ISSN 10681620, Russian Journal of Bioorganic Chemistry, 2010, Vol. 36, No. 7, pp. 816–824. © Pleiades Publishing, Ltd., 2010. Original Russian Text © D.N. Olennikov, N.K. Chirikova, L.M. Tankhaeva, 2010, published in Khimiya Rastitel’nogo Syr’ya, 2009, No. 4, pp. 89–98.
REVIEW ARTICLE
Phenolic Compounds of Scutellaria baicalensis Georgi D. N. Olennikov, N. K. Chirikova, and L. M. Tankhaeva Institute of General and Experimental Biology, Siberian Branch, Russian Academy of Sciences, str. Sakh’yanovoi 6, UlanUde, 670047 Russia email:
[email protected] Received April 1, 2008
Abstract—Information on the phenolic compounds from the Baikal scullap Scutellaria baicalensis Georgi, family Lamiaceae, is given. Structures of 125 compounds are presented, and information is given on their dis tribution in the aboveground and belowground parts of the plant and in the tissue culture of the root. There are generalized data on the use of TLC and HPLC methods for the separation of the main components of Scutellaria baicalensis. Keywords: Scutellaria baicalensis, phenolic compounds, chromatography DOI: 10.1134/S1068162010070046
The chemical composition of the plant of the genus Scutellaria L. is versatile. Up to now, derivatives of phenylethyl alcohol, phenolic acids, iridoids, cle rodans, steroids, and triterpenoids, cardenolides, cou marins, tannins, essential oils, and flavonoids were isolated from the species of this genus [1]. Among the various classes of natural compounds listed, the group of phenolic compounds is notewor thy. This is due to their anomalously high content and extraordinarily high structural diversity. The phenolics are represented by flavones, flavanoids, flavonols, phenylpropanoids, chalcones, isoflavones, biflavones, and lignoflavonoids. The biological activity of the Scutellaria prompted continuing interest in this topic and an evergrowing number of scientific publications. A great number of publications has been accumulated on the isolation, identification, structure elucidation, and biological activity of phenol compounds from var ious species of Scutellaria L. According to the data of Malikov and Yuldashev [2], currently over 200 phe nolic compounds from 65 species of the Scutellaria genus have been studied. Flavones are foremost among phenols in their dis tribution in Scutellaria L. Baicalin, chrisin, vogonin, scitellarein, oroxylin, apigenin, and luteolin are the most frequently occurring compounds. The history of flavonoid study in S. baicalensis began in 1923 when Shibata et al. isolated and charac terized baicalin from the roots of this plant [3]. An analysis of scientific information shows that 125 com pounds of phenol nature have been found to date in this species. Eightyone flavone derivatives have been isolated from S. baicalensis: 55 aglycones and 26 glycosides (4 Cglycosides among them, f 1). All of the compounds can be divided according to the character of substitu
tion of the main flavone skeleton into 5 groups: di, tri, tetra, penta, and hexasubstituted. Disubstituted flavones are represented by 5,7sub stituted chrisini and its two glycosides. Trisubstituted flavones (the second group by size) is divided into 4 types: baicalein (5,6,7), norvogonin (5,7,8), 2'hydroxychrisini (5,7,2'), and apigenin (5,7,4'). The group of tetrasubstituted is the most abundant (30 compounds). The flavones of S. baicalensis, like the whole genus and the Lamiaceae family, are characterized by the presence of 2'methyl and glycosyl substituents that belong to the group of flavone derivatives. Andersen and Markham believe [22] that these substituents (2',6'OH, MeO, and glycosyl) are characteristic tax onomic features on the level of both the Scutellaria, and the Lamiaceae family as a whole. Note also the presence of 2'6'hydroxylated, methoxylated, and gly cosylated flavones and also rare 3' and 5'substituted flavones in S. baicalensis. The carbohydrate part of the S. baicalensis Ogly cosides is represented by glucuronic acid (13 com pounds), glucose (9 compounds), and rutinose (1 compound). Uronides are also a special feature of the Scutellaria L. components. Four Cglycosides iso lated are the derivatives of chrisin, 6C and 8C gluco sides, 6Cglucoside8Carabinoside and 6Carabi noside8Cglucoside. A comparison of the compositions of the above ground and underground parts of the plant showed a great structural diversity of the compounds isolated from the roots of S. baicalensis; 67 substances were isolated from the aboveground parts and 14 from the underground parts.
