Abstract: The present research dealt with the evaluation of the anti-diarrheal activity of two therapeutic diets in model of castor oil induced diarrhea in rats. Formula I contain modest amount of skimmed milk, formula II was lactose restricted diet. Both formulae contain cereals, legumes, honey and edible source having anti-diarrheal activity. The nutritional value of the two formulated therapeutic diets was evaluated in normal growing rats in comparison to reference formula, milupa special formula, in addition to control balanced diet (contain 10% protein supplemented from casein). The evaluation of nutritional value depended on determination of total food intake, body weight gain, food efficiency ratio and protein efficiency ratio. Nutritional status of rats fed different diets was also evaluated through determination of certain biochemical parameters such as percentage haematocrit, blood haemoglobin concentration, plasma total protein, albumin, iron, phosphorus, zinc, magnesium, retinol and β-carotene. Results showed that anti-diarrheal activity of formula I was superior compared to formula II. Milupa and formula I have higher values of protein efficiency ratio and food efficiency ratio than control casein diet which were significant in case of Milupa. However diet II showed comparable values to control. Biochemical parameters showed higher values of plasma total protein, magnesium and retinol of rats fed Milupa diet. Feeding diet II produced significant increase of plasma iron magnesium and retinol. However, only significant increase of plasma magnesium has been observed when feeding diet I.
Introduction
Diarrhea, particularly persistent diarrhea is among the most important causitive factors of malnutrition (Boehm, et al., 1998). However, Javaid et al. (1991) reported that malnutrition itself contributes in the high incidence of diarrhea. So, it was of importance to formulate new therapeutic diets having both anti-diarrheal activity and simultaneously supplement the body with protein, energy and certain essential micronutrients needed during diarrheal episodes to improve the nutritional status. Micronutrients that have been postulated to have potential beneficial importance during diarrhea are zinc, vitamin A and folic acid (Fuchs, 1998; Gracey, 1999; Shoda et al., 2002). Plants constituents that have been reported to have anti-diarrheal effects are pectin, tannins and ß-carotene (Balbaa et al., 1981). Pectin has the property of conjugating toxins and enhancing the physiologic functions of the digestive tract through its physical, chemical and antibacterial properties. It is of value in the treatment of intestinal disorders such as diarrhea and dysentery. Pectic substances (Whistler and Smart, 1953) occur without exception in all higher plants. They are found most abundantly in the primary cell wall and in the intercellular layer, but secondary walls contain small amounts. Some plant juices are quite rich in these substances. Pectins are more abundant in fruits and roots.
Carrot and soybean have been reported to contain pectin (Konno et al., 2002; Fransen et al., 2000). Carrot is rich in ß-carotene (Chindavijack et al., 1996) which is a precursor of vitamin A and has been reported to have a beneficial effect towards diarrhea (Gracey, 1999). Tannins present in Ceratonia siliqua and Ciser arietinum (Priolo et al., 2000; Al-Adawy, 2002) have been reported to have antibacterial and antiviral activity, thereby may have beneficial effects during infective diarrhea. Cicer arietinum, Ceratonia siliqua, carrot and rice have been used in Egypt since ancient times to treat diarrhea in Folk medicine. Some authors showed honey to have anti-diarrheal effect and to improve blood haemoglobin of anaemic patients (Hassan and Shawky, 1997). The anti-diarrheal effect of honey might be due to its anti-microbial activity (El-Fikky and Ibrahim, 1997) and through improvement of immune function (Melentkova et al., 1993).
The present research was postulated to make use of some of the previously mentioned edible plants such as Cicer arietinum, Ceratonia siliqua, carrot, rice and soybean in two formulae for infants with diarrhea, in addition of using honey as sweetener. One formula contained chicken meat as source of animal protein (lactose restricted formula), the other formula contained modest amount of skimmed milk and fortified with zinc and vitamin A.
