Original Article · Originalarbeit Forsch Komplementmed 2014;21:184–189 DOI: 10.1159/000363709
Published online: June 16, 2014
Effects of Chronic Bryophyllum pinnatum Administration on Wistar Rat Pregnancy Jorge Kioshi Hosomi Ricardo Ghelman Marisa Pascale Quintino Eduardo de Souza Mary Uchiyama Nakamura Antonio Fernandes Moron
Keywords Bryophyllum pinnatum · Kalanchoe pinnata · Anthroposophy · Pregnancy · Wistar rats · Plant preparations
Schlüsselwörter Bryophyllum pinnatum · Kalanchoe pinnata · Anthroposophie · Schwangerschaft · Wistar-Ratten · Pflanzenzubereitungen
Summary Background: Drugs used in preterm labor (PTL) have side effects. Research into new tocolytic agents is recommended. The plant Bryophyllum pinnatum (Lam.) Oken (Kalanchoe pinnata (Lam.) Pers) is used in Anthroposophic Medicine for PTL, insomnia, and emotional disturbances. The aim of this study was to evaluate the effects of B. pinnatum mother tincture (MT) on Wistar rats and their fetuses throughout pregnancy. Method: Sixty animals were divided into 6 equal groups: controls C1 and C2 received 1 and 25 × the maximum daily dose of a 30% ethanol / water solution, serving as vehicle of B. pinnatum MT; B1 and B2 received 1 and 25 × the maximum daily dose of B. pinnatum MT; and B3 and B4 received 50 and 100 × B. pinnatum concentrate. The following parameters were assessed: weight gain; maternal and fetal mortality; implantations and resorptions; number and weight of fetuses and placentas; major external fetal malformations. Results: Rat weight gain (excluding fetal and placental weight) was higher in group B4 and lower in group B2. There were no maternal or fetal deaths and no group differences in implantations and resorptions or number and weight of fetuses and placentas. No macroscopic fetal abnormalities were observed at the 4 dosage levels investigated. Conclusion: Daily administration of B. pinnatum MT at high doses to pregnant Wistar rats interfered with maternal weight gain and did not interfere with fetal compartment.
Zusammenfassung Hintergrund: Arzneimittel, die zur Hemmung von Frühwehen eingesetzt werden, sind mit Nebenwirkungen verbunden, sodass die Erforschung neuer tokolytischer Mittel empfehlenswert ist. Die Pflanze Bryophyllum pinnatum (Lam.) Oken (Kalanchoe pinnata (Lam.) Pers.) wird in der Anthroposophischen Medizin zur Behandlung von Frühwehen, Schlaflosigkeit und emotionalen Störungen eingesetzt. Das Ziel dieser Studie war es, die Auswirkungen der B. pinnatum-Urtinktur bei Wistar-Ratten und deren Föten während der Schwangerschaft zu erforschen. Methodik: Sechzig Tiere wurden in 6 gleiche Gruppen eingeteilt: Die beiden Kontrollgruppen C1 und C2 erhielten das 1- bis 25-Fache der maximalen Tagesdosis einer Lösung aus 30% Ethanol und Wasser, die als Vehikel für die der B. pinnatum-Urtinktur diente, B1 und B2 erhielten das 1- und 25-Fache der maximalen Tagesdosis der Urtinktur und B3 und B4 erhielten die 50- und 100-fache Dosis B. pinnatum in konzentrierter Form. Folgende Parameter wurden bewertet: Gewichtszunahme, maternale und fetale Mortalität, Implantationen und Resorptionen, Anzahl und Gewicht der Föten und Plazentas, gravierende äußerliche fetale Fehlbildungen. Ergebnisse: Die Gewichtszunahme der Ratten (ohne fetales und Plazentagewicht) war in der Gruppe B4 höher und in Gruppe B2 niedriger. Es gab keine maternalen oder fetalen Todesfälle und keine Gruppenunterschiede bei den Einimpfungen und Resorptionen oder der Anzahl und dem Gewicht der Föten und Plazentas. Bei den 4 untersuchten Dosierungen wurden keine makroskopisch feststellbaren fetalen Anomalien beobachtet. Schlussfolgerung: Die tägliche hochdosierte Verabreichung der B. pinnatum-Urtinktur an trächtige Wistar-Ratten wirkte sich auf die maternale Gewichtszunahme, jedoch nicht den fetalen Raum aus.