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Table 1. Flavones of S. baicalensis Substituents in positions* Name
1
5
6
7
8
2'
3'
4'
5'
6'
2
3
4
5
6
7
8
9
10
Chrisin Chrisin 7Oglucoside Chrisin 7Oglucuronide
OH OH OH
Baicalein Baicalein 6Oglucuronide Baicalein 7Oglucoside Baicalin Baicalein 6,7diOglucuronide Baicalein 6Oglucuronide 7Osul fate 7Methoxybaicalein Oroxyline A Oroxyloside Chrisin 6Cglucoside 5Hydroxy6,7dimethoxyflavone Chrisin 8Cglucoside Vogonin Vogonoside Vogonin 5Oglucoside 7OMethylvogonin Norvogonin Norvogonin 7Oglucoside 7Methoxynorvogonin 2'Hydroxychrisine Apigenin Apigenin 7Oglucuronide
OH OH OH OH OH OH
5,8Dihydroxy6,7dimethoxyfla vone Chrysine 6Cglucoside8Cara binoside Chrysine 6C arabinoside 8C glucoside 5,7Dihydroxy6,8dimethoxyfla vone 2'Hydroxybaicalein Tenaxin II Tenaxine II 7Oglucuronide Scitellarein Scutellarin Hispidulin Dinatin Salvigenin 5,2'Dihydroxy6,8dimethoxy flavone 5,8,2'Trihydroxy7methoxyfla vone
Found in
OH OH OH OH OH OH OH OH Glc OH OH OH OH OH OH OH
Disubstituted OH Glc GlcU Trisubstituted OH OH GlcU OH OH Glc OH GlcU GlcU GlcU GlcU SO4
3' 4'
2' 8 7
A
O C
6 5
B 5' 6'
Over Under Root ground ground tissue part culture part 11
12
13
[7] [1, 2] –
[21] – [1, 2]
[19] – –
[4, 5] [1, 2] [12] [10] [1, 2] [1, 2]
[21] – – [21] – –
[17] – – [17] – –
[4] [4, 5] [10] – [1, 2] – [4, 5] [10] [8] [11] [4] [1, 2] [4] [12] – –
– – – – – – – – – – – – – – [1, 2] [1, 2]
– – – – – – [17] [17] – – – – – – – –
[10]
–
–
O
OH
MeO OH GlcU OH MeO OH Glc OH MeO GlcU MeO OH MeO MeO MeO OH OH Glc OH MeO OH OH OH OH GlcU Tetrasubstituted MeO MeO OH
OH
Glc
OH
Ara
[9]
–
[19]
OH
Ara
OH
Glc
[9]
–
[19]
OH
MeO OH
MeO
[1, 2]
–
–
OH OH OH OH OH OH OH OH OH
OH MeO MeO OH OH MeO OH MeO MeO
[1, 2] [11] [1, 2] [1, 2] [1, 2] [1, 2] [1, 2] – [1, 2]
– – – [21] [1, 2] – [1, 2] [1, 2] –
– – – – – – – – –
[7]
–
–
OH
OH MeO MeO Glc MeO
OH OH GlcU OH GlcU OH OH MeO
OH OH
OH OH OH OH OH OH MeO MeO MeO OH
MeO OH
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Table 1. (Contd.) Substituents in positions* Name
1
Found in
5
6
7
8
2'
3'
4'
5'
6'
2
3
4
5
6
7
8
9
10
Over Under Root ground ground tissue part culture part 11
12
13
Scutevulin Scullcapflavone I 5Hydroxy7,8,2'trimethoxyfla vone 2'Hydroxy5,7,8trimethoxyfla vone Isodinatin Isoscitellarein Isoscutellarin 4'Hydroxyvogonin 5,7,2'3'Tetramethoxyflavone 5,7,2'5'Tetramethoxyflavone 5,7,2'6'Tetramethoxyflavone 5,7,6'Ttrihydroxyflavone 2'O glucoside 5,7,2'Ttrihydroxy6'methoxyfla vone 5,7,6'Ttrihydroxy2'Omethox yflavone Luteolin Luteolin 7Oglucuronide
