Planta Medico 56(1990) 187

A Novel Antibacterial Diterpene from Premna schimperi Solomon Habtemariam , Alexander!. Gray2'4, Gavin W. Halbert3, and Peter G. Waterman2 2

Department of Chemistry. Addis Ababa University, P0 Box 1176, Addis Ababa, Ethiopia Phytochemistry Research Laboratories, Department of Pharmacy, University of Strathclyde, Glasgow Gil XW, Scotland, U.K. Division of Pharmaceutics, Department of Pharmacy, University of Strathclyde, Glasgow Gil XW, Scotland, U.K. Address for correspondence

Received: March 16,1989

______________

Broth E (LAB 68), Nutrient Agar (LAB 8) from LAB M Laboratories,

A novel diterpene, (5R,8R,9S, I OR)- 12oxo-ent-3,13(16)-clerodjen-15-oic acid, has been ob-

Laboratories Ltd., Merseyside, U.K. Streptomycin, Sigma Chemical Co., Poole, Dorset, U.K.

tained from the leaves of Premna schimperi (Ver-

benaceae) using an anti-microbial bioassay-guided isolation procedure. The diterperie was identified on the

basis of spectroscopic data and is active against the Gram-positive bacteria Staphylococcus aureus and Bacillus subtilis in the concentration range 20—25 tg m11.

Key words

Premna schimperi, Verbenaceae, novel clerodane diterpene, antibacterial activity.

Introduction Premna schimperi Engi. (Verbenaceae) is a

spreading shrub or small tree which is quite common in East Africa at altitudes between 1300 and 2400 m (1). In central Ethiopia it is known as "Checho" (Amharic) and is used in the treatment of inflammation and secondary infection associated with superficial wounds (S. Habtemariam,

Lancashire, U.K. Mast Disks: Chloramphenicol Disks, Mast

Plant material The leaves of Premna schimperi were collected in September 1987 from the Menagesha Forest, MenageshaAdministrative Region, Shoa Province, Ethiopia. A voucher specimen (S-

068) has been deposited at the National Herbarium of Ethiopia, Addis Ababa University.

Extraction and purefication of the antibacterial compound The dried leaves (900 g) were extracted in a Soxhiet with EtOH and the solvent removed under vacuum to yield crude extract (190 g). This extract was tested for antibacterial activity by the DDA method (300 p.g per disk). The extract was then

fractionated by vacuum liquid chromatography over silica gel G (Merck 7749) with solvents of increasing polarity; i.e. hexane, hexane: CHCI3, CHCI3, CHC13 : EtOAc; EtOAc, Me2CO and, finally,

MeOH. Fractions were concentrated and tested using the DDA method and the hexane : CHC13 and CHCI3 fractions found to be ac-

tive. These fractions were bulked and subjected to column chromatography over silica gel G eluting with hexane containing increasing amounts of EtOAc. Antibacterial activity (DDA) was identified with a single compound [Rf, 0.48 on silica gel G (Merck Art. 5735), solvent CHC13: MeOH 5: ii which gave a pink colour on

personal observation). It is also employed by farmers to heating after spraying with 1 % vanillin in concentrated H2S04. containing this spot were bulked and the resulting oil was treat common eye infections in cattle. For both human and Fractions recrystallised from benzene at 4°C to give 1 (927 mg). veterinary use the juice, or sometimes an aqueous extract, of the leaves is applied to the infected or inflamed area. The Properties of I juice is prepared by simply rubbing a few leaves between the palms of the hands until well macerated and the liquid Prisms from benzene, m.p. 96—98°C; [a]: —74° released is then squeezed onto the wound. No previous (c. 0.1, CHCI3); UVt.max(Et0H) 230nm; IRVmax(KCI)'° 3380,3190, phytochemical investigation of P. schimperi has been re- 2960, 2900, 1730, 1620, 1210, 1170, 1055, 965cm'; RIMS, MF 318.2200; C20H3003 requires 318.2195, m/z (rel. mt.) = ported and the present study was undertaken to isolate the Found 318 anti-microbial constituent(s) from this plant.

Materials and Methods M.p.'s are uncorrected. 'H-NMR (360MHz) and '3C-NMR (62.9MHz) spectra were recorded on Bruker WH-360 and WM-250 instruments, respectively, in CDCI3 using TMS as internal standard. ElMS were obtained at 70 eV.

