Toxicity studies of Cadalmin Green Algal extract (CadalminTM GAe)  
  Toxicity studies of Cadalmin Green Algal extract (Cadalmin GAe) This study was conducted to evaluate anti-inflammatory and toxicological properties of CadalminTM Green Algal extract. As part of the safety assessment of the extract, acute, sub chronic toxicity tests were conducted by the oral route in rats. Male and female rats were divided into four groups consisting of five rats each and given doses up to 2500 mg/kg BW in acute (14 day) and 2000mg/kg BW in sub chronic toxicity (90 days). During the experiment, no mortality was observed in any groups and there were no remarkable changes in general appearance, as well as in food and water consumption. No toxicity related significant (p < 0.05) changes were noted in body weights, haematological and serum biochemical parameters between the control and treated groups. No histopathological changes were observed in brain, kidneys, liver and spleen of rats. Based on these findings, it can be inferred that the CadalminTM Green Algal extract has no toxicity. These results suggest that Cadalmin Green Algal extract has significant anti-inflammatory activity, with no significant acute and sub chronic toxicity or cytotoxicity.

The LD50 values were determined in accordance with the method of Organization for Economic Cooperation and Development (OECD, Guideline for Testing of Chemicals, Guideline 425: Acute Oral Toxicity).In brief, the rats were divided randomly into three groups containing 5 nos each. After being fasted for 16 h, animals were administered different doses of Cadalmin Green Algal extract suspended in distilled water (4000, 2500 and 1500 mg/kg body weight) by oral gavage. 40 Wistar rats (20 males and 20 females) were randomly divided into 4 groups - each consisted of 5 male and 5 females. The control group received the vehicle water (1 mL), whereas the treatment group received Cadalmin GAe in different doses (2.5g/kg, 1.0g/kg & 0.5g/kg), which administered orally (once daily) for 14 days. During this study period, rats were monitored for any type of clinical symptoms, mortality, and any adverse reaction of the administered drug. The feed consumption, water consumption and body weight of rats were determined every three days. On day 15, rats were sacrificed under mild ether anesthesia; blood was collected by direct heart puncture method and taken in the EDTA and non-EDTA vials for assaying haematological parameters and serum biochemistry. Vital organs were excised, blotted and weighed. Photographs of necropsy were taken and selected organs liver, kidney, brain and spleen were dissected out and washed thoroughly in ice-cold normal saline and weights were recorded. After decapitation under the condition of anesthesia, the body surface, intracranial, intrathoracic and intra-abdominal organs as well as tissues were observed macroscopically, and the following samples of major organs and tissues like liver, spleen, kidney, brain, were preserved in neutral, phosphate-buffered 10% formalin. They were embedded with paraffin, sliced and stained with hematoxylin and eosin to perform histopathological examination. All organs and tissues taken from all animals in the vehicle control and highest dose groups were examined microscopically. The effect of the Cadalmin Green Algal extract on certain haematological parameters such as Heamoglobin (Hb), Red blood cells (RBC), Platelet count (using Haematology analyzer; Model ABX-Micro-S-60) and Total white blood cells (WBC; using haemocytometer) were analysed. For differential counts (lymphocyte, eosinophills, neutrophills, monocytes) blood was spread on a clean slide, treated with Leishman’s stain counted using a microscope (100 X). The blood samples collected with heparinized bottles were centrifuged (5000 rpm for 10 min) to obtain clear plasma for the investigation of following parameters: 1) Liver function - Serum glutamate Oxalo acetate Transaminase (SGOT) (Bergmeyer et al, 1977); Serum glutamate Pyruvate transaminase (SGPT) (Bergmeyer et al, 1980); alkaline phosphatase, ALP (Kind and King , 1954); albumin; based on its reaction with bromocresol green- Binding method (Spencer and Price, 1971), globulins, total protein by Biuret method of Henry et al. (1974) and total bilirubin (Jendrassik and Grof, 1938), 2) Kidney function – urea was determined according to Urease-Berthelot method (Weatherburn, 1967) and creatinine was estimated using Jaffe reaction (Perone et al., 1992). 3) Lipid Profile - triglycerides, cholesterol, HDL,LDL,VLDL., and 4) serum electrolytes (Na+, K+, Cl-, HCO3+).

Based on the data of acute study, repeated subchronic toxicity study (90 days) in growing rats were conducted. 40 Wistar rats (20 males and 20 females) were randomly divided into 4 groups, each consisting of 5 male and 5 female rats. Cadalmin Green Algal extract (1g) was suspended in 6 mL distilled water and different doses (2.0g/kg, 1.0g/kg & 0.5g/kg) were administered orally (once daily) for 90 days. Control received 1 mL of vehicle water every day. Rats were monitored, during this period for any type of clinical symptoms, mortality, and any adverse reaction of the administered drug. Body weight and food consumption were determined every seven days. On day 91, rats were sacrificed as described in previous section and assessment of biomarkers of safety was carried out as described. Acute toxicity test at 5000 mg/kg body weight produced no mortality after 14 days of observation which indicates that the mean lethal dose (LD50) of the extract is greater than 5000 mg/kg body weight. No treatment related signs of mortality were observed in any of the animals in any group over the administration periods during both acute (maximum dose - 2500 mg/kg BW) and subchronic (maximum dose up - 2000 mg/kg BW) toxicity studies. In addition, test groups did not exhibited any treatment-related changes in clinical signs such as mental state, external appearance and daily activities when compared with the control group. Any abnormal behavior or cases of diarrhea and soft feces were not observed during the studies. No ophthalmological abnormalities were observed in any of the treatment groups. Hence, the eyes were excluded from histological examinations. In general, dosing of Cadalmin Green Algal extract in animals did not induce any clinical signs of toxicity in both acute and subchronic regimens.

