An investigation of the toxic effects of a herbal formulation with anti-carcinogenic properties

A decoction prepared from a mixture of Nigella sativa seeds, Hemidesmus indicus root, and Smilax glabra rhizome used by some traditional medical practitioners in Sri Lanka is considered to be useful for the treatment of cancer patients. However, there is a lack of information about any adverse effects of this decoction. Experiments were carried out using Wistar rats and ICR mice as the experimental model, to evaluate any adverse effects mediated by the above decoction. Results of the investigations showed that administration of the decoction (at doses of 4g/kg body weight/day and 6g/kg body weight/day) to rats for three months had no adverse effects on the liver functions (as assessed by its effects on serum levels of alanine and aspartate aminotransferase and alkaline phosphatase) or haematological parameters (red blood cell count, white blood cell count, haemoglobin concentration, packed cell volume, mean corpuscular haemoglobin, mean corpuscular volume and mean corpuscular haemoglobin concentration). No significant pathological changes were observed in sections of the major body organs (liver, heart, lungs, stomach, duodenum and kidney) of animals treated with the decoction for three months. The investigations also demonstrated that the decoction did not have anti-ovulatory, anti-implantation, spermicidal activity. An attempt to determine the LDM concentration was unsuccessful. Even at a dose equivalent to 40 times (240g/kg/day) the normal therapeutic dose (6g/kg/day), no mortality or other toxic symptoms (loss of consciousness, salivation, muscle tremor, incoordination, hyperaesthesia, polyuria, anuria, polydipsea, defecation, piloerection, changes in locomotor activity, changes in posture, ataxia and loss of reflexes) were observed. Three month treatment with the decoction also did not produce any changes in average feed consumption, average body weight: liver weight ratios, or the general behaviour of the animals.


Experimental animals
In all experiments Wistar rats (

Determination of LD 50
To determine the dose of the decoction that would cause the death of 50% of the test animals (LD X ), 20 female and 20 male ICR mice were randomly divided into four groups of ten each (5 females and 5 males). Groups 1 to 4 were orally fed with a single dose of the decoction 6g/kg body weight/ day, 60g/kg body weight/day, 120g/kg body weight/day and 240g/kg body weight/day respectively. After single dosing with different concentrations of the decoction, mice were observed for seven days for any mortality, loss of consciousness, hyperaesthesia, salivation, muscle tremor and incoordination, polyuria, anuria, polydipsea, defecation, piloerection, and changes in posture, ataxia and loss of reflexes.

Effects on haematological parameters
Male Wistar rats (n=18,8 weeks of age) were randomly divided into three groups of six each. Group 1 was treated with 4g/kg body weight/day of the decoction for thirty days; rats in group 2 were treated with 6g/ kg body weight/day for thirty days; Group 3 (control) was fed with distilled water (3 ml) for thirty days. On the 31st day, blood was collected by tail vein bleeding for assessment of effects on haematological parameters. The white blood cell (WBC) count and differential count (DC), red blood cell (RBC) count, packed cell volume (PCV) and haemoglobin (Hb) concentrations were determined according to the methods described by the International Committee for Standardization in Haemotology (3). Using the above data, mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), and mean corpuscular haemoglobin concentration (MCHC) were calculated.

Effects on serum enzyme levels
Above experiment was continued for a further two months. At the end of the third month, rats were given ether anaesthesia and blood was collected by cardiac puncture. During this procedure the animal died and was subjected to an autopsy. Serum was analyzed for alanine aminotransferase (ALT), aspartate aminotransferase (AST) and alkaline phosphatase (ALP) according to methods described in commercially available reagent kits marketed by DMA Inc, USA.

Assessment of histological changes
At autopsy samples were taken from the heart, lungs, liver, kidneys, duodenum and stomach, and placed in 10% buffered formalin (sample volume: formalin volume=l:8) for histopathological studies. Histological changes (cell swelling, fatty change, granular and vacuolar degeneration and nuclear changes) were assessed by light microscopic examination of sections from the above organs stained with haemotoxylin and eosin.
Effects on body weight gain, feed consumption ratio and body weight: liver weight ratio Throughout the above experiment, the daily feed consumption and weekly body weight gain of each rat was recorded and average feed consumption and body weight gain was calculated. Before samples were taken from liver for histological examination, weight of each liver was recorded and body weight: liver weight ratio was calculated.

Effects on reproductive ability
Studies were done to determine effects of the decoction on reproductive ability of both male and female rats. In female rats, effects on ovulatory activity and implantation were determined while in male rats effect on spermicidal activity was determined.

