Introduction
‘Mokhyang (MH)’ is a traditional medicine which is effective for the treatment of hypochondria, accumulation-aggregation, diarrhea, intestinal convulsion and dysentery1,2). In The Korean Herbal Pharmacopoeia3), the dried root of Aucklandia lappa Decne (Aucklandiae Radix) and Inula helenium L. (Inulae Radix) are listed as genuine of MH. In Chinese Pharmacopoeia4), besides above two kinds, the dried root of Vladimiria souliei (Franch.) Ling (Vladimiriae Radix) is stated as genuine while that of Aristolochia contorta Bge. (Aristolochiae Radix) is listed as adulterant of MH5).
Aucklandiae Radix, known as Unmokhyang (Un-MH) in Korean, is called in other names such as ‘Milhyang’, ‘Chungmokhyang (Chung-MH)’ and ‘Ohyang’. Inulae Radix, named Tomokhyang (To-MH) in Korean, is also called various names such as ‘Chung-MH’ and ‘Dokhaengkeun’. Aristolochiae Radix, known as Chung-MH in Korean, has various alternate names such as Maryeongkeun, ‘To-MH’ and ‘Dokhaengkeun’6). Because of this similar herbal names and morphological features, the dried roots of 4 kinds have been confused in use and distribution in market.
Furthermore, Aristolochiae Radix is one of Aristolochia herbs containing aristolochic acid (AA) which causes nephropathy. AA-containing herbs have been a global concern after that the first side effects of slimming regimen derived from Aristolochia kinds was reported in early 1990s7). Subsequently, the Korean Food and Drug Administration (KFDA) has prohibited the medicinal use of Aristolochiae Radix because it shares the same common name, ‘Mokhyang’, with other three kinds5).
Various efforts using macroscopic, microscopic identification8) and chemical analysis9) have been performed for the discrimination of Aucklandiae Radix and Inulae Radix. However, the previous studies analyzing three genuine kinds showed only macroscopic morphological differentiation10) and pattern-recognition analysis by HPLC11). Moreover, identification of 4 kinds of MH was rarely published in using macroscopic identification12) and quantitative determination of costunolide by HPLC13).
The objective of this study is to provide identification-key of 4 kinds of MH using macroscopic and microscopic methods, and the discrimination of those herbal medicines was also performed by evaluating the amounts of chemical compounds using HPLC analysis and investigating antioxidative activities. To our knowledge, general examination on 4 kinds of MH was firstly carried.
Materials and methods
1. Herbal materials
The roots of Aucklandia lappa Decne, Inula helenium L., Vladimiria souliei (Franch.) Ling and Aristolochia contorta Bge. were gathered from various areas such as local market and habitats (Table 1), and ascertained by Prof. Young-Sung Ju, department of herbology, Woosuk University. 14 samples have been deposited in the department of herbology of Woosuk University.
2. Reagents
Costunolide and dehydrocostuslactone9,14,22) were purchased from ChromaDex (USA), Wako Pure Chemical Co. (Japan). HPLC-grade methanol, water and acetonitrile were purchased from Fisher (USA).
2,2-Diphenyl-1-picrylhydradzyl and 2,4,6-tris (2-pyridyl)-s-triazine were purchased from Sigma Chemical Co. (USA). 2,2′-Azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) diammonium salt, potassium peroxodisulfate, and FeCl3·6H2O were purchased from Kanto Chemical Co. (Japan). Acetic acid was purchased from Junsei Chemical Co. (Japan).
3. Macroscopic and microscopic morphology
Stereoscope (Carl zeiss STEMI2000, Germany) and microscope (Nikon ECLIPSE 80i, Japan) were used to observe the external and internal morphology. Samples of 4 kinds went through fixation, rapid dehydration procedures, paraffin waxing, cutting and modified Ju’s triple stain method process in orderly and then examined microscopic features by optical microscope.
4. HPLC analysis
The dried roots of 14 MH samples (coded as AL1-5, IH1-3, VS1-3, and AC1-3) were pulverized and extracted with methanol (1 g/10 mL) by ultra-sonication for 2 hours at 30°C. The supernatants were concentrated in vacuo to dryness and dissolved with methanol at the concentration of 100 mg/mL. The extracts was diluted to make a final concentration of 1 mg/mL and then passed through a 0.20 μm membrane filter prior to injection.
