RESEARCH ON ACUTE TOXICITY AND SEMI-CHRONIC TOXICITY OF MEDICINAL FUNGUS CORDYCEPS TAKAOMONTANA
Abstract and keywords
Abstract (English):
The article presents the evaluation of acute and semi-chronic toxicity of medicinal fungus Cordyceps takaomontana which extract was developed by Institute of Biotechnology of the Vietnam Academy of Science and Technology. In the course of the experiment under mice that drank water with added Cordyceps takaomontana extract, concentration of which was 975 times less than the concentration used for humans, symptoms of poisoning (signs of acute toxicity) were not revealed. Examining semi-chronic toxicity was taken under rabbits that were fed with Cordyceps takaomontana extract in doses 0.24 and 1.2 g/kg of mass once a day during 30 days. Rabbits’ general condition and mass, functions of blood, liver and kidneys, biochemical and hematological parameters have been studied; their liver and kidneys histology during and after taking Cordyceps takaomontana extract for 15 days was carried out.In the course of the study it has been found that Cordyceps takaomontana extract is absolutely not toxic. The results obtained serve a scientific justification for using fungus Cordyceps takaomontana in food and pharmaceutical production for preparing food and medicinal products.

Keywords:
medicinal fungus, Cordyceps takaomontana, acute toxicity, semi-chronic toxicity, Cordyceps takaomontana
Text
Introduction Entomogenous fungus Cordyceps takaomontana Yakushiji & Kumazawa, found in Vietnam and some Asian countries, is one of precious medicinal fungi that can synthesize some compounds with potential biological activities and many effects including additional treatment of human cancer and leukemia. This kind of fungus is exploited, planted and used widely in Korea [1] but has recently received attention in Vietnam [2, 3]. Fungus Cordyceps takaomontana was described and recorded for the first time in Vietnam by Pham Quang Thu and Nguyen Manh Ha in 2010. The fungal species is distributed in evergreen tropical broadleaf rain forest at an elevation from 800 m to 1000m above sea level of Bavi National Park, Hanoi City and Tamdao National Park, Vinh Phuc province [2, 3]. Institute of Biotechnology, the Vietnam Academy of Science and Technology has studied, exploited and developed gene source from such fungus, multiplied for creating fruit-body as medicinal materials. Therefore, the main purpose of this study was to evaluate safety of fungi Cordyceps takaomontana, through studying acute toxicity and semi-chronic toxicity. Materials and Methods Samples and animals. Solid alcohol extract from fungus Cordyceps takaomontana Yakush. & Kumaz, extraction efficiency of 15.77%. Male and female mice of Swiss albino at their 5th -7th week, weighing 20 ± 2 g were used to evaluate acute toxicity of the sample. Mature male and female rabbits of New Zealand White, weighing 20 ± 2 kg were provided by the National Institute of Hygiene and Epidemiology, used to evaluate semi-chronic toxicity of the sample. Chemicals and equipment. Equipment includes: semi-automatic photometer Humanlyzer (Germany), automatic blood analyzer SYSMEX KX21 (USA), cold centrifuge Mikro 22R made by Hettich (Germany), electronic analytical scale Precisa CA 125 SCS 9 (Switzerland), shaker; meter kits for urea, creatinine, total protein and total bilirubin made by Human (Germany); meter kits for AST, ALT made by JAS (USA); analyzers for erythrocyte, leukocyte, thrombocyte, hematocrite value, hemoglobin concentration and lymphocyte made by Diatron (Hungary). Research chemicals are pure and provided by Sigma-Aldrich Company (USA). Methods. Acute toxicity is realized under the method of Behren Karber, quoted from Biochemistry book published by the Medical Publishing House [4-8]. Evaluation