Myanamr Health Research Registration 2020; 32(2): 139-145.
DOI: DOI: https://doi.org/10.34299/mhsrj.00994
Acute Toxicity of Three Medicinal Plants Using as Antidote in Myanmar
Swe Zin Aung, Khin May Thi, Ei Ei Htway, Aye Min Maw, Moh Moh Lwin, Aye Thida Htun, Zin Nwe Soe, Rai Kit, Khin Saw Aye
Myanmar Health Sciences Research Journal, 2020; 32(2): 139-145
ABSTRACT
Snake bite and scorpion sting are the important causes of morbidity (life-threatening) and mortality for human being of rural area. In Myanmar, more than 8000 cases are reported and an estimated mortality rate was 750 to 1000 die annually each year. Regarding snake bite registry, Mandalay Region was more than Ayeyarwady (1203 vs. 122). In Myanmar Traditional Medicine and international literature, Stachytar-phetaindica (L.)Vahl(အဆိပ်တစ်ရာ), Mimosa pudica L. (ထိကရုန်း)and Amaranthusspinosus L. (ဟင်းနုနယ်ဆူးပေါက်) have been commonly used as antidotes for snake, scorpion, insect stinging evenomation and in other diseases as well. The present study, conducted from September 2017 to September 2018, was aimed to determine the acute toxicity of these three medicinal plants. Different extracts of aerial part of Stachytarpheta indica (L.) Vahl, the whole plant of Mimosa pudica L. and root of Amaranthus spinosus L. were prepared by Soxhlet extraction method with ethanol (95%). Acute oral toxicity testing on rats was performed by the main test according to OECD 425 guideline (2008). In phytochemical screening, allthree plants showed the presence of alkaloids, tannins, phenolic compounds, steroids, carbohydrates, glycosides and saponin. Protein and cyanogenic glycoside were not detected. The phytochemical compounds, flavonoids, starchs and cardiac glycosides were detected only Mimosa pudica L. and Stachytarpheta indica (L.) Vahl. From acute toxicity study, a single 5000 mg/kg dose of ethanol extracts did not show any lethal effect on the tested animals. The LD50 of the three plants were more than 5000 mg/kg Therefore, the present study proved that these three medicinal plants had not acute toxic effects. This study can give evidence for no toxic data for the three plants in community.
In this study, the percentage yield of aerial parts of Stachytarpheta indica L. (Vahl) extract (8.1%), the whole Plants of Mimosa pudica L. (16.61%) spracing and the root of Amaranthus spinosus L. (3.2%) were found. The results of phytochemical screening of three medical plants are shown in Table 1
Table 1.Results of phytochemical test on three medicinal plants
Phyto-chemical test |
Results |
||
Stachytarpheta (Aerial part) |
AmaranthusspinosusL.(Root) |
Mimosa pudicaL. (Whole plant) |
|
Alkaloids |
+ |
+ |
+ |
Carbohydrates |
+ |
+ |
+ |
Flavonoids |
+ |
_ |
+ |
Glycosides |
+ |
+ |
+ |
Phenolic compounds |
+ |
+ |
+ |
Cyanogenic glycosides |
- |
- |
- |
Proteins |
- |
- |
- |
Saponins |
+ |
+ |
+ |
Resins |
+ |
- |
- |
Starchs |
+ |
- |
+ |
Steroids |
+ |
+ |
+ |
Tannins |
+ |
+ |
+ |
Cardiac glycosides |
+ |
- |
+ |
+=Detected, -=Not detect
The three plants showed the presence of alkaloids, tannins, phenolic compounds, steroids, carbohydrates and glycosides. Protein and cyanogenic glycoside were not found in all plants.The phytochemical compounds, flavonoids, starch and cardiac glycosides were detected in Mimosa Pudica and Stachytarpheta indica (L.) Vahl. Resin was detected only in Stachytarpheta indica.
