Acylated Flavone Glycosides from the Roots of Saussurea lappa and Their Antifungal Activity

Abstract

The isolation of four novel acylated flavonoid glycosides from the roots of Saussurea lappa and their identification using a combination of 1D and 2D NMR and mass spectrometry is described. The in vitro antifungal and antibacterial activities of the isolated compounds and their mixture were tested on nine fungal and four bacterial strains, using the microdilution method. The compounds and mixture showed moderate to high antifungal activity against most of the fungi tested, compared to a miconazole standard, while only one compound and the mixture showed antibacterial activity against all strains tested.

Introduction

Saussurea lappa C.B. Clarke, syn. S. costus (Falc) Lipsch (Asteraceae) is a Himalayan species that occurs at elevations from 2,700-4,000 m in Kashmir, Lahul Valley in Himachal Pradesh and Garhwal in Uttranchal [1,2]. The roots possess carminative, analgesic, anthelmintic and emmenagogic properties, stimulate the brain and cure blood diseases and liver and kidney disorders. They are prescribed in advance stages of typhus fever, rheumatism, nervous disorders, irregular menstruation, heart diseases, to improve complexion, as a hair wash to kill lice and to turn grey hair to black [3,4,5]. It contains lappadilactone and seven sesquiterpene lactones as cytotoxic principles against selected human cancer cell lines. Lappadilactone, dehydrocostuslactone and costunolide exhibited the most potent cytotoxicity with CD50. Seven noncytotoxic compounds were also isolated, including two novel sesquiterpenes, a guaianolide type with a C17 skeleton, lappalone and 1β,6α-dihydroxycostic acid ethyl ester [6]. (E)-9-Isopropyl-6-methyl-5,9-decadiene-2-one, a terpenoid C14-ketone, lupeol palmitates, α-amyrin starate, β-amyrin from the costus leaves [7,8], 4-β-methoxydehydrocostuslactone [9], the sesquiterpenoid saussureal [10], amino acids, saussureamines A-E, the lignan glycoside (-) massoniresinol-4′′-O-β-D-glucoside [11], two guaianolides, isodehydrocostus lactone and iso-zaluzanin-C and two sesquiterpene lactones with an α,β-unsaturated aldehyde group [12] are other reported compounds from Saussurea lappa. This paper describes the isolation and characterization of four new flavonoids KSR1-KSR4 from the roots of the plant.

Results and Discussion

The compounds KSR1-KSR4 were isolated by repeated chromatography (column and HPLC) of a flavonoid fraction obtained by processing of an ethanolic extract of powdered, dry S. lappa roots.

KSR1

KSR1 was obtained as a yellow amorphous powder with negative optical rotation [[α]_D¹⁵ - 46.9°, c = 0.10; MeOH-H₂O (1:1)]. Its UV spectrum showed maxima at 378, 344, 300 and 210 nm, indicating its polyphenolic nature. The HR-FAB-MS exhibited a pseudomolecular ion [M+1]⁺ at m/z 965.32, compatible with the molecular formula C₄₅H₅₆O₂₃ (calc. 964.15333). This was in good agreement with the presence of six methyl, three methylene, 21 methine and 15 quaternary carbon resonances in its ¹³C-NMR and DEPT spectra. Its ¹H-NMR spectrum (Table 1) displayed five aromatic protons at δ_H 7.12 (d, J = 8.7 Hz), 7.44 (d, J = 2.3 Hz), 7.55 (dd, J = 8.7/2.3 Hz), 6.79 (s) and 6.98 (s), which indicate the presence of one ABX system and two non-correlated aromatic protons, respectively. This suggested that KSR1 might be a 5,6,7,3¹,4¹ or 5,7,8,3¹,4¹ pentasubstituted flavone derivative. Since the signal at δ_C 102.89 was assigned to the C-3 position in the flavone aglycone according to its ³J_HC and ²J_HC correlations in the HMBC spectrum and the published data for flavone glycosides [13], the signal at δ_C 93.70 was attributed to the A-ring. The differentiation on the basis of the A-ring methine resonance between the 5,6,7 (scutellarein-type) and 5,7,8 (isoscutellarein-type) trioxygenation patterns has been previously reported [13]. According to this finding, the presence of an aromatic methine carbon signal (δ_C 93.70) in the δ_C 90.0 – 96.0 range indicates a non-substituted C-8. Furthermore, a correlation was observed between the δ_H 3.88 methoxy proton singlet signal and C-4′ of the benzoyl group (δ_C 151.10) in the HMBC. In addition to this there is a peak at δ_C 132.7 that represents the cyclopentene ring in the compound [14] and there is an HMBC cross peak for C-1a′ of the cyclopentene ring at δ_C 113.3 and C-3′ at δ_C 152.9, which indicates the attachment of the cyclopentene ring at C-3′. A peak at [43+Na]⁺ (loss of acetic acid) in the HR-FAB-MS suggested the flavonoid having an acetate [15], which was supported by the presence of carbonyl peaks at δ_C 172.5 and δ_C 74.2 for an acetyl attached carbon in the ¹³C-NMR. In the ¹H-NMR a significant singlet peak at δ_H 2.06 for the methyl in CH₃COO- was also observed. The ¹H-NMR spectrum showed sharp singlets at δ_H 1.21 and 1.21; these two peaks represent gem-methyl groups associated with the cyclopentene ring [16]. The broad doublet peak at δ 5.02 for olefinic proton (H-2a′, J = 2.5 Hz) by HSQC. A triple doublet (ddd) appearing at δ_H 5.12 was assigned to H-3aβ′ (J_{3aβ′, 4aα′} = 12.8 Hz, J_{3aβ′, 4βa′} = 4.9 Hz, J_{3aβ′, H-2a′} = 2.5 Hz) HMBC from the already published data [16]. A triplet at δ_H 1.34 (J_{4αa′, 4βa′} = J_{4αa′, 3βa′} = 12.8 Hz) due to coupling with the H-3βa′ (HMBC) and special interactions with H-2a′ (ROE) was assigned to H-4αa′. The partially simplified multiplets between δ_H 1.94-2.0 suggested that the signals for H-4βa′ were localized in this region. Cross peaks were observed in C-3′ and C-1a′, C-3′ and C-2a′, C-2a′ and H-3βa′, H-3βa′ and H-4αa′, H-3βa′ and H-4βa′, C-3a′ to O-C=O and from all these data we deduced that the cyclopentene ring was attached at C-3′ of the flavonoid. From the above discussion we concluded that the nucleus of KSR1 was a scutellarein flavone glycoside derivative. Furthermore, the correlation observed between the methoxy proton signal as a singlet δ_H 3.88 and C-4′ of the benzoyl group δ_C 151.10 in the HMBC spectrum led us to determine the aglycone moiety as 3′-(3R-acetoxy-5,5-dimethylcyclopent-1-ene)-4′-O-methylscutellarein, as shown in Figure 1.

Figure 1. Structures of isolated compounds KSR1-KSR4.

Figure 1. Structures of isolated compounds KSR1-KSR4.

Table 1. ¹³C- (125 MHz) and ¹H-NMR (500 MHz) spectral data for KSR1* (D₂O).

