Supplementary Material

Methods

General Experimental Procedures. UV spectra were recorded with a HPLC

photodiode array detector. The 1H and 13C NMR spectra were recorded on a 600 MHz

spectrometer with a 5 mm cryoprobe. 1H chemical shifts are referenced to the residual C6D6

(δ 7.15 ppm), and 13C chemical shifts are referenced to the C6D6 (δ 128.0 ppm) solvent peak.

Reversed-phase HPLC purifications were performed using a binary HPLC pump attached to a

photodiode array detector. All solvents used for HPLC were HPLC grade and were filtered

through a 0.45 μm filter prior to use.

Collection and extraction of plant material. Dried and powdered stem bark of C.

megalobotrys (1.2 Kg) were collected around Maun, Ngamiland District in North-Western

Botswana and were botanically authenticated by Joseph Madome of the Okavango Research

Institute (ORI) Herbarium where a voucher specimen has been deposited (KM-Ks-3-2015).

The plant material was extracted twice at room temperature with 4L of 1:1 mixture of

methylene chloride-methanol for 24h. The solvent was evaporated under reduce pressure to

give 35.07 g of crude Mukungulu extract.

Fractionation of crude extract. The crude extract (35.07 g) was partitioned using

(600 mL x 3) of methanol-water-hexane (2:1:1); methanol-water-ethyl acetate (2:1:1) and

methanol-water-n-butanol (2:1:1) to give 16.79 g of hexane fraction, 1.22 g of ethyl acetate

fraction, 3.82 g of n-butanol fraction and 10.23 g of aqueous fraction.

Purification of n-hexane and ethyl acetate fractions. 16.79 g of n-hexane fraction

were chromatographed on silica gel column with mixture of hexane-ethyl acetate of

increasing polarity. Fractions of 250 mL each were collected as follow: 1-5 (Hex 100% ); 6-

12 (Hex-EtOAc 5%); 13- 20 (Hex-EtOAc 10%); 21-27 (Hex-EtOAc 15%); 28-34 (Hex-

1

EtOAc 20%); 35-45 (Hex-EtOAc 25%); 46-52 (Hex-EtOAc 30%); 53-55 (Hex-EtOAc 50%);

56-57 (MeOH 100%). The fractions were matched according to their TLC profile into eight

series: F1 (1-5); F2 (6-11); F3 (12-19); F4 (20-29); F5 (30-34), F6 (35-50); F7 (52-55); F8 (56-

57). F6 (6.55 g) was absorbed on an equivalent mass of silica gel and chromatographed over a

silica gel column [diameter: 45 mm] with pure chloroform. Fractions of 10 mL were

collected. Fractions 18-37 afforded the semi purified sub-fraction Cmhex35b (1.53 g). The

ethyl acetate fraction (1.20 g) was adsorbed on an equivalent mass of silica gel and

chromatographed on a silica gel column [diameter: 20 mm] using CHCl3/MeOH of increasing

polarity as solvent system. The sub-fraction Cmhex35b was also obtained in large quantity

(936 mg) in pure chloroform (Figure S1).

HPLC Fractionation of CmHex35b and Isolation of Namushen-1 and Namushen-

2. The fraction, CmHex35b, containing the compounds of interest was subjected to reversed-

phase C18 HPLC using a InertSustain, 5 µm, 25 x 1 cm column for 50 min under isocratic

conditions with 4:1 MeCN/H2O as eluent followed by a linear gradient to 9:1 MeCN/H2O

over an additional 20 min to give pure samples of Namushen-1 and Namushen-2.

Namushen-1 was isolated as a clear glass [UV (4:1 MeCN/H2O) λmax 201, 236 nm; 1H

and 13C NMR; positive ion HRESITOFMS [M + Na]+ m/z 625.3745 (calculated for

C35H54O8Na, 625.3716), appropriate for a molecular formula of C35H54O8, requiring 9 sites of

unsaturation]. Examination of the 1H/13C/gCOSY/gHSQC/gHMBC/tROESY NMR data

(Table S1, Figures S2-S3), the results of which are summarized in Figure S4, and

comparison with previously reported 13C NMR data (Wu et al., 2009) revealed that

Namushen-1 was a new prostratin derivative with the structure assigned in Figures S2-S3.

Namushen-2 was isolated as a clear glass [UV (9:1 MeCN/H2O) λmax 201, 236 nm; 1H

and 13C NMR; positive ion HRESITOFMS [M + Na]+ m/z 653.4057 (calculated for

C37H58O8Na, 653.4029), appropriate for a molecular formula of C37H58O8, again requiring 9

2

sites of unsaturation]. The NMR spectra of Namushen-1 and Namushen-2 were remarkably

similar (Table S1), and the structures of the two compounds were found to only differ in the

length of the alkyl chain of the lipid of the ester functionality at C-13. The structure of

Namushen-2 was assigned as shown in Figures S5-S6.