OH OH OH
OH MeO OH MeO MeO OH MeO MeO MeO
[13] [5] [1, 2]
– – –
– [19] –
MeO
MeO MeO OH
[1, 2]
–
–
OH OH
OH OH
MeO OH
OH OH
[10] [1, 2]
[1, 2] [1, 2]
– –
OH OH OH OH OH OH
GlcU OH OH MeO OH OH OH OH
OH OH OH OH OH Glc
– [1, 2] [13] [15] [5] –
[1, 2] – – – – –
– – – – – [19]
OH
OH
OH
MeO [13]
–
–
OH
OH
MeO
OH
[1, 2]
–
–
OH OH
OH GlcU
– –
[1, 2] [1, 2]
– –
5,8,2'Trihydroxy6,7dimethoxy flavone Tenaxin I 5,2',5'Trihydroxy6,7dimethoxy flavone 5,6'Dihydroxy6,7dimethoxy flavone 2'Oglycoside Hypolaetin Raderianin 5,7,6'Trihydroxy8,2'dimethoxy flavone 5,7,2'Trihydroxy8,6'dimethoxy flavone Viscidulin II Ravularin Viscidulin II 2'Oglucoside 5Hydroxy7,8,6'trimethoxyfla vone 2'Oglucuronide 5,6'Dihydroxy7,8,2'trimethoxy flavone
OH
OH OH Pentasubstituted MeO MeO OH OH
[7]
–
–
OH OH
MeO MeO MeO OH MeO MeO OH
[11] [1, 2]
– –
[19] –
OH
MeO MeO
OH
[16]
–
[19]
OH
– [1, 2] [1, 2]
[1, 2] – –
– – –
5,2',5'Trihydroxy6,7,8tri methoxyflavone 5,2'Dihydroxy6,7,8,3'tet ramethoxyflavone Scullcapflavone II 5Hydroxy6,7,8,2',6'pentame thoxyflavone
OH OH OH OH
OH OH
OH
Glc
OH OH OH
OH OH OH MeO MeO OH OH MeO MeO
OH
OH
OH
MeO OH
MeO [12]
–
–
OH OH OH OH
MeO MeO MeO MeO
MeO MeO MeO MeO
OH [13] MeO [15] OH – MeO –
– – – –
– [19] [19] [19]
OH
MeO MeO MeO
OH
[1, 2]
–
–
[12]
–
–
–
–
[20]
MeO [5] MeO [1, 2]
– –
[19] –
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OH
OH OH Glc GlcU
OH
Hexasubstituted MeO MeO MeO OH
OH
MeO MeO MeO OH
OH OH
MeO MeO MeO OH MeO MeO MeO MeO
OH
MeO
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Table 1. (Contd.) Substituents in positions* Name
1 5,6'Dihydroxy6,7,8trimethox yflavone 2'Oglycoside 5,7,3',6'Tetrahydroxy6,2' dimethoxyflavone 5,2',5'Trihydroxy7,8,6'tri methoxyflavone 5,7,2',5'Tetrahydroxy8,6' dimethoxyflavone 5Hydroxy7,8,2',5',6'pentame thoxyflavone 5,7,2',6'Tetrahydroxy8,3' dimethoxyflavone Viscidulin III Viscidulin III 2'Oglucoside 5,7,2',6'Tetrahydroxy8,6' dimethoxyflavone
Found in Over Under Root ground ground tissue part culture part
5
6
7
8
2'
3'
4'
5'
6'
2
3
4
5
6
7
8
9
10
11
12
13
OH
[16]
–
–
OH
[1, 2]
–
–
OH
MeO MeO MeO Glc
OH
MeO OH
MeO OH
OH
MeO MeO OH
OH
MeO [1, 2]
–
–
OH
OH
OH
MeO [12]
–
–
OH
MeO MeO MeO
–
–
OH
OH
MeO OH
[1, 2]
–
–
OH OH OH
OH OH OH
MeO MeO OH MeO Glc OH MeO OH OH
OH [1, 2] OH [13] MeO [1, 2]
– – –
– [19] –
MeO OH
MeO MeO [1, 2] MeO
OH
* Ara, arabinose; Glc, glucose; GlcU, glucuronic acid.