C/H	δC	δH (m, J Hz)	C/H	δC	δH (m, J Hz)
Aglycone
2	163.84		8a	105.58
3	102.89	6.79 (s)	1′	123.01
4	182.12		2′	112.86	7.44 (d, 2.3)
4a	148.78		3′	146.78
5	146.78		4′	151.10
6	130.75		5′	112.01	7.12 (d, 8.7)
7	150.87		6′	118.60	7.55 (dd, 8.7, 2.3)
8	93.70	6.98 (s)	OCH3	55.71	3.88 (s)
3′-cyclopent-1-ene
1a′	158.20		5a′	26.30
2a′	132.70	5.02 (d, 7.2 )	3a′-CO	168.3
3a′	74.20	5.12 (ddd,12.8, 4.9, 5)	CO-CH3	18.7	2.06 (s)
4aα′	29.60	1.94 (m, dd, 12.8)	5a′-αCH3	16.5	1.12 (s)
4aβ′	29.60	1.66 (m, dd)	5a′-βCH3	16.5	1.12 (s)
7-O-glucopyranosyl
1′′	97.27	5.49 (d, 7.3)	5′′	76.85	3.25 (t, 9.8)
2′′	76.53	3.81 (d, 9.4)	6′′	60.89	3.44 (dd, 12.0, 6.0)
3′′	86.63	3.73 (t, 9.4)	OCOCH3	20.38	1.94 (s)
4′′	68.14	3.44†	OCOCH3	170.04
2′′-O-rhamnopyranosyl
1′′′	101.03	5.21 (d, 1.5)	4′′′	71.97	3.18 (t, 9.8)
2′′′	70.20	3.85†	5′′′	68.89	3.64 (dd, 9.5, 6.1)
3′′′	70.20	3.37 (m)	6′′′	17.78	1.04 (d, 6.1)
3′′-O-glucopyranosyl
1′′′′	103.01	4.42 (d, 7.6)	4′′′′	69.93	3.13 (t, 9.8)
2′′′′	73.33	3.13 (t, 9.8)	5′′′′	72.91	3.85†
3′′′′	76.53	3.22 (t, 9.2)	6′′′′	62.89	4.10 (dd, 11.9, 6.7), 4.32 (brd, 9.8)

* The ¹³C- and ¹H- assignments were based on 2D NMR experiments (DQF-COSY, HMQC and HMBC); ^†Signal patterns are unclear due to overlapping; s = singlet, d = doublet, dd = doublet of doublets, t = triplet, m = multiplet, brd = broad doublet, ddd = triplet of doublets.

Table 1. ¹³C- (125 MHz) and ¹H-NMR (500 MHz) spectral data for KSR1* (D₂O).

C/H	δC	δH (m, J Hz)	C/H	δC	δH (m, J Hz)
Aglycone
2	163.84		8a	105.58
3	102.89	6.79 (s)	1′	123.01
4	182.12		2′	112.86	7.44 (d, 2.3)
4a	148.78		3′	146.78
5	146.78		4′	151.10
6	130.75		5′	112.01	7.12 (d, 8.7)
7	150.87		6′	118.60	7.55 (dd, 8.7, 2.3)
8	93.70	6.98 (s)	OCH3	55.71	3.88 (s)
3′-cyclopent-1-ene
1a′	158.20		5a′	26.30
2a′	132.70	5.02 (d, 7.2 )	3a′-CO	168.3
3a′	74.20	5.12 (ddd,12.8, 4.9, 5)	CO-CH3	18.7	2.06 (s)
4aα′	29.60	1.94 (m, dd, 12.8)	5a′-αCH3	16.5	1.12 (s)
4aβ′	29.60	1.66 (m, dd)	5a′-βCH3	16.5	1.12 (s)
7-O-glucopyranosyl
1′′	97.27	5.49 (d, 7.3)	5′′	76.85	3.25 (t, 9.8)
2′′	76.53	3.81 (d, 9.4)	6′′	60.89	3.44 (dd, 12.0, 6.0)
3′′	86.63	3.73 (t, 9.4)	OCOCH3	20.38	1.94 (s)
4′′	68.14	3.44†	OCOCH3	170.04
2′′-O-rhamnopyranosyl
1′′′	101.03	5.21 (d, 1.5)	4′′′	71.97	3.18 (t, 9.8)
2′′′	70.20	3.85†	5′′′	68.89	3.64 (dd, 9.5, 6.1)
3′′′	70.20	3.37 (m)	6′′′	17.78	1.04 (d, 6.1)
3′′-O-glucopyranosyl
1′′′′	103.01	4.42 (d, 7.6)	4′′′′	69.93	3.13 (t, 9.8)
2′′′′	73.33	3.13 (t, 9.8)	5′′′′	72.91	3.85†
3′′′′	76.53	3.22 (t, 9.2)	6′′′′	62.89	4.10 (dd, 11.9, 6.7), 4.32 (brd, 9.8)

* The ¹³C- and ¹H- assignments were based on 2D NMR experiments (DQF-COSY, HMQC and HMBC); ^†Signal patterns are unclear due to overlapping; s = singlet, d = doublet, dd = doublet of doublets, t = triplet, m = multiplet, brd = broad doublet, ddd = triplet of doublets.

On the other hand, the presence of three proton signals represented by three doublets at δ_H 4.42 (J = 7.6 Hz), 5.21 (J = 1.5 Hz) and 5.49 (J = 7.3 Hz), characteristic for the anomeric protons of two β-glucoyranosyl and one α-rhamnopyranosyl moieties indicated its triglycosidic structure. The secondary-methyl signal at δ_H 1.04 (d, J = 6.1 Hz) supported the presence of a rhamnose unit. The sugar moiety was further confirmed by the DEPT spectrum, which revealed the presence of 18 C signals, of which three at δ_C 97.27, 101.03, and 103.01 represent anomeric carbon signals, 12 methine carbon signals, two oxymethylene carbons at δ_C 60.89 and 62.89, and one methyl carbon at δ_C 17.78. The downfield shift of the C-2 signal at δ_C 82.3 of glucopyranosyl-I in the ¹³C-NMR of the glucosides was typical of an atom that has undergone a rhamnopyranosyl glycosidation and the downfield C-3 at δ_C 83.5 corresponds to the glycosidation of glucopyranosyl-II. The absence of any ¹³C-NMR glycosidation shift for the rhamnopyranosyl and the second glucopyranosyl residues suggested that these sugars are terminal. The inter-glycosidic linkages were confirmed by the HMBC correlations observed between the following protons and carbons: H-1′′′ of the rhamnose (δ_H 5.21 d) and C-2′′ of the central glucose (δ_C 76.53), and H-1′′′′ of the terminal glucose (δ_H 4.42 d) and C-3′′ of the central glucose δ_C 86.63 (Figure 2). The presence of two carbon signals at δ_C 20.38 (CH₃) and 170.04 (CO) as well as a sharp singlet in the ¹H-NMR spectrum at δ_H 1.94 (s, 3H) indicated the presence of an acetyl group. Downfield shifts of H₂-6′′′′ (δ_H 4.10 dd and 4.32 d) and an upfield shift of C-5′′′′ (δ_C 72.91) signals indicated the bonding site of the acetyl function. Additionally, HMBC correlations between the acetyl carbonyl group δ_C 170.04 and 2 protons of C-6′′′′ showed that the acetyl group was located at C-6′′′′ of the terminal glucose. The attachment of the sugar moiety to the aglycone was determined from the long-range correlation between H-1′′ of the inner glucose (δ_H 5.49 d) and C-7 of the aglycone δ_C 150.87. The HMBC connectivities are shown in Figure 2. ¹H- and ¹³C-NMR spectral data of each sugar unit and the deshielding effect of the connections were further confirmed by a comparison of its spectral data with literature values for flavonoid triglycosides [17,18,19]. From all these results, KSR1 was identified as 3′-(3R-acetoxy-5,5-dimethylcyclopent-1-ene)-4′-O-methylscutellarein 7-O-(6′′′′-O-acetyl-β-D-glucopyranosyl-(1→3)-[α-L-rhamnopyranosyl-(1→2)]-β-D-glucopyranoside. To the best of our knowledge this represents the first time that this compound has been isolated from natural sources and the trivial name saussureanoid is proposed.

