Microbiological Cytotoxic, Haematological and Histopathological Investigations of Seven Bitter Medicinal Plants of Bangladesh

Abstract

Plants are credited with many medicinal properties. Medicinal plants are alleged to possess therapeutic effects in various diseases. Seven bitter medicinal plants of Bangaldesh, viz, Andrographis paniculata N., Vinca rosea L., Adhatoda vasica N., Vitex vegundo L., Aloe indica W., Flacortia ramontchi and Nyctanthes arbortristis L. have been selected for present investigation owing to their recognised medicinal importance. The extracts of these plants are used in the treatment of bronchitis, stomachic, carminative, anthelmintic, fever, asthma, inflammation, febrifuge, gonorrhoea, antitumor, liver complaints, vomiting, leprosy, jaudice, dyspepsia, piles, antidiabelic, anti-cancer, leucoderma, lumours, gonorrhoea, rheumatism, diarrhoea, and many other diseases. These extracts are anti-asthmatic, antipasmodic, expectorent, antitussive, antifungal, antimalarial, antibacterial and antimutagenic. Our present work deals with the extraction of the above medicinal plants with ethanol and microbiological, cytotoxic, haematological and histopathological investigations of the extracts of the above plants. The leaves of the above medicinal plants were washed separately with tap-water to remove adhering dirt. They were cut into small pieces with a knife. The chopped pieces were dried in the sun for four days. The sun-dried materials were then dried in an electric oven at 40oc for about 72 hours when the leaves became almost dry. These were then removed from the oven and the dried leaves were pulverized into powder and paste with the help of a grinding machine. The whole operations for the leaves of seven medicinal plants were done separately. The powdered and pasty leaves were stored in seven separate air tight containers and kept in a cold, dark place for investigation. The powdered and pasty leaves were taken in seven separate quick-fit glass stoppered bottles. The contents of the bottles were shaken continuously with ethanol in an electric shaker for about nine hours. The bottles were allowed to stand for several days. The contents of the bottles were filtered successively in a tincture press and the filtrates were separately collected in seven glass containers. The ethanol extracts of seven medicinal plants were concentrated under reduced pressure when brownish-yellow semi-solid masses were obtained. Each of the concentrated extracts was preserved in a refrigerator for investigations. In every extraction, 250 g of powered or pasty leaves of the plants were taken in 500 ml ethanol. Antimicrobial activity of the ethanol extracts was evaluated by ‘Disc diffusion method’145. Disc diffusion method is essentially a qualitative or semi quantitative test, which indicates the sensitivity or resistance of microorganism to the test material. The microbiological investigation was done to determine the susceptibility of some pathogenic bacteria to ethanol extracts of the seven medicinal plants under investigation. Dried filter paper discs impregnated separately with the test materials were placed gently on nutrient agar plates inoculated with the test organism. The dried discs absorbed water media and the test materials diffused. As a result, there is a gradual change of test materials concentration in the media surrounding each disc. The plates were then incubated at 37.5°C for 24 hours to allow growth of the organisms. If there is any antimicrobial activity of test materials it will inhibit the growth of microorganisms and a clear zone of inhibition will be visualized surrounding the discs. The antimicrobial activity of the test materials is determined by measuring the diameter of zone of inhibition. The larger zone of inhibition is observed for more susceptible organism. The antibacterial activity of the crude ethanol extracts obtained from the leaves of seven medicinal plants (viz, Andrographis paniculata N., Vinca rosea L., Adhatoda vasica N., Vitex vegundo L., Aloe indica W., Flacortia ramontchi and Nyctanthes arbortristis L.) were tested against nine bacteria at concentration of 300 µg/disc, 400 µg/disc and 500 µg/disc. Standard antibiotic disc kanamycin (30µg/disc) was used for comparison. The diameters of the zones of inhibition derived by ethanol extracts of the leaves of the plants were measured in mm with a transparent scale and the results were recorded. The minimum inhibitory concentration (MIC) of ethanol extracts of the leaves of the above medicinal plants against four gram-positive bacteria and five gram-negative bacteria were determined by serial tube dilution technique. The diameters of the zones of inhibition derived by ethanol extract of the leaves of An. paniculata showed maximum activity against B. megaterium and Shigella dysenteriae. No activity of the extract on Sarcina lutea, Shigella shiga, Shigella sonnei was experienced. The extract of Vinca rosea L. was active against Staphylociccus aureus, B. subtilis, Sarcina lutea, Salmonella typhi, E. coli and Shigella dysenteria. The extract was found to possess the maximum activity on B.subtilis and Salmonella typhi. Activity of the extract against B. megaterium, Shigella shiga and Shigella sonnei was not found. Ad. vasica shows the maximum activity against B. megaterium and Shigella dysenteriae. The extract had no or little activity against Sarcina lutea, Shigella shiga and Salmonella typhi. V. vegundo showed the maximum activity on Staphylociccus aureus and Salmonella typhi. The extract had a little activity on Sarcina lutea, Shigella shiga and Shigella sonnei. A. vera had the maximum activity against B. megaterium and E. coli. The extract showed no activity on Sarcina lutea, Shigella shiga and Shigella sonnei. F. ramontchi showed maximum activity against