The derivatives of chrisin, apigenin, scitellarein, isoscitellarein, and luteolin were found in the above ground part; they belong to 6 substitution types of the flavone structure. Luteolin, apigenin, their glucu ronides, and hypolaetin were found only in the above ground part of the plant. The next flavonoid type with respect to the diversity of structures distributed in the plants of the Scutellaria genus are flavanones. Fortyeight compounds were isolated from 19 species of this genus, S. baicalensis containing 21 representative of the flavanoid class, derivatives of di, tri, tetra, and pentasubstituted fla vanone (Table 2). The most distributed are the types of dihydrobaicalein (5,6,7), dihydroscitellarein (5,6,7,4'), and eriodictiol (5,7,3',4'). A comparison of the structural diversity of flavones indicates a tendency to the preservation of certain sub stitution types, the representatives of which dominate: 5,7, 5,6,7, 5,7,8, 5,7,4', 5,6,7,4', 5,7,8,2', and 5,7,3',4'. Note that the transition to dihydrostructure in the case of pentasubstituted compounds is accompanied with the appearance of the only structure of flavonol type with a 3hydrohylated carbon in ring C, (2R, 3R)3,5,7,2',6' pentahydroxyflavanone which is bio synthetically related to the tetraprecursor, (2S) 5,7,2',6'tetrahydroxyflavanone. Among other compounds of phenolic nature, the following flavanols (quercetin, rutin, viscidulin I, and its 2'Oglucoside [2]), chalcones (2,6,2',4'tetrahy droxy6'methoxychalcone [13]), isoflavones (daid zein, daidzin, puerarin, and formononetin), lignofla RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY
vanods (gediotol C 4"Oglucoside and gediotol D 4" Oglucoside [2]), phenylpropanoids (2(3hydroxy4 methoxyphenylethyl)1Orhamnosyl(4Oferuloyl)) glucoside [8], acteoside, leucoceposide A, and erythroguaiacylglycerolβsiringaresinol ester 4"O glucoside [2] (Table 3). None of the mentioned com pounds had been found to date in the aboveground part. Zgorska and Hainos [21] studied the composition of phenolic acids in the aboveground part of S. baicalensis and have found there protocatechuic, vanillic, caffeic, pcoumaric, and ferulic acids; the presence of phydroxybenzoic and ferulic acids was found in the aboveground parts. Tani et al. [23] have studied the distribution of fla vones of S. baicalensis in various parts of plants and established that glycosides are mainly concentrated in the floema and xylema of roots, whereas aglycones (baicalin and baicalein) are found in the root perid erm. It should also be noted that the leaves and flowers of the plants grown in China, Korea, and Japan con tain no baicalin and baicalein. In a later work [21] devoted to the study of S. baicalensis from Poland, baicalin and baicalein were isolated from the above ground parts at the concentrations of 0.12 and 0.02%, respectively. The total content of flavanoids in the roots of S. baicalensis is 21.33–22.82% (Ukraine) [1], 5.19– 9.53% (Hong Kong), 8.11–23.14% (Japan) [14], 12.70–15.10% (Korea), 9.93–12.07% (China) [23]; in the aboveground part, it is 10.27–12.15% (Ukraine) [24]. The dominating compounds in the underground Vol. 36
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Table 2. Flavanones from S. baicalensis Substituents in position*
Found in
Name 3
5
6
7
8
2'
3'
4'
6'
under above ground ground part part
Disubstituted OH
5,7Dihydroflavanone
OH
[1, 2]
–
[1, 2]
– – – – – – – –