The plant constituents like flavonoids, tannins, polyphenols, cinamic acid derivates, hydroxyl benzonic acid dervatives, momosine alkaloid and aristolochic acid have the protein binding and enzyme inhibiting properties.4Inhibition of these compounds was generally considered to be rate limiting step in management of animal’s venom. These compounds are reputed to neutralize action of animals’ venom. They have revealed strong inhibitory potential against animal’s venom hyaluronidases.12, 19
Table 2. Clinical observation of toxic signs of three tested plants during 14 days
Observed signs |
Stachytar- |
Amara- |
Mimosa |
Skin and fur Skin changes Piloerection |
(-) (-) |
(-) (-) |
(-) (-) |
Eye lacrimation Corneal reflex Pupil reaction to light |
(-) (-) (-) |
(-) (-) (-) |
(-) (-) (-) |
Mucous membrane |
(-) |
(-) |
(-) |
Salivation |
(-) |
(-) |
(-) |
Respiratory rate |
(-) |
(-) |
(-) |
Motor activity |
(-) |
(-) |
(-) |
Paralysis of limbs |
(-) |
(-) |
(-) |
Abnormal behavioral |
(-) |
(-) |
(-) |
Tremor |
(-) |
(-) |
(-) |
Convulsion |
(-) |
(-) |
(-) |
Diarrhoea |
(-) |
(-) |
(-) |
The acute oral toxicity of ethanolic extracts of selected three medicinal plants did not show any abnormality of the skin, fur, mucous membrane, respiratory and behavior pattern Table 2.
Table 3. Result of acute toxicity study of the extracts ofthree selected medicinal plants
Test plant |
Dose |
No. death per tested |
Observed period |
Ex-pected |
Ob- |
Stachytar- |
175 |
0/1 |
14 days |
0 |
0 |
550 |
0/1 |
14 days |
0 |
0 |
|
1750 |
0/1 |
14 days |
0 |
0 |
|
5000 |
0/3 |
14 days |
0 |
0 |
|
Amaranthus |
175 |
0/1 |
14 days |
0 |
0 |
550 |
0/1 |
14 days |
0 |
0 |
|
1750 |
0/1 |
14 days |
0 |
0 |
|
5000 |
0/3 |
14 days |
0 |
0 |
|
Mimosa |
175 |
0/1 |
14 days |
0 |
0 |
550 |
0/1 |
14 days |
0 |
0 |
|
1750 |
0/1 |
14 days |
0 |
0 |
|
5000 |
0/3 |
14 days |
0 |
0 |
During the study, no deaths were observed for 14 days. The gross morphology at all dosage levels during acute toxicity testing showed no signs of toxicity. Ethanolic extracts of three selected plants showed safety dose dependant on 5000mg/kg on tested albino rats. The detail result of acute toxicity test of three medicinal plants on ratsis shown in Table 3.
According to the Research South East Asia literature, many plants have been described for traditional treatments as anti-venom.10Some herbs that have been used against venom were Leucas aspera (Willd) Link. (ပင့်ကူထိပ်ပိတ်)(Myanmar, Vietnam and India), Azadirachta indicaL. (တမာ) (Myanmar, India),Emblica officinalis Gaertn.(ဆီးဖြူ)(Myanmar, India, China),Aristolochia indicaL. (ဣဿရမူလီ) (Myanmar, India), Euonymus chinenisPrain.(မရှော့) (Vietnam, Myanmar), Curcuma aromatic Salisb(နနွင်း) (Myanmar, India, China, Thailand, pakistan, Indonesia), Stachytarpheta indica(L) Vahl. (အဆိပ်တစ်ရာ) (Myanmar, Thai), AmaranthusspinosusL. (ဟင်းနုနယ်ဆူးပေါက်) (Myanmar, Vietnam and India) and Mimosa pudicaL. (ထိကရုန်း) (India, America) which were widely used as snake bite and scorpion sting remedies.9, 11-13
Other tribal and rural populations of various regions have found their own remedies and treats poisonous animals bite.Among them, Stachytarpheta indica L. (Vahl.),Amaran-thusspinosus L. and Mimosa pudica L. are widely distributed and easily available in Myanmar. There is no evidence these three plants in traditional medicine uses in Traditional Hospital in Myanmar. But they were used as antidote by practitioners and consumers immediately at bitten places in Myanmar.
