Eriosema Kraussianum  (Fabaceae), is also known by its Zulu name of uBangalala.  The root of the plant has been traditionally chewed by Zulu warriors over centuries and is used as an aphrodisiac.  A study conducted by the University of Natal found that the compounds act as a vasodilator, boosting blood flow to the genital area and therefore enhancing sexual performance through assisting with erectile dysfunction and impotence.   In the study, key Pyrano-Isoflavones were found to have 75% activity of that of Viagra.  Eriosema extracts are also found to have a hypoglceamic effect reducing blood-glucose levels.

Active: Pyrano-isoflavones offered at 3% purity
Usage:  For erectile dysfunction and sexual impotence (safe for men and women)

  • Thermo nutrient
  • Increased metabolism to help promote weight loss
  • Natural anti-depressant
  • Increased absorption and bioavailability of nutrients
  • Topical application for treatment of vitiligo
  • Increased absorption of skin nutrients

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  • Introduction

The genus Eriosema is one genus of a collection of plants which go under the Zulu indigenous name of ‘‘uBangalala’’ in South Africa. Most of the plant species listed under this name are used for the purpose of curing or alleviating impotency (Bryant, 1983; Hutchings, 1996). Our investigations on the genus Eriosema form part of a larger programme investigating the con- stituents and uses of other indigenous plants which fall under the umbrella name ‘‘uBangalala’’. It forms part of a National Programme to research and document in- digenous knowledge systems (IKS) in South Africa. Reference to other plants in this category is made, for example, in a very recent publication dealing with the commercial market for medicinal plants in the Witwa- tersrand area of South Africa (Williams et al., 2000).

Eriosema kraussianum is a small shrublet found in grasslands rarely attaining a height of greater than 15 cm. It flowers from October to February. The plant has an extensive and well developed root system and it is this part of the plant that is used for traditional pur- poses. No chemical work has been done on the plant. If


* Corresponding author. Tel.: +27-33-260-5243; fax: +27-33-260- 5009.

E-mail address: (S. E. Drewes).


the root of the plant is damaged during collection a dark red sap forms at the point of injury. This may relate to the type of compound isolated from the plant (see later).


  1. Results and discussion

From a CH2Cl2–EtOH (1:1) extraction of the milled roots five new pyrano-isoflavones (Ingham, 1983) were isolated. They form a closely-related, and partially interconvertible, group of compounds. We have assigned the trivial names kraussianone 1, 2, 3, 4 and 5 to them. Entry into the series was by way of kraussia- none 1 which established the basic isoflavone skeleton. It crystallized as pale yellow needles amenable to X-ray analysis (Fig. 1). The molecular formula of C25H22O6 was established from HRMS (m/z M+ 418.13993, cal- culated 418.14164). From 1H NMR, 13C NMR and DEPT analysis it was clear that the compound had 13 quaternary carbons, eight CH’s (one of which was far downfield at 8 154.8) and four CH3 carbons. The 1H NMR spectrum (Table 1) gave the following analysis: two sets of methyl groups at 8 1.42 and 8 1.47, two sets

of coupled CH protons at 8 5.63 and 8 6.70 (J=10.06

Hz), and the other at 8 5.50 and 8 6.27 (J=9.60 Hz), and

singlets at 8 6.37, 6.51 and 7.90. In total there are eight


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



Fig. 1.  X-ray structure of kraussianone 1 (40% thermal ellipsoids).

Table 1

1H NMR date for compounds 1, 2, 3, 4 and 5 (500 MHz)