Trisubstituted OH OH OH OH OH OH OH OH
Dihydroxybaicalein 5,6Dihydroxy6methoxyflavanone Dihydrobaicalin Dihydrooroxylin A Dihydrooroxylin A 7Oglucoside Dihydronorvogonin 7Oglucuronide 5,8Dihydroxy7methoxyflavanone Naringenin
OH OH OH MeO MeO
OH MeO GlcU OH Glc GlcU MeO OH
3' 2' 8 7
A
B
O C
5' [11]
OH
[7] + + [1, 2] [1, 2]
6'
6 5
OH OH
4' [1, 2]
O
Tetrasubstituted Dihydroscutellarein (isocarthami din) Dihydroscutellarin 5,7,4'Trihydroxy6methoxyfla vanone 2(S)7Hydroxy5,8,2'trimethoxy flavanone Dehydroscutellarein (carthamidin) Dehydroisoscutellarin Eryodictiol Hesperetin Hesperidin 2(S)5,7,2',6'tetrahydroxyflavanone 2(S)7,2',6'trihydroxy5methoxy flavanone
OH
OH
OH
OH
[1, 2]
[6]
OH OH
OH MeO
GlcU OH
OH OH
[1, 2] [7]
[1, 2] –
[1, 2]
– [6] [1, 2] – – – – –
MeO
OH
MeO
OH OH OH OH OH OH MeO
OH GlcU OH OH Rut OH OH
OH OH
MeO
OH OH
OH OH
[1, 2] – [15] [1, 2] [1, 2] [14] [13]
OH
OH
[8]
OH OH OH
OH OH OH MeO MeO
Pentasubstituted (2R,3R)3,5,7,2',6'Pentahydroxyfla OH vanone
OH
OH
–
* Ara, arabinose; Glc, glucose; GlcU, glucuronic acid.
parts are baicalin (its content can reach up to 90% of the total flavonoid content), vogonin, and vogonoside; the populations with dominated content of baicalein (Hong Kong and Japan) occur rarer [14, 23]. The main flavanoid components of the aboveground part are scitellarin, dihydroscitellarin, and glucuronides of apigenin and luteolin [24]. The tissue culture of the S. baicalensis root and its chemical composition have been studied beginning from the 1980s. There were found 22 compounds, 17 flavones (Table 1) and 5 phenylpropanoids among them (martinoside, leucoceposide A, acteo
side, 4hydroxyβphenylethyl glucoside, and 2(3 hydroxy4methoxyphenylethyl)Orhamnosyl(4O feruloyl) glucoside [17–20]); the presence of fla vanones has not been found to date. Four substances (5,7,6'trihydroxyflavone 2'Oglycoside, viscidulin II 2'Oglycoside, 5hydroxy7,8,6'trimethoxyflavone 2'Oglucuronide, and 5,2'dihydroxy6,7,8,3'tet ramethoxyflavone) had not been found in the initial plant. The main phenol compounds in the root tissue culture are baicalin [18, 19, 48] and acteoside [20]. The main analytical methods used for the quali tative and quantitative analysis of the raw materials
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Table 3. Compounds of various classes*
R 3' R 4'
R2' HO
O R3
R 6'
OH O
HO OMe
HO
Flavanols R2' R3' Compound R3 Quercetin OH H OH Rutin Orut H OH Viscidulin I OH OH H Viscidulin I 2'Oglycoside OH OGlc H Chalcones 2,6,2',4'Tetrahydroxy6'methoxychalcone
R4' OH OH H H
R6' H H OH OH
OH OH O
R8
R7
O O
R4'
OMe HO R2
H
H OMe
Isoflavones Compound Daidzein Daidzin Formononetin Puerarin
R8 H H H Glc
R7 OH OGlc OH OH
Lignoflavanoids Compound Gediotol C 4"Oglycoside Gediotol D 4"Oglycoside erythroguaiacylglycerolβ siringaresinol ester 4"Ogly coside
R4' OH OMe OMe OH R2 H H OMe
R1 OH OH OH
O OMe R1 OH OMe OGlc
R1
HOH2C
R2
O O
OR3 OR4 OH
R4
COOH R3 COOH
R4 R3
Phenylpropanoids Compound Leucosceptoside A Martinoside 2(3Hydroxy4methoxyphe nylethyl)1Orhamnosyl(4 O feruloyl) glucoside Acteoside 4Hydroxyβphenylethyl glu coside
R1 OH OH OH
R2 OH OMe OMe
R3 Rha Rha Rha
R4 Fer Fer Caf
OH H
OH OH
Rha H
Caf H
Hydroxybenzoic acids Acid pHydroxybenzoic Protocatechic Vanillic Hydroxycinnamic acids Acid pCoumaric Caffeic Ferulic
R3 H OH OMe
R4 OH OH OH
R3 H OH OMe
R4 OH OH OH
* Glc, glucose: Rha, rhamnose; Rut, rutinose; Caf, caffeic acid; and Fer, ferulic acid.