on acute toxicity Mice are raised in 3 days before test to adapt to the testing conditions. Mice were abstained from food within 16 hour and fed with water as desired before test. They were weighed to select eligible mice. After that, exploratory research was conducted by feeding each group of mice (4 mice) with water by incremental sample dose, starting from the dose of 10-fold equivalent dose for human (3 g medicinal materials/person/day), i.e. 0.375 g and subsequent doses: 0.750 g; 1.50 g and 3.00 extract/g of weight. No mice are found dead. After that, mice were divided into lots, each lot including 10 mice. Mice were administered orally with medicine by using syringe with obtuse needle to put medicine gently into mice’s stomach. Solid extract was diluted with water in appropriate concentrations. Mice were fed with such solution with volume of 0.4-0.8 mg/20 g mice. Taking into account the test results, we found maximum dose for which no mice of the testing lot is dead (LDo) and minimum dose for which 100% mice of the testing lot is dead (LD100). If LD100 is available, 3 or 4 intermediary doses between the two above doses are tested to determine LD50. Mice were monitored and observed with their behaviors, activities, digestion, excretion and number of alive or dead mice within 72 hours. Evaluation on semi-chronic toxicity Rabbits were raised stably in 3 days before the test. After ineligible rabbits were discarded, the remaining mice were divided into 3 testing lots, including: 1. Lot 1 (pathophysiological confront lot) was used as a reference to determine environmental conditions and surrounding conditions that may affect rabbits’ health during the test. Rabbits were fed with water at a dose of 1ml/kg, once in the morning within 30 days. 2. In lot 2 (Cordyceps takaomontana extract 1), rabbits were fed with Cordyceps takaomontana extract at a dose of about 0.24 g medicinal materials/kg (equivalent to normal dose for humans with coefficient 4). Extract was diluted in water with dose of 1ml/kg, once in the morning within 30 days. 3. In lot 3 (Cordyceps takaomontana extract 2), rabbits were fed with Cordyceps takaomontana extract at a dose of about 1.20 g medicinal materials/kg (5-fold of normal dose for humans with coefficient 4). Extract was diluted in water with dose of 1ml/kg, once in the morning within 30 days. To evaluate semi-chronic toxicity, targets were monitored as follows: general situation and weight of rabbits: activities, digestion, excretion and weight of rabbits were monitored. Blood generation function was evaluated through hematological indicators: number of erythrocytes, leukocytes and thrombocytes, hemoglobin concentration, hemoglobin, % lymphocyte. Rabbit blood was put into testing vial containing anti-coagulation agent and measured with hematological parameters with automatic blood analyzer SYSMEX KX21. Liver function was evaluated through liver biochemical indicators (serum test): ALT, AST, bilirubin, total protein using meter kits for AST, ALT, bilirubin and total protein and measured with semi-automatic photometer Humanlyzer. Kidney function was evaluated through kidney biochemical indicators (serum test): creatinine and urea using meter kits for creatinine and urea and measured with semi-automatic photometer Humanlyzer. Besides, after 4 weeks of using water extracts and after 15 days of no using water, rabbits were operated on to observe all organs. Random checking was done on liver and kidney microsome structure of 30% of rabbits in each lot by cutting anatomical preparation of liver and kidney of 2-3 rabbits/testing lot after finishing use and after 15 days of stopping use. The above targets were monitored right before the date of using testing sample, after 15 days, after 30 days of using testing sample and after 