Proton 18H (mult. J in Hz) 28H (mult. J in Hz) 38H (mult. J in Hz) 48H (mult. J in Hz) 48H (mult. J in Hz) 58H (mult. J in Hz)
2 7.90(s) 7.96(s) 8.15(s) 6.23(s) 6.22(s) 6.12(s)
8 6.37(s) 6.62(s) 6.44(s) 5.93(s) 5.94(s) 5.97(s)
30 6.51(s) 6.48(s) 6.30(s) 6.37(s) 6.42(s) 6.30(s)
60 6.73(s) 6.80(s) 6.87(s) 7.03(s) 7.05(s) 6.91(s)
400 6.26(d, 9.6) 6.27(d, 10.0) 6.29(d, 9.6) 6.25(d, 10.0) 6.34(d. 9.6) 6.13(d, 10.1)
500 5.50(d, 9.6) 5.51(d, 10.0) 5.52(d, 9.6) 5.52(d, 10.1) 5.61(d, 9.6) 5.40(d, 10.1)
4000 6.70(d, 10.1) 1.68(s) 1.14(s) 6.55(d, 10.1) 6.54(d, 9.8) 1.57(s)
5000 5.63(d, 10.1) 1.80(s) 1.14(s) 5.53(d, 10.0) 5.63(d, 9.8) 1.66(s)
700  Me 1.42(s) 1.42(s) 1.35(s) 1.36(s) 1.36(s) 1.27(s)
800  Me 1.42(s) 1.42(s) 1.35(s) 1.40(s) 1.40(s) 1.31(s)
7000 Me 1.47(s) 1.40(s) 1.38(s)
8000 Me 1,47(s) 1.44(s) 1.42(s)
20  OH 8.32(s) 9.13(bs) 10.72(bs)
5 OH 12.55(s) 12.60(s) 13.15(s) 12.05(s) 12.05(s) 11.74(s)
7.OH 8.51(bs) 9.56(s) 8.90(s)
1000 3.39(d, 6.8) 2.58(t, 8.2) 3.16(d, 7.3)
2000 5.27(d, 6.8) 1.51(t, 8.2) 5.09(dd, 7.3)
Solvent (CDCl3/(CD3)2CO) DMSO-d6 (CDCl3/(CD3)2CO) (CDCl3/(CD3)2CO) CD3CN (CDCl3/(CD3)2CO)




Table 2

13C NMR data for compounds 1, 2, 3, 4 and 5 (125 MHz in CDCl2)

Proton 18C (mult.) 28C (mult.) 38C (mult.)


8C (mult.)

58C (mult.)
2 154.8(CH) 154.7(CH) 155.4(CH) 110.5(CH) 111.2(CH) 109.6(CH)
3 122.9(C) 122.2(C) 120.0(C) 78.0(C) 78.5(C) 77.7(C)
4 181.9(C) 181.7(C) 180.5(C) 192.2(C) 193.2(C) 191.1(C)
5 156.3(C) 159.2(C) 158.9(C) 158.5(C) 159.3(C) 161.5(C)
6 115.3(C) 112.5(C) 112.9(C) 103.7(C) 104.6(C) 109.5(C)
7 160.3(C) 162.3(C) 162.2(C) 163.1(C) 163.7(C) 164.9(C)
8 94.9(CH) 93.4(CH) 93.0(CH) 96.9(CH) 97.3(CH) 95.6(CH)
9 157.1(C) 155.8(C) 155.4(C) 159.2(C) 159.8(C) 157.3(C)
10 105.4(C) 104.9(C) 104.4(C) 100.2(C) 100.9(C) 99.4(C)
10 112.0(C) 112.0(C) 110.7(C) 118.5(C) 119.3(C) 118.3(C)
20 157.1(C) 156.8(C) 156.6(C) 160.4(C) 160.9(C) 160.0(C)
30 107.2(CH 106.5(CH) 103.4(CH) 99.7(CH) 100.3(CH) 99.6(CH)
40 155.5(C) 155.2(C) 153.8(C) 156.7(C) 157.4(C) 156.4(C)
50 106.0(C) 114.9(C) 112.6(C) 116.7(C) 117.7(C) 116.5(C)
60 127.2(CH) 127.6(CH) 129.5(C) 122.6(CH) 123.5(CH) 121.9(CH)
400 121.3(CH) 121.4(CH) 121.7(CH) 121.9(CH) 122.3(CH) 121.6(CH)
500 128.6(CH) 128.3(CH) 127.6(CH) 128.7(CH) 129.9(CH) 128.3(CH)
600 76.6(C) 76.5(C) 76.3(C 77.2(C) 78.1(C) 76.8(C)
700 28.1(CH3) 28.0(CH3) 27.9(CH3) 28.1(CH3) 27.3(CH3) 27.7(CH3)
800 28.1(CH3) 28.0(CH3) 27.9(CH3) 28.1(CH3) 27.4(CH3) 27.8(CH3)
100 0 21.4(CH2) 17.4(CH2) 20.8(CH2)
200 0 121.7(CH) 42.5(CH2) 121.5(CH)
300 0 132.2(C) 69.2(C) 132.1(C)
400 0 115.2(CH) 25.6(CH3) 29.3(CH3) 114.9(CH) 115.1(CH) 25.5 (CH3)
500 0 128.7(CH) 17.7(CH3) 29.3(CH3) 126.9(CH) 128.4(CH) 17.5(CH3)
600 0 78.4(C) 78.9(C) 79.9(C)
700 0 28.4(CH3) 28.5(CH3) 27.7(CH3)
800 0 28.4(CH3) 28.5(CH3) 27.8(CH3)
Solvent (CDCl3/(CD3)2CO) (CDCl3/(CD3)2CO) DMSO-d6 (CD3)2CO CD3CN (CDCl3/(CD3)2CO)


