Abstract
Bryophyllum pinnatum. Linn (Crassulaceae) is a shrub with simple or trifoliate petiolate leaves used mainly in folk medicine to alleviate pains of various intensities and etiologies. The analgesic potency of the aqueous extract of the leaves of B. pinnatum. was investigated using animal models. Results showed that the aqueous extract of B. pinnatum. was devoid of severe toxic effects (LD50 = 660.9±2.65 mg/kg body weight), increased the pain threshold in rats using the hot plate or thermal methods, inhibited or reduced phenylbenzoquinone-induced writhing or abdominal stretches in mice in a dose-dependent manner, and produced a weak or an inferior anti-inflammatory activity than aspirin. It is concluded that the aqueous extract of B. pinnatum. can demonstrate strong analgesic potency comparable in a times-and dose-dependent manner to a nonsteroidal anti-inflammatory drug.
Introduction
Human beings have used plants as medicines for several centuries; the Greeks, the Indians, the Chinese, and the Africans have cured many afflictions using plants and plant extracts. One such plant is Bryophyllum pinnatum. Linn (Crassulaceae), a fleshly shrub that is about 60–120 cm high and branched from the base with opposite, simple or trifoliate petiolate leaves. It is commonly used in traditional medicine as a painkiller, particularly in cases of earache, menstrual pains, and breast abscess. For the treatment of earache, the juice from the boiled leaves is squeezed into the affected ear; for the treatment of breast abscess, the crushed leaves are mixed with kaolin or china clay and applied externally to the entire breast. In the management of menstrual pain, kaolin mixture of the whole plant extract is applied on the waist and the umbilical region.
The plant has also been reported to be used in the treatment of headache, as a diuretic, and also for shaving (Dalziel & Hutchingson, Citation1958). It is also applied to burns and, when rubbed on bodies of febrile children, the material had an antipyretic effect (Dalziel, Citation1956). The root extract of B. pinnatum. has also been used to treat coughs and burns (Iwu, Citation1982). Among the Igbos of Nigeria, the plant has had a long history of remarkable health properties.
This study was designed to evaluate the analgesic activity of the crude extract of B. pinnatum. using animal models.
Materials and Methods
Collection and identification of the plant material
The plant was collected from Ukpor, Anambra State, Nigeria, during the dry season and was identified independently by K.K. Agwu, Chief Herbarium Officer, Department of Pharmacognosy, and by A.D. Ozioko, Botany Department, Herbarium Section, University of Nigeria, Nsukka. A voucher specimen is deposited in the University Herbarium with voucher no UNH 386.
Extraction procedures
Fresh leaves of B. pinnatum., 500 g wet-weight, were crushed with a hand mill, steeped in distilled water, and pressed mechanically using a Carver lab press in a muslin bag in order to extract the active components until the residuum was dry. Standardization of the extract of the leaves of B. pinnatum. was accomplished by evaporating to dryness using the rotary evaporator at 40°C. This gave the aqueous extract used in the study, and the yield was 35%.
Experimental animals
Analgesia experiments were performed on Wistar rats of either sex weighing 120–250 g and adult mice weighing 20–30 g. All experimental procedures followed the recommendations of the Committee for Research and Ethical issues of the International Association for the Study of Pain (Covino et al., Citation1980) and the guidelines on ethical standards for investigations of experimental pain in animals (Zimmerman, Citation1983). The number of experimental animals was kept to a minimum; they were maintained in an animal room at a constant temperature of 32°C with a 2-h alternating light/dark cycle. Twelve hours before the experiment, food was withheld, but animals had free access to water. All experiments were performed during the light phase with each animal being used only once.
Phytochemical analysis
The aqueous extract was analyzed by thin-layer chromatography (TLC) or tube test using specific reagents according to the methods described by Marini-Bettolo et al. (Citation1981).
Acute toxicity studies in mice
Adult mice of either sex weighing between 20 and 30 g were divided into 5 groups of 10, and mice in each group received the same dose intraperitoneally (i.p.) of the aqueous extract of B. pinnatum., 50–1000 mg/kg body weight. The number of deaths in each group 24 h later was recorded and the percentage mortality calculated. The percentage mortality was plotted against the log-dose and the LD50 determined. Based on the results of preliminary studies, the doses of 100, 200, and 300 mg/kg body weight of B. pinnatum. were chosen for further experiments.
