Effect of Aqueous Extract of Hibiscus sabdariffa Calyx on the Red Blood Cell Count of Male and Female Wistar Rats
Atsukwei Denen*, Ameh O Grace, Seriki A Samuel
Department of Human physiology, Faculty of Basic Medical Sciences, Bingham University, Nigeria.
This study was conducted to evaluate the effects of aqueous extract of Hibiscus sabdariffa calyx on the red blood cell count of male and female wistar rats. Different doses of aqueous extract of Hibiscus Sabdariffa L calyx were administered orally by intra Oesophageal cannulation. The rats were divided into six groups. Group 1 rats (Control Group for males) and Group 2 rats (Control group for females), were administered distilled water for fourteen consecutive days. Group 3 rats (Low dose males), and Group 4 rats (Low dose females), were all administered 5mg Hibiscus sabdariffa aqueous extract for fourteen consecutive days. Group 5 rats (High dose males) and Group 6 rats (High dose females), were administered 10mg Hibiscus sabdariffa aqueous extract for 14 consecutive days. Results showed that high doses of the aqueous extract of Hibiscus sabdariffa calyx significantly lowered the red blood cell count of both male and female rats in Groups 5 and 6 (p<0.01). However, there was no significant difference (p>0.01) in the Red Blood Cell Count of Groups 3 and 4 (Low dose males and females respectively) which were all administered 5mg of the extract. This study shows that, aqueous extract of Hibiscus Sabdariffa calyx at high doses had no beneficial effects on the red cell count of the wistar rats. Thus, consumption of Hibiscus sabdariffa in high doses can cause generalized suppression of red blood cell production.
Key words: Hibiscus sabdariffa, red blood cell, anaemia, wistar rats
The use of plants in Medicine goes as far back as thousands of years ago and still continues today. Many plants are used for the treatment of different diseases and many possess antimicrobial activities (Arora and Kaur, 1999).
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Hibiscus sabdariffa is commonly called Roselle in English. It belongs to the family Malvaceae, which is native to Old World tropics; now cultivated annually throughout the tropics. It is an annual herb or woody-based shrub plant reported native to tropical Africa and grown in warm countries including Malaysia. The Petals may be red or green, its leaves are deeply three to five lobed, long, and arranged alternately on the stems.
All the parts of Hibiscus sabdariffa are used for medicinal purposes especially in alternative medicine (Ali et al., 2005). The plant is grown for the red or green calyces or petals of its flower which are mainly used to prepare herbal drinks, beverages, jam and jellies.
Besides the pleasant aroma and beauty of the plant itself, Hibiscus sabdariffa possesses qualities of traditional medicinal plants and food. As a medicinal plant, it is used as a therapeutic, a laxative, and anti-carcinogenic, an antihypertensive, a diuretic and a cholesterol lowering medicinal plant (McKay and Blumberg 2006). Also it exhibits great antioxidant activity, lowers hepatoxicity and reduces fever. Hibiscus calyx and leaves can be used in medicinal applications as a soothing reagent for cough, poor appetite and as an anti-bacterial agent. Almost all the parts of Hibiscus sabdariffa plant are considered diuretic and anti-scorbutic in their action (McKay and Blumberg 2006).
Hibiscus seeds are used in the production of vegetable oil in China and sometimes as a substitute for coffee. Hibiscus root is edible and used as an apperceive and tonic in the Philippines(Ali et al.2005).The fibrous part of Hibiscus sabdariffa plant is used in the production of twine and cord known as “rosella hemp” (McKay and Blumberg 2006).
The vegetable is widely grown and commonly used as port herb or soup in the northern part of Nigeria. In Hausa the plant is locally called Yakuwa, the seed ‘Isontea’ while the fresh calyx is referred to as Soboroto. The Yoruba call the leaves Amukan and the flowers Ishapa. The Igbo’s call it Okworoozo. However, it is commonly called Zobo in Nigeria (Adeguloye et al., 1993).
Nigerian researchers have shown how extracts of Hibiscus sabdariffa (commonly called Zobo) enhanced recovery from liver damage, offered cardiovascular protection, could be a good sedative, improved pregnancy outcome and helped in weight loss (Adeguloye et al., 1993).
All parts of hibiscus plants are used traditionally. Due to their soothing (demulcent) and astringent properties, the flowers and leaves have been traditionally used to treat conditions such as cancer and gallbladder attacks, to lower blood pressure, to relieve dry coughs, and topically to treat skin afflictions. The root has been used as a tonic (Onyenekwe et al., 1999).