structure (Table 3). As was the case in  compound  1, there is a good NOESY correlation between H-2 and H- 60, emphasizing the fact that rotation of the  C-3–C-10 bond allows these two protons to  come  close  together. The NOESY  evidence for the close  proximity of the proton on C-2000 to the C-4000 methyl group on C-3000 clarifies the stereochemistry around the C-2000–C-3000 double bond. Proton and 13C spectra are collected in Tables 1 and 2.

The third compound, kraussianone 3, C25H26O7, with two more hydrogens than 2, and an additional oxygen, crystallized readily and was amenable to X-ray analysis. It was considerably more polar than the two foregoing compounds and was light orange in colour. In this instance spectral analysis again indicated an intact D ring, with rings A, B and C similar to those in 1 and 2. DEPT and HMQC analysis showed unambiguously that a 3, 3-dimethyl-3-hydroxybutyl side chain was attached to C-6 i.e. a hydrated 3,3-dimethylalyl side chain. The relationship of the C-1000 protons to the sur- rounding atoms is shown up well in the HMQC spec- trum. Positive correlations extend to C-7, C-5, C-6, C- 3000 and C-2000. This technique also makes it possible to distinguish between the singlet methyl groups arising from the 4000/5000 methyls and the 700/800 methyls (Tables 1 and 2). The mass spectrum exhibits a strong molecular ion peak at M+ 438 and the anticipated losses of methyl radical and subsequently water are shown by prominent peaks at m/z 423 and 405 (base peak) respectively.

Kraussianone 4, while obviously related to 1, 2 and 3, also exhibited several differences. Its molecular formula from high resolution mass spectrometry is M+ 434.13677 (C25H22O7). This is the same as for com- pound 1, except for an additional oxygen atom. Mass fragmentation gives rise to a large fragment ion at m/z 419 (loss of methyl radical) and the base peak is at m/z

219. The latter probably represents the ion 6, a fragment

typical of a retro Diels–Alder fission (Drewes, 1974).  In the 1H NMR spectrum of 4, H-2, typically reso-

nating at ca. 8 8.0 in 1, 2 and 3, is absent. In its place a ‘‘new’’ singlet peak is observed at 8 6.22 assigned to H-

2. In the 13C spectrum the peaks normally associated


Table 3

HMQC correlations for kraussianone 2

Proton                                                          Coupling to

H-2                                                                     C-4(C=O), C-9, C-3, C-10

H-30                                                                                                                                         C-20, C-40, C-10

H-60                                                                                                                                         C-20, C-40, C-400

H-8                                                                     C-4, C-7, C-9, C-10, C-6

H-400                                                                                                                                       C-40, C-60, C-50, C-600

H-500                                                                                                                                      C-600 , C-50

H-1000                                                                                                                                   C-7, C-5, C-300 0, C-2000 ,C-6

H-4000                                                                                                                                   C-3000 , C-2000

H-5000                                                                                                                                   C-3000 , C-2000

700 , 800  Me                                                              C-500 , C-400 , C-600

Fig. 2. NOESY correlations for kraussianones 1–6.                                                                                                                                                        

with C-600 and C-6000 (at about 8 77 and 8 79) are still present, but there is an additional peak in the same chemical shift region (8 78.01) belonging to C-3. Fol- lowing detailed analysis of DEPT, HSQC, HMQC and NOESY spectra, the structure shown in 4 was proposed. A possible pathway involving ring closure of the phe- nolic hydroxyl at C-20 (see structure 1) at position C-2 (in ring B) by a conjugated mechanism, followed by hydroxylation at C-3 (of ring B) is postulated.  Ring closure thus leads to the formation of an  additional furan ring F, in the system. The compounds lisetin (Falshaw et al., 1966) and milletin (Raju et al., 1981) have systems of this nature.