Each of reference standards were accurately dissolved in HPLC grade methanol to yield a concentration of 100 μg/mL. Working calibration solutions were prepared by serial dilution of the stock solutions with HPLC grade methanol to yield concentration of 1–50 μg/mL for costunolide and 0.5–25 μg/mL for dehydrocostuslactone.
Samples were analyzed by Agilent 1200 series with multiwavelength detector (Agilent, USA). Acquired data was processed using Agilent Chemstation (Agilent 1200 series). The separation was conducted on an Phenomenex Gemini NX C18 (3.0 × 150 mm, 5 μm) on the conditions of flow rate at 0.8 mL/min, column temperature at 35°C and UV wavelength at 225 nm15). The mobile phase consisted of deionized water (A) and acetonitrile (B) as a gradient elution: 0min, 60 % B; 29min, 80% B; 30min, 60 % B.
5. Antioxidant activity assays
UV/Vis Spectrophotometer and DU 730 (Beckman Coulter, USA) were used for antioxidant activity tests. The DPPH assay was performed according to the method of Blois16). The ABTS assay was modified from the method of Thaipong et al17). The ABTS+ solution was diluted with methanol to obtain 0.99 ± 0.05 units at 734 nm. The scavenging activity of DPPH and ABTS+ were calculated as follows: scavenging activity % = [(A0 − Ac) / A0] × 100, where A0 is absorbance of the control and Ac is absorbance of the sample. For Frap assay, the procedure followed the method of Thaipong et al17) using a trolox (0.05–0.4mg/mL) standard curve. Results were expressed in mM Fe2/μg free mass.
Results
1. Macroscopic morphology of 4 kinds of ‘Mokhyang’
In original plants, the leaf arrangement of Aucklandia lappa, Inula helenium and Aristolochia contorta is alternate although it of Vladimiria souliei is rosulate. Leaves of 4 kinds were various in the shape. While Aristolochia contorta showed climbing stem, racem inflorescence and capsule fruit, other kinds showed straight stem, capitulum inflorescence and achene fruit (Fig. 1 and Table 2). On this, macroscopic identification-key of 4 kinds ‘Mokhyang’ is as follow (Table 3).
In herbal medicine, the roots of 4 kinds were cylindrical shape, however, those were divided in two categories by the thickness and branch of root. The top of Aucklandiae Radix and Inulae Radix were dented, whereas that of Vladimiriae Radix contained black and sticky glue-like substance. Beside Aucklandiae Radix, Inulae Radix and Vladimiriae Radix had dense longitudinal wrinkles and distinct features on outer surface besides, Aristolochiae Radix had fine longitudinal wrinkles and other outer feature was indistinct. On the cross section, 4 kinds were classified through two structures; oil spot and pith (Fig. 2, Table 4). On this, macroscopic identification-key of herbal medicine of 4 kinds ‘Mokhyang’ is as follow (Table 5).
2. Microscopic morphology of 4 kinds of ‘Mokhyang’
In common, cork is made up with several rows of cells. In phloem, Aucklandiae Radix and Vladimiriae Radix had denser ray cells than others and both had alternate arrangement of fibre bundles and sieve tube group but differed in the number of arrangement. Ring-shaped cambium was distinct in all 4 kinds. In xylem, depending on kinds, singly scattered or grouped vessels were arranged radially. Aucklandiae Radix and Vladimiriae Radix were tetrarch protoxylem, while Inulae Radix and Aristolochiae Radix were polyarch protoxylem. Only in Aristolochiae Radix, numerous starch granules were presented in parenchymatous cells. Oil cavities were scattered in phloem and xylem of all 4 kinds and especially in Vladimiriae Radix it also existed in pith (Fig. 3, Table 6). On this, microscopic identification-key of herbal medicine of 4 kinds ‘Mokhyang’ is as follow (Table 7).
