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RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 36 No. 7 2010. PHENOLIC COMPOUNDS OF Scutellaria baicalensis GEORGI. 817. Table 1. Flavones ...
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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