The flowers contain substantial quantities of flavonoids and proanthocyanidins, which are associated with anti-oxidant, antipyretic, analgesic and spasmolytic activities (Onyenekwe et al.,1999).
MATERIALS AND METHOD
The fresh samples of Hibiscus sabdariffa calyx was obtained from Nyanya market a suburb of the Federal Capital Territory, Abuja, Nigeria.
Preparation of plant extract
It was then air dried at room temperature (36-37 degree Celsius), and grounded into powder using the Warring commercial blender. 200 g of the powder was dissolved in 1500ml of distilled water, it was stirred thoroughly, twice everyday for 3 days after which it was decanted, filtered using filter paper, and the residue discarded. The filtrate was then evaporated to dryness in an oven at boiling point of 100 degree Celsius, the resulting powder of the extract was scrapped from the crucible and then stored in capped bottles (Adegunloye et al., 1993).
The extract of Hibiscus sabdariffa was then dissolved in distilled water to make a stock solution (e.g., 2.5g of the extract of Hibiscus sabdariffa, dissolved in 500ml of distilled water to give a stock solution of 5mg/ml).The stock solution was preserved by refrigeration throughout the 14 days of administration.
Treatment of animals
Thirty Wister rats weighing between 180 to 230g were used throughout this investigation for uniform results. They were purchased from the animal breeding Unit of the National Institute of Pharmaceutical Research and Development (NIPRD) Idu, Abuja, Nigeria.
The animals were housed in clean steel cages and kept at room temperature. The wistar rats were fed twice daily with clean water and pelleted standard laboratory feed. They were allowed to acclimatize for 28 days prior to the commencement of the experiment. After the 28 days of acclimatization, the animals were divided into six groups of five each as shown below.
GROUP 1 (Control male group); comprising of five males were given 0.5ml of distilled water daily throughout the 14 days of oral administration
GROUP 2 (Control females); comprising of five females were given 0.5ml of distilled water daily throughout the 14 days of oral administration.
GROUP 3 (Low dose males); comprising of five males were administered 5mg of the Hibiscus sabdariffa calyx’s extract (orally) daily for 14 days.
GROUP 4 (Low Dose Females’ Group); comprising five females were administered 5mg of the Hibiscus sabdariffa calyx’s extract (orally) daily for 14 days.
GROUP 5 (High dose males); Comprising of five males were all given 10mg of the Hibiscus sabdariffa calyx’s extract (orally) daily for 14 days.
GROUP 6 (High dose females); Comprising of five females were all given 10mg of the Hibiscus sabdariffa calyx’s extract (orally) daily for 14 days.
Blood collection and storage
After the 14 days of oral administrations of the Hibiscus sabdariffa calyx extracts by intra oesophageal cannulation, blood samples were collected by severing the tip of the tail of each animal in all the groups including the control groups on day 15 of the experiment. To prevent coagulation, the blood samples were collected in EDTA bottles.
Red blood cell count procedures
The red blood cell count were estimated using haemocytometer method as described by Schalm et al., (1975
Results were expressed as Mean ± Standard Error of Mean. Statistical analysis was carried out using student’s t-test and P<0.01 was considered significant.
Results of total Red blood cell count
The mean value of red blood cell of Group 1 rats (Control males) was 8.90 ± 0.195, while that of Group 2 rats (Control females) was 8.70 ± 0.173. There was an extremely significant difference in the mean red blood cell values of rats in Group 1 (Control males) which had mean red blood cell value of 8.90 ± 0.195, and Group 5 (High dose males) which had mean red blood cell value of 5.90 ± 0.070, (p < 0.01). These rats were administered 10mg of the extract of Hibiscus sabdariffa calyx for 14 days. There was also an extremely significant difference in the mean Red blood cell values of Group 2 rats (Control females), with mean red blood cell value of 8.70 ± 0.173 and Group 6 rats (High dose females) which had mean red blood cell value of 5.90 ± 0.070,( p > 0.01). These rats were administered 10mg of Hibiscus sabdariffa calyx for 14 days.