3. HPLC analysis of 4 kinds of ‘Mokhyang’
1) Linearity and sensitivity
The coefficient of correlations (r2) of two standard compounds were > 0.999, suggesting good linear relationship between the peak area (y) and the concentration (x). The limit of quantification (LOQ) was calculated as the concentration of each analyte where the signal/noise(S/N) ratio is ≥ 10, respectively. The LODs and LOQs for each standard compounds were as follows: costunolide, 0.49 and 1.64 μg/mL; dehydrocostuslactone, 0.30 and 1.00 μ g/mL, which showed a high sensitivity at the chromatographic condition (Table 8).
2) Sample analysis
The representative chromatograms of the samples and standard compound are shown in Fig. 4. Evaluating the phytochemical equivalency of MH using two STD peaks, all 4teen samples were divided into three groups. One (AL1-5, VS1-3) containing costunolide and dehydrocostuslactone both were Aucklandiae Radix and Vladimiriae Radix. The other (IH1-3) containing dehydrocostuslactone only was Inulae Radix. Another (AC1-3) containing none of them was Aristolochiae Radix.
The quantity of each compound present in samples was measured and the results are summarized in Table 9. Among two standard compounds, costunolide was the most abundant compound. The content of costunolide in Vladimiriae Radix (157.78 ± 38.96 μg/mL; average value of VS1-3) was higher than that in Aucklandiae Radix (86.75 ± 30.41 μg/mL; average value of AL1-5),
The content of dehydrocostuslactone of Aucklandiae Radix and Vladimiriae Radix were 63.60 ± 19.68 μ g/mL and 98.87 ± 59.50 μg/mL in average, lower than that of costunolide. In Inulae Radix, dehydrocostuslactone (5.91 ± 2.60 μg/mL; average value of IH1-3) showed lowest content among three and costunolide was not detected. Dehydrocostuslactone was detected in all three genuine herbs while costunolide was detected only in Aucklandiae Radix and Vladimiriae Radix. The order of the contents of two compounds was Inulae Radix < Aucklandiae Radix < Vladimiriae Radix. On the other hand, costunolide and dehydrocostuslactone were not detected in Aristolochiae Radix.
4. Antioxidant activity of 4 kinds of ‘Mokhyang’
The absorbance profiles of DPPH, ABTS and FRAP assay are shown in Fig. 5. In DPPH assay, the scavenging effects was highest in Vladimiriae Radix (VS1, 90.09 ± 0.19%), followed by Aristolochiae Radix (AC1), Aucklandiae Radix (AL1) and Inulae Radix (IH1). In ABTS assay, the scavenging activity of Vladimiriae Radix (99.20 ± 0.22%) was as effective as ascorbic acid (100 ± 0.11%), which was followed by Aristolochiae Radix. While Aucklandiae Radix and Inulae Radix showed low scavenging activity less than 50%. In FRAP, Vladimiriae Radix displayed FRAP value (103.40 ± 2.02 mM Fe2/μg) 2-fold higher than others and followed by Aristolochiae Radix, Aucklandiae Radix and Inulae Radix.
Discussion
The dried root of 4 species of herbal medicines, Aucklandia lappa, Inula helenium, Vladimiria souliei and Aristolochia contorta, were called ‘Mokhyang (MH)’ in the markets, which causes much confusion in using MH. It is because that they share some similar morphological characteristics and have the synonyms of herbal names in various ancient literatures.
Since its first record in ‘Shennong’s Classic of Materia Medica’ at Han dynasty, MH was recorded in various names as ‘Milhyang’ in ‘Myungyuibyulrok’ and ‘Ohyang’ in ‘Rakbusizib’. Another alternate name of MH, Chung-MH was first used in ‘Bonchogyeong-jipju’ saying MH with blue root top was wonderfully efficacious. In Tang dynasty, because of shortage of genuine MH, Aristolochiae Radix known as ‘Maduryungkeun’ were substituted and called as ‘Tochung-MH’ which is lately called as Chung-MH. In the result, Chung-MH which was only another name of MH before Myung dynasty became a common herbal name of Aristolochiae Radix after Myung dynasty and used as a substitute of MH18).
