Phytomedicine 9: 52–55, 2002 © Urban & Fischer Verlag http://www.urbanfischer.de/journals/phytomed Phytomedicine Estrogenic and cholinergic properties of the methanol extract of Ruellia praetermissa Sceinf. ex. Lindau (Acanthaceae) in female rats A. M. Salah1, J. Gathumbi2, W. Vierling3 and H. Wagner4 1 National Center for Natural Products Research, Institute of Pharmaceutical Sciences, School of Pharmacy, University of Mississippi, USA 2 Department of Veterinary Pathology, Faculty of Veterinary Medicine, University of Nairobi, Kenya 3 Institute of Pharmacology and Toxicology, Technical University of Munich, Germany 4 Institute of Pharmacy, Center of Pharma Research, Pharmaceutical Biology, University of Munich, Germany Summary In search for alternative drugs with pharmacological profile to replace hormone replacement therapy, the effects of MeOH extract of Ruellia praetermissa on the uterus and gestation in rats was investigated. 350 mg/kg/day of extract from days 1–9, 1–17 and 9–17 respectively, resulted in increase of the number of implantation sites (56 to 64) and the percentage of implantation (68.6 ± 2.7 to 90.5 ± 0.5%). There was also an increase in body weight (1–9 and 1–17) followed by a slight decrease (154 ± 15.5 to 125 ± 10) in the body weight at term. The number and the mean value of corpora lutea per female decreased from 25.4 ± 1.6 to 14.00 ± 1.6. The extract produced dose-related contraction of the isolated uterine muscle in vitro comparable to ACh. Atropine in doses from 3.4 × 10–6 to 3 × 10–3 µM antagonized the response of the uterus to ACh at 2 µM. It induced an increase (0.03 ± 0.002 to 0.34 ± 0.001 g) of the uterine weight comparable to that produced by using 3 µM estradiol (0.03 ± 0.001 to 0.35 ± 0.005 g). It could therefore be postulated that this extract possesses estrogenic and possible cholinergic effects. The estrogenic effect could have been generated by plant sterols (β-sitosterol and stigmasterol) and flavonoids (luteolin and apigenin) while cholinergic effect could be due to iridoid glycosides. Key words: Ruellia praetermissa, estrogen effect, cholinergic effect j Introduction Hormone replacement therapy with estrogen may cause severe side effects with regards to risk of cancer. In this light alternative treatments with compounds or plant extracts having more specific pharmacological profile is required. In view of this need, Ruellia praetermissa Sceinf. ex. Lindau (Acanthaceae) was chemically analyzed and pharmacologically investigated for its effect in the gestation of female rats. It is a wild herb and indigenous to central and south eastern Asia, and also widespread in tropical and subtropical Africa. In Kenya, and other south eastern African countries, it is applied to relief pain (Gelfand et al., 1985). In Cameroon, traditional healers apply it as phytomedicines to remedy gastro-intestinal problems and gynaecology complaints especially in connection with gestation and post-delivery haemorrhages. Some chemical compounds have been reported in related species: apigenin 7β-glucoronide in R. tuberosa (Wagner et al., 1972), Apigenin, luteolin, and their respective glycosides in R. prostrata (Sankara and Nair, 0944-7113/02/09/01-052 $ 15.00/0 Estrogenic and cholinergic properties of the methanol extract of Ruellia praetermissa 1972), Tetramethylpultrescine in R. rosea (Siegeried et al., 1985), iridoid glycosides in other Acanthaceae (Fischer et al., 1988) and plant sterols (Lupeol, stigmasterol and β-sitosterol) in R. brittoniania (Ahmad et al., 1990). R. prostata is reported to have antifertility effect in albino rats (Kirtikar and Basu, 1935). A decoction of the roots of R. suffrusticosa mixed with liquid copal produced abortive effect when administered to pregnant women (Harborne and Baxter, 1993). Little information is known about R. praetermissa and this work is therefore aimed at validating the claims for its medicinal values. Our earlier