; Park, H. J. Biol. Pharm. Bull. 2006, 29, 967-970. https://doi.org/10.1248/bpb.29.967
  6. Jung, H. J.; Nam, J. H.; Choi, J.; Lee, K. T.; Park, H. J. Biol. Pharm. Bull. 2005, 28, 101-104. https://doi.org/10.1248/bpb.28.101
  7. Yamagishi, T.; Zhang, D. C.; Chang, J. J.; McPhail, D. R.; McPhail, A. T.; Lee, K. H.; Phytochemistry 1988, 27, 3213-3216. https://doi.org/10.1016/0031-9422(88)80028-1
  8. Kim, Y. H.; Kang, S. S. Arch. Pharm. Res. 1993, 16, 109-113. https://doi.org/10.1007/BF03036856
  9. Seto, T.; Tanaka, T.; Tanaka, O.; Naruhashi, N. Phytochemistry 1984, 23, 2829-2834. https://doi.org/10.1016/0031-9422(84)83023-X
  10. Ohtani, K.; Miyajima, C.; Takahashi, T.; Kasai, R.; Tanaka, O.; Hahn, D. R.; Naruhashi, N. Phytochemistry 1990, 29, 3275-3280. https://doi.org/10.1016/0031-9422(90)80199-Q
  11. Lei, Z.; Jervis, J.; Helm, R. F. J. Agric. Food Chem. 2001, 49, 1165-1168. https://doi.org/10.1021/jf000974a
  12. Ono, M.; Tateishi, M.; Masuoka, C.; Kobayashi, H.; Igoshi, K.; Komatsu, H.; Ito, Y.; Okawa, M.; Nohara, T. Chem. Pharm. Bull. 2003, 51, 200-202. https://doi.org/10.1248/cpb.51.200
  13. Chen, J. H.; Xia, Z. H.; Tan, R. X. J. Pharm. Biomed. Anal. 2003, 32, 1175-1179. https://doi.org/10.1016/S0731-7085(03)00160-2
  14. Jung, H. J.; Nam, J. H.; Lim, S. C.; Kim, W. B.; Park, H. J. Kor. J. Plant Res. 2006, 19, 563-572.
  15. Wang, B. G.; Jia, Z. J. Phytochemistry 1998, 49, 185-188. https://doi.org/10.1016/S0031-9422(97)01057-1
  16. Hussein, S. A. M.; Ayoub, N. A.; Nawwar, M. A. M. Phytochemistry 2003, 63, 905-911. https://doi.org/10.1016/S0031-9422(03)00331-5