1078 0009-3130/13/4806-1078 ©2013 Springer Science+Business Media New York

Chemistry of Natural Compounds, Vol. 48, No. 6, January, 2013 [Russian original No. 6, November–December, 2012]

FLAVONOIDS AND PHENYLPROPANOIDS FROM SEVERALSPECIES OF Bupleurum GROWING IN BURYATIA

D. N. Olennikov* and V. V. Partilkhaev UDC 582.893:547.814.5:547.587.5

The genus Bupleurum L. is a monophyletic genus of the family Umbelliferae that comprises up to 190 species,representatives of which are broadly distributed in the northern hemisphere and are used traditionally in European and Asiancountries as drugs [1]. Thus, the aerial part of several Bupleurum species (zi ra ser po) is used in Tibetan medicine forgastrointestinal diseases, disorders of circulatory organs, and as a cholegogic agent [2]. In Russia, B. multinerve is apharmacopoeial species, the preparation “Buplerin” of which is recommended for prevention and treatment of capillary toxicosisand hemorrhages of various etiologies [3]. About 250 compounds representing classes such as triterpene glycosides,sesquiterpenes, flavonoids, and polyynes were isolated from plants of this genus [1, 3]. Considering the satisfactory rawmaterial reserves of Bupleurum species in Buryatia and their unknown chemical compositions, we studied phenolic compoundsfrom five Bupleurum species.

Aerial parts of Bupleurum were collected in regions of the Republic of Buryatia during mass flowering. These wereB. bicaule Helm, Ivolginsk village (Ivolginsk region, July 21, 2010, 51°74�57�� N, 107°27�77�� E); B. longifolium subsp.aureum (Fischer ex Hoffm.) Soo, Tankhoi village (Kabansk region, Aug. 17, 2011, 51°54�63� N, 105°11�23�� E); B. multinerveDC., Tataurovo village (Pribaikalsk region, July 30, 2010, 51°12�67�� N, 107°43�53�� E); B. scorzonerifolium Willd., Shartykeivillage (Dzhidinsk region, Aug. 11, 2011, 50°30�62�� N, 104°44�82�� E); and B. triradiatum Adam ex Hoffm., Barguzin village(Barguzinsk region, Aug. 7, 2010, 54°55�15�� N, 110°67�66�� E). The species were determined by Cand. Pharm. Sci.G. V. Chekhirova (IGEB, SB, RAS, Ulan-Ude). Samples of raw material are preserved in the herbarium of the IGEB, SB,RAS (No. AP/h-37/11-02/0710, AP/h-29/18-07/0811, AP/h-22/18-02/0710, AP/h-37/31-14/0811, AP/h-45/16-18/0810).

Extraction and Fractionation. Ground raw material of B. longifolium subsp. aureum (515 g) was extracted withEtOH (96, 70, and 40%) on a boiling-water bath (1:15, 5�). The alcohol extract was concentrated to an aqueous residue thatwas extracted with hexane, CHCl3, EtOAc, and BuOH. Phenolic compounds in the EtOAc (7.7 g, 1.5% of air-dried rawmaterial mass) and BuOH (95.8 g, 18.6%) fractions were studied. Fractions were separated after preliminary solid-phaseextraction over a polyamide cartridge (1:35) preconditioned with EtOH (96%, 500–700 mL) and H2O (800–1200 mL) byelution successively with H2O (simple phenolic acids), EtOH (40 and 96%) (flavonoids), and NH3 (0.5%) in EtOH(phenylpropanoids). Then, fractions were chromatographed over Sephadex LH-20 (4 � 50 cm) using a gradient of EtOH:H2O(96:4�0:100). Subfractions were rechromatographed over SiO2 (2 � 45 cm, CHCl3:EtOH eluent, 100:0�70:30) and by TLC(SiO2, EtOAc:CH2Cl2:AcOH:HCOOH:H2O, 10:2.5:1:1:1). As a result, the EtOAc fraction (7 g) afforded quercetin (64 mg,1) [4]; isorhamnetin (29 mg, 2) [5]; isoquercitrin (quercetin-3-O-glucoside, 127 mg, 3) [6]; avicularin* (quercetin-3-O-arabinoside, 18 mg, 4) [7]; isorhamnetin-3-O-glucoside* (94 mg, 5) [8]; 1-O-caffeylglycerin* (14 mg, 6); 1,3-di-O-caffeylglycerin* (23 mg, 7) [9]; and caffeic* (51 mg, 8) [4] and 3-O-caffeylquinic acids* (112 mg, 9) [10]. The BuOH fraction(40 g) yielded 2-O-caffeylquinic* (18 mg, 10); 4-O-caffeylquinic* (24 mg, 11) [11]; 5-O-caffeylquinic* (57 mg, 12); and1,3-di-O-caffeylquinic acids* (31 mg, 13) [10]; rutin (quercetin-3-O-rutinoside, 1.65 g, 14); narcissin (isorhamnetin-3-O-rutinoside, 1.72 g, 15) [6]; and quercetin-3-O-�-glucopyranosyl-(1�2)-�-glucopyranoside* (22 mg, 16) [12]. Recrystallizationfrom MeOH of the aqueous effluent from the polyamide cartridge of the BuOH fraction afforded colorless crystals of acompound (0.92 g) that was identified using IR and 13C NMR spectroscopy as ribitol [13].