3

Table S1. 13C and 1H NMR Data for Namushen-1 and Namushen-2 recorded in C6D6 and

comparison with the C-1 to C-20 13C NMR data for Prostatin recorded in CDCl3 (Wu et al.,

2009).

Namushen-1 Namushen-2 Prostratin

Positio

n #

C H, (J in Hz) C H, (J in Hz) C

1 160.4 7.42 (bs) 160.4 7.42 (bs) 160.6

2 132.9 / 132.9 / 132.9

3 208.1 / 208.1 / 209.2

4 73.8 / 73.8 / 73.8

5 38.8 2.25 (bd, 18.9)

2.37 (bd, 18.9)

38.8 2.23 (bd, 19.2)

2.37 (bd, 19.2)

38.7

6 140.6 / 140.6 / 140.4

7 129.0 5.60 (bd, 5.4) 129.0 5.60 (bd, 4.9) 130.4

8 38.9 3.11 (bt, 5.4) 38.9 3.11 (bt, 4.9) 39.1

9 76.1 / 76.1 / 76.0

10 56.2 3.43 (bs) 56.2 3.43 (bs) 56.2

11 36.6 2.05b 36.6 2.04b 36.6

12 32.4 1.78 (dd, 16.5,

13.7)

2.05b

32.4 1.78 (dd, 16.6,

13.7)

2.04b

32.3

13 63.5 / 63.5 / 63.8

14 31.6 1.20 (d, 5.4) 31.6 1.20 (d, 4.9) 32.8

15 26.9 / 26.9 / 22.5

16 69.3 4.17 (d, 11.3) 69.3 4.17 (d, 11.4) 23.2

4

4.21 (d, 11.3) 4.21 (d, 11.4)

17 11.5 1.18 (s) 11.5 1.18 (s) 15.3

18 18.8 0.96 (d, 5.9) 18.8 0.96 (d, 5.9) 18.8

19 10.0 1.58 (dd, 2.9,

1.3)

10.0 1.58 (bd, 1.7) 9.9

20 67.8 3.59 (d, 12.8)

3.64 (d, 12.8)

67.8 3.58 (d, 12.9)

3.63 (d, 12.9)

67.9

1’ 175.7 / 175.6 /

2’ 34.6 2.10 (m) 34.6 2.10 (m)

3’ 25.0 1.51 (m) 25.0 1.52 (m)

4’ 29.57 1.18b 29.4 1.20b

5’ 29.36a 1.16-1.27 29.61a 1.18-1.30

6’ 29.64a 1.16-1.27 29.82a 1.18-1.30

7’ 29.74 1.16-1.27 30.02a 1.18-1.30

8’ 32.2 1.24b 30.02a 1.18-1.30

9’ 23.1 1.30 (m) 29.77a 1.18-1.30

10’ 14.3 0.91 (t, 7.3) 32.3 1.18-1.30

11’ / - 23.1 1.31 (m)

12’ / - 14.4 0.92 (t, 6.9)

1” 175.9 / 175.9 /

2” 41.3 2.33 (qdd, 7.0,

7.0, 7.0)

41.3 2.33 (qdd, 7.1,

7.1, 7.1)

5

3” 27.2 1.40 (dqd, 13.6,

7.0, 7.0)

1.72 (dqd, 13.6,

7.0, 7.0)

27.3 1.40 (dqd, 14.4,

7.1, 7.1)

1.72 (dqd, 14.4,

7.1, 7.1)

4” 11.8 0.88 (t, 7.0) 11.8 0.88 (t, 7.1)

5” 16.9 1.12 (d, 7.0) 16.9 1.12 (d, 7.1)

aAssignments within a column are interchangeable. bMultiplicity not determined due

to overlapping signals/chemical shifts determined from 2D data.

6

Figure S1. TLC profile of the sub-fraction CmHex35b (mixture of Namushen-1 and 2).

7

Figure S2. 1H NMR Spectrum of Namushen-1 recorded at 600 MHz in C6D6.

8

Figure S3. 13C NMR Spectrum of Namushen-1 recorded at 150 MHz in C6D6.

9

Figure S4. Selected gCOSY60, gHMBC and tROESY correlations used to assign the

structure of Namushen-1.

10

Figure S5. 1H NMR Spectrum of Namushen-2 recorded at 600 MHz in C6D6.

11

Figure S6. 13C NMR Spectrum of Namushen-2 recorded at 150 MHz in C6D6.

12