© 2014 S. Karger GmbH, Freiburg 1661-4119/14/0213-0184$39.50/0 Fax +49 761 4 52 07 14 [email protected] www.karger.com
Accessible online at: www.karger.com/fok
Jorge Kioshi Hosomi, MS Alameda Ministro Rocha Azevedo 770 São Paulo, SP 01410–000, Brazil [email protected]
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Department of Obstetrics, Escola Paulista de Medicina, Universidade Federal de São Paulo (EPM-UNIFESP), São Paulo, Brazil
Preterm labor (PTL) is a major public health issue, affecting 5–11% of pregnancies [1, 2]. In view of the social, economic, and emotional burden of prematurity, both researchers and clinicians have sought to understand its pathophysiology and develop new treatment and prevention strategies [3]. Research into the development of new tocolytic agents has been recommended, as have efforts toward optimized use of currently available agents [4, 5]. In this endeavor, consideration of resources employed by complementary and alternative medicine practices, also known as integrative medicine [6], is warranted. Since the 1970s, Anthroposophic Medicine (AM), a system of complementary medicine developed by Rudolf Steiner and Ita Wegman [7], has used preparations of the plant Bryophyllum pinnatum (Lam.) Oken in the treatment of PTL in Germany and Switzerland [8]. B. pinnatum inhibits myometrial contractility in humans [8], and was found to be better tolerated than beta-agonists for treatment of PTL [9] in a randomized controlled trial of 67 matched pairs of pregnant women. B. pinnatum is employed in folk medicine for the treatment of wounds, arthralgia, and other painful conditions [10]. Despite its widespread native use in several regions of the world (India, China, Australia, Hawaii, Africa, and Tropical Americas), there has been relatively little research into this plant [8]. Organic acids, hydrocarbons, phenolic compounds, flavonoids, steroids, and compounds of other classes have been isolated from its leaves [11–13]. Experimental studies have shown that B. pinnatum leaf extract has antinociceptive (analgesic), anti-inflammatory, antihistamine, immunomodulatory, antidiabetic, sedative, muscle relaxant, hepatoprotective, and antimicrobial properties [10, 14–16]. In Brazil, research into B. pinnatum has captured the attention of government agencies. Since the implementation of the National Policy for Integrative and Complementary Practices (Política Nacional de Práticas Integrativas e Complementares) into the Unified Health System in 2005, initially including the subject areas Medicinal Plants and Herbal Medicine, Homeopathy, Traditional Chinese Medicine/Acupuncture and Anthroposophic Medicine, the Brazilian Ministry of Health has developed a Proposal for Medicinal Plants and Herbal Medicine in the Unified Health System (Proposta para Plantas Medicinais e Fitoterapia no Sistema Único de Saúde) designed to expand the range of treatment options available to system users, ensuring access to medicinal plants within a framework of safety, efficacy, and quality [17]. In Germany, B. pinnatum is prescribed in various preparations [18]. In Brazil, preparations are manufactured in 2 pharmaceutical forms: oral formulation of the mother tincture (MT) and anthroposophic dilutions [19]. As the MT B. pinnatum is indicated for the treatment of sleep disorders, anxiety, agitation, fears, exhaustion, asthenia,
B. pinnatum on Wistar Rat Pregnancy
recurrent inflammatory conditions, and PTL [19], it may be an alternative for management of sleep disorders, anxiety, and muscle aches that often afflict women during pregnancy. In a study on the most common non-obstetric complaints reported by Brazilian pregnant women, Guerreiro da Silva found that 30% of them experienced sleep disturbances [20], 56% anxiety [21], and 77% low back pain [22]. Although these symptoms are extremely common during pregnancy, they are often neglected by physicians during prenatal care, as most drugs available for their management are of questionable safety (pregnancy category C) or pose a risk to the fetus (pregnancy category D). These medicines should only be used when their benefits outweigh their risks [23]. One of the basic criteria defined by the World Health Organization for the medicinal use of herbal preparations is evidence of their non-toxicity [24]. Currently available studies on B. pinnatum in pregnancy were all conducted at more advanced stages of the gestational period. If appropriate evidence was available, the indications of this herbal medicine could be extended to non-obstetric symptoms occurring in earlier stages of pregnancy. A review of literature did not yield any studies on its safety during pregnancy, not even in experimental models. Therefore, the objective of this investigation was to assess the potential undesirable effects of B. pinnatum on Wistar rats and their offspring throughout pregnancy.
Materials and Methods This was a longitudinal, prospective, randomized controlled animal study. The study sample comprised adult virgin female Wistar EPM-1 rats (Rattus norvegicus albinus; age ca. 90 days, body weight 200–250 g). The study protocol was approved by the Universidade Federal de São Paulo Research Ethics Committee (judgment no. 0857/09), and procedures were carried out in compliance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. The study design followed the research protocol developed at the Obstetric Department of this university [25–27]. During the adaptation period (15 days), animals were housed 5 to a cage, at a controlled temperature of approximately 22 °C, under artificial fluorescent lighting and a standard 12/12 h light/dark cycle (lights on at 7 a.m., lights out at 7 p.m.). All animals were provided access to a balanced diet (rat chow) and water ad libitum. The feed used was Nuvilab CR1TM (Nuvital) containing ground whole corn, soybean meal, wheat bran, calcium carbonate, dicalcium phosphate, sodium chloride, vitamin and mineral premix, and amino acids. After the adaptation period, animals were mated on a 1 male to 2 females basis during the lights-out period (7 p.m. to 7 a.m.). Onset of gestation (day 0) was diagnosed by detection of sperm in the vagina. The preparation used for this experiment was MT of B. pinnatum, provided by Weleda in Brasil. A voucher specimen of B. pinnatum is deposited at the herbarium of the Zurich Succulent Plant Collection (Sukkulenten-Sammlung Zürich, Switzerland) under no. ZSS 29717. The vehicle of B. pinnatum MT is a 30% ethanol solution. Due to the presence of alcohol in the preparation, we chose to use the pure vehicle (30% ethanol solution) as control. So as to ascertain the effects of high-dose B. pinnatum administration, we produced a concentrate of B. pinnatum MT by rotary evaporation to
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Introduction
Table 1. Experimental doses and total flavonoid and ethanol content in each group
Group Dose (mL/kg/day) Total flavonoid dose, mg/kg/daya Total ethanol dose, mg/kg/dayb
30% ethanol (vehicle)
B. pinnatum MT
B. pinnatum Conc.
C1 0.067 — 16
B1 0.067 1.7 17
B3 1.675 57.3 133
C2 1.675 — 396
B2 1.675 41.9 430
B4 3.35 114.6 265
C1 and C2 = vehicle control (1× and 25 × the maximum dose of pure vehicle); B1 and B2 = 1× and 25 × the maximum daily dose of B. pinnatum MT; B3 and B4 = 50 × and 100 × the maximum daily dose in B. pinnatum Conc. a Method: spectrophotometry. Total flavonoids expressed in quercetin. b Method: European Pharmacopoeia, ed 6.