Figure 2. Significant HMBC correlations for KSR1. Arrows point from H to C.

Figure 2. Significant HMBC correlations for KSR1. Arrows point from H to C.

KSR2

KSR2 was obtained as a white amorphous powder, with negative optical rotation [[α]_D²¹ – 31.1°, c = 0.48; MeOH-H₂O (1:1)]. The HR-FAB-MS of KSR2 exhibited a pseudo molecular ion [M+1]⁺ at m/z 1269.39, compatible with the molecular formula C₅₃H₇₂O₃₅ (calc.1268.12). This was in good agreement with the presence of three methyl, four methylene, 36 methine and 10 quaternary carbon resonances in its ¹³C-NMR and DEPT spectra (Table 2). Positive results of the HCl-Mg reaction and the Molisch reaction indicated it to be a flavonoid glycoside. Its UV spectrum showed λ_max at 378, 344, 300 and 210 nm, indicating its polyphenolic nature and addition to this in methanol it showed an absorptions at 262 (band II) and 346 (band I) nm. The bathochromic shift (48 nm) of band I with aluminium chloride-HCl and bathochromic shift (6nm) of band II with sodium acetate indicted KSR2 was a 5-hydroxy-3-O-substituted flavonol with the possible presence of two free hydroxy groups at C-7 and C-4′ [20]. The FABMS spectrum of KSR2 gave peaks at m/z [286+Na] (aglycone), [257+Na] (aglycone-CO-H) suggesting that the aglycone was kaempferol, which was confirmed by the presence characteristic signals of the kaempferol nucleus [21,22]; in the ¹H-NMR spectrum, two doublets at δ_H 6.20 and 6.40 (J = 2.1 Hz) were assigned to the H-6 and H-8 protons respectively, and a pair of A₂B₂ aromatic system protons at δ_H 6.93 and 7.77 (J = 8.4 Hz) were assigned to H-3′, H-5′ and H-2′, H-6′, respectively (Table 2). A series of peaks are seen in the ¹H-NMR that are a duplicate of those assigned to the glycosidic part attached at C-7 in KSR1 [23] and a cross peak is observed in the HMBC (Figure 3) for C-7 and C-1′′. From these we concluded that the same trisaccharide chain -O-(6′′′′-O-acetyl-β-D-glucopyranosyl-(1→3)-[α-L-rhamnopyranosyl-(1→2)]-β-D-glucopyranoside is attached at the C-7 position in KSR2. This was also confirmed by its hydrolysis (cf. Figure 1). KSR2 was proven to be a C-3 substituted monodesmosidic flavonol glycoside by comparing its ¹H-NMR and ¹³C-NMR spectra with literature values. The presence of three proton signals represented by δ_H 5.30 (d, J = 7.7 Hz), 4.42 (s) and 4.34 (d, J = 7.8 Hz) indicated its triglycosidic structure. The secondary-methyl signal at δ_H 1.16 (s) supported the presence of a rhamnose unit. Presence of 18 C signals and three anomeric carbon signals at δ_C 102.3, 100.03, 104.6 [24]. The data is shown in Table 3. From the HMBC experiment correlations were found between the rhamnopyranosyl methyl protons (δ_H 1.16, s) and two carbon atoms δ_C 82.3, 66.8 assignable to the C-4a′′ and C-5a′′ of the rhamnose (Figure 3).

Figure 3. Significant HMBC Correlations for KSR2. Arrows point from H to C.

Figure 3. Significant HMBC Correlations for KSR2. Arrows point from H to C.

The downfield C-4a′′ signal at δ_C 82.3 was typical of an atom that has undergone a glycosidation. In HMBC correlations cross peaks were found between the rhamnopyranosyl anomeric proton δ_H 4.42 and the methylene carbon δ_C 65.4 assignable to the C-6a′ of the galactose, and between C-4a′′ of the rhamnose and the anomeric proton δ_H 4.34 (d, J = 7.8 Hz) of the glucose residue. Corresponding carbon signals δ_C C-1a′′′ 104.6, C-2a′′′ 74.6, C-3a′′′ 76.7, C-4a′′′ 70.2, C-5a′′′ 77.0 and C-6a′′′ 61.3 (Table 3) together with proton signals of the same spin system given by the TOCSY spectrum, proved the existence of a terminal glucopyranosyl unit. The remaining six carbon signals of the saccharide part, together with proton signals of the same spin system given by TOCSY spectrum, were in good agreement with a galactopyranosyl unit with C-6a′ substituted by a rhamnopyranosyl unit. Glycosidation shifts of the rhamnopyranosyl C-4a′′ δ_C 82.3, C-3a′′ δ_C 70.0, and C-5a′′ δ_C 66.8 (Table 3) were observed by comparing with the ¹³C-NMR data of kaempferol 3-O-α-L-rhamnopyranosyl-(1→6)-β-D-galactopyranoside [24,25,26,27]. The long range HMBC correlations are shown in Figure 3. As a result, we concluded that KSR2 could be unambiguously identified as kaempferol-3-O-β-D-glucopyransoyl-(1→4)-α-L-rhamnopyranosyl-(1→6)-β-D-galactopyranoside 7-O-(6′′′′-O-acetyl-β-D-glucopyranosyl-(1→3)-[α-L-rhamnopyranosyl-(1→2)]-β-D-glucopyranoside.

Table 2. ¹³C- (125 MHz) and ¹H-NMR (500 MHz) spectral data for the aglycone and trisaccharide moieties attached at the C-7 position in KSR2, 3 and 4 * (D₂O)