The aim of the present research is to study the anti-diarrheal activity of the newly formulated therapeutic diets in rats. The aim includes evaluation of the nutritional value of the therapeutic diets in rats through determination of body weight gain, protein efficiency ratio and food efficiency ratio with the assessment of different biochemical parameters reflecting nutritional status.
Materials and Methods
I. Biological Evaluation of the nutritional value of the therapeutic diets and the nutritional status of rats fed these diet
Materials
Therapeutic diets: In a previous work (Soliman et al., 2001) we have formulated and prepared two therapeutic diets for diarrhea, the constituents and chemical composition of which are present in Table 1. These diets were biologically evaluated in rats, in the present study. Milupa HN25 special formula (made in Germany by Milupa GmbH and Co, KG) was used as reference formula, its chemical composition is shown in Table 1 as written on packing. Some of the chemical constituents are not appeared on packing such as Fe, Zn and ash.
Experimental diets: Appropriate amounts of each of the previously prepared therapeutic diets and reference formula were taken so as to contain 10 g protein. The fat content was completed by corn oil to 10%, fibres were completed to 3% by cellulose. An amount of 3.5% salt mixture (Briggs and Williams, 1963), 1% water soluble vitamin mixture (the oil soluble vitamins were given weekly to rats separate from the diet) (Morcos, 1967) and 24.1 g% sucrose were added to each diet. The diets were completed by corn starch to 100%. A control diet containing 10% protein supplemented from casein was prepared. The composition of the different experimental diets are shown in Table 2.
Animals: White male and female albino rats of 50–60 g body weight were used in the present experiment. The animals were kept individually in wire bottomed cages at room temperature of 25±2°C.
Methods: This experiment was designed to evaluate the protein efficiency ratio of the formulated therapeutic diets in comparison to reference diet (milupa) and a casein diet as control. In addition of assessing their effect on body weight, food efficiency ratio and biochemical parameters reflecting nutritional status.
Rats were divided into 4 groups each of 8 rats. The rats of each group were fed one of the prepared experimental diets present in Table 2 for 4 weeks. During the period of the experiment, the food intake was calculated and the rats were weighed twice weekly. Growth curves were drawn representing the relationship between the rats body weight and time. After the end of experimental period, biological changes including food intake, body weight gain, food efficiency ratio (body weight gain/total food intake) and protein efficiency ratio (body weight gain/total protein intake) were calculated. Growth rates of rats of different groups were calculated as g/g body weight/day. At the end of experiment rats were fasted 16–18 h. The blood samples were drawn for determination of haemoglobin concentration (Betke and Savelsberg, 1956) and percentage haematocrit (Strumia et al., 1954). Plasma was separated from heparinized blood by centrifugation for determination of plasma total protein (Rheinhold, 1953), albumin (Doumas et al., 1972), iron (Tabacco et al., 1981), phosphorus (Gamst and Try, 1980), calcium (Gitelman, 1967), zinc (Homsher and Zak, 1985), magnesium (Gindler and Heath, 1971), retinol and ß-carotene (Neeld and Pearson, 1963). The results were statistically analysed using student's t-test.
II. Anti-diarrheal test (Iwao and Terada, 1962; Niemegeers et al., 1981)
Materials
| • | Rats: Female albino rats weighing 138.5 g±4.143 SE were used in the present experiment. |
| • | Therapeutic diets: Formula I and formula II shown in Table 1. |
| • | Ceratonia siliqua pods without seeds, reduced to very fine powder. |
| • | Roasted Cicer arietinum reduced to very fine powder. |
| • | Carrot, dried and grinded to fine powder. |
| • | Castor oil. |
Methods: Rats were used after overnight food deprivation. For the experiment the rats were housed in individual cages with no access to drinking water. Ratswere divided into two division, the first contain 2 group (gp1, gp2), the second contain 3 groups (gp3, gp4, gp5). Each group included 6 rats. Four grams of either formula I or II (after adding very little equal amount of water) were given orally by gavage to each rat of gp1 and gp2 respectively. While, 2.5 g of Ceratonia siliqua, Cicer arietinum and carrot were given orally as previous to each rat of gp3, gp4 and gp5 respectively. One hour later, 1 ml of castor oil was administered orally to each rat. Stools were collected on non wetting paper sheets of uniform weight up to 24 h after administration of the castor oil. Every 15 min during the first 8 h, urine is drained off by gravity and the net stool weight, termed early diarrheal excretion, was recorded.
