CC BY
9[9] Koo J.C., Lee S.Y., Chun H.J., Cheong Y.H., Choi J.S., Kawabata S., Miyagi M., Tsunasawa S., Ha K.S., Bae D.W., Han C.D., Lee B.L., Cho M.J. Two hevein homologs isolated from the seed of Pharbitis nil L. exhibit potent antifungal activity. Biochim Biophys Acta. -1998 Jan. -1382(1). -P.80-90
[10] Xu J.S. One case of Pharbitidis Semen poisoning. China's basic medical. -1994. -4(4). -P 108
Identification of the metabolites of Baicalin in rat urine by UPLC/ESI-TOF/MS coupled with MetaboLynx XS automated data analysis
Na Wanga, Fang Lua, Xuzhao Lia, Wanru Donga, Na Zhang a, Changfeng Liu a* Shumin Liua'b*
a Traditional Chinese medicine toxicology laboratory, Institute of Traditional Chinese Medicine, Heilongjiang University of Chinese Medicine, He Ping Road 24, Harbin 150040, China. b The center of GLP, Heilongjiang University of Chinese Medicine, He Ping Road 24, Harbin 150040, China.
Abstracts:To illustrate the main biotransformation pathways of Baicalin in vivo, we elucidated the metabolic profile of Baicalin in rat's urine. The urine was collected from each animal within 12 h after administrating orally Baicalin and distilled water (100mg/kg), and analyzed by ultra-performance liquid chromatography/electrospray ionization time-of-flight mass spectrometry (UPLC/ESI-TOF-MS) at the positive ion mode scanning coupled with MetaboLynx XS (version4.1) automated data analysis method. A total of 26 metabolites were detected, 13 of which were identified. A fairly comprehensive metabolic pathway was proposed for Baicalin include dehydroxylation, methylation, hydroxylation and glucuronidation after deglycosylation. These results are important for understanding the material basis and clinical mechanism of Baicalin for drug discovery, design and clinical application.
Keywords: Baicalin; metabolites; UPLC/ESI-TOF/MS; MetaboLynx XS
Drug metabolism (biotransformation) can contribute significantly to the overall therapeutic and adverse effects of drugs. As part of any drug discovery activity, it is important to analyze the metabolic profile of the parent drug.Baicalin (Fig.1), formulated as 7-D-glucuronic acid-5,6-dihydroxy-flavone, is a major bioactive constituent of Scutellariae Radix (root of Scutellaria Baicalensis Georgi) which has been known to have a multitude of pharmacological properties, such
1 2 2 3 4
as neuroprotective , anti-diogenic , anti-inflammatory , anticancer anti-bacterial, antioxidative , antiviral5 and anti-HIV 6, et al. The metabolism of Baicalin has been preliminarily investigated. However, only a fewmetabolites including BBaicalein, oroxylin A, and their glucuronides were identified . Thus, those works only partly understood the metabolic performance of Baicalin.Herein, ultra-performance liquid chromatography/electrospray ionization time-of-flight mass spectrometry (UPLC/ESI-TOF/MS) followed by multiple mass defect filtering was applied to analyze the common and uncommon metabolites of Baicalin in rat urine for the universal understanding of the metabolism of Baicalin.
Materials and methods
Standard of Baicalin was purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). Formic acid (HPLC grade) was purchase from DIKMA Technologies Inc. (Lake Forest, CA, USA). Acetonitrile (HPLC grade) was purchase from Fisher (USA). The distilled water was purchased from Watson's Food & Beverage Co., Ltd. (Guangzhou, China). Leucine enkephalin was purchased from Sigma-Aldrich (St. Louis, MO, USA).Male Wistar rats (220±20 g in weight) were kept in the breeding room with temperature (25°C), humidity (60 ± 5%) and under 12:12-h light-dark cycle conditions,divided into 2 groups
with 8 ones in each: the control and Baicalin groups. Each rat in the Baicalin group was administrated orally with the aqueous solution of Baicalin with the concentration of 100mg/kg. Each in the control group was administrated orally with the distilled water. The urine was collected from each animal over the course of 12 h after dosing. An aliquot of 5^L of supernatant was injected to UPLC/ESI-TOF-MS analyze. The ethical approval for the experiment was given by the Legislation on the Protection of Animals Used for Experiment Purposes (Directive 86/609/EEC).