Table 2. Effects of B. pinnatum or vehicle administration during Wistar rat pregnancy in the study groups Group
Weight Baseline Gestation day 20 Fetal Placental Fetal and placental Exclusive dam gain n Fetuses Placentas Implantations Resorptions Macroscopic fetal malformations
p value
C1
B1
C2
B2
B3
B4
200.5 (45) 343.5 (61) 44.1 (15.4) 6.45 (0.5) 50.4 (16.9) 80.1 (37.9)
211.0 (43) 316.5 (49) 41.6 (15.8) 6.6 (0.8) 48.4 (16.6) 64.1 (47.2)
213.5 (21) 337.5 (30.2) 48.6 (34.9) 6.95 (1.4) 55.3 (36.7) 65.0 (34.9)
237.5 (13) 321.5 (38) 47.2 (12.9) 6.45 (2.2) 51.8 (13.7) 50.4 (64)
236.0 (32) 349.5 (44) 46.5 (9.0) 6.6 (0.8) 52.7 (10.7) 77.3 (27.4)
231.0 (29) 382.0 (28) 47.8 (8.7) 6.85 (1.5) 54.9 (8.6) 97.0 (48.6)
11.5 (4) 11.5 (4) 11.5 (4) 0 (0) 0 (0)
11.0 (1) 11.0 (1) 11.0 (1) 0 (0) 0 (0)
12.0 (2) 12.0 (2) 12.5 (3) 0 (0) 0 (1)
10.5 (4) 10.5 (4) 10.5 (4) 0 (0) 0 (0)
11.5 (3) 11.5 (3) 12.0 (2) 0 (1) 0 (1)
11.5 (2) 11.5 (2) 11.5 (2) 0 (0) 0 (0)
0.03a < 0.01b 0.34 0.67 0.39 0.01c 0.84 0.84 0.60 0.25 < 0.01d
Data expressed as median (interquartile range; in brackets). C1 and C2 = vehicle control (1 × and 25 × the maximum dose of pure vehicle); B1 and B2 = 1 × and 25 × the maximum daily dose of B. pinnatum MT; B3 and B4 = 50 × and 100 × the maximum daily dose in B. pinnatum Conc. a C1, C2 B1, B2, B3, B4. b B4 > C1, B1, C2, B2. c B4 > B1, C2, B2; C1 > B2. d C2 > C1, B1, B2, B3, B4.
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incision from xiphoid to pubic symphysis), and hysterotomy. The following parameters were recorded: weight gain; maternal and fetal mortality; number of implantations and resorptions; number and weight of fetuses and placentas; major external fetal malformations. At the end of the experiment, all animals were euthanized by terminal anesthesia. Quantitative variables were expressed as medians and interquartile ranges (IQR), and categorical variables as absolute (n) and relative (%) frequencies. The non-parametric Kruskal-Wallis test for independent samples was performed, and Dunn’s multiple comparison test used to assess between-group differences. The significance level was set at 0.05 (_ = 5%). All calculations were performed in the SPSS for Windows 15.0 software environment.
Results Descriptive statistics (medians and IQR) are shown in table 2. Comparative analysis of baseline rat weight showed a difference among the study groups (p = 0.03). Animals in groups C1 and C2 had similar weights, and were leaner than animals in the other groups; all other groups had similar baseline
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half volume in a RotaVapor® device, at 70 °C and a pressure of 600 mm Hg. The resulting product had an alcohol content of 10%, and was denoted B. pinnatum Conc. for the purposes of this study. On day 0 of gestation, 60 rats were randomly allocated to the following 6 groups: 2 control groups, C1 and C2; and 4 experiment groups, B1, B2, B3, and B4. Rats in group C1 received a maximum daily dose equivalent of vehicle (30% ethanol in water), and those in group C2, the equivalent to 25 × the maximum daily dose of vehicle. Rats in group B1 received the usual maximum daily dose of B. pinnatum MT (0.067 mL/kg/day) and those in group B2 the equivalent to 25 × the maximum daily dose (1.675 mL/kg/day). Animals in groups B3 and B4 were given B. pinnatum Conc. at a dose equivalent to 50 and 100 × the maximum daily dose of MT, respectively. The maximum daily dose was derived by calculating the equivalent to the recommended human dose (20 drops 4 times a day for a 60 kg patient = 4 mL/60 kg = 0.067 mL/kg). Table 1 lists the administered doses and the total flavonoid and ethanol content of each dosage. Flavonoid and ethanol content was measured by the Quality Control Department of Weleda in Brasil. Doses were diluted in water q.s.p. 1 mL and administered by gavage, through a metal feeding needle, once daily in the morning, from gestational day 0 to gestational day 20. At term (day 20), dams were anesthetized with xylazine hydrochloride 10 mg/kg and ketamine hydrochloride 100 mg/kg by intraperitoneal injection, underwent laparotomy (midline
We chose to assign 2 control groups (C1 and C2) due to the pharmaceutical form of the study product (MT, hydroalcoholic extract 30% for oral administration). Animals in control groups C1 and C2 were administered pure vehicle (30% ethanol in water) so as to provide comparative data on the potential effect of the drug vehicle on experimental group results, as alcohol intake is known to interfere with pregnancy in a dosedependent manner [28]. The choice of commercially available B. pinnatum MT as the pharmaceutical formulation for this experiment was prompted by its status as the only available extract obtained under strictly controlled conditions of plant cultivation, manufacturing practices, and quality control. We also took into account that using a hydroalcoholic extract in this experiment would contribute to the existing literature on herbal medicines made from B. pinnatum. The use of MT provides several advantages. Its manufacturing processes are standardized, it is readily available, and has a long shelf life [29]. However, in any experiment consisting of high-dose administration of herbal medicines prepared in alcoholic vehicles, one must control for the potential effects of alcohol content. Therefore, experimental group B2 (25 × the maximum daily dose of B. pinnatum MT) was paired with control group C2 (25 × the maximum dose of vehicle). In groups B3 and B4, which received 50 and 100 × the maximum daily dose of B. pinnatum MT, alcohol content was artificially reduced by concentrating the MT by rotary evaporation. Comparative analysis of median dam weight in the 6 study groups (C1, B1, C2, B2, B3, B4) revealed significant differences in baseline weight: rats in groups C1 and C2 were leaner
