0031-9422/02/$ – see front matter # 2002 Published by Elsevier Science Ltd. PII : S0 031 -9 422 (0 2)000 35 -3

CH signals. Two phenolic OH signals appeared at 8 8.32 and 8 12.55 (obviously H – bonded). The main features of the 13C spectrum (Table 2) were two high field quar- tets at 8 76.6 and 8 78.4, five quartets in the region 105– 123 ppm, a further five quartets, obviously bonded to oxygen functionalities clustered between 150 and 161 ppm, and a carbonyl group at 181.9 ppm.

From  HMQC  spectra   the   remaining   correlations could be established. Of particular significance was the chemical shift position of H-2 and its linkage to C-4, H- 9 (across oxygen), H-20, C-3 and C-10. Other  correla- tions include: H-8 to C-7, C-9, C-10; H-30 to C-40, C-20, C-10; H-60 to C-20, C-40, C-3, C-400; H-4000 to C-7, C-6000,

C-5. The far downfield OH group (at 8 H 12.56) is undoubtedly attached to C-5 [see also X-ray confirma- tion (Fig. 1) of this] and the second OH (8 H 8.32) at C- 20.

NOESY correlations are shown in Fig. 2. The corre- lations confirm the observation from the X-ray analysis (Fig. 1) that rotation about the C-10 – C-3 bond allows H-2 and H-60 to approach one another closely.

Examination of the spectral data for 2 (C25H24O6) suggested a close similarity to 1, except that either ring

E or ring D was in the open chain form i.e. with a 3,3- dimethylallyl side chain on one of the aromatic rings. The latter side chain is an obvious feature of the 1H NMR spectrum with resonances at 8 3.39 (J=6.8 Hz), 8 5.27 (J=6.8 Hz), 8 1.68 and 8 1.80 for H-1000, H-2000, H-

4000   and H-5000   (resp.). The proton and 13C spectra are

summarized in Tables 1 and 2. The structure shown for 2, in which the side chain is attached at C-6 of ring A, while ring D is unaltered compared with kraussianone 1, is based on analysis of the 1H and 13C spectra, and particularly the correlations which were forthcoming from the HMQC and NOESY spectra. The molecular formula C25H24O6, with two more hydrogens than compound 1, can be accommodated by a free OH at C- 7, and an additional hydrogen in the dimethylallyl side chain. The HMQC spectra show correlations of H-500 with C-50, C-600 and C-700/800. H-400 correlates with C-60, C-50 and C-600. This clearly establishes the relationship of rings C and D with one another. There is a strong correlation of H 4000/5000 with C-3000 and C-2000 while H-1000 is linked to C-7, C-5, C-3000 and C-2000, thus proving the nature and position of this side chain. The remaining correlations  are  all  in  agreement  with  the  proposed

(CD3)2CO as solvent, CD3CN was examined as alter-

H-5

C-5 , C-6

00                                                                                                                                                 0                  00

native with excellent results. In this solvent H-2 and H-

400   are  well  separated  (Table  1)  and  the  overlapping

doublets  of  H-5000   and  H-500   are  also  separated  as  two

distinct doublets. In addition in CD3CN there is the additional advantage  that C-3 is not ‘‘hidden’’  under overlying chloroform peaks. In this solvent, apart from

Table 4

Comparison of chemical shifts (d) of atoms at positions 2, 3 and 4 in compounds 1 and 4

H-700  (Me)                                                            C-500, C-600

H-800  (Me)                                                            C-500, C-600

H-4000  (Me)                                                           C-2000 , C-3000

H-5000  (Me)                                                           C-2000 , C-3000

Table 6

Percentage carvenosal smooth muscle relaxation by kraussianones 1, 2

and Viagra at 78 ng/ml                                                                                       Replicate     Kraussianone 1            Kraussianone 2            Viagra

10.8                                 6.2                                   0

1. 154.8 (sp2 carbon)                       110.5 (sp3 carbon)                                                                                                                                      
2. 122.9 (sp2 carbon)                       78.0 (sp3 carbon)

1. 181.9 (C=O, doubly conjugated)

192.2 (C=O, singly conjugated)

1   Proposed  structure  subsequently  confirmed  by  X-ray  analysis (January 2002).

proposed structure (Table 2). The long-range connec- tions obtained from the HMQC spectrum are very convincing and are depicted in Table 5. The clear con- nectivity of H-2 with both C-9 and C-20 as well as C-10 serves to confirm its assigned position in the structure.

1. Biological activity

In order to examine the properties of the kraussia- nones for the traditional usage as agents to cure impo- tence (‘‘uBangalala’’) a standard procedure (Levin et al., 1997) for examining the smooth muscle relaxation of rabbit penile muscle was employed. This test is based on the veno-occlusive mechanism (Godschalk et al., 1997) which depends on facilitating increased arterial  flow into the penis (via relaxation of the relevant smooth muscles) and decreased venous outflow so that pressure within the penis rises and it becomes rigid. In order to compare the activity of the new compounds, sildanafil (Viagra), was used as the reference substance. The test is effectively a procedure which measures male erectile dysfunction.

At a concentration of 78 ng/ml the results shown (Table 6) were obtained (the effect obtained is dose- dependent). To our pleasant surprise kraussianone 1 showed an activity close to that of Viagra, thus living up to the plant’s traditional use. At this preliminary stage the mode of action and many other details are unknown and await further investigations. Compounds 3, 4 and 5 did not show relaxation of smooth muscle.

