I N H I B I T I O N OF MITOCHOXDRIAL FUNCTION I N T E R M I T E QUEEN OVARY BY RABDOSIA D I T E R P E N O I D S Is.40
KUBO*
Dir,ision of E n t o m o l o g y and Parasitology, Vnirversity of California, Berkeley, California 94720
NABIL ABO-KHATIFA Internutional Centre of Insect P h y si o l o g y and Ecology, P . 0. Box 30778 Xahirobi, K e n y a
TAKASHI KUBOTA School of Medicine, K i n k i r n i v e r s i t y , Sayamu-cho, Osuka 589, J a p a n hfAKOTO
TASIGL-CHI
Faculty of Science, O s u k e C i t y l n i c , e r s i f y , Sugimoto-cho, Sumigoshi-ku, Osaka 558, J a p a n .iBSTR.\CT.-The effect of diterpenoids from plants of t h e genus Rabdosia (Labiatae) on t h e energy-linked functions in mitochondria from termite queen ovary h a s been investigated polarographically with an oxygen electrode. Some Rabdosia diterpenoids inhibited oxidative phosphorylation and s'ignificantly depressed t h e r a t e of respiration in s t a t e 3 and in 2,4dinitrophenol uncoupled mitochondria (state l U ) without depressing t h e respiration r a t e in s t a t e 4.
We have previously reported that the diterpenes from the plants of the genus Rabdosia' (Labiat.ae) possess bot,h specific antimicrobial activity against, grampositive bacteria (1) and cytotoxic activity against KB tissue culture ( 2 ) . These
same compounds have been shown to exhibit a weak inhibitory effect on oxidative phosphorylation in mitochondria isolated from rat liver and silkworm midgut (3). The present work is an investigation of the inhibit,ory effect of Rabdosia diterpenoids on respiratory function in mit,ochondria isolat.ed from the ovary of a termite queen of Macrotermes subhyaliiws. X 4 T E R I h L S X S D METHODS DITERPESES.-The Rabdosin diterpenoids used in this s t u d y were all analytically pure samples obtained from our previous studies. T h e selection of these compounds was based mainly upon the availability of t h e samples as awl1 as their characteristic structure types. IssEcT.-Termite queens of JI. szibhyalinus were collected from termite mounds located in t h e Kajiado area near S a i r o b i , Kenya. Among the 100 species of African termites, Jf. subhyalinus w a s selected for research material because, although it is not considered an economic pest, it is readily available from the large mounds (up t o five meters tall above ground).
PREPAR.ATIOS of ~iITOcHOsDRI.4Ls r s m s s I o s s . - T h e queen ovaries replete with eggs (15 g) were dissected and homogenized. T h e mitochondrial suspensions were prepared from t h e homogenate a s previously described (4, 5 ) . ME.~SL-REMEST OF OXIDATIVE PHOSPHORTLATI0S.-The reaction mixture of mitochondria contained 2% m l f sucrose, 30 mhI K2HP04,15 m l f KC1, 2 m l f E D T A , 5 mhl lfgC19, 50 m h l Tris-HC1, test compounds, 1 mg of mitochondrial protein and 20 mM succinate as a respiratory substrate at p H 7 . 4 . During t h e course of the reaction, 0.2 pmole ADP was added t o t h e mixture. h Clark oxygen electrode coupled t o a recording system was used t o measure oxygen consumption polarographically at 26". The measurement was repeated five times. Oxidative phosphorylation activity w a s calculated from the oxygen electrode tracing and expressed as a 'This genus was previously known as Zsodon.
593
( H . H a m , J . J u p . Bot., 47, 193 (19T2) )
[Vol. 44, No. 5
Journal of Natural Products
594
respiratory control ratio and an ADP/O ratio. t h e ADP/O ratio x the s t a t e 3 respiration.
The phosphorylation rate F a s calculated a s
RESULTS AND DISCUSSION As shov-n in table 1, the Rabdosia diterpenes, isodonal (1) (6), enemin (2) (7), oridonin (3) (S), and nodosin (4) (9), exhibited an inhibitory effect on the mitochondrial oxidative phosphorylation. Interestingly, (2) and (4) lowered the respiratory control ratio and phosphorylation rate but did not affect the ADP/O ratio at 100 and 250 ph1, respectively. T ~ B L1.E Effect of Rabdosza diterpenoids on some energy-linked functions in mitochondria isolated from termite queen ovary.
i Compounds
Cone.
rJf
Sone 100 100 100
1
2 3 4
Respirators rates
I
Respiratory control ratio
State
148 117 114 117
9-3 6+3 61.2 6-2
2 1 6 9
33 6+1 44 2+1 33 61.1 32 4-0 41 2-1
5 8 1 9 9
181 41 103 106 61
4-3 2-3 6-2 0+2 8-2
5d 0 4 1 2
4 43 2 66 3 41
3 63 2.30
ADP/O
1 72-0 1 03-0 1 621.0 1 33-0
16d 08 21 15
1 i91.0 11
SExpressed as atoms O/mg proteinimin. bIn the presence of 2,4-dinitrophenol a t 150rhl. synthesized/mg protein/min. dhlean-SE.
Phosphorylation ratec
256
121 186 156 170
CExpressed as nmoles ATP
These compounds (1 to 4) significantly depressed the rate of respiration in state 3 and in state 4U (2,4-dinitrophenol uncoupled mitochondrial), though hardly affecting that in state 4. Thus, they seem to act neither as an uncoupler, e . g . , 2,4-dinitrophenol, nor as a phosphorylation inhibitor, e . g . , oligomycin, because the former markedly stimulates the respiratory rate in state 3 and in state 4,
1
2,
RI=OH, R2=H
3
while the latter jnhibits only the respiratory rate in state 3 but shows no effect in state 4 or state 4U. The Rabdosia diterpenoids seem to have an action on the respiratory chain enzymes similar to that of cyanide and antimycin A. Received 2’7 October 1980.
Sep-Oct 19811 Kubo et sl. : Inhibition of 3litochondrial Function
595
L I T E 11 ATU 11 E C I T E D 1. I . Kubo, 11.Taniguchi, T . Satomura and T. Kubota, d g r i c . Biol. C hem ., 38, 1261 (1974). 2. 11.Tamaguchi, 11.Taniguchi, I. Kubo and T. Kubota, d g r i c . Bio2. Chem., 41, 2475 (1977). 3. 11.Taniguchi, 31. Tamaguchi, I. Kubo and T. Kubota, A g r i c . B i o l . C h e m . , 43, 71 (1979). 1. S . Abo-Khatwa and R . 11.Hollingworth, P e st i c . B i o c h e m . PhTsiol., 3, 358 (1973). 5 . S . rlbo-Khatwa, L i f e S c i e n c e s, 18, 329 (1977). 6. I. Kubo, T . Kamikawa and T. Kubota, Tetruhedron, 30, 615 (1974).
7 . T. Kubota, T. Matsuura, T. Tsutsui, S.Uyeo, H. Irie, .4. S u m a t a , T. Fujita and T. Suzuki,
T et r a h ed r o n , 22, 1659 (1966). 8. E. Fujita, T. Fujita, H. Katayama, 31.Shibuya and T. Shingu, J . C h e m . Soc. C., 1674 (1974). 9. E. Fujita, T. Fujita and 11.Shibuya, Chem. P h a r m . Bull., 16, 3 9 (1968).