Analgesic study (hot-plate method)
The hot-plate method of Woolfe and MacDonald (Citation1964) was employed with modifications, using a Pan lab 475 hot plate kit at a constant temperature of 45°C. Adult Wistar rats of either sex weighing between 200 and 250 g were used. The rats were randomly divided into 5 groups of 6 rats per group. Groups 1–3 received 100, 200, and 300 mg/kg body weight of the aqueous extract, respectively, while groups 4 and 5 were given 100 and 25 ml/kg body weight of acetylsalicylic acid (positive control) and normal saline (negative control), respectively.
Phenylbenzoquinone-induced writhing in mice
This method is based on that described by Otterness and Bliven (Citation1985) for measuring the analgesic potency of nonsteroidal anti-inflammatory drugs. It was derived from the writhing model (Siegmund et al., Citation1957). Male mice of about 4 weeks of age and weighing 20–25 g were fasted for 24 h before the experiment but had free access to water. This fasting period maximizes the pain response to the irritant agent. The mice were all randomized into five groups of four mice per group and were given the drug solution orally by gavage, that is, aqueous extract at doses of 100, 200, and 300 mg/kg body weight to the first three groups, respectively, while the control animals (positive) received 100 mg/kg body weight of aspirin and the negative control received normal saline 25 ml/kg body weight. Thirty minutes after oral administration, 2 mg/kg of phenylbenzoquinone was administered intraperitoneally. After a 5-min interval, the number of abdominal stretches was counted during the next 5-min. Analgesic efficacy was expressed as the percentage decrease in abdominal stretches of treated versus untreated animals.
Carrageenan-induced rat paw edema
Male rats weighing 120–200 g fasted overnight to increase absorption were randomized in groups of five: A, B, C, D, and E. The aqueous extract of B. pinnatum. was administered orally to groups A, B, and C at doses of 100, 200, 300 mg/kg body weight, respectively, while the control animals, groups D and E, received 100 mg/kg of aspirin body weight and 25 ml/kg body weight of normal saline, respectively. All animals had access to food and water ad libitum..
One hour after the oral administration of the aqueous extract of B. pinnatum., inflammation was induced by subplantar injection of 0.1 ml carrageenan solution (1% in normal saline) into the right-hand footpad. The degree of inflammation was assessed by measuring the volume of the animals right foot by means of a water plethysmograph (Ugo Basile) at 1-, 3-, and 6-h intervals after inducing the inflammation. The volume of untreated left hind limb of each animal was also measured as a reference. Throughout the study, the animals were kept at room temperature to promote edema formation.
Analgesic/anti-inflammatory effect was expressed as the mean percentage inhibition of inflammation compared with the control 3 h after induction of inflammation for each treatment group.
Drugs used
The following drugs that were used in this study were purchased from the Sigma Chemical Company (St. Louis, MO, USA): phenylbenzoquinone, acetylsalicylic acid, and carrageenan. All drug solutions were prepared fresh prior to use.
Statistical analysis
Data are expressed as means±SEM. Differences between groups (extract-treated and controls) were considered to be significant at p < 0.05 using ANOVA.
Results
Preliminary phytochemical analysis by thin-layer chromatography using specific reagents (Marini-Bettolo et al., Citation1981) showed that the extract was rich in alkaloids, flavonoids, glycosides, lipids, saponins, steroids, and so on.
Acute toxicity studies in mice
The LD50 value after i.p. administration of aqueous extract of B. pinnatum. in mice was 660.9 ± 2.65 mg/kg body weight. It was also observed during the study that the extract did not produce any external symptoms such as wet fur, and the animals moved freely.
Hot plate (thermal pain)
shows the results of the analgesic testing of the extract of B. pinnatum. using the hot plate method. The analgesic effect of the extract had a maximum percentage increase of 193.5% at a dose of 300 mg/kg, while at 100 and 200 mg/kg, the extract gave percentage increases of 88.5 and 175.0%, respectively. For each dose of the extract, the analgesic activity increased with time up to 90 min after which the effect declined. A similar pattern was observed with acetylsalicylic acid (aspirin), the positive control. Using the Student's t.-test and a 95% confidence limit, there were no significant differences between the pretreatment reaction time (PTRT) and after treatment reaction time (ATRT) when normal saline was given.