15 days of stopping testing sample. Statistical analyses Research data were processed statistically under T-test method with Avant - Après comparison. Changes in biochemical and hematological indicators as well as pathologic histology of rabbits before using testing sample (1st time) and upon using testing sample of the lot of rabbits using testing sample and the experimental lot of rabbits in 15 days after using (2nd time), 30 days (3rd time) and 15 days after stopping use (4th time) were also compared. Results and Discussion Evaluation on acute toxicity. We have prepared the sample and fed 5 lots of mice with respective doses of 5.00; 10.00; 18.25; 28.50 and 36.50 g of solid extract/kg mice weight. In all lots in which mice were administered only once, the results are the same. After administration, mice still operate normally. No abnormal expression in behaviors or digestion was found. After 72 hours of monitoring, all mice of testing lots were alive and sound (Table 1). Table 1 Acute toxicity of solid extract of Cordyceps takaomontana No. Testing dose, g solid extract/kg Number of testing mice, pcs Number of dead mice 1 5.00 10 0 2 10.00 10 0 3 18.50 10 0 4 28.50 10 0 5 36.50 10 0 According to the traditional medicine documents on using Entomogenous fungus, for normal people, dose is about 3 g/kg, converted to 0.6 g/kg (with coefficient 10) for mice. We have done experiments on mice with the highest dose of 36.50 g solid extract/kg mice weight (equivalent to 584 g/kg mice weight). This testing dose is 973-fold converted dose, but mice are still healthy and do not express abnormality. Therefore, we stop testing at this dose and conclude that Cordyceps takaomontana extract causes no acute toxicity. Evaluation on semi-chronic toxicity. To evaluate semi-chronic toxicity, test is conducted on 4 testing lots of rabbits, each lot including 10 rabbits: Lot 1 (pathophysiological confront lot); lot 2 (testing Cordyceps takaomontana extract with dose of 0.24 g medicinal materials/kg rabbit weight); lot 3 (testing Cordyceps takaomontana extract with dose of 1.20 g medicinal materials/kg rabbit weight). General state and weight of testing animal. In the lots of using testing sample and control lot, digestion and excretion of rabbits were still normal and no rabbit was found dead. Weight of rabbits after administration in all lots has increased, as compared with weight before test. Such increase between confront lot and testing lot was the same. The difference is of no statistical significance but the difference is of statistical significance next time compared with the initial time in the same lot. It shows that testing sample causes no effect to the weight of rabbits. This weight gain is a normal physiological one (Table 2). Table 2 Weight of testing rabbits at monitoring times No. 1st time 2nd time 3rd time 4th time Lot 1 1.9 ± 0.0 2.0 ± 0.0* 2.2 ± 0.1* 2.2 ± 0.0* Lot 2 1.9 ± 0.0 2.1 ± 0.0* 2.1 ± 0.1* 2.1 ± 0.1* Lot 3 1.9 ± 0.0 2.1 ± 0.1* 2.2 ± 0.1* 2.2 ± 0.1* * Different weights are of statistical significance with P < 0.05 compared with first time in the same lot. Hematological indicators Monitoring results of hematological indicators of rabbits during research are shown in Table 3. Table 3 Hematological indicators of testing lots of rabbits* Indicators 1st time 2nd time 3rd time 4th time Leucocyte, 103/mm3 Lot 1 7.2 ± 0.6 7.2 ± 0.6 8.4 ± 0.9 8.9 ± 0.7 Lot 2 7.2 ± 0.4 7.3 ± 0.6 8.9 ± 0.6 8.8 ± 0.8 Lot 3 6.7 ± 0.5 7.0 ± 0.6 8.0 ± 0.4 7.3 ± 0.9 Erythrocyte, 106/mm3 Lot 1 5.44 ± 0.17 5.38 ± 0.2 5.19 ± 0.24 5.27 ± 0.22 Lot 2 5.13 ± 0.17 5.23 ± 0.13 4.91 ± 0.14 5.03 ± 0.18 Lot 3 5.14 ± 0.15 4.97 ± 0.22 5.02 ± 0.13 5.26 ± 0.21 Thrombocyte, 106/mm3 Lot 1 412 ± 39 324 ± 35 366 ± 32 445 ± 62 Lot 2 304 ± 39 341 ± 45 383 ± 42 388 ± 76 Lot 3 389 ± 41 368 ± 53 