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Table 4. Conditions of TLC separation of the S. baicalensis components Moving phase*
Detection*
Separated components, references
1
2
3
Silica gel Benzene–ethylformiate (4 : 1)
Vapor NH3
Baicalin [25]
CHCl4.3–MeOH (40 : 1); cyclohexane–EtOAc (5 S. : 1)baicalensis components UV: λ = 254 nm; 5% phosphomo Baicalin [26] Table Conditions of TLC separation of the lybdic acid, 5% vanillin Separated components, Moving phase* (1 : 5 : 1) UV: λ = 365Detector* nm Baicalin [27] 36% AcOH; CHCl3–benzeneMeOH references UV: λ = 280 nm2
Toluene ethylacetate–HCOOH (10 1 : 15 : 11)
Baicalin [28] 3
Ethylacetate–EtOH (3 : 2); ethylacetate–MeOH–HCOOH (10 : 1 : 1) Silica gel UV: λ = 278 nm
Baicalin [29]
UV: λ =NH 2544nm Vapour UV: λ 5% UV: λ= = 254 365,nm; 2545% nm;ФМК, 1% FeCl 3 vanillin UV: λ = 365 nm UV: λ = 280 nm 10% λ H= 5% FeCl3 2SO 4; nm UV: 278 UV: λ 254 nm 10% H= 2SO4; 5% vanillin UV: λ = 365, 254 nm; 1% FeCl3
Baicalin [27] Baicalin [28] Baicalin Baicalin [32] [29] Baicalin [30] [33] Baicalin [31]
UV: 282 nm 10% λ H= 2SO4; 5% FeCl3 UV: λ 290 10% H= 5% vanillin 2SO 4; nm
Baicalin Baicalin [34] [32] Baicalin [35] [33]
UV: λ = 282 nm UV: λ = 280 nm UV: λ = 290 nm UV: λ = 365 nm; 2% FeCl3
Baicalin [34] Baicalin [36] Baicalin [35] Scutellarin [37]
UV: λ = 280 nm UV: UV: λ λ= = 365 365 nm; nm; 2% 2% FeCl FeCl3 3
Baicalin [36] Scutellarin Scutellarin [37] [38]
UV: λ = 410 nm UV: λ = 365 nm; 2% FeCl3 UV: λ = 410 nm
Scutellarin [39] Scutellarin [38] Scutellarin [39]
10% H2SO4 10% H2SO4
Scutellarin [40] Scutellarin [40]
UV: λ = 254 nm
Scutellarin [41]
Benzene–MeOH–butanone–HCOOH (14 : 2 : 3 : 1)
UV: λ = 254 nm
Baicalin, Baicalein, Vogonin, Vogonoside [42]
33% AcOH
UV: λ = 365 nm
Baicalin [43]
30% AcOH
UV: λ = 365 nm
Scutellarin [44]
5% Sodium dodecylsulfate–7.5% Triton X100–HCOOH (2 : 1 : 1)
UV: λ = 365 nm
Scutellarin [45]
Ethylacetate–Me Benzene–EtFm (4 : 1)–HCOOH–H2O (5 : 3 : 1 : 1) 2CO CHCl3–MeOH (40 : 1); cyclohexane–EtOAc (5 : 1) Ethylacetate–EtOH–H 2O (8 : 2 : 1); light petroleum–ethylacetate (9 : 1); 36% AcOH; CHCl3 –benzene –MeOH (1 : 5 : 1) CHCl3–MeOH–MeO (5 : 1); ethylacetate Toluene œAtAc –HCOOH (10 : 15 : 11) : 1); cyclohexane–ethylacetate CHCl 3–MeOH EtAc –EtOH (3 :(32); EtAc –MeOH –HCOOH (10 (3 : 1::1) 1) : 3 : 13–MeOH–H : 1) EtAc –3 –MeOH–AcOH Me2CO –HCOOH (20–H : 3 2: O 1);(5CHCl CHCl 2O (14 : 6 : 1); EtAc –EtOH –H2O (8 : 2 : 1); petroleum –AtAc (9 : 1); Hexane–ethylacetate (9 : 1) CHCl3 – MeOH – MeO (5 : 1); EtAc Ethylacetate–MeOH–NH 3 (17 : 2 : 1)–EtAc (3 : 1) CHCl3 –MeOH (3 : 1); cyclohexane Benzene–ethylacetate–HCOOH : 1 : 0.05); CHCl CHCl 3 –MeOH –AcOH (20 : 3 : 1);(3 3 –MeOH –H2O (14 : 6 : 1); hexane – EtAc (9 : 1) Toluene–ethylacetate–HCOOH (10 : 15 : 6) EtAc –MeOH –NH3 (17 : 2 : 1) AmOH–MeOH–HCOOH–H2O (7 : 1 : 1 : 1) Benzene – EtAc – HCOOH (3 : 1 : 0.05); – H: 26) O (5 : 3 : 1 : 1); Ethylacetate–butanone–HCOOH toluene – EtAc –HCOOH (10 : 15 AmOH –MeOH –HCOOH –H2O (7 : 1 : 1 (15 : 1) : 5 : 1) petroleum–ethylformiate–HCOOH Light butanone – HCOOH – H O (5 : 3 : EtAc – 1) Ethylacetate–BuOH–HCOOH–H22O (5 : 3 :11::1); petroleum – EtFm – HCOOH (15 : 5 : 1) BuOH–AcOH–H2O (7 : 1 : 2); EtAc – BuOH –HCOOH – H2O (5 : 3 : 1 : 1) (7 : 3 : 1 : 1) CHCl BuOH3–ethylacetate–HCOOH–MeOH – AcOH – H2O (7 : 1 : 2); (7 : 3 : 1 : 1) CHCl 3 –EtAc –HCOOH –MeOH Buthylacetate–HCOOH–H 2O (6 : 1 : 1); BuAc – HCOOH – H2O (6 : 1 : 1); ( 5 : 3 : 1 : 2) CHCl3–ethylacetate–HCOOH–MeOH CHCl3 – EtAc – HCOOH –MeO ( 5 : 3 : 1 : 2) AmOH–MeOH–HCOOH–H2O (7 : 1 : 1 : 1)
Baicalin [30] [25] [26] Baicalin Baicalin [31]
Polyamide
*AcOH, acetic acid; AmOH, amyl alcohol; BuOH, butyl alcohol; CHCl3, chloroform; EtOAc, ethyl acetate; EtOFm, ethyl formate; EtOH, ethyl alcohol; HCOOH, formic acid; MeOH, methanol; NH3, 25% ammonia; SDS, sodium dodecylsulfate. ** UV, irradiation by UV light of the corresponding wavelength; phosphomolybdic acid. RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY
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Table 5. Conditions of HPLC separation of the S. baicalensis compounds Stationary phase
Mobile phase
Conditions
Separated compounds, references
μBondapak C18 (300 × 3.9 mm)
Tetrahydrofurane – dioxane – MeOH –AcOH – 5% H3PO4 – H2O (145 : 125 : 50 : 20 : 2 : 658)
ν = 1.5 ml/min; Baicalein, baicalin, vogonin, vogonoside [18] T = 50°C; λ = 274 nm
Capcell Pak C18 (250 × 4.6 mm)
1 M tetrabutylammonium chlo ride – MeCN (4 : 1 1 : 4)
ν = 0.6 ml/min Baicalein, baicalin, vogonin, vogonoside, 5,2' dihydroxy6,7,8,3'tetramethoxyflavone, T = 40°C; 3,5,7,2',6'pentahydroxyflavanone, scullcapfla λ = 280 nm vones I and II, acteoside, 5,2',6'trihydroxy6,7 dimethoxyflavone 2'glucoside, 5,2',6'trihy droxy6,7,8trimethoxyflavone 2'glucoside, 7 methoxybaicalin, 5,7,2',6'tetrahydroxyflavon, 5,7,2',5'tetrahydroxy8,6'dimethoxyflavon [20]
Cosmosil 5C18MS; 0.3% H3PO4 – MeCN Cosmosil 5C18AR (90 : 10 55 : 45) (150 × 4.6 mm)
ν = 1 ml/min; T = 20°C; λ = 254 nm