Table 1.. Analgesic activity (hot-plate method) of the aqueous extract of B. pinnatum..
Writhing pain induced in mice
Five minutes after i.p. injection of phenylbenzoquinone in mice, the number of writhings was counted for 5 min. The mean value was 16 constrictions with a range of 5–35. After oral administration of B. pinnatum. extract, there was a significant reduction (inhibition) of writhing compared with the control. Potent analgesic activity was demonstrated by the extract with about 80% inhibition at the highest dose (300 mg/kg) and 60% inhibition at the least dose of 100 mg/kg. There was a clear dose-effect relationship ().
Table 2.. Analgesic effects of aqueous extract of B. pinnatum. and acetylsalicylic acid against writhing test in mice and carrageenan-induced rat paw edema.
Rat paw edema
Parallel analgesic activity was demonstrated by the aqueous extract of B. pinnatum. with a dose-dependent anti-inflammatory activity similar to acetylsalicylic acid but lower in intensity and efficacy. Acetylsalicylic acid (aspirin) inhibited inflammation by 54% at 100 mg/kg body weight, whereas the aqueous extract achieved about 18% inhibition at 300 mg/kg body weight (). The aqueous extract of B. pinnatum. did not prevent the formation of edema but facilitated the use of the leg, thus demonstrating analgesic activity, whereas in the control (saline-treated group) there was total or complete demobilization.
Discussion
The index normally used in assessing analgesic activity in experimental animals is failure of the animals to respond to pain within a given period (Beckett & Casy, Citation1962). In the current investigation, the indices for assessing analgesic activity were the prolongation of response of rats exposed to heat stimuli (hot plate or thermal pain) maintained at 45°C, decrease in writhing or abdominal stretches in mice induced by phenylbenzoquinone, and drug-induced rat paw edema.
Data obtained from the acute toxicity studies in mice showed that the LD50 was 660.9 ± 2.65 mg/kg body weight, and at the doses investigated the extract of B. pinnatum. was devoid of toxic effect such as wet fur. Furthermore, at the dose level investigated, the extract demonstrated high analgesic activity by decreasing or inhibiting the writhing or abdominal stretches in mice induced by phenylbenzoquinone. The analgesic potency of the plant extract is equivalent to that obtained with acetylsalicylic acid (3:1) in this study or as described by Chau et al. (Citation1993). However, on a weight-for-weight basis, aspirin is only 1.3-times more potent than the extract as an analgesic while the standard drug is yet a stronger anti-inflammatory agent than the extract (3:1), whereas the equianalgesic dose of the extract is three-times the dose of the standard ().
The greater anti-inflammatory activity demonstrated by aspirin might be due to its capacity to inhibit peripheral prostaglandin biosynthesis, which also may contribute to its analgesic action. Furthermore, the vasodilatation caused by aspirin might contribute to the reduction in inflammation and pain due to antagonistic effects of aspirin to brandykinin, a potent algesic agent. These mechanisms seem to be lacking in the aqueous extract of B. pinnatum., hence its inferior or weak anti-inflammatory effect in rat paw edema.
The efficacy and potency of the extract as an analgesic were demonstrated in all the models used. shows that the analgesia produced by the extract of B. pinnatum. was dose- and time-dependent, maximizing at 90 min postadministration after which there was a gradual return of pain reflex. Again, the analgesic effect was dose-dependent reaching a maximum at 300 mg/kg body weight.
Taken together, these results have demonstrated that the extracts of B. pinnatum. are a potent analgesic by oral route comparable to acetylsalicylic acid, a standard nonsteroidal anti-inflammatory drug. The use of B. pinnatum. has continued to be a popular natural remedy throughout the tropics where it grows.
In conclusion, the results presented in this study confirm the popular use of B. pinnatum. against pain associated with local reactions and conditions. The aqueous extract revealed potent and significant analgesic properties against all the models studied and minor anti-inflammatory activity against carrageenan-induced rat paw edema. The elucidation of the exact mechanism of action requires further investigation, and studies are currently in progress to isolate and identify the components responsible for the analgesic effects demonstrated by the aqueous extract of B. pinnatum..
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