Body weight, feed intake and water consumption of the rats were not affected during both acute and long term studies as the feed intake of untreated control and treated groups decreased steadily.

The weights of liver, kidney, spleen and brain recorded at the end of acute study (day 15) did not show significant differences (p > 0.05) among the groups. Similarly, no significant differences were observed (p > 0.05) in the organ weights of rats treated with Cadalmin GAe at the end of subchronic study (day 91). At necropsy, gross appearances of the internal organs of all animals were examined. Necropsy of the animals after sacrifice did not show any morphological changes in neither the tissues nor any gross pathological abnormalities during both acute and subchronic toxicity studies. There were no macroscopic findings considered to be related to the treatment of Cadalmin Green Algal extract. Gross examination of vital organs such as brain, kidney, spleen and liver of rats and microscopic examination of tissue sections prepared from these organs did not observed any histopathological alteration in any rats treated with Cadalmin Green Algal extract during acute and subchronic toxicity studies. The treated section of brain showed normal glial cells. Astrocytes also appeared normal. Interstitial tissue of brain also showed normal appearance compared to control rats. Portion of cerebellum also appeared normal. The section of kidney showed normal glomeruli with normal bowman’s capsule. Glomeruli showed normal cellularity. Renal tubules appeared normal. Interstitial tissue also appeared normal. The section of spleen showed normal lymphoid follicles with germinal centres. Sinusoidal spaces are dilated and they were lined by normal endotheilial cells. Some areas showed haemorrhage congestion and there were many siderophages. The section of liver tissue showed normal portal triads. Biliary duct were normal. A few lymphocytic collections were seen in the portal area which was normal. Central venous systems also appeared normal. Hepatocytes showed normal morphology and they were arranged in cords. Sinusoidal space and Kupffer cells also appeared normal.
  Haematological parameters and differential counts after acute (14 days) and subchronic toxicity (90 days) studies indicated that feeding of Cadalmin Green Algal extract even at a dose upto 2500 m g/kg BW (acute) and 2000mg/kg BW (subchronic) did not induce significant change (P > 0.05) in hematological parameters such as hemoglobin, RBC and platelet count. In acute study, a significant reduction (p< 0.05) of WBC count was observed for high and low dose groups (in both male & female). In addition, the WBC of all dose groups in subchronic study showed significant reduction compared with that of control rats (p< 0.05). There were no treatment-related significant adverse effects of Cadalmin Green Algal extract on other liver functioning parameters (total protein, albumin, bilirubin and globulin) in male and female rats (both acute and subchronic). The serum creatinine level did not show significant difference (p > 0.05), indicating no expressive changes in the general metabolism after consumption of Cadalmin Green Algal extract by rats. The electrolytes, sodium, potassium, chlorine and bicarbonate did not modify significantly (p > 0.05), indicating no expressive changes in the general metabolism after consumption of Cadalmin Green Algal extract by rats. The total cholesterol in the females of the highest dose group (2000 mg/kg) significantly decreased (p < 0.05) compared with control group.

As it is observe to be slow acting, long-term use of Cadalmin Green Algal extract may be required in treatments of arthritis related diseases. The results demonstrate a lack of toxicity following oral administration of Cadalmin Green Algal extract at a dose as high as 5000 mg/kg in the acute toxicity study and the repeated oral administration of the extract at a dose of 2000 mg/kg/day, the highest dose tested in the 90-day oral toxicity study. Cadalmin Green Algal extract had no effect on the normal growth of rats. Organ weights are widely accepted in the evaluation of toxicity related studies (Wooley, 2003). No significant differences were recorded in the weights of the organs indicating that the acute/subchronic oral administration of Cadalmin Green Algal extract did not detrimentally affect the wet weight, organ-to-body weight ratio and the colour of organs. The histopathological examination of selected organs (heart, liver, brain and kidneys) from treated and control animals showed normal architecture, suggesting that daily oral administration of Cadalmin Green Algal extract for 90 days caused no detrimental changes or morphological disturbances.

In the acute study for 14 days in growing rats, Cadalmin Green Algal extract failed to induce any significant alterations in parameters that indicate safety such as feed intake, growth, organ weight, histology, hematological indices, serum and liver biochemical parameters. Further study for 90 days among growing rats also appeared to be safe as growth of rats was normal and analysis of other safety parameters after terminal autopsy did not reveal significant difference among control and treated rats. Based on these observations, it was concluded that Cadalmin Green Algal extract is safe to consume without any adverse effects in the body.