It is of interest to note that the pyranoisoflavones isolated from gorse (Ulex eurapaeus, Russell et al., 1990) were examined for their insect feeding deterrent activity. The prenylated chromones from Eriosema tuberosum (Ma et al., 1995, 1996a,b) were all found to be active as antifungal reagents. Ma et al. (1995), however, make the interesting observation that ‘‘Indians around Kunana, Venezuela, use the root decoction of E. rufum against sterility in women and give it to accelerate delivery in childbirth’’. Our present finding highlighting the activity of Eriosema kraussianum in the sexual/reproductive field is thus not without precedent in other parts of the world, and emphasizes the crucial role which accurate indigenous knowledge can play in uncovering new uses of pharmaceutical products.

1. X-ray structures of 1 and 2

The X-ray structure of 1 is depicted in Fig. 1. Four observations can be made:

1. two molecules are found in each unit cell,
2. hydrogen bonds exist between the C-5 hydroxyl group and the C=O on C-4 as well as a similar

H- bond between the 20- hydroxyl group and the C-4  carbonyl,

1. the dihedral angle between C-2, C-3, C-10 and C-

60  is 48.2o  (molecule A). When the ring C rotates

about  the  C-3–C-10   intercarbon  bond  to  the

alternative     conformation     the    corresponding dihedral angle is -49.4o  (molecule B).

Semi-empirical quantum mechanics calculations (AMI gas phase) indicate that the most stable con- formation of the molecule differs only little from that assumed in the solid (crystal) state. It is of interest to note, that, in keeping with the above observations a strong NOE effect is seen between H-2 and the hydro- gen attached to C-60, thus confirming the close proxi- mity of these two atoms.

The X-ray structure of 3 is shown in Fig. 3. Again there are two molecules in the unit cell, and the con- formations are identical. More advanced molecular simulations for both 1 and 3 are underway and these results will be published elsewhere.

1. Experimental

1. General

1H and 13C spectra were recorded on a Varian 500 spectrometer. High resolution mass spectra were mea- sured on a Kratos MS 80 RF double-focussing magnetic sector instrument at 70 eV. X-ray studies were carried out on a Nonius CAD 4 diffractometer with graphite monochromated MoKa  radiation.

1. Plant material

Eriosema kraussianum N. E. Br was collected in October 1999 from open veld adjacent to the National Botanical Gardens in Pietermaritzburg. Flowering material was identified by Dr. T. Edwards, curator of the Bews Herbarium at the University of Natal, Pieter- maritzburg. A voucher specimen (S. E. D. No. 7) of the whole plant was deposited in the Herbarium.

1. Extraction and isolation

Plant material (rootstock, 670 g) from Eriosema kraussianum was finely milled and extracted with CH2Cl2 for 9 days to give a brown powder (4.1 g). Subsequent extraction  with CH2Cl2/EtOH (50:50) afforded a further 5.6 g of material.

The CH2Cl2 extract showed (on a TLC plate run in CH2Cl2 as solvent) five UV fluorescent bands (staining various shades of blue with acid/anisaldehyde reagent) at Rf values of 0.65, 0.39, 0.17, 0.08 and 0.00. These spots   were   subsequently   identified   (see   below)   askraussianones 1, 4, 2, 5 and the more polar kraussia-

none 3, respectively.

Separation of the crude CH2Cl2 extract (3 g) on a silica gel column (Merck 09835) using CHCl3/CH2Cl2 (1:1) gave several fractions. From the first and second fractions two compounds were isolated, 1 (60 mg) and 4

(4.7 mg). The latter was obtained in pure form only after additional separation on a silica gel column eluted with EtOAc/petroleum ether bp 40–60 oC (1:1).

In order to obtain the next compound 2 the CH2Cl2/ EtOH (1:1) extract (see above) was utilized. The extract

(5.65 g) was first partitioned between water and EtOAc, and the organic phase (3 g) then fractionated on a silica gel column using gradient elution with hexane/EtOAc. The first fractions provided a mixture of 1 and 4 (220 mg) and subsequently the major compound 2 (700 mg crude pro- duct, giving 220 mg of fairly pure material) was eluted. High purity  was only  achieved after several additional separations on a chromatotron with CH2Cl2 as solvent.

For the last two compounds, 3 and 5, a more polar

elution  system  was  employed.  Crude  CH2Cl2   extract

(3.7 g) was fractionated using a succession of solvents: CH2Cl2, then CH2Cl2/MeOH (94:6) and finally Et2O. The mustard-yellow ether fractions were pooled, con- centrated to a small volume and hexane added. Orange crystals of 3 (40 mg) formed slowly and were subjected to X-ray analysis.

To obtain 5 the following procedure gave the best yield: CH2Cl2 extract (2.87 g) was fractionated succes- sively with CHCl3, CH2Cl2 and Et2O (dry-packed silica gel column). A portion of the ether eluent was purified on a chromatotron (hexane/Et2O gradient) and afforded more 3 (90 mg). The remainder of the ether solution

(1.65 g) was fractionated on the chromatotron CH2Cl2/ ether (80:20) to yield 5 (30 mg) as an oil.

1. Kraussianone (1). 5,20-Dihydroxy-[(600,00-

dimethylpyrano     (200,300:40,50)][(6000,6000-dimethylpyrano

(2000,3000:7,6)]-isoflavone

Yellow crystals mp 185–187 oC, no optical rotation.

1H  and  13C  spectral  data  (500  and 125  MHz  resp.,

bath. The compounds to be analysed were added after a stable contraction baseline had been obtained. The final compound concentration in the perfusion bath was 78 ng/ml. The same procedure (and concentration) was repeated for the positive control, Viagra. In these experiments the stimulation frequency used for rabbit strips was 9 Hz.

Acknowledgements

Financial support form the National Research Foun- dation and the University of Natal Research Fund is acknowledged. Diana McCann from the University Research Office is thanked for helpful advice for filing of a provisional patent (RSA Patent Application No. 2001/4350, 28 May 2001).

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