Aristolochic acid (AA)-induced nephropathy was firstly reported in 1991, that is, anemia and kidney disorder caused by AA-containing botanical products7). A previous founding on side effects of Aristolochiae Radix reported contained AAs were carcinogenic agents of upper urinary tract19). The cytotoxic effects of other components; aristololactam IVa and 7-methoxy-aristololactam IV, in Aristolochiae Radix were demonstrated in human proximal tubular epithelial cell line NK-2 in vitro. However, their cytotoxicity was similar or even more toxic than AA20). Therefore, KFDA prohibited distribution of Aristolochiae Radix in local market from June first, 20055). UK, Belgium and Taiwan also prohibited Aristolochia contorta, and US Food and Drug Administration (FDA) prohibited using all AA-containing botanical medicines21).
Even though MH is commonly used for treating various problems in digestive system 4 kinds of MH have been used for slightly different medicinal purposes. Aucklandiae Radix primarily helps chest pain. Inulae Radix and Vladimiriae Radix have been used in treatment of dyspepsia, stomach pain, vomiting and diarrhea. Aristolochiae Radix, which is now substituted with Inulae Radix, has been used mainly in hypochondria, dysentery and tenesmus6). Because of their differential medicinal efficacy, it is necessary to identify genuine herbs for better beneficial treatment.
Macroscopic identification of original plants was introduced because growers of traditional medicines usually classify kinds based on features of original plants and it is also helpful to consumers when identifying unprocessed herbal medicine. In macroscopic discrimination, 4 kinds of original plants were differentiated by the types of stem, phyllotaxy and leaf shape and 4 kinds of herbal medicines were classified by the size, oil spots, color and root top. To be precise, in microscopic discrimination phloem and central cylinder were used as major features.
In previous studies, chemical analysis of Aucklandiae Radix and Inulae Radix by high performance liquid chromatography (HPLC)9) have been published. Analyzing three genuine kinds showed pattern-recognition analysis by HPLC11). Also, evaluating 4 kinds of MH by HPLC was reported using quantitative contents of costunolide only13).
A HPLC method was developed and applied to analyze two different standard compounds in 4 kinds of MH simultaneously. The samples from the same kinds showed similar chemical constituents and relative proportions of each compound but discrepancies in contents of each compound were observed. It is presumably due to the difference of agricultural environments. Aristolochiae Radix could be easily differentiated from the genuine kinds of MH by the facts neither of standard compounds were detected and it had low similarity in fingerprint. Auckladiae Radix and Vladimiriae Radix showed high standard compound contents and similar fingerprint and the content of costunolide in Vladimiriae Radix was higher than that in Aucklandiae Radix. Besides Inulae Radix showed low quantities of dehydrocostuslactone and low fingerprint similarity among those three herbs.
In contrast, previous study showed that the content of costunolide in Aucklandiae Radix was higher than that in Vladimiriae Radix13). Moreover, both costunolide and dehydrocostuslactone were undetected in Inulae Radix22). More samples should be analyzed to precise whether Inulae Radix could be substituted for MH. Even though further researches need to be done, difference in contents of major biologically active compounds in 4 kinds of MH could be reasonable for the variation of therapeutic efficacy.
Comparing 4 kinds of MH on antioxidant activity also showed difference. Vladimiriae Radix was the most effective antioxidant. The antioxidant activity ranking of samples detected by DPPH, ABTS and FRAP assay was strongly positively correlated as shown in Fig. 5. The difference between antioxidant capacities of Vladimiriae Radix and other kinds was greater in FRAP assay.
Conclusion
The classical identification of traditional medicines with various original plants has been mainly conducted by comparing morphological features in elucidated form. The newly established identification keys in this study based on macroscopic and microscopic identification, and histochemical and phytochemical methods would provide more practical and accurate authentication of traditional medicine. In addition, this study provides comprehensive view on dicriminating Aucklandiae Radix, Inulae Radix, Vladimiriae Radix and Aristolochiae Radix for the first time. Our data could be helpful to ensure eligible therapeutic use and the safety control in local market by distinguishing 4 kinds of Mokhyang.