B. subtilis and Shigella sonnei. The activity of the extract against Sarcina lutea, Shigella shiga and Salmonella typhi was not observed. N. arbortristis was very active against B. subtilis and Salmonella typhi. The activity of the extract against B. megaterium, Sarcina lutea and Shigella shiga was not found. The Minimum Inhibitory Concentration (MIC) of the crude ethanol extract was determined by Serial Tube Dilution Technique against nine test organisms mentioned earlier. Nutrient broth medium and culture media were prepared earlier following the standard methods143,145. Fresh culture of the pathogenic microorganisms, preparation of test samples, placement of the discs and incubation were achieved accordingly. Kanamycin (30 µg/disc) was used as standards disc. Crude ethanol extracts of the leaves of the above medicinal plants were transferred in seven separate vials containing 2% DMSO solution (2ml). This was mixed well to achieve sample solutions having concentration 1024 µg/ml. Nine sterilized test tubes containing 1 µg/ml, 2 µg/ml, 4µg/ml, 8µg/ml, 16µg/ml, 32µg/ml, 64µg/ml, 128µg/ml and 256µg/ml sample solutions were prepared for each of the 7 ethanol extracts by SDT148. Nine test tubes containing ethanol extracts of Andrographis paniculata N. having the above concentrations were considered for determining the MIC against Bacillus megaterium. Three test tubes containing media (CM), media plus sample (CMS) and media plus inoculums (CMI) were also maintained for the experiments. Diluted inoculums (10µl) were added to each of the nine test tubes and mixed well. One ml of the sample was added to CMS and mixed well. 10µl of inoculums of Bacillus megaterium was added to CMI to observe the growth of the organisms in the media. CM containing media was used the check the sterility of the solution. Twelve test tubes were incubated at 37.5oC for 24 hours. After incubation, the test tubes were examined for visible growth of the organisms. The lowest concentration of the test material at which there was no visible growth was recorded as the MIC of the test material for the test organisms. The first sign of inhibition of test organisms by the ethanol extract of Andrographis paniculata N. was visualized in the test tubes and recorded. Similar 8 experiments were performed for the same extract against remaining 8 bacteria and MIC of the particular extract against above nine bacteria were recorded. Same experimental procedures were followed for the determination of MIC of remaining 6 extracts (viz, extracts of Vinca rosea L., Adhatoda vasica N., Vitex vegundo L., Aloe indica W., Flacortia ramontchi and Nyctanthes arbortristis L.) against nine bacteria mentioned above. Thus, the MIC of seven ethanol extracts of the leaves of above seven plants against nine bacterial strains were determined and the results were recorded in 63 tables. Thus, the MIC values of ethanol extracts of the leaves of the above medicinal plants were determined. It was evident from the results that all the extracts had notable antimicrobial activities against most of the test organisms. The extract of the plants had MIC values ranging from 32 to 128 mg/ml. All plant extracts showed no MIC against Shigella Shiga only. These extracts had notable activities on other bacterial strains mentioned above. Appreciable MIC of the extracts has been enunciated in the results. The first sign of inhibition of organisms was seen in the test tube against the respective extracts having lower concentrations than those cited in the results had no inhibitory activities on the organisms. The results revealed that the ethanol extracts of the plants under present investigation possess appreciable antibacterial activities. The MIC values of the extracts have remarkable significance about the therapeutic effects of the active principles associated with the leaves of the plants. Cytotoxic effects of ethanol extracts of the plants were investigated. Brine Shrimp Lethality bioassay150 indicates cytotoxicity and a wide range of pharmacological activities of the compounds as well. It utilizes a large number of organisms for statistical validation and a relatively small amount of sample. Generally the median effective dose (ED50) values for cytotoxicity’s are one tenth of median Lethal Concentration (LC50) values in the Brine Shrimp bioassay.151-155 This can be accompanied by testing in vitro lethality in a simple zoologic organism, viz, Brine Shrimp nauplii, Leach (Artemia Salina). In this method, bioactivity can be found not only for natural product extracts but also for pure compounds. Brine Shrimp Lethality test is a positive correlation between Brine Shrimp toxicity and cytotoxicity. The crude ethanol extracts of seven medicinal plants under present investigation represented positive response to Brine Shrimp Lethality Bioassay. Lethal concentration (LC50) of Brine Shrimp Lethality was determined to evaluate the cytotoxic effect of ethanol extracts. LC50 of Brine Shrimp Lethality was obtained from a plot of percentage of mortality versus concentration of the extracts on the graph. The results, presented in the tables and graphs, established a positive correlation between Brine Shrimp Lethality and cytotoxicity. Brine Shrimp Lethality bioassay resembled that the ethanol extracts the leaves of Andrographis paniculata N. is more cytotoxic (LC50= 30 µg/ml) than those of other extracts. The very low LC50 indicated the high toxic effect of the extract of the leaves of the plant. Low cytotoxic effect of each of Vitex vegundo L. and Nyctanthes arbortristis L. (LC50= 72 µg/ml) was observed. The other ethanol extracts exhibited moderate cytotoxic effects. Thus, it may be concluded that the ethanol extracts of the leaves of seven medicinal plants under present investigation have cytotoxic effects. Haematological