Institute of General and Experimental Biology, Siberian Branch, Russian Academy of Sciences, 670047, Ulan-Ude,Ul. Sakh�yanovoi, 6, fax: (3012) 43 47 43, e-mail: oldaniil@rambler.ru. Translated from Khimiya Prirodnykh Soedinenii,No. 6, November–December, 2012, pp. 950–953. Original article submitted July 23, 2012.

������� Here and further, asterisks denote compounds isolated from this species for the first time.

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1-O-Caffeylglycerin (6). C12H14O6. UV spectrum (MeOH, �max, nm): 232, 301sh, 325. FAB-MS (m/z): 253[M H], 179, 161, 135. 13C NMR spectrum (125 MHz, MeOH-d4, , ppm): glycerin: 63.7 (CH2, C-3), 65.8 (CH2, C-1), 71.3(CH, C-2); caffeate: 114.5 (CH, C-8�), 114.9 (CH, C-2�), 116.4 (CH, C-5�), 123.3 (CH, C-6�), 127.4 (C, C-1�), 146.9 (C, C-4�),148.1 (CH, C-7�), 150.1 (C, C-3�), 168.8 (C, C-9�).

1,3-Di-O-caffeylglycerin (7). C21H20O9. UV spectrum (MeOH, �max, nm): 228, 300sh, 326. FAB-MS (m/z): 415[M H], 253, 179, 161, 135. 13C NMR spectrum (125 MHz, MeOH-d4, , ppm): glycerin: 66.1 (CH2, C-1, C-3), 71.3 (CH,C-2); caffeate: 114.3 (CH, C-8�, C-8��), 115.0 (CH, C-2�, C-2��), 116.4 (CH, C-5�, C-5��), 123.5 (CH, C-6�, C-6��), 127.3 (C,C-1�, C-1��), 147.1 (C, C-4�, C-4��), 148.3 (CH, C-7�, C-7��), 150.0 (C, C-3�, C-3��), 169.2 (C, C-9�, C-9��).

An analogous methodical approach was used to study the other Bupleurum species. The results are given below.B. bicaule (52 g): 1 (2 mg), 2 (9 mg), 3 (2 mg), 5* (4 mg), 6* (1 mg), 7* (5 mg), 9* (14 mg), 11* (4 mg), 13* (6 mg),

14 (22 mg), 15 (45 mg), 3,5-di-O-caffeylquinic acid* (2 mg, 17) [10], ribitol* (114 mg).B. multinerve (64 g): 1 (11 mg), 2 (7 mg), 3 (18 mg), 4* (5 mg), 5* (6 mg), 7* (5 mg), 8* (1 mg), 9* (27 mg),

12* (4 mg), 13* (5 mg), 14 (32 mg), 15 (21 mg), ribitol (118 mg).B. scorzonerifolium (217 g): 1 (5 mg), 2 (7 mg), 3 (1.28 g), 5* (108 mg), 7* (37 mg), 9 (264 mg), 10* (10 mg),

11* (2 mg), 12* (5 mg), 13* (33 mg), 14 (32 mg), 15 (14 mg), hyperoside* (quercetin-3-O-galactoside, 9 mg, 18) [7], cacticin*

(isorhamnetin-3-O-galactoside, 25 mg, 19) [14].