studies have shown that Ruellia praetermissa possesses a purgative effect in vitro (Salah et al., 2000) and a moderate ACE inhibition (Salah et al., 2001). j Materials and Methods Plant Material The plant was collected at Elemighong Cameroon in 1998. The specimen was identified and authenticated by Kofany of Cameroon National Herbarium Obili, Yaounde where a voucher specimen number 43596 was deposited. Extraction and chemical analysis of the plant extracts Sun dried leaves of the plant were pulverized and 250 g sohxlet extracted for 24 h with methanol p.a and the extracted fraction was recovered by rotavaporization. After the removal of chlorophyll, 1 mg/ml of the chlorophyll-free plant material was analyzed by TLC 53 and HPLC for their chemical constituents and used for pharmacological investigation. The Animals Albino rats bred in the animal house of the Institute of Pharmacology and Toxicology of the Technical University of Munich, Germany were used for in vitro studies. Others bred in the Animal house of the Department of Veterinary Pathology of the University of Nairobi, Kenya, were used for in vivo studies. The experimental procedure Vaginal discharges were observed daily and after two consecutive 4-day cycles, pre-estrus females were caged overnight together with fertile mature males. Mating was confirmed by observing the presence or absence of spermatozoa in the vaginal smears the following morning. The date was noted as day 1 of pregnancy. Five mated female rats were caged together in groups and were assigned to experiment A. • Experiment A: A dose of 350 mg/kg/day of the methanol extracts of Ruellia praetermissa was administered from every morning from day 1–9 (group 1), 9–17 (group 2), and 1–17 (group 3). Control received no treatment. The animals were sacrificed on day 18 and their genital tracts inspected. The number of life/death fetuses, the weight of each litter, gross malformation and the number of corpora lutea were recorded. • Experiment B: Daily (1–17) dose (350 mg/kg/day) was administered to 3 groups of virgin female rats (n = 5). Control group was treated with 3 µM estradiol. The Table 1. Reproductive and fetal parameters of the pregnant rats receiving 350 mg/kg/day of the MeOH extract of Ruellia praetermissas by forced administration in different stages of gestation. 1 2 3 4 5 6 7 8 9 10 11 Parameters of investigation Group 1: Control (3 ml of Water) MeOH extract of Ruellia praetermissa (350 mg/kg/day) ––––––––––––––––––––––––––––––––––––––––––––––––––– Group 2 Group 3 Group 4 Day 1–9 Day 9–17 Day 1–17 Pregnant female rats Average weight Day (g) Day 18 Weight gain Number of corpora lutea Mean of corpora lute/female Number of death fetuses Number of implantation sites Mean number of fetus/female Mean weight of the fetuses (g) Number of resorption sites Mean number of resorption sites Percentage of implantation (5) 5 210 ± 10.0 365 ± 14.8 154 ± 15.5 79 25.4 ± 1.6 55 56 11.0 ± 0.7 1.8 ± 0.2 2 0.4 ± 0.2 5 222 352 132 ± 15.7 82 16.4 ± 1.2 56 60 11.2 ± 1.0 1.6 ± 0.02 3 0.6 ± 0.4 5 210 ± 10.0 340 ± 12.5 130 ± 9.4 80 16 ± 1.5 56 58 11.1 ± 10 1.5 ± 0.3 3 0.4 ± 0.5 5 210 ± 10 335 ± 10 125 ± 10 85 14 ± 1.6 54 64 10.1 ± 0.3 1.6 ± 0.4 2 0.3 ± 0.3 68.6 ± 27 88.6 ± 5.9 70.0 ± 0.5 90.5 ± 0.5 54 A. M. Salah et al. animals were sacrificed successively on days 4, 9, 15 and 18. The uteri were removed and the weights recorded. • Experiment C: Non pregnant mature female rats were administered with 0.1 mg/kg of estradiol benzoate intraperitoneally a day prior to the experiment. Uterine strips obtained from these female rats were mounted vertically for isometric force recording in a two-chambered 50 ml bath containing a constantly aerated (5% CO2 in O2 ) 2.0 µM Ca2+ Krebs-Hanseleit solution at 35 °C. The set-up was allowed to equilibrate for 1 h. The effect of increasing concentration on the isolated uterus was investigated by cumulative addition of the extract in comparison