Organisms: Staphylococcus aureus (NCTC

6751), Bacillus subtilis (NCTC 8326), Escherichia coli (NCTC

(11), 190 (90), 188 (14), 175 (100), 147(17), 122 (10), 113 (4), 93(11), 55 (42); 1H-NMR (360MHz) b = 1.78(311, s, Me-20), 0.83 (3H, d, J= 6.7 Hz, Me-17), 0.96 (3H, s, Me-19), 1.53 (3H, s, Me-18), 1.68 (111, dd, J= 12.1, 1.7Hz, 11-10), 1.88 (1H, m, H-8), i.92(2H, m, CH2-2), 2.59, 2.82 (2H, ABq, J= 15.5 Hz, CH2-11), 3.27 (2H, s, CH2-14), 5.11 (111, m, H-3), 5.87 (1H, hr. s, 11-16), 6.14 (1H, s, H16), 10.45 (1H, hr. s, COOH); 3C-NMR (62.9 Hz) h: sat 38.5, 42.3,

143.4, 143.8, 176.1, 201.2; d at 37.0, 46.8, 120.6; tat 19.7, 26.6, 27.6, 36.3, 37.3, 43.5, 126.6; q at 16.6, 17.6, 18.0, 20.0. Methylation of 1 (25 mg) with 2% KOH in MeOl-I (2 ml) yielded 1 methyl ester; 1H-NMR (250MHz) b = 3.95 (3H, S. OMe), 2.90 (2H, s, CH214).

Downloaded by: Chinese University of Hong Kong. Copyrighted material.

9001), Pseudomonas aeruginosa (NCTC 6750). Media: Nutrient

Abstract

188 Planta Medica 56(1990)

A lOml agar pour plate containing 0.1 ml of an overnight broth culture was prepared and filter paper disks (What-

man No. 1; 5mm diameter) containing the dried extract/fraction applied to the surface. For each organism the experiment was performed in triplicate and standard disks of chloramphenicol were used as a positive control. The plates were incubated at 37°C and the resulting zones of inhibition measured.

Antibacterial properties of 1 Determination of minimum inhibitory concentration (MIC) (7): To nutrient broth (2 ml) 16 l.il or 20 .sl ofa 1 %w/v sol-

ution of the test compound dissolved in 20% w/v EtOH was added. Doubling serial dilutions were aseptically prepared from this broth

and then inoculated with bacteria to a final concentration of 106 mL. Each organism was tested in duplicate and control broths containing solvent (20 % w/v EtOH) or streptomycin were also prepared. After overnight incubation at 37°C the MIC was read. Broths with no visible growth were subcultured into 10 ml of fresh broth and incubated overnight at 37°C to check for the presence of viable organisms.

Other features of the 'H-NMR spectrum were a broad singlet at = 10.45 ppm for an acid proton which, after methylation by methanolic KOH, was converted to the methyl ester (singlet = 3.95 ppm). Resonances for other side chain protons indicated three isolated methylene moieties; at ö = 5.87, 6.14 (=CH2), 3.27 (singlet),

and 2.82, 2.59 ppm (J =

15.5 Hz).

These could be

rationalised as CH2-CO-C(=CH2)-CH2-COOH which is sub-

stantiated by m/z = 113 where fission occurs a to the carbonyl.

On the basis of these data the isolated compound was assigned structure 1, in which the A/B ring is trans-fused. This is further supported by the 13C-NMR spec-

trum (4, 5). The relative stereochemistry was determined by a series of nuclear Overhauser experiments (Table 1) which confirmed the stereochemical relationship between the methyl substituents and also suggested the preferred conformation of the side chain (Fig. 1). The negative [aID was indicative of the absolute stereochemistry depicted in 1.

Determination of bactericidal properties of 1: Solutions of 1 at 25 and 50 ig mt1 were prepared in 10 ml of saline (0.9% w/v in H20) and inoculated with S. aureus to a concentration of 106 organisms mt1. The suspension was incubated at 25°C and at various times, up to 2 h, 1 ml samples were counted after suitable dilution in saline using a surface plate technique.

Results and Discussion Isolation and identification of the antibacterial compound

Table 1 Enhancements from nuclear Overhauser experiments on 1. Proton irradiated

Proton(s) enhanced (%)

Me-20 (b = 0.78)

H-i i(a)a (3.5), H-il (b) (2.5), Me-17 (2) Me-i9 (2)

Me-i7 (b = 0.83) Me-i9 (b = 0.96)

H-il (a)a (1), H.8 (1) Me-18 (1), Me-20 (2)

For distinction between H-I 1(a) and H-I 1(b), see Fig. 1.

An ethanolic extract of the powdered leaf of P. schimperi was found to be antibacterial when tested by the disk diffusion assay (DDA) (2, 3) against a range of micro-organisms. The concentrated extract was fractionated by vacuum chromatography (VLC) over silica gel eluting with solvents of increasing polarity. Fractions eluted with hexane : chloroform (1 1) and chloroform were found

COOH

H2C

0 H

to be active (DDA) against Staphylococcus aureus and Bacillus subtilis and were further subsequently treated by column chromatography over silica gel eluting with hexane containing increasing amounts of ethyl acetate.