investigation deals with the composition, formation, destruction, production and conservation of blood and related substances in blood, blood plasma and tissue fluids. Hence, the dietary effects of ethanol extracts of the medicinal plants under present investigation supplemented separately with formulated cereal at 5% level have been investigated in the present study. A survey of 28 consecutive days on young male albino rats, body weight ranging 52-56 g was performed. The dietary effects of the extracts at 5% level with formulated cereal on the composition of red cells like erythrocytes and white cells, viz, leucocytes and platelets (thrombocytes) were investigated in this experiment. Its effects on the composition of white cells like esnophil, neutrophil, monocytes, basophil and lymphocytes were investigated. Haematological parameters, i,e, haemoglobin, red blood cells (RBC), erythrocyte sedimentation rate (ESR), cholesterol, white blood cells (WBC) and total count of WBC of the rats under experimental and control groups were investigated after feeding the extracts, separately fortified with the formulated cereal at 5% level for a period of 28 consecutive days. Histopathological as well as toxicological effects of the ethanol extracts were investigated on livers, lungs, kidneys, hearts and spleens of the experimental rats. These were achieved by observing the changes in the cellular structures of these organs. The present investigation included the identification and toxicological effects in those organs of the experimental rats. These were achieved by observing any changes in the cellular structures (degradation and regeneration) of organs of the experiment rats after taking fortified cereal for a period of 28 consecutive days. Dietary effects of feeding on the experimental rats during the experimental period were evaluated by careful observation of the depression of central nervous system, excitation, muscular reflexes and behavior of the rats. Haematological and histopathological investigations of the ethanol extracts of the plants supplemented separately with formulated cereal at 5% level on young male albino rats after feeding for 28 consecutive days both for the experimental rats and control group were performed. During this experiment, the effect of feeding the supplemented cereal on gain in body weights of the rats during the experimental period was investigated simultaneously. A control group of the rats for formulated cereal (without having any extracts) was also maintained for gain in body weight. Initial body weight of the rats, gain in body weight during the experiment of feeding and gain in body weight per one gram of food intake by each of the rats were recorded. The results resembled that there was a close similarity of the diets in gain in body weight of the rats. It appeared from the results that the diets had almost equal impact in gain in body weight. The results were computed to show the gain in body weight for one gram of food intake by 16 young male albino rats for feeding eight diets. Analysis of variance table and a difference table for multiple comparison of mean gain in body weight per one gram of food intake were computed to show which of the diets was statistically more significant for gain in body weight and for the normal development of the rats. It was evident from the tables that all the diets had similar impacts for normal development and maintenance of the rats. None of the diets was statistically significant in gain in body weight of the rats. Separate tables were prepared for Dan can’s New Multiple Range Test for protection level α = 0.01 and α = 0.05. The tables failed to identify which of the diets was statistically significant at 1% or 5% levels of significance. The tables also showed that the treatments and errors of the diets had no Least Significance Range (LSR) in comparison with their respective Standard Significance Range (SSR).162 Total count of RBC and WBC, differential count of WBC and percentage of haemoglobin in the blood of the rats were investigated and the results were summarized. Comparing these values with those of reference values for haematological parameters it resembled that the changes in the parameters remained within the normal limit. No abnormalities in haematological investigations were detected. During the experimental period, the rats under investigation were found to restore no signs of muscular numbness of the legs, salivation, excitation, weakness, diarrhoea, etc. and no reflex abnormalities were detected. Thus, it appeared that the dietary effects of feeding the ethanol extracts of the plants fortified at 5% level with formulated cereal for a period of 28 consecutive days had no adverse action on both control and experimental rats. At the end of the feeding experiment for a period of 28 consecutive days, the rats were sacrificed under mild anesthesia with ether-chloroform and their livers, lungs, kidneys, hearts and spleens were preserved, processed, sliced and the tissues were mounted on glass slides and viewed under microscope to find any histopathological change in the organ. Microscopic view of sliced tissues of the organs on glass slides were recorded. On gross necropsy evaluation, all the organs under examination exhibited normal color. No abnormal histopathological change in the cellular structure in the tissues was detected. This implied from microscopic view of those organs that the effect of feeding the ethanol extract at 5% level with the formulated cereal for 28 consecutive days had no adverse effect on cellular structure of those organs of the experiment rats and no morphological changes occurred therein. From the foregoing evidences, it appeared that the ethanol extracts of the above plants, fortified with formulated cereal at 5% level was non-toxic.