| Table 1: | Chemical composition of the newly formulated therapeutic diets for diarrhea and reference formula (milupa) per 100 g |
| *RE = Retinol equivalent | |
| Ingredients of milupa: | Lactose reduced skimmed milk powder, precooked rice, banana powder, maltodextrin, sodium caseinate, apple powder, vegetable fat, starch, calcium carbonate, vitamins and sodium chloride |
| Ingredients of formula I: | Roasted Cicer arietinum (chick pea), soybean powder, precooked wheat, Ceratonia siliqua (Carob), skimmed milk (all the ingredients were dried and reduced to powder form), zinc sulphate, vitamin A and honey |
| Ingredients of formula II: | Precooked chicken meat (breast without skin), soybean powder, precooked rice, carrot (all the ingredients were dried and reduced to powder form) and honey (lactose restricted formula) |
| Table 2: | Composition of the different experimental diets (g/100 g diet) |
| *Composition of : 47.619 g of formula I 42.667 g of formula II 57.143 g of milupa | |
The diarrhea-free period is defined as the time between castor oil administration and the occurrence of the first diarrheal output. Stool occurring between 8 and 24 h after castor oil administration are called late diarrheal excretion. Evaluation of anti-diarrheal effect correlates with the decrease of 24 h stool output (Stool weight) and with increase of diarrhea-free period. Inhibitors of prostaglandin biosynthesis increase the diarrhea free period but do not affect early diarrheal secretion (Niemegeers et al., 1981).
Parameters of rats of gp1 and gp2 were compared with each other. Parameters obtained of rats of gp3, gp4 and gp5 were compared with each others. Statistical analysis of student's t-test was applied to the results.
Results and Discussion
Table 3 showed that rats fed diet I showed significant increase in final body weight (P < 0.05), body weight gain (P < 0.01) and total food intake (P < 0.001) with remarkable increase in both protein efficiency ratio and food efficiency ratio compared with control. Feeding milupa diet produced significant increase in final body weight (P < 0.05), body weight gain (P < 0.025), food efficiency ratio (P < 0.025) and protein efficiency ratio (P < 0.05) with remarkable non significant increase in total food intake. Rats fed diet II showed significant increase in total food intake with non significant change in body weight gain, food efficiency ratio and protein efficiency ratio. Growth rates of rats were only significantly higher when feeding either milupa (P < 0.01) or diet I (P < 0.001) compared to control. Growth rate of rats fed diet II showed non significant change compared to control.
Although, protein efficiency ratio of milupa diet is higher than that of diet I and II and significantly higher than control casein, however the significant increase of total food consumed by rats of diet I and II may show important advantages when given to infants with diarrhea. Since those patients usually suffer anorexia (Fuchs, 1998) because this effect reflect the palatability of diet I and II which may show better taste than milupa.
Growth curves of rats (Fig. 1) showed that rats fed diet II showed gradual increase in body weight during the experimental period which did not show non significant change when compared to control casein i.e. comparable to casein diet. Growth curves of rats fed diet I also showed gradual increase in body weight which was significantly higher than control after 15 days, 21 days, 24 days and 28 days of the start of experiment. Milupa diet produced significant increase in body weight after 21 days, 24 days and 28 days compared to control casein.