329 ± 56 312 ± 54 Continuation Table 3 Indicators 1st time 2nd time 3rd time 4th time Hemoglobin, g/dl Lot 1 11.1 ± 0.3 10.8 ± 0.2 10.7 ± 0.3 10.3 ± 0.3 Lot 2 11.0 ± 0.3 10.9 ± 0.2 10.3 ± 0.3 10.3 ± 0.3 Lot 3 10.7 ± 0.3 10.4 ± 0.4 10.2 ± 0.3 10.5 ± 0.4 Hematocrite, % Lot 1 33.3 ± 0.5 32.6 ± 0.9 32.3 ± 1.2 32.1 ± 0.8 Lot 2 34.1 ± 1.2 33.2 ± 0.8 30.9 ± 0.6 30.6 ± 1.5 Lot 3 33.7 ± 0.7 32.6 ± 1.2 32.4 ± 0.8 33.0 ± 1.3 Lymphocyte, % Lot 1 56.6 ± 1.9 59.5 ± 2.8 51.6 ± 5.4 50.5 ± 3.2 Lot 2 56.7 ± 2.6 61.5 ± 4 50.1 ± 2.9 52.4 ± 5.9 Lot 3 58.1 ± 4.8 59.3 ± 5.9 51.4 ± 4.7 53.5 ± 4.3 * P (confront - test) in all lots is greater than 0.05, P (Avant - Après) in all lots is greater than 0.05. Table 3 showed that after 15 days, 30 days of administration and 15 days after stopping administration, the number of erythrocytes, leukocytes and thrombocytes, hemoglobin concentration, hematorite value in both lots of Cordyceps takaomontana extract (dose of 0.24 g and 1.20 g medicinal materials/kg) has no significant difference compared with the confront lot at the same time as well as compare with before test (P > 0,05). Liver function indicators. The results in Table 4 showed that after 15 days, 30 days of using testing sample and 15 days of stopping use, all tests for liver function evaluation including bilirubin concentration, protein concentration, enzyme activity AST and ALT of serum in both lots of Cordyceps takaomontana has no significant difference compared with the confront lot at the same time as well as compare with before test (P > 0,05). Table 4 Biochemical indicators of liver function of testing lots* Indicators Lot 1st time 2nd time 3rd time 4th time ALT, U/l 1 58.2 ± 7.1 62.5 ± 3.4 68.7 ± 2.2 71.5 ± 4.6 2 63.5 ± 5.4 69.1 ± 2.5 71.8 ± 4.2 71.5 ± 4.5 3 59.8 ± 5.3 70.2 ± 2.7 66.5 ± 4.1 68.5 ± 2 AST, U/l 1 39.1 ± 2 40.8 ± 3.1 40.7 ± 3.5 47.4 ± 7.0 2 36.7 ± 1.6 40.2 ± 1.8 36.4 ± 3.2 38.6 ± 4.3 3 37.6 ± 2.4 41.7 ± 2 35.9 ± 3.2 45 ± 5.9 Bilirubin, µmol/l 1 1.81 ± 0.35 1.63 ± 0.11 1.59 ± 0.3 1.80 ± 0.21 2 1.59 ± 0.18 1.68 ± 0.16 1.69 ± 0.27 1.92 ± 0.26 3 2.07 ± 0.32 1.68 ± 0.2 1.78 ± 0.32 1.68 ± 0.42 Total Protein, g/l 1 57.1 ± 1.4 59.4 ± 1.1 58.4 ± 0.7 60.9 ± 1.8 2 55.0 ± 1.2 57.3 ± 0.7 56.8 ± 0.6 59.4 ± 2.3 3 57.5 ± 0.8 56.4 ± 1.6 55.9 ± 1.1 59.9 ± 1.2 * P (confront - test) in all lots is greater than 0.05, P (Avant - Apres) in all lots is greater than 0.05. Kidney function indicators. After 15 days, 30 days of using testing sample and 15 days of stopping use, two tests for liver kidney evaluation including urea concentration and creatinine concentration of serum in both lots of Cordyceps takaomontana extract has no significant difference compared with the confront lot at the same time as well as compare with before test (P > 0,05) (Table 5). Table 5 Biochemical indicators of kidney function of testing lots* Indicators Lot 1st time 2nd time 3rd time 4th time Urea, mg/dl 1 7.5 ± 0.4 6.7 ± 0.5 7.0 ± 0.3 7.1 ± 0.6 2 7.6 ± 0.3 7.0 ± 0.4 7.7 ± 0.4 7.7 ± 0.8 3 7.8 ± 0.4 7.0 ± 0.5 7.3 ± 0.4 7.0 ± 0.4 Creatinine, µmol/l 1 7.5 ± 0.4 6.7 ± 0.5 7.0 ± 0.3 7.1 ± 0.6 2 7.6 ± 0.3 7.0 ± 0.4 7.7 ± 0.4 7.7 ± 0.8 3 7.8 ± 0.4 7.0 ± 0.5 7.3 ± 0.4 7.0 ± 0.4 *P (confront - test) in all lots is greater than 0.05, P (Avant - Après) in all lots is greater than 0.05. Histological testing result. After rabbits were fed with testing sample for 30 days, 3 rabbits were selected at random. These rabbits were operated on to observe general organs and check liver and kidney microsome structure. Remaining rabbits were abstained from using testing sample and raised for more 15 days. After 15 days of stopping extract, all rabbits were operated on to observe general organs and check liver and kidney microsome structure with 3 rabbits of each lot. Research result shows that there are no pathological