**13.4.** *Calotropis procera* **(Aiton)**

**13.3.** *C. occidentalis* **L**

74 Herbal Medicine

*13.3.1. Animal toxicity*

*13.3.2. Human toxicity*

shown some level of toxicity [55, 56].

adverse effects occurred in a dose-dependent manner [57].

were observed in chicks treated with 0.3 and 0.5% of *Cassia* beans.

*C. occidentalis* is an annual shrub found in many African and Asian countries. Its leaves and roots are used in some traditional herbal medicines, but its pods or beans are avoided or used sparingly [52]. In Ghana, however, the roasted seeds are used as a beverage in the treatment of hypertension [53]. Many popular herbal tonics and medicines for liver disorders contain the leaves or roots of the plant. *C. occidentalis* has also been used in the treatment of scabies, snake and scorpion bites, diabetes, edema, fever, inflammation, rheumatism and ringworm. It is widely used

The fresh or dried/roasted seeds have demonstrated toxicity in several animal studies [54]. Toxicity in animals is usually seen on the kidney, liver, skeletal muscle and the heart. Grazing animals such as cattle, sheep, horses and goats have shown toxicity upon the ingestion of large amount of the seed pods, the most poisonous even though all parts of the plants have

Although the toxicity of the plant has been demonstrated in different animal species, the toxicity of the pod and bean is dose-dependent: low doses result in mild liver damage and myodegeneration while higher doses cause fatal hepatic degeneration followed by myodegeneration [56]. As the amount of *Cassia* in the animal's diet increases, muscle degeneration becomes a predominant characteristic of the poisoning and cause of the clinical signs. Roasting the seeds from the pod has, however, been shown to reduce the toxicity. Studies in rats [57] and chicken [58] fed a ration with *C. occidentalis* seeds at different concentrations showed histopathological and biochemical changes in muscles, liver and central nervous system. Barbosa-Ferreira et al. [57] in a study involved Wistar rats in four groups of 10 animals each, three of them fed rations containing 1, 2 and 4%, respectively, of *C. occidentalis* seeds, and the control fed normal commercial ration for a period of 2 weeks; rats in the experimental groups showed lethargy, weakness, among other adverse reactions. Histopathological study showed fiber degenerations in the skeletal and cardiac muscles. In the liver parenchyma, vacuolar degeneration was observed and, in the kidney, mild necrosis in the proximal convoluted tubules. All the

Haraguchi et al. [58] studied the chronic effect of varying concentrations of *C. occidentalis* seeds in broiler chicks. All birds were killed on day 49 of age. Low doses of seeds showed no significant variation in biochemical parameters compared to the control group. Degenerative changes in striated skeletal muscles particularly pectoral as well as the liver and myocardium

Studies have shown that the ingestion of *C. occidentalis* can cause severe purging possibly due to the anthracene glycoside content [59]. Whereas this may produce great discomfort and pain in adults, in a child, this can be fatal; thus, while few pods might not have any ill effect

for the treatment of bacterial and fungal infections and to boost the immune system.

The plant is widely distributed in Asia, tropical and subtropical Africa [64]. In ancient Egypt, it was recommended for the treatment of nodular leprosy [64]. In Indian traditional medicine, the decoction is used for the treatment of asthma, dysentery, rheumatism, fever, painful muscular spasm and as a purgative and expectorant [65], and as a proteolytic enzyme for the coagulation of cow milk in Ghana [66]. The extract from the plant has been reported to possess antibacterial, nematocidal and larvicidal [67] and anticancer [68] properties. The flower of the plant has been shown to possess potent antimicrobial and anti-inflammatory activities [69].


*4.1. Cell Lines*

HOG cell line, established from a surgically removed human oligodendroglioma by Dr. A. T. Campagnoni (University of California, UCLA, Berkeley, CA, USA) [73] was kindly provided by Dr. J. A. López-Guerrero (Universidad Autónoma de Madrid, Madrid, Spain) [74]. Cells were grown in DMEM, low glucose, pyruvate, HEPES (22320- 022, Invitrogen, Paisley, Scotland, UK), 100 units/mL penicillin/streptomycin (17-602E, Invitrogen, Paisley, Scotland, UK), and 10% (*v*/*v*) heat inactivated fetal bovine serum (FBS) (10270-106, Invitrogen, Paisley, Scotland, UK).

Human neuroblastoma SH-SY5Y cell line was obtained from Sigma (ref 94030304, Sigma-Aldrich, St. Louis, MO, USA) and was grown in DMEM supplemented with 2 mM L-glutamine (61965-059, Invitrogen, Paisley, Scotland, UK), 100 units/mL penicillin/streptomycin (17-602E, Invitrogen, Paisley, Scotland, UK), 1% nonessential amino acids (11140-035, Invitrogen, Paisley, Scotland, UK), and 10% (*v*/*v*) heat inactivated FBS (10270-106, Invitrogen, Paisley, Scotland, UK).

Cells were maintained at 37 ◦C in a humidified atmosphere of 5% CO2 and were passaged when they were 80–90% confluent, i.e., approximately twice a week for no more than 20 passages.


3.2.1. Exploratory Factor Analysis

Exploratory factor analysis was conducted based on 98 observations for two dimensions: accessibility and effectiveness. Variables D4 and D5 did not meet the normality assumption. They were therefore removed from the EFA model. Variable D1 did not load correctly on the expected factors—accessibility. Eventually, eight variables: D2, D3, D6, D7, E1, E2, E3, and E4 were left. The principal component analysis (PCA) and promax rotation with Kaiser normalisation were used to extract two components (Table 5). The Kaiser-Meyer-Olkin measure of sampling adequacy (KMO) equalled 0.73 > 0.6. The KMO value considered as correct is 0.6. Bartlett's test of sphericity provided a significant result (χ2 = 201.125; df = 28, *p* < 0.0001). The probability *p* should be smaller than 0.05, thereby indicating that the values are correct and the sample size is sufficient for the factor analysis.

**Table 5.** Pattern matrix for the EFA model.

Extraction Method: Principal Component Analysis. Rotation Method: Promax with Kaiser Normalization, a. Rotation converged in 3 iterations.

PCA retained two factors with eigenvalues greater than 1. The total variance explained by the EFA model was equal to 56% (Table 6), which should be greater than 50% [78]. For eight variables, the factor loadings ranged from 0.638 to 0.837 and are greater than the recommended 0.35 cut-off point [79]. A reliability analysis showed that the extracted model was acceptable since the Cronbach's alpha coefficients for accessibility (0.666) and effectiveness (0.663) were greater than 0.6 [80]. Those values (Table 6) allowed for further factor analysis [65,81].

**Table 6.** Eigenvalues and total variance explained by the EFA model.

Extraction Method: Principal Component Analysis.