| Table 3 : | Nutritional parameters of different experimental groups. (Values are means±S.E) |
| Values significantly differ from control :a : P < 0.05, b: P < 0.025, c : P < 0.01, e : < 0.001 | |
| Table 4: | Biochemical parameters of rats of the different experimental groups (Values are means±SE) |
| Values significantly differ from control : a : P < 0.05, b : P < 0.025, d : P < 0.005, e : P < 0.001 | |
| Table 5: | Parameters reflecting anti-diarrheal effect of formula I and II, Ceratonia siliqua, carrot and Cicer arietinum (Values are mean±SE) |
| Significant value: ***: P < 0.01 | |
Biochemical parameters (Table 4) showed that feeding milupa diet produced significant increase in plasma total protein (P < 0.005) which coincided with its significant high protein efficiency ratio i.e. improve protein status which was not reflected by significant increase in albumin, so it may be reflected in globulin fraction. Significant increase in plasma magnesium and retinol were also noticed (P < 0.005 and P < 0.025 respectively).
Feeding diet II showed significant increase in plasma iron (P < 0.05) compared to control. Although diet I contain higher contents of iron than diet II, however diet II has a beneficial effect in elevating plasma iron than diet I which might be due to presence of tannins rich sources in diet I such as Ceratonia siliqua and chick pea (Priolo, et al., 2000 and Al-Adawy, 2002), that may hinder iron absorption (Roe, 1978) however, this did not produce any reduction on plasma iron of rats fed diet I which may be due to presence of honey that have been reported to improve iron status (Hassan and Shawky, 1997). However, the presence of Ceratonia siliqua and Cicer arietinum are essential as they have beneficial anti-diarrheal effect (Loeb, et al., 1989; Garcia and Harum, 1975).
Plasma retinol showed significant higher levels (P < 0.05) when diet II was fed. Significant higher levels of plasma magnesium have been noticed in rats fed diet I and II (P < 0.005 and P < 0.05 respectively). The increased magnesium level might have beneficial effects in infants with diarrhea since infants with chronic diarrhea are usually magnesium depleted (Mittal et al., 1990). No other significant changes have been noticed in other biochemical parameters.
The induction of diarrhea with castor oil results from the action of ricinoleic acid formed by hydrolysis of the oil (Iwao and Terada, 1962; Watson and Gordon, 1962). Ricinoleic acid produces changes in the transport of water and electrolytes resulting in a hyper-secretory response (Ammon et al., 1974). In addition to hyper secretion, ricinoleic acid sensitizes the intramural neurons of the gut, Recently Uchida et al. (2000) suggest the involvement of nitric oxide from nerves on the diarrhea induced by castor oil in rats i.e. nitric oxide is involved in the mechanism of castor oil-induced diarrhea.
Table 5 showed formula I more superior than formula II when testing anti-diarrheal activity in castor oil model in rats. This evaluation based on that diarrhea free period of rats given formula I (6.3±1.078 h) was significantly higher than that of formula II (2.007±0.609 h.) (P < 0.01), in addition of early diarrheal excretion and stool out put after 24 h of rats given formula I were less than that of formula II.
| Fig. 1: | Growth curves of the different experimental groups Values are significantly differ from control a: P<0.05 b: P<0.025 c: P<0.01 |
However, late diarrheal excretion is higher when formula I was given compared to formula II, which may refer to that the onset of anti-diarrheal activity of formula I was faster than formula II and that formula II had delayed onset of action.
When comparing rats given Ceratonia siliqua, carrot or Cicer arietinum, it was noticed that diarrhea free period was the highest and stool out put after 24 h was the least after giving Cicer arietinum, followed by Ceratonia siliqua then carrot i.e. Cicer arietinum was the best anti-diarrheal agent followed by Ceratonia siliqua then carrot. However, it was noticed that Cicer arietinum although superior as anti-diarrheal agent and though increased diarrheal free period, the early diarrheal excretion was high compared to rats given Ceratonia siliqua and carrot which may let us suggest that Cicer arietinum may have prostaglandin inhibiting activity as referred by Niemegeers et al. (1981). The presence of Cicer arietinum and Ceratonia siliqua in formula I which were superior as anti-diarrheal agent than carrot may reflect the superiority of formula I compared formula II that contain carrot.