than those in all other groups. Due to between-group differences in weight at baseline and throughout pregnancy, we conducted a subanalysis of exclusive dam weight gain (total weight gain minus fetal and placental weight) at gestational day 20. At term, dams in group B4 had the greatest body mass gain. There was no significant difference in pooled fetal and placental weight (total litter weight). Weight change from baseline was greatest in groups B4 and C1, and body weight was lower in B2 dams than in C1 dams. Therefore, despite lower weight at baseline in groups C1 and C2, this difference did not influence fetal and placental weight at term. Subanalysis of exclusive dam weight gain enabled assessment of how much body mass was actually gained by dams regardless of offspring weight. Administration of B. pinnatum MT at a dose 25 × greater than the maximum daily dose (group B2) had no effect on fetal and placental weight. Breast, uterine, and adipose tissues account for most weight gained during pregnancy [30]. As there were no significant differences among study groups in fetal and placental weight, we believe the difference observed is attributable to less body fat accumulation in group B2. To date, no studies have confirmed any weight-reducing effects of B. pinnatum in obese subjects. However, due to its antianxiety effect [15], B. pinnatum may have reduced the avidity of feeding behaviors. Another hypothesis for the lesser weight gain observed in group B2 is a potential effect of ethanol present in B. pinnatum MT. As alcohol is a non-nutritive energy source, rats in this group may have exhibited decreased food intake [31]. Paradoxically, dams in group B4 (100 × the usual daily dose) exhibited the greatest weight gain as compared with groups B1, C2, and B2. There were no significant differences on comparison with groups C1 and B3. Hypothetically, dams in groups B3 and B4 would have experienced lower exclusive (non-placenta, non-fetal) weight gain if body mass followed a dosedependent trend, as suggested by less weight gain in group B2 than in group B1. As groups B3 and B4 received a concentrate in which alcohol content had been reduced by heating, we propose 2 explanatory hypotheses for these findings: 1. Higher doses of B. pinnatum (50 and 100 × the maximum dose) may interfere with the orexin/hypocretin system; 2. Heating may have altered the chemical composition of the original extract. It is widely known that the composition and concentration of active ingredients in herbal medicines depend on the methods of extraction, including the choice of vehicle, pH, temperature, extraction time, and other factors [32]. A recent study [33] found that cultivation and collection conditions as well as extraction methods influence the antileishmanial flavonoid content of B. pinnatum preparations. We raise the hypothesis that high-dose B. pinnatum therapy may interfere with regulation of the orexin/hypocretin system. The neuropeptides orexin A and orexin B (also known as hypocretins 1 and 2) are produced by neurons in the hypothalamus and play a major role in the regulation of feeding and wakefulness [34]. Leptin, a modulator of energy balance, is one
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Discussion
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weights. Between-group differences in dam weight at gestational day 20 were also significant (p < 0.01). Dams in group B4 were significantly heavier than those in groups C1, B1, C2, and B2, with no significant difference as compared to group B3. There were no differences in fetal and placental weight (p = 0.39). Comparison of exclusive weight gain, however (difference between rat weight at baseline and weight at gestational day 20, after subtracting the weight of the fetus and of the placenta) showed a significant difference among groups (p = 0.01). Dams in group B4 gained more weight than those in groups B1, C2, and B2, with no significant differences from groups C1 and B3. Furthermore, body weight was greater in dams from group C1 as compared with group B2. There were no between-group differences in number of fetuses and placentas, number of implantations and resorptions, or fetal and placental weight. There were no instances of maternal mortality or stillbirth. No significant external fetal malformations were observed in pups delivered from dams in any of the 4 dosage groups, except for control group C2 (livid pups).
of the main components of this system. Disruption of the circadian rhythm of leptin encourages feeding and reduces energy expenditure, leading to obesity, as observed in human subjects with narcolepsy [35, 36]. B. pinnatum is used in the treatment of sleep disorders and possesses neurosedative properties [15, 37]. Therefore, the sedative action of B. pinnatum and the association between B. pinnatum administration and weight gain observed in our experiment may suggest interference with the orexin system. Research into the interactions between diet, energy metabolism, and regulation of sleep and wakefulness is still incipient [38], and investigation of the mechanisms of action of B. pinnatum constituents only began in recent years. We did not find any significant differences among the study groups in the number of fetuses, number of placentas, number of implantations and resorptions, or total litter weight and placental weight. Fetal macroscopic changes were observed in control group C2, but not in group B2 (same ethanol content), suggesting a protective effect of B. pinnatum against damage caused by ethanol. Study Contribution This study assessed a herbal preparation used in AM that may be beneficial in the treatment of preterm labor, sleep disorders, and emotional disturbances (such as anxiety and fear) in pregnant women. We evaluated the potential harmful effects of B. pinnatum, a medicinal plant used widely by folk medicine in several countries, when administered to pregnant Wistar rats. The pharmaceutical form tested herein (MT) is standardized, easy to prepare, and has a long shelf life, thus facilitating access to the product by low-income populations. We were intrigued by the finding that B. pinnatum interfered with dam weight gain without affecting fetal or placental weight.
Study Limitations Reduction in ethanol content by rotary evaporation for preparation of B. pinnatum Conc. may have altered the chemical composition of the MT. Therefore, the chemical profile of the concentrated preparation may differ from that of the original extract, which would, in turn, introduce bias to our comparison of dosages. As the animals had free access to food, the amount ingested was not controlled. Perspectives In vitro and animal studies of the effects of B. pinnatum, including biochemical analyses and tissue microscopy, are warranted. Further studies are required to assess potential mechanisms of the effect of B. pinnatum on weight gain regulation during pregnancy.
Conclusion Daily administration of B. pinnatum MT to Wistar rats during pregnancy interfered with maternal weight gain. It was not associated with maternal or fetal death, did not produce macroscopic fetal malformations, and did not interfere with fetal or placental weight or number of implantations or resorptions.
Acknowledgments This study was supported by Associação Beneficente Mahle (ABM).
Disclosure Statement No conflict of interests exists.