Stereochemistry of 1 based on nuclear Overhauser effect experiments. Fig. 1

°CH37 --CH3

10

8 19CH3 H3 C 1

Antibacterial activity chromatographed

with a single compound which gave a pink colour with vanillin-sulphuric acid spray reagent. Fractions containing this

CH2COOH H2

compound were bulked and concentrated to give an oil which was recrystallised from benzene. High resolution

C0

Hlb -.-1

—H

ElMS revealed an M for C20H3003 with fragments at m/z = 190 (C14H22) and 113 (C5H503), indicative of a diterpene

with a side chain bearing three oxygens. The JR spectrum revealed carbonyl absorption bands indicative of carboxylic acid and a,13-unsaturated ketone. The 1H-NMR spectrum showed resonances for four methyls (one secondary and one olefinic) which, together with a 1H double-doublet for an axial H-b proton and an olefinic proton with coupling

CH3

Table 2 Minimum inhibitory concentration (MIC, ig mL1) of 1 on Staphylococcus aureus and Bacillus subtilis.

indicative of a -CH2-CH=C(Me)- system, suggested a clerodane nucleus substituted with a double bond between C-3 and C-4. This hypothesis was substantiated by the m/z = 190 fragment.

MIC

S. aureus

r Streptomycin sulphate

20 6.3

B. subtilis

25 12.5

a Inactive, up to 100 g mI, against the Gram-negative organisms Pseudomonas aeruginosa and Escherichia co/i.

Downloaded by: Chinese University of Hong Kong. Copyrighted material.

Disk diffusion assay (DDA)

Solomon Habtemariam et al.

Planta Medica 56(1990) 189

A NovelAntibacterialDiterpene from Premna schimperi Figure 2.

3°.

Figure 3.

7 E

E a)

og a,N

E

20

a)

E .2 C

/7

10

0)

Q)

4

> 3 2

1

10

100

1000

0

1

2

Time (hours).

Log Concentration (jig/disk). Fig. 2 Activity of plant extracts, 1, and chloramphenicol in the disk diffusion

Fig. 3 Bactericidal effect of 1 on Staphylococcus aureus.U = 1 at 25 ig

assay against Staphylococcus aureus. S = chloramphenicol, A = 1,U

ml', = 1 at 50 .tg m1'.

chloroformfraction from vacuum chromatography, = crude ethanol extract.

References

Study of antibacterial activity

The results for the DDA method, with the crude extract, the chloroform eluate from VLC, pure I and chioramphenicol (as standard), against S. aureus are presented in Fig. 2. There is clearly increasing activity with increasing purification of the active compound, culminating

1 Demissew, S. (1988) Revision of the plant family Verbenaceae for the 2

with pure 1, and the similarity of the slopes of the plots suggests that I is largely or completely responsible for the activity in each fraction. Compound I proved to be active

(MIC) below 25 .tg m11 against the Gram-positive test organisms (Table 2) but was inactive against Gram-negative organisms, even at concentrations of 100 j.tg m11. Against Gram-positive bacteria no viable organisms could be recovered on subculture of test broths above the measured MIC. Further studies demonstrated that 1 was rapidly bactericidal against S. aureus at concentrations of25 and 50 .tg mt1 (Fig. 3).

Most antibiotics in clinical use exhibit activity below the level of 10 jig ml1 but compounds with activity

only at the level of 100 jig mt1 can be used routinely, provided they are non-toxic (6). It remains to be seen whether

1 will be too toxic for general use but, meantime, the employment of extracts of P. schimperi to treat infections caused by S. aureus, the common cause of eye and skin infections in rural Ethiopia, has been validated.

Acknowledgements One of us (S. H.) thanks the Ethiopian Science and Technology Commission for financial support. Dr. P. Bladon, Department of Chemistry, University of Strathclyde (ElMS) and Dr. I.

H. Sadler, Department of Chemistry, University of Edinburgh (NMR) are thanked for spectral data.

Flora of Ethiopia. The National Herbarium, Addis Ababa University, Manuscript for inclusion in Volume 4. Hartman, M. A. (1968) Miniaturized Microbiology Methods, Academic Press, New York, p. 101. Ahmad, A., Khan, K. A., Ahmad, V. U., Qazi, S. (1986) Planta Med. 52, 285.

Bohlmann, F., Zdero, C., King, T. M., Robinson, H. (1981) Phytochemistry 20, 1657.

Lopes, L. M. X., Boizani, V. S., Trevisan, L. M. W. (1987) 6

Phytochemistry 26, 2781. Korolkovas, A., Burckhalter, J. H. (1976) Essentials of Medicinal Chemistry. John Wiley and Sons, New York, p. 541. Schwarz, L. H., Brown, B. A. (1954) Antibiotics and Chemotherapy 4, 333.

Downloaded by: Chinese University of Hong Kong. Copyrighted material.

0

A novel antibacterial diterpene from Premna schimperi.

A novel diterpene, (5R, 8R, 9S, 10R)-12-oxo-ent-3,13(16)-clerodien-15-oic acid, has been obtained from the leaves of Premna schimperi (Verbenaceae) us...
240KB Sizes 0 Downloads 0 Views