TABLE 1. Content of Phenolic Compounds in Morphological Groups of B. bicaule and B. longifolium subsp. aureum, mg/ga

B. bicaule B. longifolium subsp. aureum Compound

flowers leaves stems herb flowers leaves stems herb

Caffeic acid (8) 3-O-Caffeylquinic acid (9) 4-O-Caffeylquinic acid (11) 5-O-Caffeylquinic acid (12) 1,3-Di-O-caffeylquinic acid (13) 1,3-Di-O-caffeylglycerin (7) Quercetin (1) Isoquercitrin (3) Hyperoside (18) Avicularin (4) Rutin (14) Isorhamnetin (2) Isorhamnetin-3-O-glucoside (5) Cacticin (19) Narcissin (15) Identified: phenylpropanoids flavonoids including aglycons glycosides monosides biosides quercetin derivatives isorhamnetin derivatives Total content: phenylpropanoids flavonoids

– Tr.b

– – – –

0.53 Tr. – –

2.34 2.18 0.59

– 30.74

Tr.

36.38 2.71

33.67 2.77

66.16 2.87

33.51

1.03 38.12

– 1.65

– 1.12 0.32 0.14 0.18 0.15

– –

4.89 0.46 0.16

– 11.41

3.23 17.58 0.64 16.94 0.77 16.30 5.22 12.03

3.18 19.24

– 0.53 0.41 0.18 0.17

– Tr.

0.08 – –

1.67 0.09 0.12

– 8.37

1.29 10.33 0.09 10.24 0.29 10.04 1.75 8.58

1.53 10.52

– 0.97 Tr.

0.57 Tr.

0.08 0.21 0.11

– –

3.64 0.80 0.22

– 18.14

1.62

23.12 1.01

22.11 0.33

21.78 3.96

19.16

2.14 24.32

– 2.67

– – –

Tr. 2.75 1.09

– Tr.

26.84 4.48 0.59

– 55.35

2.67 91.10 7.23 83.87 6.16 82.19 30.68 60.42

2.84 94.67

0.34 8.80 0.62 0.66 0.59 0.43 0.19 0.62

– Tr.

31.22 0.16 0.21

– 15.39

11.44 47.79 0.35 47.44 0.99 46.61 32.03 15.76

14.04 56.33

– 0.20

– – –

Tr. 0.06 0.09

– –

4.32 0.03 Tr. –

4.87

0.20 9.37 0.09 9.28 0.12 9.19 4.47 4.90

0.36 11.65

0.19 3.96 0.49 0.42

– 0.20 0.70 0.50

– –

19.02 0.94 0.31

– 19.20

5.26 40.67 1.64 39.03 0.81 38.22 20.22 22.25

7.32 42.18

______aOf air-dried raw material mass; b <0.005 mg/g.

HO

HO

O

O

OHO

OOH

OH

HO

HO

O

O

OHOH

1'2' 7' 1 3

8'

6

1 31'' 2''1'

8'

7'2'

7

1080

B. triradiatum (185 g): 1 (52 mg), 2 (41 mg), 3 (29 mg), 5 (27 mg), 7* (14 mg), 9* (65 mg), 10* (3 mg), 14 (860 mg),15 (735 mg), 16* (9 mg), ribitol* (105 mg).

The studied species afforded 19 compounds as a result of chromatographic separation. These included 10 flavonoids(1–5, 14–16, 18, 19) and 9 phenylpropanoids (6–13, 17) (Tables 1 and 2). The flavonoids were mono- and biosides ofquercetin (1) and isorhamnetin (2) such as isoquercitrin (3), avicularin (4), isorhamnetin-3-O-glucoside (5), rutin (14), narcissin(15), quercetin-3-O-�-glucopyranosyl-(1�2)-�-glucopyranoside (16), hyperoside (18), and cacticin (19). The presence ofpreviously observed compounds 1–3, 5, 14, and 15 [1, 3] as markers for the genus Bupleurum was noted in all species. Theless distributed compounds were avicularin (4, B. longifolium subsp. aureum, B. multinerve), which was found earlier inB. falcatum subsp. cernuum [15]; cacticin (19, B. scorzonerifolium), which was observed in B. rotundifolium [16] andB. spinosum [17]; and quercetin-3-O-�-glucopyranosyl-(1�2)-�-glucopyranoside (16, B. longifolium subsp. aureum,B. triradiatum) and hyperoside (18, B. scorzonerifolium); which were also identified for the first time in the genus Bupleurum.