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There was no significant difference between the mean Red blood cell values of Group 1 rats (Control males) which had mean red blood cell value of 8.90 ± 0.195, and Group 3 rats (Low dose males) administered 5mg of Hibiscus sabdariffa calyx for 14 days, which had mean red blood cell value of 8.62 ± 0.444, (p > 0.01). There was also no significant difference between the mean red blood cell value of Group 2 rats (Control females) which had mean red blood cell value of 8.70 ± 0.173 and Group 4 rats (Low dose females) administered 5mg aqueous extract of Hibiscus sabdariffa calyx for 14 days which had mean red blood cell value of 8.53 ± 0.321 , (p>0.01)
In this study, aqueous extract of Hibiscus sabdariffa calyx was administered orally at different doses.There was a significant difference in the mean red blood cell count values of rats in Group 5 (High dose males) when compared with rats in Group 1 (Control males), (p < 0.01).There was also a significant difference in the mean red blood cell count values of rats in Group 6 (High dose females), when compared with Group 2 (Control females), (p < 0.01).Groups 5 and 6 rats were each administered 10mg Hibiscus sabdariffa calyx’s extract.
There was no significant difference in the mean red blood cell count of Group 3 rats (Low dose males) when compared with Group 1 rats (Control males), (p>0.01). There was also no significant difference in the mean red blood cell count values of rats in Group 4 (Low dose females) when compared with rats in Group 2 (Control females), ( p > 0.01). Groups 3 and 4 rats were each administered 5mg Hibiscus sabdariffa calyx’s extract. Decrease in red blood cell counts may be due to decreased red blood cell production or increased removal of red blood cells from circulation. This results in anemia and may be caused by the direct toxic effect of aqueous extract of Hibiscus sabdariffa calyx on the bone marrow of red blood cell precursors (Fakeye et al., 2009)
This findings does not agree with the nutritionist in Central American markets that found the plant high in Calcium, Niacin, riboflavin and Iron (Gilani et al., 2009). Ordinarily, the plant being rich in Calcium, Niacin, Riboflavin and Iron, should increase red blood cell count values, but it rather decreased the red blood cell count values. This may be due to the degree of toxicity of the plant being high enough to suppress the positive effects of Calcium, Niacin, Riboflavin and Iron on the bone marrow of red blood cell precursors, thus causing a decrease rather than an increase in the red blood cell count of the wistar rats.
In the course of the 14 days of administration of Hibiscus sabdariffa calyx extract, a significant reduction in the weights of the rats was observed; this may be due to the diuretic properties of the plant (McKay et al.2010).
There were slight differences in the red blood cell count of male rats in Groups 1, 3 and 5 when compared with the female rats in Groups 2, 4 and 6. Hormonal differences have been linked to the difference in the red blood cell count of males and females. It has been assumed that females have high oestrogen level which suppresses action of erythropoietin, hence females have lower red blood cell count. Also, testosterone present in males have been assumed to have an accelerating effect on erythropoietin, hence, males have a slightly higher red blood cell count, though it has not been scientifically proven.
Apart from Hibiscus sabdariffa, other substances have been shown to exert different effects on red blood cell count. For example, researches have shown that spinach extract contains a high level of Iron and hence increases red blood cells in circulation (Koren, 2007). It has been discovered that garlic extract due to its mineral constituents e.g. Calcium and Iron, increases red blood cell count of mammals (including humans) (Oluwole, 2001). Accutane also known as isotretinoin, a drug used to treat severe acne or certain other skin conditions and cancer decreases red blood cell count , also alcohol (e.g. dry gin) has been discovered to reduce red blood cell count (Bunn et al., 2011).
The results of this work have shown that oral administration of aqueous extract of Hibiscus sabdariffa calyx in high dose decreased the red blood cell count of Wistar rats. This result suggest that excess consumption of Hibiscus sabdariffa calyx may lead to anaemia.
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Table 1: Effect of oral administration of aqueous extract of Hibiscus sabdariffa calyx on red blood cell count after 14 days of administration.
Group Amount of extract Red blood cell count
Group1(Control males) – 8.90 ± 0.195
Group 2(Control females) – 8.70 ± 0.173
Group 3(Low dose males) 5 8.62 ± 0.444
Group 4(Low dose females) 5 8.53 ± 0.321
Group 5(High dose males) 10 5.90 ± 0.070*
Group 6(High dose females) 10 5.90 ± 0.070*
Values are Mean ± S.E.M (n=5): * asterisks indicate significant difference (p<0.01) when compared with the controls.
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