C/H	KSR2		KSR3		KSR4
	δC	δH (m, J)	δC	δH (m, J)	δC	δH (m, J)
Aglycone
2	156.7		156.5		156.7
3	133.5		133.1		133.5
4	177.5		177.3		177.5
4a	104.0		104.1		104.0
5	161.3		161.2		161.3
6	99.0	6.20 (s)	98.8.	6.22 (s)	99.0	6.20 (s)
7	164.6		164.1		164.6
8	93.9	6.42 (s)	93.7	6.41 (s)	93.9	6.42 (s)
8a	156.6		156.3		156.6
1′	121.0		121.6		121.0
2′	131.1	8.05 (d, 8.6)	131.1	8.04 (d, 8.6)	131.1	8.05 (d, 8.6)
3′	115.2	6.87 (d, 8.6)	115.2	6.9 (d, 8.6)	115.2	6.87 (d, 8.6)
4′	160.1		160.3		160.1
5′	115.2	6.87 (d, 8.6)	115.3	6.9 (d, 8.6)	115.2	6.87 (d, 8.6)
6′	131.1	8.05 (d, 8.6)	131.2	8.04 (d, 8.6)	131.1	8.05 (d, 8.6)
7-O-glucopyranosyl
1′′	97.27	5.49 (d, 7.3)	98.20	5.46 (d, 7.3)	97.30	5.49 (d, 7.3)
2′′	76.53	3.81 (d, 9.4)	76.55	3.84 (d, 9.4)	76.50	3.87 (d, 9.4)
3′′	86.63	3.73 (t, 9.4)	86.62	3.72 (t, 9.4)	86.77	3.76 (t, 9.4)
4′′	68.14	4.08 (dd, 3.3, 1.8)	68.15	4.68 (dd, 3.3, 1.8)	68.14	4.07 (dd, 3.3, 1.8)
5′′	76.85	3.25 (t, 9.8)	76.85	3.52 (t, 9.8)	76.88	3.30 (t, 9.8)
6′′	60.89	3.44 (dd, 12.0, 6.0)	60.89	3.46 (dd, 12.0, 6.0)	60.86	3.43 (dd, 12.0, 6.0)
OCOCH3	20.38	1.94 (s)	20.38	1.95 (s)	20.50	2.00 (s)
OCOCH3	170.04		170.50		170.60
2′′-O-rhamnopyranosyl
1′′′	101.03	5.21 (d, 1.5)	101.01	5.20 (d, 1.5)	101.00	5.25 (d, 1.5)
2′′′	70.20	3.85†	70.24	3.85†	70.20	3.85†
3′′′	70.20	3.37 (m)	70.20	3.37 (m)	70.20	3.38 (m)
4′′′	71.97	3.18 (t, 9.8)	71.99	3.20 (t, 9.8)	72.03	3.19 (t, 9.8)
5′′′	68.89	3.64 (dd, 9.5, 6.1)	68.88	3.60 (dd, 9.5, 6.1)	68.89	3.64 (dd, 9.5, 6.1)
6′′′	17.78	1.04 (d, 6.1)	17.80	1.06 (d, 6.1)	17.77	1.05 (d, 6.1)
3′′-O-glucopyranosyl
1′′′′	103.01	4.42 (d, 7.6)	103.05	4.46 (d, 7.6)	103.04	4.45 (d, 7.6)
2′′′′	73.33	3.13 (t, 9.8)	73.33	3.15 (t, 9.8)	73.35	3.17 (t, 9.8)
3′′′′	76.53	3.24 (t, 9.2)	76.53	3.22 (t, 9.2)	76.57	3.26 (t, 9.2)
4′′′′	69.93	3.16 (t, 9.8)	69.93	3.14 (t, 9.8)	69.90	3.13 (t, 9.8)
5′′′′	72.91	3.85(m)	72.96	3.89 (m)	72.98	3.88(m)
6′′′′	62.89	4.15 (dd, 11.9, 6.7) 4.35 (brd, 9.8)	62.89	4.10 (dd, 11.9, 6.7) 4.36 (brd, 9.8)	62.86	4.10 (dd, 11.9, 6.7) 4.38 (brd, 9.8)

* The ¹³C- and ¹H- assignments were based on 2D NMR (DQF-COSY, HMQC and HMBC) experiments; ^†Signal patterns are unclear due to overlapping; s = singlet, d = doublet, dd = doublet of doublet, t = triplet, m = multiplet, brd = broad doublet, ddd = triplet of doublets.

Table 2. ¹³C- (125 MHz) and ¹H-NMR (500 MHz) spectral data for the aglycone and trisaccharide moieties attached at the C-7 position in KSR2, 3 and 4 * (D₂O)

C/H	KSR2		KSR3		KSR4
	δC	δH (m, J)	δC	δH (m, J)	δC	δH (m, J)
Aglycone
2	156.7		156.5		156.7
3	133.5		133.1		133.5
4	177.5		177.3		177.5
4a	104.0		104.1		104.0
5	161.3		161.2		161.3
6	99.0	6.20 (s)	98.8.	6.22 (s)	99.0	6.20 (s)
7	164.6		164.1		164.6
8	93.9	6.42 (s)	93.7	6.41 (s)	93.9	6.42 (s)
8a	156.6		156.3		156.6
1′	121.0		121.6		121.0
2′	131.1	8.05 (d, 8.6)	131.1	8.04 (d, 8.6)	131.1	8.05 (d, 8.6)
3′	115.2	6.87 (d, 8.6)	115.2	6.9 (d, 8.6)	115.2	6.87 (d, 8.6)
4′	160.1		160.3		160.1
5′	115.2	6.87 (d, 8.6)	115.3	6.9 (d, 8.6)	115.2	6.87 (d, 8.6)
6′	131.1	8.05 (d, 8.6)	131.2	8.04 (d, 8.6)	131.1	8.05 (d, 8.6)
7-O-glucopyranosyl
1′′	97.27	5.49 (d, 7.3)	98.20	5.46 (d, 7.3)	97.30	5.49 (d, 7.3)
2′′	76.53	3.81 (d, 9.4)	76.55	3.84 (d, 9.4)	76.50	3.87 (d, 9.4)
3′′	86.63	3.73 (t, 9.4)	86.62	3.72 (t, 9.4)	86.77	3.76 (t, 9.4)
4′′	68.14	4.08 (dd, 3.3, 1.8)	68.15	4.68 (dd, 3.3, 1.8)	68.14	4.07 (dd, 3.3, 1.8)
5′′	76.85	3.25 (t, 9.8)	76.85	3.52 (t, 9.8)	76.88	3.30 (t, 9.8)
6′′	60.89	3.44 (dd, 12.0, 6.0)	60.89	3.46 (dd, 12.0, 6.0)	60.86	3.43 (dd, 12.0, 6.0)
OCOCH3	20.38	1.94 (s)	20.38	1.95 (s)	20.50	2.00 (s)
OCOCH3	170.04		170.50		170.60
2′′-O-rhamnopyranosyl
1′′′	101.03	5.21 (d, 1.5)	101.01	5.20 (d, 1.5)	101.00	5.25 (d, 1.5)
2′′′	70.20	3.85†	70.24	3.85†	70.20	3.85†
3′′′	70.20	3.37 (m)	70.20	3.37 (m)	70.20	3.38 (m)
4′′′	71.97	3.18 (t, 9.8)	71.99	3.20 (t, 9.8)	72.03	3.19 (t, 9.8)
5′′′	68.89	3.64 (dd, 9.5, 6.1)	68.88	3.60 (dd, 9.5, 6.1)	68.89	3.64 (dd, 9.5, 6.1)
6′′′	17.78	1.04 (d, 6.1)	17.80	1.06 (d, 6.1)	17.77	1.05 (d, 6.1)
3′′-O-glucopyranosyl
1′′′′	103.01	4.42 (d, 7.6)	103.05	4.46 (d, 7.6)	103.04	4.45 (d, 7.6)
2′′′′	73.33	3.13 (t, 9.8)	73.33	3.15 (t, 9.8)	73.35	3.17 (t, 9.8)
3′′′′	76.53	3.24 (t, 9.2)	76.53	3.22 (t, 9.2)	76.57	3.26 (t, 9.2)
4′′′′	69.93	3.16 (t, 9.8)	69.93	3.14 (t, 9.8)	69.90	3.13 (t, 9.8)
5′′′′	72.91	3.85(m)	72.96	3.89 (m)	72.98	3.88(m)
6′′′′	62.89	4.15 (dd, 11.9, 6.7) 4.35 (brd, 9.8)	62.89	4.10 (dd, 11.9, 6.7) 4.36 (brd, 9.8)	62.86	4.10 (dd, 11.9, 6.7) 4.38 (brd, 9.8)

* The ¹³C- and ¹H- assignments were based on 2D NMR (DQF-COSY, HMQC and HMBC) experiments; ^†Signal patterns are unclear due to overlapping; s = singlet, d = doublet, dd = doublet of doublet, t = triplet, m = multiplet, brd = broad doublet, ddd = triplet of doublets.