Both the dosed data file and the control data file were input in the Accurate Mass Filter Tool for filtering. After processing, the output files were used to compare the differences between the dosed and control traces for searching metabolites of Baicalin (Table 1).
oh o
HO^ ^o
Figure 1. Structure of Baicalin.
Table 1. List of expected metabolites for which narrow window extracted ion chromatograms are generated in phase I and phase II of Baicalin
Mass(mDa) Formula Description Phase
0 - Parent 1
2.0157 +H2 Reduction 2
14.0157 +CH2 Methylation 1
15.9949 +O Hydroxylation 2
30.0106 +O+CH2 Hydroxylation+ Methylation 1
31.9898 +O2 2*Hydroxylation 1
47.9847 +O3 3*Hydroxylation 2
79.9568 +SO3 Sulfate conjugation 2
95.9517 + SO4 Hydroxylation+ Sulfate 2
176.0321 +C6H8O6 Glucuronide conjugation 2
192.027 + C6H8O7 Hydroxylation+ Glucuronide conjugation 2
352.0642 + C12H16O12 2*Glucuronide conjugation 2
-15.9949 -O Dehydroxylation 1
Metabolite peaks were assigned by MS/MS analysis or interpreted with available biochemical databases, such as HMDB (http://www.hmdb.ca/) ; MassBank (http://www.massbank.jp/) and the fragmentation patterns of flavonoid such as Retro-Diels-Alder(RDA)reaction(Fig. 1). Results and discussion
Detection of urinary samples and screening of Baicalin metabolites
After pretreatment, rat urinary samples were detected by UPLC-TOF/MS in positive ESI modes to obtain the full-scan mass chromatograms with accurate mass measurement. The UPLC-ESI MS total ion current chromatograms in positive ion mode are shown in Fig. 2b.
Metabolite data from the UPLC-TOF MS system identified using Metabolynx XS in
MS2. TheMetaboLynx output browser was able to show the metabolites of Baicalin by using the modified MetaboLynx screening routine (Fig. 3).Considering the output browser report results may be pseudomorph, this study modified the metabolite name using MS2(Table 2. ).
100
Huangqin_huangqingan_urine_6
2.50 359.1232
232.1289
9.88 448.3050
1 : TOF MS ES+ TIC 5.82e5
15.93 496.4199
16.83 696.5299
Hu
abngq 100n
2.50 huangqingan_
2.50 357.1002
in
5.00 urine 6-NEG
7.50
10.00
12.50
15.00
17.50
2.36 172.9880
1 : TOF MS ES-TIC 8.75e5
2.80 385.1400
4.97 919.2057
1 1 .60 453.2857
0-1........................
2.50 5.00
Huangqin_yaocai_urine_kongbai-1 100^ 2 48 -
l00H 180.0633 2.36 340.1021
7.50
10.00
12.50
15.00
17.50 1:
TOF MS ES+ TIC 7.1 2e5
15.11 388.3936
1 6.67 1 136.8833
2.50 5.00 7.50 10.00 12.50 15.00 17.50
Figure 2.The total ion chromatograms of urine: (a) urine after oral administration of Baicalin in ESI+mode ; (b)urine after oral administration of Baicalin in ESI- mode and; (c) blank sample in ESI+mode.