to 2 µM ACh. The organ bath was incubated with 3 µM atropine followed by cumulative addition of the plant extract. A recovery period of 30 min was allowed for the organ between each drug challenge, after wash. The results were analyzed using Graph Pad Prism software. The results were expressed as the mean value ± SE with a value of p < 0.05 considered significant. j Results and Discussion Chemical studies showed that the extracts contain flavonoids aglycons (luteolin and apigenin) and their respective glycosides. Triterpenes (campestrol, stigmasterol, β-sitosterol, lupeol), and iridoid glycosides (Salah, 1999) The effect of forced p.o. administration of 350 mg/kg/day of plant extract at various stages of gestation is shown in Fig. 1 and Tab. 1. The body weight slightly Table 2. Contraction produced by ACh and MeOH extracts of Ruellia praetermissa on isolated uterus of rat. Increase in concentration Response of the contraction of isolated rat uterus –––––––––––––––––––––––––––––––––––– ACh (×10–2 µM) MeOH extract of R. praetermissa (×10 µg/ml) 0 2 4 8 16 32 0 5 ± 0.35 28 ± 0.60 52 ± 0.50 75 ± 0.72 100 ± 0.82 0 0 ± 0.61 24 ± 0.74 41 ± 0.80 62 ± 85 84 ± 1.23 Table 3. Contraction produced by ACh (1.6 × 10–3 µM) and MeOH extracts (350 µg/ml) of Ruellia praetermissa on isolated uterus of rat in presence of increasing concentrations of atropine antagonizing the effect. Fig. 1. The Body weights of pregnant female rats, treated with 350 mg/kg/day with MeOH extract of Ruellia praetermissa at various periods of pregnancy. ■ control; ▲ group 1; ▼ group 2; ◆ group 3. Concentration (×10–2 µM) of atropine Mean response of the contraction of isolated rat uterus –––––––––––––––––––––––––––––––––––– ACh MeOH extract of R. praetermissa (1.6 × 10–6 µM) (350 µg/ml) 0 2 4 8 16 32 76 ± 0.75 68 ± 0.67 53 ± 0.78 34 ± 0.56 21 ± 0.45 7 ± 0.41 61 ± 0.98 45 ± 0.88 31 ± 0.73 22 ± 0.65 10 ± 0.48 0 ± 0.00 Table 4. Effect of MeOH extract (350 mg/kg/day) of Ruellia praetermissa pretreatment on the uterine muscle weight in rats. Duration of treatment No. of Rats Uterine Muscle Weight (g) –––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––– Control (0.9 M NaCl) Estradiol 17β (3 µM) Plant Extract 4 9 15 18 15 15 15 15 0.016 ± 0.001 0.018 ± 0.008 0.022 ± 0.009 0.021 ± 0.005 0.3 ± 0.001 0.29 ± 0.003 0.36 ± 0.001 0.35 ± 0.005 0.03 ± 0.002 0.27 ± 0.004 0.33 ± 0.001 0.34 ± 0.001 Estrogenic and cholinergic properties of the methanol extract of Ruellia praetermissa increased during the first 6 days (Exp. 2). There was a significant decrease in the body weight and the number and the mean values of corpora lutea in groups 2, 3 and 4. The decrease in weight was more remarkable from the 16th day but did not induce resorption. There was an increase in the percentage of implantation and slight increase in the implantation sites in groups 2 and 4. The extract and ACh produced a concentration dependent increase in contraction of the isolated uterus (Tab. 2). Atropine (3.4 × 10–6 to 3 × 10–3 µM) antagonized (concentration-dependent) the maximal response of the isolated uterus to ACh (1.6 × 10–2 µM) and the plant extract (350 µg/ml) (Tab. 3). Daily administration of the plant extract (350 mg/kg/day) and estradiol 17β (3 mM) significantly increased the weight of the uterus (p < 0.05) in a time-dependent manner (Tab. 4). These observations indicate that Ruellia praetermissa possesses direct influence on the uterine physiology during gestation in rats. The plant extract appears to activate the myometrial cells membrane muscarinic receptors resulting in a uterotonic effect by a mode of action possibly via the cholinergic system. This effect is observed in vivo and in vitro only when the plant extracts are applied at the second phase of gestation. Initial increase in the weight of the pregnant rats treated with the extract in early pregnancy signified a possible estrogenic effect confirmed by the gain in uterine weight comparable