Effects on ovulatory activity
Wistar female rats (n=18,14 to 16 weeks of age) with normal oestrus cycle (average 4-6 days of pro-oestrus, oestrus, metoestrus and dioestrus) were divided into three groups of six each. Group 1 and 2 were given decoction doses 4g/kg body weight/day and 6g/kg body weight/day respectively for 18 days. Group 3 was considered as the control group and 3ml of distilled water was given daily for 18 days. Vaginal smears were examined daily to check the stage of oestrus they were in. Persistent dioestrus, non-cyclic and lengthy oestrus cycles were considered as toxic signs. Different stages of the oestrus cycle were determined according to Soejartoef al (4).

Effects on implantation
Regularly cyclic female virgin rats (14 to 16 weeks of age) were mated. Presence of copulation plugs or sperms in the vaginal smear on the following morning were regarded as confirmation of mating. The day copulation plugs or sperms appeared was assumed the day one of pregnancy. Pregnant female rats (n=15) were randomly divided into three groups of five each. Groups 1 and 2 were given decoction doses 4g/kg body weight/day and 6g/kg body weight/day respectively from days 1 to 7 of pregnancy. Group 3 was given 3ml of distilled water from days 1 to 7. Autopsies were performed on the 10th day and the number of implantation sites, the number of live/dead foetuses and the number of corpora lutea of pregnancy were recorded (4).

Effects on spermicidal activity
Wistar male rats (n=18, 14 to 16 weeks of age) were randomly divided into three groups of six each. Groups 1 and 2 were given the decoction in doses of 4g/kg body weight/day and 6g/kg body weight/day respectively, for three months. Group 3 was considered as the control group and 3ml distilled water was given for three months. At the end of treatment, each male rat was mated individually with a female rat in oestrus (14 to 16 weeks of age). Presence of copulation plugs or sperms in the vaginal smear on the following morning were regarded as evidence of mating. After 18 to 21 days of gestation period, the number of mated female rats: number of unmated female rats, number of female rats who delivered pups, and the litter size of each female rat were recorded. All these parameters were considered as a reflection of the reproductive ability of the particular male rat, which mated each female rat (4).

Statistical analysis
Results are expressed as mean ± standard error of mean (mean ± SEM). Statistical analysis was done using the student's t test. P<0.05 was considered as significant.

LD^ determination
No deaths were observed in the seven days following a single oral administration of several different doses of the decoction (60, 120, and 240 mg/kg body weight). Even the highest dose (240 mg/kg body weight did not produce any apparent adverse effects. Therefore, these results did not allow the calculation of the LD".

Anti-ovulatory activity
As can be seen in Table 4, treatment with either of the two doses of decoction did not result in any alteration of the oestrus cycle, nor did the animals become persistently dioestrus.
Effects on implantation Table 5 shows that oral administration of 4g/kg body weight/day of decoction did not significantly alter the number of pregnant animals, the number of non pregnant animals, the number of implantation sites, the number of corpora lutea of pregnancy and the number of resorptions per total number of implantation sites (P>0.05).

Spermicidal activity
Results obtained are presented in Table 6. No significant differences were found after treatment with the decoction for three months in the number of pups delivered, or the litter size (P>0.05). The decoction therefore does not appear to have any significant effect on spermicidal activity of male rats.   According to Nagaratnam et al (12), alkaloids in various decoctions consumed by people in Sri Lanka may cause liver damage, leading to hepatocellular carcinoma. However, the decoction used in the present study protected against chemically induced hepatocarcinogenesis (1) and did not cause any liver damage, as assessed by lack of alterations in the serum enzyme levels AST, ALT and ALP and liver histopathology, even after three months of continuous treatment. Whether the difference in effects is due to species difference or due to difference in the chemical components of the decoction used in the study requires investigation.
Zaoui et al (13) had reported that fixed oil of N. sativa could cause leukocyte counts to decrease and PCV and haemoglobin levels to increase in rats. However, present study with decoction containing H. indicus and S. glabra in addition to N. sativa, revealed that none of the haematological parameters that were tested were altered even after three months of continuous treatment. The same investigation by Zaoui et al also showed that fixed oil of N. sativa caused a slowing of body weight gain in rats. However, in the present study no changes in body weight gain was observed during the experimental period. Whether these differences are due to protective effects of components in H. indicus and S. glabra is not clear.
It is well known that several commonly used plant based drugs have adverse effects on fertility, as well as teratogenic effects (14).
Morphine and caffeine given to male rats before mating decreases the number of pups and frequently lead to death (15). In the present study where the effects of the decoction on ovulatory activity, implan tation and spermicidal activity were investigated, no alterations were found in the length of oestrus cycle, the interval between cycles, gestation period and number of pups per litter or any other parameter investigated.
The general well being of the animals also did not change. All the animals remained in good health throughout the experimental period. This study has shown that this decoction did not produce any significant adverse effects on body systems in rats and mice.