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18 Simões-Wüst AP, Jeschke E, Mennet M, Schnelle M, Matthes H, von Mandach U: Prescribing pattern of Bryophyllum preparations among a network of anthroposophic physicians. Forsch Komplementmed 2012;19:293–301. 19 Gardin NE, Schleier R: Medicamentos Antroposóficos: Vademecum. São Paulo, João de Barro, 2009, p 285. 20 da Silva JB, Nakamura MU, Cordeiro JA, Kulay LJ: Acupuncture for insomnia in pregnancy – a prospective, quasi-randomised, controlled study. Acupunct Med 2005;23:47–51. 21 Bosco Guerreiro da Silva J: Acupuncture for mild to moderate emotional complaints in pregnancy – a prospective, quasi-randomised, controlled study. Acupunct Med 2007;25:65–71. 22 Guerreiro da Silva JB, Nakamura MU, Cordeiro JA, Kulay L Jr: Acupuncture for low back pain in pregnancy – a prospective, quasi-randomised, controlled study. Acupunct Med 2004;22:60–67. 23 Buhimschi CS, Weiner CP: Medications in pregnancy and lactation: part 1. Teratology. Obstet Gynecol 2009;113:166–188. 24 World Health Organization: The Promotion and Development of Traditional Medicine – Technical Report Series. Geneva, WHO, 1978.
Published online: June 16, 2014
Effects of Chronic Bryophyllum pinnatum Administration on Wistar Rat Pregnancy Jorge Kioshi Hosomi Ricardo Ghelman Marisa Pascale Quintino Eduardo de Souza Mary Uchiyama Nakamura Antonio Fernandes Moron
Keywords Bryophyllum pinnatum · Kalanchoe pinnata · Anthroposophy · Pregnancy · Wistar rats · Plant preparations
Schlüsselwörter Bryophyllum pinnatum · Kalanchoe pinnata · Anthroposophie · Schwangerschaft · Wistar-Ratten · Pflanzenzubereitungen
Summary Background: Drugs used in preterm labor (PTL) have side effects. Research into new tocolytic agents is recommended. The plant Bryophyllum pinnatum (Lam.) Oken (Kalanchoe pinnata (Lam.) Pers) is used in Anthroposophic Medicine for PTL, insomnia, and emotional disturbances. The aim of this study was to evaluate the effects of B. pinnatum mother tincture (MT) on Wistar rats and their fetuses throughout pregnancy. Method: Sixty animals were divided into 6 equal groups: controls C1 and C2 received 1 and 25 × the maximum daily dose of a 30% ethanol / water solution, serving as vehicle of B. pinnatum MT; B1 and B2 received 1 and 25 × the maximum daily dose of B. pinnatum MT; and B3 and B4 received 50 and 100 × B. pinnatum concentrate. The following parameters were assessed: weight gain; maternal and fetal mortality; implantations and resorptions; number and weight of fetuses and placentas; major external fetal malformations. Results: Rat weight gain (excluding fetal and placental weight) was higher in group B4 and lower in group B2. There were no maternal or fetal deaths and no group differences in implantations and resorptions or number and weight of fetuses and placentas. No macroscopic fetal abnormalities were observed at the 4 dosage levels investigated. Conclusion: Daily administration of B. pinnatum MT at high doses to pregnant Wistar rats interfered with maternal weight gain and did not interfere with fetal compartment.
Zusammenfassung Hintergrund: Arzneimittel, die zur Hemmung von Frühwehen eingesetzt werden, sind mit Nebenwirkungen verbunden, sodass die Erforschung neuer tokolytischer Mittel empfehlenswert ist. Die Pflanze Bryophyllum pinnatum (Lam.) Oken (Kalanchoe pinnata (Lam.) Pers.) wird in der Anthroposophischen Medizin zur Behandlung von Frühwehen, Schlaflosigkeit und emotionalen Störungen eingesetzt. Das Ziel dieser Studie war es, die Auswirkungen der B. pinnatum-Urtinktur bei Wistar-Ratten und deren Föten während der Schwangerschaft zu erforschen. Methodik: Sechzig Tiere wurden in 6 gleiche Gruppen eingeteilt: Die beiden Kontrollgruppen C1 und C2 erhielten das 1- bis 25-Fache der maximalen Tagesdosis einer Lösung aus 30% Ethanol und Wasser, die als Vehikel für die der B. pinnatum-Urtinktur diente, B1 und B2 erhielten das 1- und 25-Fache der maximalen Tagesdosis der Urtinktur und B3 und B4 erhielten die 50- und 100-fache Dosis B. pinnatum in konzentrierter Form. Folgende Parameter wurden bewertet: Gewichtszunahme, maternale und fetale Mortalität, Implantationen und Resorptionen, Anzahl und Gewicht der Föten und Plazentas, gravierende äußerliche fetale Fehlbildungen. Ergebnisse: Die Gewichtszunahme der Ratten (ohne fetales und Plazentagewicht) war in der Gruppe B4 höher und in Gruppe B2 niedriger. Es gab keine maternalen oder fetalen Todesfälle und keine Gruppenunterschiede bei den Einimpfungen und Resorptionen oder der Anzahl und dem Gewicht der Föten und Plazentas. Bei den 4 untersuchten Dosierungen wurden keine makroskopisch feststellbaren fetalen Anomalien beobachtet. Schlussfolgerung: Die tägliche hochdosierte Verabreichung der B. pinnatum-Urtinktur an trächtige Wistar-Ratten wirkte sich auf die maternale Gewichtszunahme, jedoch nicht den fetalen Raum aus.