Phenylpropanoids were represented by various derivatives of caffeic acid (8), e.g., mono- and disubstituted esters ofquinic acid [2- (10), 3- (9), 4- (11), and 5-O-caffeylquinic (12); 1,3- (13) and 3,5-di-O-caffeylquinic acids (17)] and glycerin[1-O-caffeyl- (6) and 1,3-di-O-caffeylglycerin (7)]. The compound 3-O-caffeylquinic acid (9) was found previously inB. falcatum [18], B. komarovianum [3], B. longiradiatum, and B. scorzonerifolium [18]; 5-O-caffeylquinic acid (12), inB. falcatum [18]; 1-O-caffeylglycerin (6), in B. chinense [19]. Compounds 7, 10, 11, 13, and 17 were detected in this genus forthe first time.

TABLE 2. Content of Phenolic Compounds in Morphological Groups of B. multinerve, B. scorzonerifolium,B. triradiatum, mg/g

B. multinerve B. scorzonerifolium B. triradiatum Compound

1 2 3 4 1 2 3 4 1 2 3 4

8 9

11 12 13 7 1 3

18 4

14 2 5

19 15

Identified: phenylpropanoids flavonoids including aglycons glycosides monosides biosides derivatives of quercetin derivatives of isorhamnetin Phenylpropanoids* Flavonoids*

– 1.84

– 0.16

– 0.98 2.29 2.45

– –

21.87 2.47 2.45

– 49.96

2.98 81.49 4.76 86.25 7.37 71.85 26.61

54.88

3.04 82.64

Tr. 7.73 Tr. Tr.

0.81 0.71 1.00 2.08

– 0.40

34.14 0.21 0.44

– 9.29

9.25

47.56 1.21

48.77 2.73

43.83 37.62

9.94

11.24 51.14

– 1.94

– Tr. Tr. Tr.

0.36 1.11

– Tr.

12.65 0.20 0.29

– 10.12

1.94

24.73 0.56

24.17 1.60

22.77 14.12

10.61

2.19

27.18

– 5.31

– Tr. Tr.

0.68 1.16 2.15

– 0.19 30.91 0.69 0.84

– 18.98

5.99 54.92 1.85 53.07 3.18 49.89 34.41

20.51

6.39 55.27

– 8.57

– –

5.01 0.77 Tr.

21.53 0.98

– 2.51 Tr.

1.64 1.12 0.80

14.35 28.58

Tr. 28.58 25.27 3.31 25.02

3.56

16.27 32.37

– 23.27 2.45 1.94 2.81 1.24 Tr.

11.35 – –

Tr. –

0.89 3.09

–

31.71 15.33

Tr. 15.33 15.33

Tr. 11.35

3.98

33.14 28.50

– 3.61 0.41 0.63 0.74 0.38 Tr.

3.87 – –

0.30 –

0.95 0.72

–

5.77 5.84

– 5.84 5.54 0.30 4.17

1.67

6.84 11.54

– 13.48 1.28 1.08 3.12 0.75 Tr.

15.34 0.53

– 1.39 Tr.

1.31 1.94 Tr.

19.71 20.51

Tr. 20.51 19.12 1.39

17.26

3.25

22.87 23.20

– 7.24

– – –

0.95 6.20 0.79

– –

37.69 7.89 0.44

– 38.52

8.19 91.53 14.09 77.44 9.12 76.21 44.68

46.85

9.14

102.04

– 3.85

– – –

0.46 2.26 0.39

– –

19.74 1.68 0.30

– 9.16

4.31 33.53 3.94 29.59 2.37 28.90 22.39

11.14

5.70 42.20

– 2.01

– – –

Tr. 0.22 0.33

– –

7.14 0.68 Tr. –

12.85

2.01 21.22 0.90 20.32 1.01 19.99 7.69

13.53

2.67 23.59

– 4.19

– – –

0.42 2.88 0.55

– –

20.36 3.06 0.26

– 18.93

4.61 46.07 5.94 40.13 0.81 39.29 23.79

22.25

5.37 47.52

______1, flowers; 2, leaves; 3, stems; 4, herb.*Total content.

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The five-atom alcohol ribitol (adonitol) was isolated as a ballast compound from the four species in yields of 0.06–0.43% (of air-dried raw material mass). It was found earlier in B. longifolium subsp. aureum [20] and B. multinerve [21] andwas found for the first time in B. triradiatum and B. bicaule.