Allocation of the 1H and 13C resonances for 4 (run in

CDCl3/(CD3)2CO) are shown in Tables 1  and 2. The spectral evidence for allocation of all carbons and hydrogens shown in structure 4 is unambiguous. The evidence presented below relates specifically to atoms at C–2 and C-3 (ring F) and C–4 (ring B) since the situa- tion here is unlike that found in compounds 1, 2 and 3. It is instructive to compare the proton and 13C chemical shifts of these atoms in 4, with those in compound  1 (Table 4).

Since H-2 is part of an acetal system in 4 (as opposed to an sp2 system as in 1) the observed upfield shift is expected. The carbon shifts of C–2 and C–3 reflect accurately the changed magnetic environment in com- pound 4, and the downfield shift of the carbonyl group in 2 is in line with the chemical shift for a less con- jugated carbonyl. Cross peaks in the HMQC spectrum link H-2 with C-20, C-9, C-10, C-3 and C-4 as can be anticipated. Apart from the obvious NOESY correla- tions, the only other strong correlation  detected  was that between H-60 and H-400.

Since  the  proton  resonance  for  H-2  (singlet)  in  4

overlapped with the doublet due to H-400  in CDCl3/


the cross coupling peaks seen for H-2 (see above), additional long-range couplings could be detected. Thus, C-3 (at 8 78.8) is cross-coupled to H-60 and the phenolic OH at 8 12.6 cross couples to C-5. This is sig- nificant since it demonstrates that there is only one phenolic OH in 4, and that it resides at C-5.

The final compound in the series is kraussianone 51

chemically closely-related to kraussianone 4. This was an important find in the plant since it gave us the opportunity to examine the structural detail of a second representative of the ‘‘furano’’ isoflavone series. It dif- fers from the first compound in the series kraussianone 4, only by having the ‘‘open chain’’ system attached to ring A. The proton NMR of 5 (Fig. 1) shows a striking resemblance to that of 4. The major differences are the appearance of an additional OH- signal at 8 8.90, a tri- plet at 8 5.09, a doublet at 8 3.16 and a simpler methyl resonance region. This is indicative of a compound identical to kraussianone 4, but with a 3, 3-dimethylallyl side chain at C-6 instead of ring E. The molecular for- mulae was found to be C25H24O7, i.e. two hydrogens more than 4. The additional H’s are due to the free phenolic group at C-7 (explaining the new peak at 8 8.90) and the methylene group in the dimethylallyl side chain. The 13C spectrum is in good accord with the

Table 5

HMQC correlations for kraussianone 5

Proton                                                          Coupling to

H-2                                                                         C-20, C-9, C-10

H-30                                                                                                                   C-20, C-40, C-10, C-50

H-60                                                                                                         C-20, C-40, C-400, C-30 , C-3

H-8                                                                     C-7, C-9, C-6, C-10

H-1000                                                                                                             C-7, C-5, C-300 0, C-2000 ,C-6

H-2000                                                                                                                  C-5000 , C-4000,1000

H-400                                                                                                                C-40, C-60, C-50, C-30, C-600


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




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




0H NMR Compound




1                                                  4                                            3 85





H-2                           7.90                                             6.22                                       5 100



13C NMR                                                                                                                   s.d.




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


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

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




Fig. 3.  X-ray structure of kraussianone 3 (40% thermal ellipsoids).


CDCl3/(CD3)2CO) in Tables 1 and 2, respectively; H–R EI–MS m/z 418.13993 M+, calcd. for C25H22O6, 418.14164,   EI–MS   m/z   (rel.   int.):   418[M+]   (25),

403(100), 203(15), 194(21), 185(10); IRvmax (KBr) cm-1:

3035,  1650,  1541  and  1132.  The  X-ray  structure  is

shown in Fig. 1.

    1. Kraussianone (2). 5,7,20-Trihydroxy-6-(3,3-

dimethylallyl)-[(600,600-dimethylpyrano(200,  300:40,50)]- isoflavone

White crystals, mp 162–168 oC, no optical rotation. 1H and 13C NMR (500 and 125 MHz resp., CDCl3/ (CD3)2CO spectra data in Tables 1 and 2, respectively; H–R EI–MS m/z 420.15703 M+ calc. for C25H24O6, 420.15729,  EI–MS  m/z  (rel.  int.):  420  [M+]  (28),

405(100), 377(6), 349(23), 201(18), 165(15).