© 2014 S. Karger GmbH, Freiburg 1661-4119/14/0213-0184$39.50/0 Fax +49 761 4 52 07 14 [email protected] www.karger.com
Accessible online at: www.karger.com/fok
Jorge Kioshi Hosomi, MS Alameda Ministro Rocha Azevedo 770 São Paulo, SP 01410–000, Brazil [email protected]
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Department of Obstetrics, Escola Paulista de Medicina, Universidade Federal de São Paulo (EPM-UNIFESP), São Paulo, Brazil
Preterm labor (PTL) is a major public health issue, affecting 5–11% of pregnancies [1, 2]. In view of the social, economic, and emotional burden of prematurity, both researchers and clinicians have sought to understand its pathophysiology and develop new treatment and prevention strategies [3]. Research into the development of new tocolytic agents has been recommended, as have efforts toward optimized use of currently available agents [4, 5]. In this endeavor, consideration of resources employed by complementary and alternative medicine practices, also known as integrative medicine [6], is warranted. Since the 1970s, Anthroposophic Medicine (AM), a system of complementary medicine developed by Rudolf Steiner and Ita Wegman [7], has used preparations of the plant Bryophyllum pinnatum (Lam.) Oken in the treatment of PTL in Germany and Switzerland [8]. B. pinnatum inhibits myometrial contractility in humans [8], and was found to be better tolerated than beta-agonists for treatment of PTL [9] in a randomized controlled trial of 67 matched pairs of pregnant women. B. pinnatum is employed in folk medicine for the treatment of wounds, arthralgia, and other painful conditions [10]. Despite its widespread native use in several regions of the world (India, China, Australia, Hawaii, Africa, and Tropical Americas), there has been relatively little research into this plant [8]. Organic acids, hydrocarbons, phenolic compounds, flavonoids, steroids, and compounds of other classes have been isolated from its leaves [11–13]. Experimental studies have shown that B. pinnatum leaf extract has antinociceptive (analgesic), anti-inflammatory, antihistamine, immunomodulatory, antidiabetic, sedative, muscle relaxant, hepatoprotective, and antimicrobial properties [10, 14–16]. In Brazil, research into B. pinnatum has captured the attention of government agencies. Since the implementation of the National Policy for Integrative and Complementary Practices (Política Nacional de Práticas Integrativas e Complementares) into the Unified Health System in 2005, initially including the subject areas Medicinal Plants and Herbal Medicine, Homeopathy, Traditional Chinese Medicine/Acupuncture and Anthroposophic Medicine, the Brazilian Ministry of Health has developed a Proposal for Medicinal Plants and Herbal Medicine in the Unified Health System (Proposta para Plantas Medicinais e Fitoterapia no Sistema Único de Saúde) designed to expand the range of treatment options available to system users, ensuring access to medicinal plants within a framework of safety, efficacy, and quality [17]. In Germany, B. pinnatum is prescribed in various preparations [18]. In Brazil, preparations are manufactured in 2 pharmaceutical forms: oral formulation of the mother tincture (MT) and anthroposophic dilutions [19]. As the MT B. pinnatum is indicated for the treatment of sleep disorders, anxiety, agitation, fears, exhaustion, asthenia,
B. pinnatum on Wistar Rat Pregnancy
recurrent inflammatory conditions, and PTL [19], it may be an alternative for management of sleep disorders, anxiety, and muscle aches that often afflict women during pregnancy. In a study on the most common non-obstetric complaints reported by Brazilian pregnant women, Guerreiro da Silva found that 30% of them experienced sleep disturbances [20], 56% anxiety [21], and 77% low back pain [22]. Although these symptoms are extremely common during pregnancy, they are often neglected by physicians during prenatal care, as most drugs available for their management are of questionable safety (pregnancy category C) or pose a risk to the fetus (pregnancy category D). These medicines should only be used when their benefits outweigh their risks [23]. One of the basic criteria defined by the World Health Organization for the medicinal use of herbal preparations is evidence of their non-toxicity [24]. Currently available studies on B. pinnatum in pregnancy were all conducted at more advanced stages of the gestational period. If appropriate evidence was available, the indications of this herbal medicine could be extended to non-obstetric symptoms occurring in earlier stages of pregnancy. A review of literature did not yield any studies on its safety during pregnancy, not even in experimental models. Therefore, the objective of this investigation was to assess the potential undesirable effects of B. pinnatum on Wistar rats and their offspring throughout pregnancy.
Materials and Methods This was a longitudinal, prospective, randomized controlled animal study. The study sample comprised adult virgin female Wistar EPM-1 rats (Rattus norvegicus albinus; age ca. 90 days, body weight 200–250 g). The study protocol was approved by the Universidade Federal de São Paulo Research Ethics Committee (judgment no. 0857/09), and procedures were carried out in compliance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. The study design followed the research protocol developed at the Obstetric Department of this university [25–27]. During the adaptation period (15 days), animals were housed 5 to a cage, at a controlled temperature of approximately 22 °C, under artificial fluorescent lighting and a standard 12/12 h light/dark cycle (lights on at 7 a.m., lights out at 7 p.m.). All animals were provided access to a balanced diet (rat chow) and water ad libitum. The feed used was Nuvilab CR1TM (Nuvital) containing ground whole corn, soybean meal, wheat bran, calcium carbonate, dicalcium phosphate, sodium chloride, vitamin and mineral premix, and amino acids. After the adaptation period, animals were mated on a 1 male to 2 females basis during the lights-out period (7 p.m. to 7 a.m.). Onset of gestation (day 0) was diagnosed by detection of sperm in the vagina. The preparation used for this experiment was MT of B. pinnatum, provided by Weleda in Brasil. A voucher specimen of B. pinnatum is deposited at the herbarium of the Zurich Succulent Plant Collection (Sukkulenten-Sammlung Zürich, Switzerland) under no. ZSS 29717. The vehicle of B. pinnatum MT is a 30% ethanol solution. Due to the presence of alcohol in the preparation, we chose to use the pure vehicle (30% ethanol solution) as control. So as to ascertain the effects of high-dose B. pinnatum administration, we produced a concentrate of B. pinnatum MT by rotary evaporation to
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Introduction
Table 1. Experimental doses and total flavonoid and ethanol content in each group
Group Dose (mL/kg/day) Total flavonoid dose, mg/kg/daya Total ethanol dose, mg/kg/dayb
30% ethanol (vehicle)
B. pinnatum MT
B. pinnatum Conc.
C1 0.067 — 16
B1 0.067 1.7 17
B3 1.675 57.3 133
C2 1.675 — 396
B2 1.675 41.9 430
B4 3.35 114.6 265
C1 and C2 = vehicle control (1× and 25 × the maximum dose of pure vehicle); B1 and B2 = 1× and 25 × the maximum daily dose of B. pinnatum MT; B3 and B4 = 50 × and 100 × the maximum daily dose in B. pinnatum Conc. a Method: spectrophotometry. Total flavonoids expressed in quercetin. b Method: European Pharmacopoeia, ed 6.