Application of HPLC established that, as a rule, narcissin (15) (B. bicaule) or 15 and rutin (14) (B. longifolium subsp.aureum, B. multinerve, B. triradiatum) dominated the aerial part of six studied species. The exception was B. scorzonerifolium,the predominant components in the herb of which were isoquercitrin (3) and 3-O-caffeylquinic acid (9). The contents ofidentified phenylpropanoids were from 1.62 (B. bicaule) to 19.71 mg/g (B. scorzonerifolium); flavonoids, from 20.51(B. scorzonerifolium) to 54.92 mg/g (B. multinerve) (Table 1). Glycoside forms of the flavonoids always prevailed over theaglycons. The ratio of flavonoid glycoside types favored biosides (85–94%) except for B. scorzonerifolium, in whichmonoglycosides dominated by 93%.

Pure compounds were distributed unevenly among morphological groups. However, the greatest structural variationof phenylpropanoids was noted in leaves; of flavonoids, in flowers. Marker components of the genus Bupleurum couldaccumulate in both flowers and leaves. The greatest contents of 3-O-caffeylquinic acid (9, 23.27 mg/g) and 1,3-di-O-caffeylglycerin (13, 1.24 mg/g) were found in leaves of B. scorzonerifolium; isoquercitrin (3, 21.53 mg/g), in flowers ofB. scorzonerifolium; isorhamnetin-3-O-glucoside (5, 2.45 mg/g), in flowers of B. multinerve; quercetin (1, 6.20 mg/g),isorhamnetin (2, 7.89 mg/g), and rutin (14, 37.69 mg/g), in flowers of B. triradiatum; narcissin (15, 55.35 mg/g), in flowers ofB. longifolium subsp. aureum.

The studies showed that the accumulation of phenolic compounds was characteristic of several Bupleurum speciesgrowing in Buryatia. Thus, they could be viewed as potential regional substitutes for the pharmacopoeial raw-material speciesB. multinerve, which is traditionally collected in the Altai.

TLC was performed on Sorbfil PTSKh-AF silica gel plates (Imid Ltd.); CC, over Sephadex LH-20 (Pharmacia),silica gel 100/400 (Woelm), and polyamide (Woelm). Spectrophotometry studies were carried out on an SF-2000spectrophotometer (OKB Spektr). Mass spectral analysis used an MAT 8200 high-resolution mass spectrometer (Finnigan).13C NMR spectra were recorded on a VXR 500S NMR spectrometer (Varian). The total content of flavonoids was determinedby a differential spectrophotometric method calculated for the dominant flavonoid (isoquercitrin, rutin, narcissin) [22]; ofphenylpropanoids, by a spectrophotometric method according to Arnow calculated as caffeic acid [23].

HPLC. An accurately weighed sample (1.0 g) of ground raw material was extracted successively with EtOH (96, 70,and 50%; 30 mL) on a boiling-water bath for 45 min. The EtOH extracts were filtered into a 100-mL volumetric flask andadjusted to the mark with EtOH (70%). An aliquot of the resulting solution was filtered through a Millipore membrane filter(0.45 �m) and used for the analysis without dilution. The chromatographic separation used a Millichrom A-02 microcolumnliquid chromatograph (Ekonova), ProntoSIL-120-5-C18 AQ column (2 � 75 mm, � 5 �m, Metrohm AG), mobile phase eluentA (4.1 M LiClO4 in 0.1 M HClO4) and H2O (5:95); eluent B, CH3CN. Gradient elution conditions were 0–12 min, lineargradient of 7–50% B in A; 12–14 min, linear gradient of 50–100% B in A; 14–15 min, isocratic mode of 100% B in A; mobilephase flow rate 200 �L/min; column temperature 40°C. The volume of the injected sample was 2 �L. The detector wavelengthswere 330 (phenylpropanoids) and 360 nm (flavonoids).

ACKNOWLEDGMENT

The work was supported financially by Project RGNF No. 12-06-12026.

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