Expected let abolit es - Hu£meqin_huanEqingaii_uriiie_2_IDF_5ü1 parent C15H1DC5 (116/248 entries)
St... I Ma.55_I Metabolite Name
Time
254.0579 Dehydroxylation
270.0528 270.0528 284.0685 / 284.0685 300.0634 / 314.0790 / 334.0147 350.0096 364.0253 / 430.0900 430.0900 / 446.0849 / 446.0849 446.0849 460.1006 / 460.1006 460 1006 476.0955 526.0417 / 606.1221 / 622.1170 / 636.1326 638.1119 652.1276 / 798 1491 y 254.0579
Farent Farent Hethylati on Hethylati on
Hydroxylation + methylation
Hethylation + Hydroxylati-;n + niethylation
Sulfate conjugation \ Dehydroxylati'-n
Sulfate conjugation
Hethylation + Sulfate conjugation
Glucuronide conjugation t Dehydroxylation
Glucuronide conjugation + Dehydroxylation
Glucuronide conjugation
Glucuronide conjugation
Glucuronide conjugation
Hethylation + Glucuronide conjugation
Hethylation + Glucuronide conjugation
Hethylation + Glucuronide conjugation
Hethylation + Hydroxylation t glucuronide conjugation
Sulfate conjugation + Glucuronide conjugation
2 x Glucuronide conjugation + Dehydroxylat:. on
2 x Glucuronide conjugation
Hethylation + 2 x Glucuronide conjugation
Hydroxylat.ion +..2. x Glucuronide conjugation
Hydroxylati on + methylation + 2 x Glucuronide conjugation
Glucuronide conjugation + 2 x Glucuronide conjugation
Dehydroxylation_
■ ■■■ilia iïi /1 F i'Uf.d mDa Tine Area Abs
C15H100.; ¿55.0660 0 3 1.1 1J 04 1709 50
C15H1005 27!.0602 -0.4 -1.6 6.86 85.30
C15H1005 271.0611 0.5 l.'fi 7.97 139. 30
C16H1205 285.0766 0.3 1.1 10.94 3281.30
C16H1205 285.0760 -0.3, -L.0 11.56 8518.50
C16H1206 301.0710 -0.2 -0.6 7.;i 29.10
C17H1406 315.0862 -0.6 -2.0 11.28 42.10
C15H1007S ■335.0221 4 3 8.64 133.20
C15H1008S 351.0173 -0.1 -0.4 8.81 142.50
C16H1208S 365.0331 0.0 0.0 8.67 636.70
C21H18010 43?. 0955 -2.Ü - 3 4 's| 418 .3
C21H18010 431.0964 -1.4 -3.2 5.03 2776.70
C21H18011 447.0932 0.5 1.1 3.93 3338.10
C21H18011 447.0949 2.2 4.9 4.87 603 50
C21H18011 447.0914 -1.3 -2.9 5.32 9565.30
C22H20011 461.1Ü75 -0.9 r-1 9 4.51 557.10
C22H20011 46i 1058 -2.6 -5.6 4 .:7 12845.70
C22H20011 46;. :.059 -2.5 -5.4 5.51 2121.00
C22H20012 477.1017 -3.3 5.15 680.60
C21H18014S 527.0482 -1.3 -2.5 5.68 180.00
C27H26016 60- !.298 -0.1 -0.1 2.93 329.20
C27H26017 623.1284 3.6 5.8 3.19 11252.40
C28H28017 63". 1.425 2.1 3.2 3.29 58.70
C27H26018 639.1206 0.9 1 4 2.41 65.60
C28H28018 653.1396 4.2 6.5 3.41 338.60
C33H34023 799.! 611 4.2 5.3 2.72 56.50
C15H1004 255.0749 9.2 36.2 1.00 136.30
KT
Figure 3. Expected metabolites list showed by MetaboLynx output browser.