changes in general organs such as heart, lungs, liver, spleen, kidney and digestion system in 3 testing lots under surgery. In particular, we consider microsome morphology of liver and kidney and gain the following result: Microsome morphology of liver. In the confront lot, liver cell has even dimensions, no degradation or necrosis cavities. Its periportal space has no inflammation but has hepatohemia. In both lots of using Cordyceps takaomontana extract at dose of 0.24 g and dose of 1.20 g medicinal materials/kg, after 30 days of using medicine and after 15 days of stopping use, liver cells are normal, even in dimensions, free from degradation and necrosis. Its periportal space has no inflammation, but has hepatohemia (Fig. 1-6). Fig. 1. Image of rabbit liver cell in lot 1 after 30 days of testing (HE. 400) Fig. 2. Image of rabbit liver cell in lot 1 after 15 days of stopping test (HE. 400) Fig. 3. Image of rabbit liver cell in lot 2 after 30 days of testing (HE. 200) Fig. 4. Image of rabbit liver cell in lot 2 after 15 days of stopping test (HE. 200) Fig. 5. Image of rabbit liver cell in lot 3 after 30 days of testing (HE. 400) Fig. 6. Image of rabbit liver cell in lot 3 after 15 days of stopping test (HE. 400) Such hepatohemia at the periportal space occurs in both pathological confront lot and two lots of using testing sample. This phenomenon is often seen when rabbit is killed but struggles very hard till its death. This indicates that this is not the effect of using testing sample and it is normal. Image of kidney microsome In the confront lot, glomerulus is even in dimensions and free from sclerosis. Kidney tube has no injury. Interstitial tissue has no inflammation and nephrohemia. In both lots of using Cordyceps takaomontana extract at dose of 0.24 g and dose of 1.20 g medicinal materials/kg, after 30 days of using and 15 days of stopping, it is found that glomerulus is even in dimensions and free from sclerosis, interstitial tissue has no inflammation, kidney tube has no injury and nephrohemia (Fig. 7-12). Fig. 7. Image of rabbit kidney cell in lot 1 after 30 days of testing (HE. 400) Fig. 8. Image of rabbit kidney cell in lot 1 after 15 days of stopping test (HE. 400) Fig. 9. Image of rabbit kidney cell in lot 2 after 30 days of testing (HE. 400) Fig. 10. Image of rabbit kidney cell in lot 2 after 15 days of stopping test (HE. 400) Fig. 11. Image of rabbit kidney cell in lot 3 after 30 days of testing (HE. 400) Fig. 12. Image of rabbit kidney cell in lot 3 after 15 days of stopping test (HE. 400) Conclusion According to the research results of acute toxicity and semi-chronic toxicity of Cordyceps takaomontana fungus extract, it is possible to conclude that the extract of this fungus is safe. In terms of acute toxicity, mice are fed with a dose of about 975-fold of the dose used for humans but mice have no poisoning symptom. This indicates that Cordyceps takaomontana extract is safe in terms of acute toxicity. In terms of semi-chronic toxicity, when rabbits are fed with Cordyceps takaomontana extract at dose of 0.24 g and dose of 1.20 g medicinal materials/kg weight/day in 30 days, there is no statistically significant effect on the general state and weight of rabbits as well as their blood generation function, liver function and kidney function on biochemical, hematological indicators as well as liver and kidney histology during administration as well after administration for 15 days. As such, testing sample is not toxic during 30 days of administration in tested doses. These results are an important scientific basis for subsequent researches and applications on the possibility of using Cordyceps takaomontana to prepare functional food or medicinal products for community health.
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