KSR3

KSR3 was obtained as a white amorphous powder, with negative optical rotation [[α]_D²¹ – 31.1°, c = 0.48; MeOH-H₂O (1:1)]. Its HR-FAB-MS exhibited a pseudo molecular ion [M+1]⁺ at m/z 1285.38 compatible with the molecular formula C₅₆H₆₈O₃₄ (calc. 1284.12). This was in good agreement with the presence of two methyl, four methylene, 37 methine and 13 quaternary carbon resonances in its ¹³C-NMR spectra (Table 2). Positive results of the HCl-Mg reaction and the Molisch reaction indicated it to be a flavonoid glycoside. Its UV spectrum showed λ_max at 378, 344, 300 and 210 nm, indicating its polyphenolic nature. In the ¹H-NMR of KSR3 a set of signals identical to those of the parent nucleus of KSR2 was found, as shown in Table 2. In addition to this there are two β-glucopyranosyl units from the anomeric signals at δ_C 101.5 and 105.6 as well as the other signals characteristic for glucopyransoyl units as shown in Table 3 [28]. From ¹H-NMR spectral data of KSR3 the δ_H 5.11 (d, J = 7.6 Hz) of the anomeric proton one of the two glucopyranosyl units indicated its β configuration. Moreover, the coupling constant (15.6 Hz) of the two doublets at δ_H 5.59 and 7.30 (each 1H) indicated the trans configuration of the caffeoyl moiety (fission) in the ¹³C-NMR spectrum of compound 3 the downfield shift of C-2a′′ δ_C 82.2 together with the upfield shift of C-1a′′ δ_C 101.5 (Table 3) proved the attachment of the terminal glucosyl unit to this position [29]. Furthermore, the signal at δ_C 64.6 was assigned to the C-6a′ or C-6a′′ in these two glucosyl units to which the caffeoyl moiety is attached [30] this can be determine the attachment site of the caffeoyl moiety little amount of the KSR3 was acetylated and subjected to EI-MS spectral analysis according to the procedure established in reference [31] which revealed a characteristic peak at m/z 331 (18%) corresponding to tetraacetylated glucose as a cationic fragment with a molecular formula [C₁₄H₁₉O₉]⁺ as shown in Figure 4. This significant peak proved the presence of an unsubstituted terminal glucosyl unit [31]. Consequently, the attachment site of the caffeoyl moiety must be at C-6a′ of the glucopyranosyl. The connectivity is shown in Figure 5, as also conformed by HMBC and consequently KSR3 was identified as kaempferol-3-O-β-D-glucopyranosyl(1→2)-β-D-(6a′-O-caffeoyl)-glucopyranoside 7-O-(6′′′′-O-acetyl-β-D-glucopyranosyl-(1→3)-[α-L-rhamno-pyranosyl-(1→2)]-β-D-glucopyranoside.

Figure 4. EI-MS fragmentation of KSR3.

Figure 4. EI-MS fragmentation of KSR3.

Figure 5. Significant HMBC correlations of KSR3. Arrows point from H to C.

Figure 5. Significant HMBC correlations of KSR3. Arrows point from H to C.

Table 3. ¹³C- and ¹H-NMR spectral data for side groups attached at the C-3 position in compounds KSR2, KSR3 and KSR4 * (D₂O, ¹³C:125 MHz; ¹H: 500 MHz).

KSR2
C/H	δC	δH (m, J Hz)	C/H	δC	δH (m, J Hz)
3-O-galactopyranosyl
1a′	102.3	5.30 (d, 7.7 )	4a′	68.1	3.83 (dm, 3.3)
2a′	71.2	3.57 (dd, 9.8,7.4)	5a′	73.6	3.47 (ddm, 7.5, 5)
3a′	73.1	3.51 (dd, 9.8, 3.3)	6a′	65.4	3.23 (dd, 11.7, 7.5) 3.62 (dd, 11.7, 5)
4a′′-O-glucopyranosyl
1a′′′	104.6	4.34 (d, 7.8)	4a′′′	70.2	3.02 (m)
2a′′′	74.6	2.99 (dd, 9.8, 7.7)	5a′′′	77.0	3.55 (m)
3a′′′	76.7	3.15 (dd, 8.7, 7.4)	6a′′′	61.3	3.44 (dd, 11.5, 5.2) 3.67 (d, 10.9)
6a′-O-rhamnopyranosyl
1a′′	100.0	4.42 (brd, 1.8)	4a′′	82.3	3.37 (d, 9.5)
2a′′	70.2	3.48 (dd, 3.3, 1.8)	5a′′	66.8	3.47 (dq, 9.5,6.2)
3a′′	70.7	3.56 (dd, 9.5, 3.3)	6a′′	17.9	1.16 (s)
KSR3
C/H	δC	δH (m, J Hz)	C/H	δC	δH (m, J Hz)
3-O-glucopyranosyl
1a′	101.5	4.30 (d, 7.5)	4a′	70.0	3.03 (dm, 3.9)
2a′	82.2	3.61 (dd, 8.9, 7.5)	5a′	75.8	3.55 (ddm, 8.7, 5.8)
3a′	75.7	3.15 (8.6, 3.5)	6a′	64.6	3.44 (dd, 11.6, 7.5) 3.67 (dd, 11.7, 5)
6a′-O-caffeoyl
αa′′	114.5	5.59 (d, 15.6)	4a′′	146.9
βa′′	146.4	7.30 (d, 15.6)	5a′′	116.3	6.62 (d, 8.1)
1a′′	127.4		6a′′	122.8	6.69 (d, 8.1)
2a′′	115.0	6.79 (bs)	COO	169.1
3a′′	149.3	6.75 (d, 8.7)
2a′-O-glucopyranosyl
1a′′	105.6	4.81 (d, 7.8)	4a′′	71.7	3.53 (d, 8.9)
2a′′	74.7	3.19 (dd, 8.1,3.3)	5a′′	77.7	3.23 (m)
3a′′	76.7	3.38 (dd, 8.9,3.4)	6a′′	61.7	3.78 (m) 3.80 (m)
KSR4
C/H	δC	δH (m, J Hz)	C/H	δC	δH (m, J Hz)
3-O-rhamnopyranosyl
1a′	100.1	5.60 (d, 1.7)	4a′	70.9	3.64 (t, 9.7)
2a′	70.8	5.83 (d, 1.7)	5a′	72.1	3.56 (m )
3a′	72.9	5.30 (dd, 9.7, 3.4)	6a′	17.7	1.06 (d, 6.3)
2a′-O-E-p-coumaroyl
αa′′′	114.8	6.28 (d, 15.9)	4a′′′	161.5
βa′′′	147.6	7.60 (d, 15.9)	5a′′′	116.8	6.75 (d, 8.7)
1a′′′	127.0		6a′′′	131.3	7.37 (d, 8.4)
2a′′′	131.3	7.37 (d, 8.4)	COO	168.4
3a′′′	116.8
3a′-O-E-p-coumaroyl
αa′′′	114.3	6.36 (d, 15.9)	4a′′′	161.3
βa′′′	146.9	7.62 (d, 15.9)	5a′′′	116.7	6.80 (d, 8.7)
1a′′′	126.9		6a′′′	131.1	7.44 (d, 8.4)
2a′′′	131.1	7.44 (d, 8.4)	COO	167.7
3a′′′	116.7	6.80 (d, 8.7)

* The ¹³C- and ¹H- assignments were based on 2D NMR (DQF-COSY, HMQC and HMBC) experiments; s = singlet, d = doublet, dd = doublet of doublet, t = triplet, m = multiplet, brd = broad doublet, ddd = triplet of doublets.