Table 2. Effects of B. pinnatum or vehicle administration during Wistar rat pregnancy in the study groups Group
Weight Baseline Gestation day 20 Fetal Placental Fetal and placental Exclusive dam gain n Fetuses Placentas Implantations Resorptions Macroscopic fetal malformations
p value
C1
B1
C2
B2
B3
B4
200.5 (45) 343.5 (61) 44.1 (15.4) 6.45 (0.5) 50.4 (16.9) 80.1 (37.9)
211.0 (43) 316.5 (49) 41.6 (15.8) 6.6 (0.8) 48.4 (16.6) 64.1 (47.2)
213.5 (21) 337.5 (30.2) 48.6 (34.9) 6.95 (1.4) 55.3 (36.7) 65.0 (34.9)
237.5 (13) 321.5 (38) 47.2 (12.9) 6.45 (2.2) 51.8 (13.7) 50.4 (64)
236.0 (32) 349.5 (44) 46.5 (9.0) 6.6 (0.8) 52.7 (10.7) 77.3 (27.4)
231.0 (29) 382.0 (28) 47.8 (8.7) 6.85 (1.5) 54.9 (8.6) 97.0 (48.6)
11.5 (4) 11.5 (4) 11.5 (4) 0 (0) 0 (0)
11.0 (1) 11.0 (1) 11.0 (1) 0 (0) 0 (0)
12.0 (2) 12.0 (2) 12.5 (3) 0 (0) 0 (1)
10.5 (4) 10.5 (4) 10.5 (4) 0 (0) 0 (0)
11.5 (3) 11.5 (3) 12.0 (2) 0 (1) 0 (1)
11.5 (2) 11.5 (2) 11.5 (2) 0 (0) 0 (0)
0.03a < 0.01b 0.34 0.67 0.39 0.01c 0.84 0.84 0.60 0.25 < 0.01d
Data expressed as median (interquartile range; in brackets). C1 and C2 = vehicle control (1 × and 25 × the maximum dose of pure vehicle); B1 and B2 = 1 × and 25 × the maximum daily dose of B. pinnatum MT; B3 and B4 = 50 × and 100 × the maximum daily dose in B. pinnatum Conc. a C1, C2 B1, B2, B3, B4. b B4 > C1, B1, C2, B2. c B4 > B1, C2, B2; C1 > B2. d C2 > C1, B1, B2, B3, B4.
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incision from xiphoid to pubic symphysis), and hysterotomy. The following parameters were recorded: weight gain; maternal and fetal mortality; number of implantations and resorptions; number and weight of fetuses and placentas; major external fetal malformations. At the end of the experiment, all animals were euthanized by terminal anesthesia. Quantitative variables were expressed as medians and interquartile ranges (IQR), and categorical variables as absolute (n) and relative (%) frequencies. The non-parametric Kruskal-Wallis test for independent samples was performed, and Dunn’s multiple comparison test used to assess between-group differences. The significance level was set at 0.05 (_ = 5%). All calculations were performed in the SPSS for Windows 15.0 software environment.
Results Descriptive statistics (medians and IQR) are shown in table 2. Comparative analysis of baseline rat weight showed a difference among the study groups (p = 0.03). Animals in groups C1 and C2 had similar weights, and were leaner than animals in the other groups; all other groups had similar baseline
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half volume in a RotaVapor® device, at 70 °C and a pressure of 600 mm Hg. The resulting product had an alcohol content of 10%, and was denoted B. pinnatum Conc. for the purposes of this study. On day 0 of gestation, 60 rats were randomly allocated to the following 6 groups: 2 control groups, C1 and C2; and 4 experiment groups, B1, B2, B3, and B4. Rats in group C1 received a maximum daily dose equivalent of vehicle (30% ethanol in water), and those in group C2, the equivalent to 25 × the maximum daily dose of vehicle. Rats in group B1 received the usual maximum daily dose of B. pinnatum MT (0.067 mL/kg/day) and those in group B2 the equivalent to 25 × the maximum daily dose (1.675 mL/kg/day). Animals in groups B3 and B4 were given B. pinnatum Conc. at a dose equivalent to 50 and 100 × the maximum daily dose of MT, respectively. The maximum daily dose was derived by calculating the equivalent to the recommended human dose (20 drops 4 times a day for a 60 kg patient = 4 mL/60 kg = 0.067 mL/kg). Table 1 lists the administered doses and the total flavonoid and ethanol content of each dosage. Flavonoid and ethanol content was measured by the Quality Control Department of Weleda in Brasil. Doses were diluted in water q.s.p. 1 mL and administered by gavage, through a metal feeding needle, once daily in the morning, from gestational day 0 to gestational day 20. At term (day 20), dams were anesthetized with xylazine hydrochloride 10 mg/kg and ketamine hydrochloride 100 mg/kg by intraperitoneal injection, underwent laparotomy (midline
We chose to assign 2 control groups (C1 and C2) due to the pharmaceutical form of the study product (MT, hydroalcoholic extract 30% for oral administration). Animals in control groups C1 and C2 were administered pure vehicle (30% ethanol in water) so as to provide comparative data on the potential effect of the drug vehicle on experimental group results, as alcohol intake is known to interfere with pregnancy in a dosedependent manner [28]. The choice of commercially available B. pinnatum MT as the pharmaceutical formulation for this experiment was prompted by its status as the only available extract obtained under strictly controlled conditions of plant cultivation, manufacturing practices, and quality control. We also took into account that using a hydroalcoholic extract in this experiment would contribute to the existing literature on herbal medicines made from B. pinnatum. The use of MT provides several advantages. Its manufacturing processes are standardized, it is readily available, and has a long shelf life [29]. However, in any experiment consisting of high-dose administration of herbal medicines prepared in alcoholic vehicles, one must control for the potential effects of alcohol content. Therefore, experimental group B2 (25 × the maximum daily dose of B. pinnatum MT) was paired with control group C2 (25 × the maximum dose of vehicle). In groups B3 and B4, which received 50 and 100 × the maximum daily dose of B. pinnatum MT, alcohol content was artificially reduced by concentrating the MT by rotary evaporation. Comparative analysis of median dam weight in the 6 study groups (C1, B1, C2, B2, B3, B4) revealed significant differences in baseline weight: rats in groups C1 and C2 were leaner