    1. Kraussianone (3). 5,7,20-Trihydroxy-6-(3-hydroxy-

3-methylbutyl)-[(600,600-dimethylpyrano  (200,300:40,50)]- isoflavone

Orange yellow crystals, softening 156 oC melting 218– 220 oC, no optical rotation. 1H and 13C NMR spectra (500 and 125 MHz resp., DMSO-d6) are in Tables 1 and 2, respectively; H–R EI–MS m/z 438.16801 M+ calc. for C25H26O7=438.16785, EI–MS m/z (rel. int.): 438 [M+] (54),  423(58),  405(100),  349(83),  185(36),  175(23),

165(15). The X-ray structure is shown in Fig. 3.

    1. Kraussianone (4). 5b,7-Dihydroxy-2,2,10,10- tetramethyl-5b,13a-dihydro-2H,6H,10H-chromeno [60,70:4,5]furo[2,3-b]pyrano[3,2-g]chromene-6-one


In the text this compound as well as kraussianone 5, retain the numbering used for the other pyrano-iso- flavones in order to simplify cross-referencing.

Pale yellow oil, no optical rotation. 1H and 13C NMR

spectra (500 and 125 MHz resp., (CDCl3/(CD3)2CO) and CD3CN) are shown in Tables 1 and 2, respectively; H–R    EI–MS    m/z     434.13677    M+       calc.    for


C25H22O7=434.13655,  EI–MS  m/z  (rel.  int.):  434 [M+] (38), 419(84), 219(C12H11O4)(100), 201(60), 187(13).

    1. Kraussianone  (5).  5b,7,9-Trihydroxy-2,2-dimethyl- 8-(3-methyl-2-butenyl)-5b,11a-dihydro-2H,6H- chromeno[60,70:4,5]furo[2,3b]chromen-6-one


Oil, no optical rotation. 1H and 13C NMR spectra (500 and 125 MHz resp., (CDCl3/(CD3)2CO) are shown in Tables 1 and 2 respectively; H–R EI–MS m/z 436.15272 M+ calc. for C25H24O7=436.15220, EI–MS m/z   (   436   [M+]   (68),   421(94),   403(8),

221(C12H13O4)(97), 201(100), 187(16), 183(14), 165(62).

    1. X-ray data


Details regarding the crystal structure of compounds

1 and 3 are collected in Table 7. See also Figs. 1 and 3.

    1. Measurement of rabbit corpus cavernosum relaxation


The bioassay was done as described (Levin et al., 1997) with some minor changes. Strips (12 mm long and 1–2 mm thick) of rabbit corpus carvernosal smooth muscle were dissected and mounted in an organ-bath chamber containing Krebs–PSS solution with the fol- lowing composition: NaCl=7.01 g/l, KCl=0.34 g/l, KH2PO4=0.1  g/l,  NaHCO3=1.99  g/l,  CaCl2=0.2  g/l,

MgSO4=0.3 g/l and glucose=1.8 g/I. One end of the muscle was secured to the inside case of the perfusion bath and the other end to the thin wire connected to a Harvard isotonic force transducer for isotonic tension measurements. Changes in isotonic tension were recor- ded on a computerised calibration program. The corpus cavernosum muscle was perfused with 2 ml Krebs–PSS buffered saline and oxygenated with 95% O2 and 5% CO2 for 5 min to ascertain a stable baseline recording. This was followed by perfusion with 2 ml of CaCl2 in Krebs–PSS (17.8 mg/ml) for muscle contraction. Base- line tension was set at the point of maximal contraction following the addition of CaCl2  into the experimental


Table 7X-ray data for compounds 1 and 3 (Figs. 1 and 3)
Molecular formula Compound 1 Compound 3


Crystals (space group) Triclinic P1 Triclinic P1-bar
Unit cell dimensions (Ao ) a=8.0583(10)






Z 1


Volume (Ao 3) 1023.34(18)


Crystal size (mm)Unique reflections collected 0.70 X 0.60 X 0.404471/4336 [R(int)=0.0029] 0.55 X 0.35 X 0.308377/7498  [R(int)=0.0245]
Final R indices [I > 2 sigma (I)] R1=0.0356, wR2=0.1038 R1=0.0729, wR2=0.2138
R indices (all data) R1=0.0399, wR2=0.1106 R1=0.0940, wR2=0.2275

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.



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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