a
0
c
Table 2. Information of identified metabolites in ESI+ mode
No tR (min) M+H (m/z) MS/MS (m/z) Formula Metabolite
1 4.57 447.09 103;168;271; 447 C21H18O11 Baicalin
2 8.02 271.06 102;123;169; 211;253;271 C15H10O5 Baicalein
3 11.09 255.06 103;129;153 237;255 C15H10O4 chrysin
4 5.09 431.10 105;153;255 431 C21H18O10 chrysin-7-glucuronide
5 10.99 285.08 105;168;179 252;270;285 C16H12O5 Wogonin
6 11.62 285.08 105;168;267 270;285 C16H12O5 Oroxylin-A
7 5.81 491.12 101;198;255 285;300;315 447;493 C23H22O12 5, 7- dihydroxy-6, 8- dimethoxy flavone-7 - O - glucuronide
8 4.51 461.11 113;159;271 285;461 C22H20O11 Oroxylin A-7-glucuronide (5, 7 -dihydroxy - 8 - methoxy flavone - 7 - O -glucuronide
9 4.99 461.11 113;159;175 270;285;461 C22H20O11 Oroxylin A -5-O-glucuronide (5, 7 -dihydroxy - 6 - methoxy flavone - 5 - O -glucuronide
10 5.589 461.10 105;179;252 270;285 C22H20O11 Wogonoside
11 4.86 477.10 101 ;177;285 301;477 C22H20O12 5, 6, 7 - trihydroxy - 8 - methoxy flavone - 7 - O - glucuronide
12 3.40 653.14 103;301;371 477;653 C28H28O18 5, 6,7 - trihydroxy - 8 - methoxy flavone - 6, 7 - di-O - glucuronide
13 3.188 623 159;177;271 447;623 C27H26O17 Baicalein 6,7-di-O-glucuronide
UPLC/ESI-TOF/MS provides faster separation with increased resolution and sensitivity for analysis and elucidation of drug metabolism. The MetaboLynx XS (version4.1) software package requires minimal operator intervention and is capable of batch processing of samples, automated
o
detection and identification of drug metabolites . Therefore, they were combinated for rapid and sensitive analysis of the metabolites of Baicalin in this study. There are many isomerides of them because of the specific structure of flavonoids. Further research on the structures of metabolites is needed to separate the metabolites from biofluid and tissues (urine and liver, e.g.) and character the composition and molecular structures by color reactions and spectral analysis methods as FTIR, UV-Vis, MS, 1 H NMR and 13C-NMR.
Conclusions
In summary,we detected 26 metabolites, 13 of which were identified in this study (Table 2). These results indicate that the major in vivo metabolic processes associated with Baicalin include dehydroxylation, methylation, hydroxylation and glucuronidation after deglycosylation. A fairly comprehensive metabolic pathway was proposed for Baicalin(Fig. 4). The results are important for
understanding the material basis and the clinical mechanism of Baicalin for drug discovery, design and clinical application.
Figure 4. Potential metabolic biotransformation pathway and the metabolites of Baicalin. (A) dehydroxylation; (B) glucuronidation; (C) methylation; and (D) hydroxylation+ methylation.
Acknowledgements
This work was supported by a grant from the National Basic Research Program of China
(Grant No. 2007CB512608 and 2013CB531804).
References
1. Cao Y, Li G, Wang YF, Fan ZK, Yu DS, Wang ZD and Bi YL. Neuroprotective effect of baicalin on compression spinal cord injury in rats. Brain research 2010;1357:115-123.
2. Lee H, Bae S, Kim K, Kim W, Chung SI and Yoon Y. Beta-catenin mediates the anti-adipogenic effect of baicalin. Biochemical and biophysical research communications 2010;398:741-746.
3. Li-Weber M. New therapeutic aspects of flavones: The anticancer properties of scutellaria and its main active constituents wogonin, baicalein and baicalin. Cancer treatment reviews 2009;35:57-68.
4. .Kowalczyk E, Krzesinski P, Kura M, Niedworok J, Kowalski J and Blaszczyk J. Pharmacological effects of flavonoids from scutellaria baicalensis. Przeglad lekarski 2006;63:95-96.