than those in all other groups. Due to between-group differences in weight at baseline and throughout pregnancy, we conducted a subanalysis of exclusive dam weight gain (total weight gain minus fetal and placental weight) at gestational day 20. At term, dams in group B4 had the greatest body mass gain. There was no significant difference in pooled fetal and placental weight (total litter weight). Weight change from baseline was greatest in groups B4 and C1, and body weight was lower in B2 dams than in C1 dams. Therefore, despite lower weight at baseline in groups C1 and C2, this difference did not influence fetal and placental weight at term. Subanalysis of exclusive dam weight gain enabled assessment of how much body mass was actually gained by dams regardless of offspring weight. Administration of B. pinnatum MT at a dose 25 × greater than the maximum daily dose (group B2) had no effect on fetal and placental weight. Breast, uterine, and adipose tissues account for most weight gained during pregnancy [30]. As there were no significant differences among study groups in fetal and placental weight, we believe the difference observed is attributable to less body fat accumulation in group B2. To date, no studies have confirmed any weight-reducing effects of B. pinnatum in obese subjects. However, due to its antianxiety effect [15], B. pinnatum may have reduced the avidity of feeding behaviors. Another hypothesis for the lesser weight gain observed in group B2 is a potential effect of ethanol present in B. pinnatum MT. As alcohol is a non-nutritive energy source, rats in this group may have exhibited decreased food intake [31]. Paradoxically, dams in group B4 (100 × the usual daily dose) exhibited the greatest weight gain as compared with groups B1, C2, and B2. There were no significant differences on comparison with groups C1 and B3. Hypothetically, dams in groups B3 and B4 would have experienced lower exclusive (non-placenta, non-fetal) weight gain if body mass followed a dosedependent trend, as suggested by less weight gain in group B2 than in group B1. As groups B3 and B4 received a concentrate in which alcohol content had been reduced by heating, we propose 2 explanatory hypotheses for these findings: 1. Higher doses of B. pinnatum (50 and 100 × the maximum dose) may interfere with the orexin/hypocretin system; 2. Heating may have altered the chemical composition of the original extract. It is widely known that the composition and concentration of active ingredients in herbal medicines depend on the methods of extraction, including the choice of vehicle, pH, temperature, extraction time, and other factors [32]. A recent study [33] found that cultivation and collection conditions as well as extraction methods influence the antileishmanial flavonoid content of B. pinnatum preparations. We raise the hypothesis that high-dose B. pinnatum therapy may interfere with regulation of the orexin/hypocretin system. The neuropeptides orexin A and orexin B (also known as hypocretins 1 and 2) are produced by neurons in the hypothalamus and play a major role in the regulation of feeding and wakefulness [34]. Leptin, a modulator of energy balance, is one
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weights. Between-group differences in dam weight at gestational day 20 were also significant (p < 0.01). Dams in group B4 were significantly heavier than those in groups C1, B1, C2, and B2, with no significant difference as compared to group B3. There were no differences in fetal and placental weight (p = 0.39). Comparison of exclusive weight gain, however (difference between rat weight at baseline and weight at gestational day 20, after subtracting the weight of the fetus and of the placenta) showed a significant difference among groups (p = 0.01). Dams in group B4 gained more weight than those in groups B1, C2, and B2, with no significant differences from groups C1 and B3. Furthermore, body weight was greater in dams from group C1 as compared with group B2. There were no between-group differences in number of fetuses and placentas, number of implantations and resorptions, or fetal and placental weight. There were no instances of maternal mortality or stillbirth. No significant external fetal malformations were observed in pups delivered from dams in any of the 4 dosage groups, except for control group C2 (livid pups).
of the main components of this system. Disruption of the circadian rhythm of leptin encourages feeding and reduces energy expenditure, leading to obesity, as observed in human subjects with narcolepsy [35, 36]. B. pinnatum is used in the treatment of sleep disorders and possesses neurosedative properties [15, 37]. Therefore, the sedative action of B. pinnatum and the association between B. pinnatum administration and weight gain observed in our experiment may suggest interference with the orexin system. Research into the interactions between diet, energy metabolism, and regulation of sleep and wakefulness is still incipient [38], and investigation of the mechanisms of action of B. pinnatum constituents only began in recent years. We did not find any significant differences among the study groups in the number of fetuses, number of placentas, number of implantations and resorptions, or total litter weight and placental weight. Fetal macroscopic changes were observed in control group C2, but not in group B2 (same ethanol content), suggesting a protective effect of B. pinnatum against damage caused by ethanol. Study Contribution This study assessed a herbal preparation used in AM that may be beneficial in the treatment of preterm labor, sleep disorders, and emotional disturbances (such as anxiety and fear) in pregnant women. We evaluated the potential harmful effects of B. pinnatum, a medicinal plant used widely by folk medicine in several countries, when administered to pregnant Wistar rats. The pharmaceutical form tested herein (MT) is standardized, easy to prepare, and has a long shelf life, thus facilitating access to the product by low-income populations. We were intrigued by the finding that B. pinnatum interfered with dam weight gain without affecting fetal or placental weight.
Study Limitations Reduction in ethanol content by rotary evaporation for preparation of B. pinnatum Conc. may have altered the chemical composition of the MT. Therefore, the chemical profile of the concentrated preparation may differ from that of the original extract, which would, in turn, introduce bias to our comparison of dosages. As the animals had free access to food, the amount ingested was not controlled. Perspectives In vitro and animal studies of the effects of B. pinnatum, including biochemical analyses and tissue microscopy, are warranted. Further studies are required to assess potential mechanisms of the effect of B. pinnatum on weight gain regulation during pregnancy.
Conclusion Daily administration of B. pinnatum MT to Wistar rats during pregnancy interfered with maternal weight gain. It was not associated with maternal or fetal death, did not produce macroscopic fetal malformations, and did not interfere with fetal or placental weight or number of implantations or resorptions.
Acknowledgments This study was supported by Associação Beneficente Mahle (ABM).
Disclosure Statement No conflict of interests exists.
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