Table 3. ¹³C- and ¹H-NMR spectral data for side groups attached at the C-3 position in compounds KSR2, KSR3 and KSR4 * (D₂O, ¹³C:125 MHz; ¹H: 500 MHz).

KSR2
C/H	δC	δH (m, J Hz)	C/H	δC	δH (m, J Hz)
3-O-galactopyranosyl
1a′	102.3	5.30 (d, 7.7 )	4a′	68.1	3.83 (dm, 3.3)
2a′	71.2	3.57 (dd, 9.8,7.4)	5a′	73.6	3.47 (ddm, 7.5, 5)
3a′	73.1	3.51 (dd, 9.8, 3.3)	6a′	65.4	3.23 (dd, 11.7, 7.5) 3.62 (dd, 11.7, 5)
4a′′-O-glucopyranosyl
1a′′′	104.6	4.34 (d, 7.8)	4a′′′	70.2	3.02 (m)
2a′′′	74.6	2.99 (dd, 9.8, 7.7)	5a′′′	77.0	3.55 (m)
3a′′′	76.7	3.15 (dd, 8.7, 7.4)	6a′′′	61.3	3.44 (dd, 11.5, 5.2) 3.67 (d, 10.9)
6a′-O-rhamnopyranosyl
1a′′	100.0	4.42 (brd, 1.8)	4a′′	82.3	3.37 (d, 9.5)
2a′′	70.2	3.48 (dd, 3.3, 1.8)	5a′′	66.8	3.47 (dq, 9.5,6.2)
3a′′	70.7	3.56 (dd, 9.5, 3.3)	6a′′	17.9	1.16 (s)
KSR3
C/H	δC	δH (m, J Hz)	C/H	δC	δH (m, J Hz)
3-O-glucopyranosyl
1a′	101.5	4.30 (d, 7.5)	4a′	70.0	3.03 (dm, 3.9)
2a′	82.2	3.61 (dd, 8.9, 7.5)	5a′	75.8	3.55 (ddm, 8.7, 5.8)
3a′	75.7	3.15 (8.6, 3.5)	6a′	64.6	3.44 (dd, 11.6, 7.5) 3.67 (dd, 11.7, 5)
6a′-O-caffeoyl
αa′′	114.5	5.59 (d, 15.6)	4a′′	146.9
βa′′	146.4	7.30 (d, 15.6)	5a′′	116.3	6.62 (d, 8.1)
1a′′	127.4		6a′′	122.8	6.69 (d, 8.1)
2a′′	115.0	6.79 (bs)	COO	169.1
3a′′	149.3	6.75 (d, 8.7)
2a′-O-glucopyranosyl
1a′′	105.6	4.81 (d, 7.8)	4a′′	71.7	3.53 (d, 8.9)
2a′′	74.7	3.19 (dd, 8.1,3.3)	5a′′	77.7	3.23 (m)
3a′′	76.7	3.38 (dd, 8.9,3.4)	6a′′	61.7	3.78 (m) 3.80 (m)
KSR4
C/H	δC	δH (m, J Hz)	C/H	δC	δH (m, J Hz)
3-O-rhamnopyranosyl
1a′	100.1	5.60 (d, 1.7)	4a′	70.9	3.64 (t, 9.7)
2a′	70.8	5.83 (d, 1.7)	5a′	72.1	3.56 (m )
3a′	72.9	5.30 (dd, 9.7, 3.4)	6a′	17.7	1.06 (d, 6.3)
2a′-O-E-p-coumaroyl
αa′′′	114.8	6.28 (d, 15.9)	4a′′′	161.5
βa′′′	147.6	7.60 (d, 15.9)	5a′′′	116.8	6.75 (d, 8.7)
1a′′′	127.0		6a′′′	131.3	7.37 (d, 8.4)
2a′′′	131.3	7.37 (d, 8.4)	COO	168.4
3a′′′	116.8
3a′-O-E-p-coumaroyl
αa′′′	114.3	6.36 (d, 15.9)	4a′′′	161.3
βa′′′	146.9	7.62 (d, 15.9)	5a′′′	116.7	6.80 (d, 8.7)
1a′′′	126.9		6a′′′	131.1	7.44 (d, 8.4)
2a′′′	131.1	7.44 (d, 8.4)	COO	167.7
3a′′′	116.7	6.80 (d, 8.7)

* The ¹³C- and ¹H- assignments were based on 2D NMR (DQF-COSY, HMQC and HMBC) experiments; s = singlet, d = doublet, dd = doublet of doublet, t = triplet, m = multiplet, brd = broad doublet, ddd = triplet of doublets.

KSR4

KSR4 was obtained as a white amorphous powder, with negative optical rotation [[α]_D²¹ – 32.1⁰, c = 0.45; MeOH-H₂O (1:1)]. The HR-FAB-MS exhibited a pseudomolecular ion [M+1]⁺ at m/z 1237.38 compatible with the molecular formula C₅₉H₆₄O₂₉ (calc. 1236.12). This was in good agreement with the presence of 3 methyl, 2 methylene, 38 methine and 16 quaternary carbon resonances in its ¹³C-NMR spectra Table 2, Table 3. The characteristic UV spectrum of KSR4, a shoulder at λ_max = 268 nm and a broad band at λ_{max =} 312 nm, suggested a diacylated glycoside [32]. The ¹H-NMR spectrum displayed the typical signal pattern of KSR2 (Table 2) nucleus as in previous compounds, in addition to two p-coumaroyl moieties, as indicated by the presence of two pairs of doublets with a relatively large coupling constant (15.9 Hz) [33]. The first pair at δ_H 6.63 and 7.62 and the second at δ_H 7.60 and 6.36, indicated two pairs of trans configured ethylene protons. Additionally, two A₂B₂ systems, each integrating for two protons, were found at δ_H 7.44, 6.80 and 6.75, 7.37, respectively. This was also supported by a fragment at M⁺ [164+Na] in HR-FAB-MS. It conform the presence of E-di-p-coumaroyl derivatives in the compound. ¹H-NMR showed the presence of three downfield rhamnose proton signals at δ_H 5.30 (dd, J = 9.7, 3.4 Hz), 5.60 (d, J = 1.7 Hz) and 5.82 (d, J = 1.7 Hz) which could be ascribed to H-3a′, H-1a′ and H-2a′. This assignment was deduced by the correlation of H-1a′ with H-2a′, H-2a′ with H-1a′ and H-3a′, in ¹H-¹H COSY and cross peaks are also observed in HMBC between δ_H 5.60 H-1a′ of rhamnose and C-3 of kaempferol. The connectivity is showed in Figure 6. The downfield shift of H-2a′ and H-3a′ of rhamnose, relative to the corresponding positions in literature proved their acylation by the two P-coumaric acid units. The acylation at C-2a′ and C-3a′ positions is further confirmed by the upfield shift of the adjacent carbons in ¹³C of C-1a′ and C-4a′ relative to the corresponding carbons of kaempferol-3-O-α-L-rhamnoside [34]. The assignments of the chemical shifts of the carbons were deduced by 2D NMR experiments (Table 3). Therefore, KSR4 was identified as kaempferol-3-O-α-L-(2a′,3a′-E-di-p-coumaroyl)-rhamnoside 7-O-(6′′′′-O-acetyl-β-D-glucopyranosyl-(1→3)-[α-L-rhamnopyranosyl-(1→2)]-β-D-glucopyranoside.

Figure 6. Significant HMBC correlations of KSR4. Arrows point from H to C.