The choice of the different ingredients of formula I (roasted Cicer arietinum, soybean, precooked wheat, Ceratonia siliqua, skimmed milk, zinc, vitamin A and honey) and formula II (Precooked chicken meat, soybean, precooked rice, carrot and honey) based on that most ingredients have been shown previously to have beneficial role towards diarrhea.
Sotelo et al. (1987) reported that both soy formula and chick pea proved promising in children with lactose intolerance with diarrhea, however diarrhea was better controlled by chick pea. In addition, chick pea flour has been used in the treatment of acute diarrhea of the infants by Garica and Harum (1975). Tannins in chick pea (Al-Adawy, 2002) may be one of the active constituents that have anti-diarrheal activity. However, Ouhida et al. (2002) reported that soybean is rich in pectin. Nakamura et al. (2001) proved that soybean soluble polysaccharides extracted from soybean cotyledons have a pectin-like structure. These constituent may be the cause of anti-diarrheal activity of soybean.
Mixed diets containing carrot flour and wheat flour versus lactose-free, soy protein formula showed comparable results in management of diarrhea (Alarcon, et al., 1991). The presence of pectin in carrot (Messiaen and Van Cutsem, 1999) may be one of the cause of beneficial effect of carrot as anti-diarrheal agent. In addition carrot can replace enteral loss of sodium and potassium and contains sufficient glucose as free sugars to ensure optimum sodium and water absorption in the jejunum (Hascke et al., 1980).
Tannins in carob pulb (Priolo et al., 2000) may have the anti-diarrheal effect, however Sahle et al. (1992) reported decreased protein digestibility and food efficiency on inclusion of carob pod in meal. While in the present study, formula I that contain carob had food efficiency and growth rate more than casein control diet which might be due to presence of other constituent in the formula such as zinc, honey, skimmed milk and vitamin A. In spite of the reported disadvantage of carob, Loeb et al. (1989) proved that infants with acute diarrhea of bacterial and viral origin were treated using tannin-rich carob pod powder that normalized defecation and body weight and produced cessation of vomiting. It has been also reported that children of acute diarrhea given ORS + carob juice showed shorter duration, reduction of stool out put and ORS requirement compared to ORS and did not lead to any metabolic problems. Thereby carob juice has a role in the treatment of children's diarrhea (Serairi-Beji et al., 2000). It has been also shown that successful preventive treatment of infantile diarrhea was verified by using formula containing semi-skimmed milk with 2% carob flour (Bustamante, 1970).
As a matter of fact, adherence of enterobacteria on intestinal epithelial cells is considered a major pathogenic mechanism of infantile diarrhea. The cell free carbohydrate fraction of carrot soup and a 2% solution of carob were able to block hemagglutination and adherence of Escherichia coli. The active blocking agent was found in the oligosaccharide fraction of carob which may also explain the therapeutic effectiveness of carob as diarrheal remedy (Guggenbichler, 1983).
It has been reported that feeding full strength animal milk during diarrhea may have adverse effect (Bhutta et al., 1997). However, modest amount of skimmed milk with cereals reported to have beneficial role in management of diarrhea (Bhan et al., 1996). So we made use of this report in the present study to formulate formula I.
The use of honey as sweetener may have a role in management of diarrhea since honey contain fructose and glucose that readily absorbed without the need of disaccharidases that usually decreased during diarrhea (Khoshoo and Bhan, 1990). Honey also contain oligosaccharides that may stimulate activity of one or limited number of colonic bacteria that may improve the health (Gibson and Roberfroid, 1995).
Nutritional parameters showed that diet II was comparable to control casein and was better in elevating food intake. Diet I was superior to control and to diet II concerning food intake, body weight gain, growth rate and protein and food efficiency ratio.
The overall biochemical status reflected that diet I and II were comparable to casein diet and may be superior in elevating plasma iron, magnesium and retinol levels of rats fed diet II and concerning only magnesium when diet I was fed. The anti-diarrheal activity of formula I was superior to formula II. Cicer arietinum was the best anti-diarrheal agent followed by Ceratonia siliqua then carrot.