Develosil ODS5 (200 × 6 mm)
Tetrahydrofurane – dioxane – MeOH – AcOH – 5% H3PO4 – H2O (145 : 125 : 50 : 20 : 2 : 658)
ν = 1.5 ml/min; Baicalein, baicalin, baicalein7glucoside, vogo nin, vogonoside, dihydroxyoroxylin A, oroxylin A, T = 50°C; oroxylin A7glucuronide, scullcapflavone II, λ = 274 nm 5,7,2',6'tetrahydroxyflavanone, chrisin [14]
Develosil ODS5 (200 × 6 mm)
Tetrahydrofurane – AcOH – 5% ν = 1.5 ml/min; Baicalin, baicalein7glucoside, vogonoside, H3PO4 – H2O (95 : 10 : 1 : 394) T = 50°C; oroxylin A7glucuronide [14] λ = 274 nm
Baicalin, vogonin [46]
Eurospher 100 C18 MeCN – 0.1% TFA (250 × 20 mm) (1 : 4 4 : 1)
ν = 6 ml/min; λ = 276 nm
Eurospher 100C (250 × 4 mm)
MeCN; 0.1% TFA
ν = 1 ml/min; Acteoside, baicalein, baicalin, vogonin, vogono λ = 220, 380 nm side, chrisin [48]
Hypersil C18 (250 × 4.6 mm)
MeOH – 0.04% H3PO4 (23 : 27) ν = 1 ml/min λ = 280 nm
LiChrospher100 RP18 (250 × 10 mm)
MeCN – 0.1% TFA (1 : 9 9 : 1)
ν = 6 ml/min λ = 276 nm
Baicalein, baicalin, vogonin, vogonoside [47]
Luna 5u C18(2) (250 × 4.6 mm)
MeOH – 1% TCA (56 : 44)
ν = 1 ml/min; λ = 320 nm
Baicalin [50]
ODS Hypersil (250 × 4.6 mm)
MeOH – 0.001 M H3PO4, λ = 254, 280, pH = 3 (22 : 78); MeCN – 0.001 M 270, 320 nm H3PO4, pH = 3 (13 : 87)
ODS Hypersi (250 × 4.6 mm)
MeCN; 0.1% H3PO4
TSK gel LS410 (150 × 4 mm)
5 mM tetraphenylammonium bromide in MeCN – H2O (32 : 68), pH = 4
λ = 280 nm
Baicalein, baicalin, vogonin, vogonoside [47]
Baicalin [49]
Baicalein, baicalin, scutellarein, chrisin; protocate chuic, phydroxybenzoic, vanillic, caffeic, pcou maric, ferulic acid [21] Baicalein, baicalin, vogonin [49] Oroxylin A, oroxylin A7glucuronide [49]
YMOPak ODS MeOH – 0.1 M A132 (150 × 6 mm) phosphate bufer (1 : 1)
λ = 280 nm
Baicalin, vogonin [49]
YMOPak ODS MeOH – 0.1 M A132 (150 × 6 mm) phosphate bufer (17 : 8)
λ = 280 nm
Baicalein, vogonoside [49]
Zorbax C8 (250 × 4 mm)
0.5 M H3PO4 –Me2NH (100 : 7); ν = 1 ml/min; MeCN 30% 100% λ = 254 nm
Baicalein, baicalin, vogonin, vogonoside [23]
Notes: * AcOH, acetic asid; MeCN, acetonitrile; MeOH, methanol; TBAC, tetrabutylammonium chloride; THF, tetrahydrofurane; TPAB, tetraphenylammonium bromide; TFA, trifluoroacetic acid; TCA; trichloroacetic acid. ** ν, rate of moving phase; T, column temperature; λ, wavelength of spectrophotometric detector. RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY
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OLENNIKOV et al.
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