Figure 6. Significant HMBC correlations of KSR4. Arrows point from H to C.

Biological activity

From Table 4, it can be seen that KSR4 possessed the highest antifungal potential, while KSR3 shows medium activity. Compounds KSR1 and KSR2, showed good activity, while the EtOH extract showed medium activity. The results of individual compounds are correlated with those obtained for the mixture of all four compounds. The mixture shows better activity than individual compounds. All the compounds and extracts tested show greater antifungal activity than miconazole, a commercial fungicide, which was used as a control. These results were obtained in duplicate or triplicate and the mean of the results taken. As for the antibacterial potential, only KSR4 and mixture of four compounds are active against all bacterial strains tested.

Table 4. Minimum inhibitory concentrations (MICs) and minimum fungicidal concentrations (MFCs), nmol/mL. MICs and MFCs are presented as mean values.

Fungi	EtOH MIC (MFC)	KSR1 MIC (MFC)	KSR2 MIC (MFC)	KSR3 MIC (MFC)	KSR4 MIC (MFC)	R1 MIC (MFC)	M1 MIC (MFC)
A. niger	1.2 (1.2)	2.5 (2.5)	0.6 (0.6)	7.5 (7.5)	0.6 (0.6)	3.0 (8.0)	0.3 (0.3)
A. ochraceus	1.2 (1.2)	2.5 (2.5)	0.6 (0.6)	7.5 (7.5)	0.6 (0.6)	3.0 (8.0)	0.3 (0.3)
A. versicolor	0.6 (1.2)	2.5 (2.5)	0.3 (0.6)	7.5 (7.5)	0.6 (1.2)	4.0 (8.0)	0.3 (0.3)
A. flavus	1.2 (1.2)	2.5 (2.5)	0.6 (1.2)	7.5 (7.5)	0.3 (0.6)	1.0 (8.0)	0.3 (0.3)
P. ochrochloron	1.2 (2.4)	5.0 (5.0)	1.2 (2.4)	10.0 (12.0)	0.6 (1.2)	2.0 (10.0)	0.45 (0.45)
P. funiculosum	1.2 (2.4)	1.25 (1.25)	1.2 (2.4)	10.0 (12.0)	0.6 (1.2)	4.0 (10.0)	0.45 (0.45)
T. viride	1.2 (2.4)	5.0 (7.5)	1.2 (1.2)	10.0 (15.0)	1.2 (2.4)	4.0 (4.0)	0.45 (0.45)
C. cladosporioides	3.45 (3.75)	2.4 (2.4)	12.0 (10.0)	8.0 (4.0)	5.0 (5.0)	0.06 (0.06)	1.25 (1.25)
A. alternata	5.0 (5.0)	7.5 (7.5)	6.0 (6.0)	3.0 (8.0)	10.0 (12.0)	1.0 (1.0)	1.25 (1.25)

R1: miconazole; M1: Mixture of KSR1,2,3 and 4; all the tests were run in duplicate or triplicate.

Table 4. Minimum inhibitory concentrations (MICs) and minimum fungicidal concentrations (MFCs), nmol/mL. MICs and MFCs are presented as mean values.

Fungi	EtOH MIC (MFC)	KSR1 MIC (MFC)	KSR2 MIC (MFC)	KSR3 MIC (MFC)	KSR4 MIC (MFC)	R1 MIC (MFC)	M1 MIC (MFC)
A. niger	1.2 (1.2)	2.5 (2.5)	0.6 (0.6)	7.5 (7.5)	0.6 (0.6)	3.0 (8.0)	0.3 (0.3)
A. ochraceus	1.2 (1.2)	2.5 (2.5)	0.6 (0.6)	7.5 (7.5)	0.6 (0.6)	3.0 (8.0)	0.3 (0.3)
A. versicolor	0.6 (1.2)	2.5 (2.5)	0.3 (0.6)	7.5 (7.5)	0.6 (1.2)	4.0 (8.0)	0.3 (0.3)
A. flavus	1.2 (1.2)	2.5 (2.5)	0.6 (1.2)	7.5 (7.5)	0.3 (0.6)	1.0 (8.0)	0.3 (0.3)
P. ochrochloron	1.2 (2.4)	5.0 (5.0)	1.2 (2.4)	10.0 (12.0)	0.6 (1.2)	2.0 (10.0)	0.45 (0.45)
P. funiculosum	1.2 (2.4)	1.25 (1.25)	1.2 (2.4)	10.0 (12.0)	0.6 (1.2)	4.0 (10.0)	0.45 (0.45)
T. viride	1.2 (2.4)	5.0 (7.5)	1.2 (1.2)	10.0 (15.0)	1.2 (2.4)	4.0 (4.0)	0.45 (0.45)
C. cladosporioides	3.45 (3.75)	2.4 (2.4)	12.0 (10.0)	8.0 (4.0)	5.0 (5.0)	0.06 (0.06)	1.25 (1.25)
A. alternata	5.0 (5.0)	7.5 (7.5)	6.0 (6.0)	3.0 (8.0)	10.0 (12.0)	1.0 (1.0)	1.25 (1.25)

R1: miconazole; M1: Mixture of KSR1,2,3 and 4; all the tests were run in duplicate or triplicate.

Experimental

General

¹H- and ¹³C-NMR spectra were recorded on a Varian Inova-500 instrument using the solvent peak (δ 4.75 or 3.31 for D₂O-d₂ or DMSO-d₆ respectively or the CH₃-1, 2 peak δ 61.5) containing TMS as the internal standard. HSQC, HMBC, HMQC and NOESY spectra were recorded on a Bruker Avance–400 instrument. HR-FAB-MS (were recorded in positive model on a JEOL JMS-AX505W instrument using a bis(hydroxyethyl) disulfide matrix. Optical rotations were measured in MeOH with a Perkin-Elmer 241 Polarimeter. Column chromatography (CC) was carried out on Kieselgel 60 (63-200 mesh) Merck, Fluka or Lichroprep RP-18 (40-63 µm) Merck. HPLC was performed with a C-18 Dionex Vydac column (201SP510, 250×10 mm. 5µm) unsing a binary eluent [solvent A. H₂O (pH 2.4 with 0.025% TFA): solvent B. MeCN] and a flow rate of 3 mL min^-1.

Plant Material:

Saussurea lappa roots were obtained from the local market of Ujjain in October, 2003, and identified by Dr. P. Ramakrishna, IEMPS. A voucher sample is kept at the School of Studies in Chemistry of Vikram University. Dried and powered roots (1 kg) were macerated in EtOH for 17 h and then boiled for 3 h. The extract was filtered, evaporated and freeze-dried to give a residue (106 g) which was suspended in EtOH (400 mL). The ethanolic solution was added to acetone (2 L) and the precipitate was filtered and dried over KOH in vacuo. This dried precipitate (57 g) was dissolved in pure H₂O and dialysed against H₂O in seamless cellulose tubing under agitation for 48 h. The contents of the tubes were freeze-dried to afford 32 g of flavonoid mixture (3 % yield). Chromatography on a Merck Lobar Lichroprep RP-18 (size B) column eluting with CHCl₃/MeOH gave four fractions: 1-4. Fraction 2 (25 g) was chromatographed and eluted with H₂O-MeOH (2:7) to give two fractions, the smaller of which (1.2 g) was further purified by HPLC with 45% CH₃CN to give KSR1 (70 mg). The large fraction (19.1 g) was further separated using 50%, 80% and 100% ratios of H₂O-MeOH. The subfractions were purified by HPLC (45% CH₃CN) to afford KSR2 (60 mg), KSR3 (72 mg) and KSR4 (78 mg).