5. Chu ZY, Chu M and Teng Y. Effect of baicalin on in vivo anti-virus. Zhongguo Zhong Yao Za Zhi 2007;32:2413-2415.
6. Li BQ, Fu T, Dongyan Y, Mikovits JA, Ruscetti FW and Wang JM. Flavonoid baicalininhibits hiv-1 infection at the level of viral entry. Biochemical and biophysical research communications 2000;276:534-538.
7. Li C, Zhang L, Lin G and Zuo Z. Identification and quantification of baicalein, wogonin, oroxylin a and their major glucuronide conjugated metabolites in rat plasma after oral administration of radix scutellariae product. Journal of pharmaceutical and biomedical analysis 2011;54:750-758.
8. Lu F, Sun Q, Bai Y, Bao S, Li X, Yan G and Liu S. Characterization of eleutheroside b metabolites derived from an extract of acanthopanax senticosus harms by high-resolution liquid
chromatography/quadrupole time-of-flight mass spectrometry and automated data analysis. Biomedical chromatography 2012;26:1269-1275.
The Observation on the Effect of Xuanbei Xiaocuo Soup Works on the 78 Cases of Phlegm Stagnation Type Acne
Wang Ruoxin
Heilongjiang University of Chinese Medicine, Graduate Academy, Surgery of Traditional Chinese Medicine Department, Graduate Student of 2011, Heilongjiang, Haerbin,China 150040)
Abstract
Goal: Observing the effect of Xuanbei Xiaocuo Soup on Phlegm Stagnation Type Acne (PSTA).Method: Dividing the78 patients of PSTA into 2 groups randomly, the 39 patients in the therapy group were treated with Xuanbei Xiaocuo Soup; and the other 39 ones in the control group were treated with Zhongjiefeng Fensan pills, and the observation on the two groups started after 30 days. Result: The therapy group's effective rate is 94.87%, and the control group's is 82.05%, the
difference between the two groups is very obvious (P<0.05). Conclusion: Xuanbei Xiaocuo Soup is a very effective method for curing PSTA, which should be wildly clinical applied.
Key words: Xuanbei Xiaocuo Soup, Phlegm Stagnation Type, acne, curative effect.
Acne is usually occurred around the puberty, which is a type of pilosebaceous chronic inflammatory disease; this type of disease happens to the males more often, and happens to the females earlier, which serious symptom is forming hollow or hypertrophic scar, and its deep-seated inflammation can create massive abscess. The scar may remain even after the successful curing, the curing process is very long, which would strongly influence the life quality of patients. The writer of this article has been treated 62 cases with Xuanbei Xiaocuo Soup, and the curative effect is very satisfactory.
1.Materials and Methods
1.1 Common materials
All the cases are collected from the dermatology clinic of Heilongjiang University of Chinese Medicine's first affiliated hospital between January 2012 and March 2013, which are all matched with the diagnostic standard according to Clinical Dermatology(Ed., Zhao Bian) and Diagnostic and therapeutic effect evaluation criteria of diseases and syndromes in traditional Chinese medicine. All the patients are categorized into severe acne, which equals the third level of the forth level according to the Acne Pillsbury Classification, at the same time the patients, who are pregnant, breast feeding, having sensitive constitution, allergic to the related medicine, having systematic problems on cardio-vascular system, hemopoietic system, and immune deficiency disorder, are excluded. There are totally 78 cases, which include 55 male patients and 23 female patients, whose age are between 17 and 35 years old; their courses of disease are between 15 days and 6 years. The skin eruptions are majorly distributed on faces, which may spread to backs and chests. The clinical feature of skin rash is usually repeatedly presented as red or dark red protuberance, abscess, cyst, and cicatrix. The two groups do not have obvious difference in gender, age, course of disease, and the order of severity on skin eruptions (P>0.05), which means they are comparable.
1.2 Treatment Methods
The control group would be treated with Zhongjiefeng Fensan Pill, each time 4 pills, three times daily, orally.
Enforcement Standards: The State Food and Drug Administration Standard
Drug standard number: YBZ07152006