to animals treated with 3 µM estradiol 17β (Tab. 4). The extract is possibly acting by facilitating the synthesis of endogenous estradiol which influences the stimulation of the growth of the uterine endometrium. In earlier stages of gestation and the contractility of the uterine myometrium at term by modifying the binding sites of Ca2+ and increasing the sensitivity of the uterine muscles to excitable hormones such as oxytocin. The diversity of the chemical constitution of the extract could be responsible at least in part for effects observed. β-Sitosterol and stigmasterol (plant sterols known to generate synthesis of steroid hormones in vivo) could be responsible for the estrogenic effect of the extract which facilitates the synthesis of endogenous estrogens. Flavonoids (luteolin and apigenin) possessing low estrogenic effect (Spilkova and Hubik, 1992) could have also contributed to the effect. Iridoid glycosides, having cholinergic effect (Harborne and Baxter, 1993) might have contributed to the contractile activity of the extract on the isolated uterus. Ruellia suffrusticosa mixed with liquid copal has been reported to produce an abortive effect when administered to pregnant women (Kritikar and Basu, 1935). Our findings preliminary justify the use of Ruellia praetermissa in traditionally herbal medicine in Africa to regularize pregnancies threatened with miscarriages in the earlier stages. This is explained by its ability to 55 stimulate the growth of the uterine endometrium in the early stages of gestation. It also explains the frequent use of this plant prior to birth to accelerate parturition and placental expulsion since it produces contractility of the uterine myometrium, at term. j References Admad V., Choudhary M., Igbal M., Akhtar M., Farid M., Ahmed M., Ghazalah R., Usmanghani K., and Clardy J. αD-galactospyranosyl glycerol hexaacetate from Ruellia brittoniana. J. Nat. Prod. 53: 960–963. Fischer H., Jensen W., Rosendal S., and Juhl B. N. Chemotaxonomy of Acanthaceae Iridoids and quaternary Amines. Phytochemistry 27: 2581–2589, 1988. Gelfand M., Mavi S., Drummond R. B., and Ndemera B. The traditional medical practitioner in Zimbabwe: His Principles of Practice and Pharmacopoeia. Mambo Press 411 p, 1985. Harborne B. J. and Baxter H. Phytochemical Dictionary. A handbook of bioactie compounds from plants. Taylor Frost, London. 191 pp, 1993. Kirtikar K. R. and Basu B. D. Indian Medicinal Plants Vol. III, 1866, 1935. Salah A. M. Anti-inflammatory and Cardiovascular effects of some chemically active principles from Ruellia praetermissa. Doctorate Thesis: 75–93, 1999. Salah A. M., Dongmo A. B., Kamanyi A., Bopelet M. and Wagner H. Angiotensin-Converting Enzyme-Inhibitory Effect by Ruellia pratermissa. Pharmaceutical Biology 39: 16–19, 2001. Salah A. M., Dongmo A, B., Kamanyi A., Bopelet M., Vierling W. and Wagner H. In vitro purgative effects of Ruellia pratermissa Sceinf. ex. Lindau (Acanthaceae). J. of Ethnopharmacol. 72: 269–272, 2000. Sankara S. S. and Nair A. G. R. Flavonoids of Ruellia prostrata and Berleria cristata. J. Indian Chem. Soc. 49: 823–826, 1972. Siegeried J., Groeger D. and Reiner R. Tetramethylputrescine from young plants of Ruellia rosea. Phytochemistry 14: 2635–2636, 1985. Spilkova J. and Hubik J. Biologische Wirkungen von Flavonoiden II*. Flavonoidwirkungen in Pharmazie in unserer Zeit/21. Jahrg. 174–182, 1992. Wagner H., Danninger H., Iyenga M. A., Seligmann O., Farkas L., Sankara S. S. and Nair A. G. R. Synthesis of Glucuronides in the Flavonoid Series, III 1, 2 Isolation of Apigenin-7β-glucuronide from Ruellia tuberosa L. and its synthesis. Chem. Ber. 104: 2681–2687, 1972. j Address A.M. Salah, National Center for Natural Products Research, Research Institute of Pharmaceutical Sciences, School of Pharmacy, University of Mississippi, University, MS 38677, USA Tel.: ++1-662-915-7151; Fax: ++1-662-915-7989; e-mail: salah_anchang_ma@hotmail.com