Hydrolysis of the isolated compounds

A few mg of the glcosides were refluxed with 10% HCl in 50% methanol for 3 hrs. The aglycones and sugar fractions were identified by chromatographic comparison with authentic samples. Kamepferol as the aglycone and glucose, rhamnose, and galactose were identified through GC-MS analysis of their alditol peracetate derivatives.

Acetylation of KSR3

Pyridine (10 mL) and acetic anhydride (10 mL) were added to KSR3 (9 mg). The resulting mixture was heated at 120°C for 4 hours after which it was cooled to room temperature. Water was added and the solution was extracted with dichloromethane. Evaporation of the solution furnished a residue which was put on the top of a silica gel chromatography column eluted with CHCl₃-MeOH-H₂O (30:20:1). The product was further purified by semi-preparative HPLC with 37-38% B in 30 min to give acetylated KSR3 (4.3 mg).

3′-(3R-acetoxy-5,5-dimethylcyclopent-1-ene)-4′-O-methylscutellarein 7-O-(6′′′′-O-acetyl-β-D-gluco-pyranosyl-(1→3)-[α-L-rhamnopyranosyl-(1→2)]-β-D-glucopyranoside (KSR1). C₄₅H₅₆O₂₃, yellow amorphous powder (from MeOH:D₂O), mp 227-229⁰C; [α]_D¹⁵ - 46.9⁰C (c = 0.10; MeOH-H₂O (1:1)); UV_max(MeOH): 378, 344, 300 and 210 nm; IR ν_max (KBr) cm-1: 3410, 2925, 1710(C=O), 1656 (C=O), 1606, 1502, 1446, 1361, 1178, 1070, 1026; ¹H and ¹³C-NMR (D₂O-d₂) data are listed in Table 1. HR-FAB-MS: m/z 987.1515 [M+Na]⁺ (calcd. for C₄₅H₅₆O₂₃ 964.1533).

Kaempferol-3-O-β-D-glucopyransoyl-(1→4)-α-L-rhamnopyranosyl-(1→6)-β-D-galactopyranoside 7-O-(6′′′′-O-acetyl-β-D-glucopyranosyl-(1→3)-[α-L-rhamnopyranosyl-(1→2)]-β-D-glucopyranoside (KSR2). C₅₃H₇₂O₃₅, pale yellow paste (from MeOH: D₂O, mp 207-209⁰C; [α]_D²¹ – 31.1⁰, c = 0.48; MeOH-H₂O (1:1); UV_max (MeOH): 374, 345, 302 and 212 nm; IR ν_max (KBr) cm-1: 3410, 2925, 1721 (C=O), 1606, 1504, 1447, 1359, 1208, 1178, 1086, 1014; ¹H and ¹³C NMR (D₂O-d₂) data are given in Table 2 and Table 3. HR-FAB-MS: m/z 1291.3915 [M+Na]⁺ (calculated. for C₅₃H₇₂O₃₅ 1268.3933)

Kaempferol-3-O-β-D-glucopyranosyl(1→2)-β-D-(6a′-O-caffeoyl)-glucopyranoside 7-O-(6′′′′-O-acetyl-β-D-glucopyranosyl-(1→3)-[α-L-rhamnopyranosyl-(1→2)]-β-D-glucopyranoside (KSR3). C₅₆H₆₈O₃₄, white amorphous powder(from MeOH: D₂O), mp 275-280 ⁰C. [α]_D²¹ – 35.1⁰, c = 0.45; MeOH-H₂O (1:1); UV_max (MeOH): 378, 344, 300 and 210 nm; IR ν_max (KBr) cm-1: 3410, 2925, 1721 (C=O), 1656 (C=O), 1606, 1580, 1504, 1447, 1359, 1208, 1178, 1086, 1014, 808, 757, 705; ¹H and ¹³C NMR (D₂O, ¹³C: 125 MHz; ¹H: 500 MHz) data are given in Table 2 and Table 3. HR-FAB-MS: m/z 1307.1915 [M+Na]⁺ (calcd. for C₅₃H₇₂O₃₅ 1284.3943).

Kaempferol-3-O-α-L-(2a′,3a′-E-di-p-coumaroyl)-rhamnoside 7-O-(6′′′′-O-acetyl-β-D-glucopyrano-syl-(1→3)-[α-L-rhamnopyranosyl-(1→2)]-β-D-glucopyranoside (KSR4). C₅₉H₆₄O₂₉ Obtained as a white amorphous powder (from MeOH: D₂O), mp 284-290⁰C. [α]_D²¹ – 33.1⁰, c = 0.47; MeOH-H₂O (1:1); UV_max (MeOH): 378, 344, 315, 300, 293, 220 and 210 nm; IR ν_max (KBr) cm-1: 3448, 2922, 1721 (C=O), 1656 (C=O), 1606, 1580, 1504, 1447, 1359, 1208, 1178, 1075, 833, 808, 757, 705; ¹H and ¹³C NMR (D₂O, ¹³C: 125 MHz; ¹H: 500 MHz) data are given in Table 2 and Table 3. HR-FAB-MS: m/z 1259.3415 [M+Na]⁺ (calcd. for C₅₉H₆₄O₂₉ 1236.3543).

Bioassays:

Nine fungi were used for the bioassays: Aspergillus niger (ATCC 6275), Aspergillus Ochraceus (ATCC 12066), Aspergillus versicolor (ATCC 11730), Aspergillus flavus (ATCC 9643), Penicilium ochrochloron (ATCC 9112), Penicillium funiculosum (ATCC 36839), Trichoderma viride (IAM 5061), Cladosporium cladosporioides (ATCC 13276) and Alternaria alternate (DSM 2006). The strains were obtained from the National Chemical Laboratory (NCL), Pune, India. The micromycetes were maintained on malt agar (MA) and the cultures were stored at +4°C and subcultured once a month [35]. To investigate the antifungal activity of the compounds the modified microdilution technique was used [36,37]. The fungal spores are washed from the surface of agar plates with sterile 0.85% saline containing 0.1% Tween 80 (v:v). The spore suspension was adjusted with sterile saline to a concentration of approximately 1.0 × 10⁵ in a final volume of 100µL per well. The inocula were stored at +4°C for further use. Dilutions of the inocula were cultured on solid MA to verify the absence of contamination and to check the validity of the inoculum.

Minimum inhibitory concentrations (MICs) which inhibited fungal growth, were established by a serial dilution technique using 96-well microliterplates. Extracts of compounds investigated were dissolved in malt medium broth with fungal inoculum to achieve concentrations of 0.03-4 µg/mL. The microplates were incubated for 72h at 28°C. The lowest concentrations without visible growth (at the binocular microscope) were defined as MIC. The minimum fungicidal concentrations (MFCs), were determined by serial sub-cultivation of 2µL into microliter plates containing 100 µL of broth per well and further incubation for 72 h at 28°C. The lowest concentration which killed the 99.5% of the original inoculum, spores and mycelium of the fungi, was defined as the MFC. Commercial fungicides, miconazole and bifonazole, were used as control (0.03-5 µg/mL). The antimicrobial activity of the compounds against the Gram-Positive bacterial Staphylococcus aureus (ATCC 25923) and Bacillus subtilis BBL 12084, the Gram-negative bacteria Esherichia coli (ATCC 35128) and Pseudomonas aeruginose (ATCC 27853) was evaluated suing the microdulution technique. Streptomycin (solution of 1 mg/mL in H₂O) was used as standard form the antibacterial activity.