Stachytarpheta indica L. (Vahl.) is an invasive herb species found in many countries,commonly called as Indian snake weed or portor weed.14This plant is being traditionally used in wound healing, skin disease, asthma and anti-venom in Myanmar.15AmaranthusspinosusL. is widely distributed throughout the tropics and warm temperate regions of Asia. It is known as prickly amaranthus in English.16 The plant is also used as green vegetables and cultivated in all over South East Asia. In Indian traditional system of medicine (Ayurveda), this plant is used in the treatment of internal bleeding, fever, burning sensation, leprosy, bronchitis, piles, eucorrhoea, snake bite, and as laxative, diuretic, stomachic.17, 18The whole plant and root of this plant are used for the treatment of snake-bite.16Previous studies showed this plant extract was used for anti-inflammatory effect, immune modulatory effect, anthelmintic, antidiabetic, antihyperli-pidemic and spermatogenic effect.1, 16
Mimosa pudicaL. is grown naturally throughout Asia in moist waste ground, lawns or open plantations. It is known as sensitive plant. Mimosa pudica has been extensively studied for myotoxicity and toxic enzymes of Monocled cobra (Najakaouthia) venom.19, 20 All parts of this plant are used in the treatment of dysentery, vaginal and premenstrual syndrome, hemorrhoids, inflammations, muscular pain, convulsions, snake bite, burning sensation, asthma, leucoderma and internal bleeding.18-20 According to the Unani system of medicine, it is useful in blood impurities and biliousness, piles, jaundice, leprosy.21 In Western medicine, Mimosa pudica root is used for treating insomnia, premenstrual syndrome and hemorrhoids.19
They are administered in most disease conditions over a long period of time without proper dosage monitoring and consideration of toxic effects that might result from such prolonged usage.The toxic effects may take place prior to the binding of the toxicants to the vital organs such as liver and kidneys.Hence, evaluation of toxic properties of a substance is crucial when considering for public health protection because exposure to toxic substances can be hazardous and results in adverse effects on human being.In practice, the evaluation of toxicity typically includes acute, sub-acute, sub-chronic,chronic, carcinogenic and reproductive effects.22Acute toxicity is a single dose of a substance, or multiple doses given within 24 hours, and the determination of gross behavior and LD50. Usually the LD50 fora particular substance is the amount that can be expected to cause death in half of animals.23
Acute toxicity studies of Stachytarpheta indica (L.) Vahl.,Amaranthus spinosus L. and Mimosa pudica L. have been conducted but there is no reported toxic data in Myanmar.The present study aimed to investigate the acute oral toxicity of three selected medicinal plants using as antidote in Myanmar.
Plant collection and identification
Aerial parts of Stachytarpheta indica L.(Vahl) during the month of September to October, root of Amaranthus spinosusL and the whole plants of Mimosa pudicaL.during the month of October to November were collected from Mandalay Region. Stachytar-phetaindica L. (Vahl) and Mimosa pudicaL. are well grown in the rainy season. During these months active phytotherapeutic materials are plantiful in these plants. Identification of plants was performed by using Floral of Ceylon, India.24
Plant extraction
The collected plants were washed with distilled water, and cut and dried under shade area about two weeks.The dried plant samples were grinded by blender to get powder and extracted with solvent. Dried powder of Stachytarpheta indica L. (Vahl) was extracted with 95% ethanol by Soxhlet’s extraction method for 24 hours (4 times for 6 hours). The extract was dried in the digital water bath at 50ºC and stored in desiccator. The extracts of Amaranthus spinosus L. and Mimosa pudica L. were done by above procedure.
Phytochemical studies
The phytochemical screening was performed by Unani formulation, (India), 1986 and N- Raaman (2006).25
Acute toxicity testing
Acutetoxicity study was carried out by the main test of OECD 425 guideline (2008). Eighteen healthy young adult Wistar albino rats (200±20gm, either sex) were used in this study with the exclusion criteria of nulliparous and females were non-pregnant.They were maintained fed with standard pellet diet with distilled water. The rats were fasted overnight prior to dosing but water was not withheld. After fasted, the animals were weighed and the calculated amount of plants extracts were administered orally.The dose ranges of 175 mg/kg, 550 g/kg,1750 mg/kg and 5000mg/kg were used according to (test toxicity on animals as per) guidelines.The up and down procedure (dosing) were adopted based on toxic system of each dose according to guideline.
Each animal was observed at least once during the first 30 minutes after dosing for toxic signs and special attention was given during the first 4hours and every one-hour interval during the first 24 hours. The observation included changes in skin and fur, eyes and mucous membranes and also respiratory, autonomic and behavior pattern. Attention was given to observe of tremors, convulsions, salivation, diarrhea, lethargy, sleep and coma.23The animals were also observed for toxic signs and mortality for 48hours.And then, they were monitored for any abnormal changes throughout the study period (14 days).Then, the gross morphology was seen at all dosage levels during acute toxicity testing. Toxic signs and body weight were recorded for each animal to calculateLD50.
Data analysis (Statistical analysis)
The LD50 value was calculated by AOT425 Stat P gm software (version: 1.0) after all doses of main test recommendation was complete.Statistiacal estimate were calculated based on long-term outcomes.
In previous research, tannin compound of M.pudica was more effective in neutralizing N.kaouthia venom than commercial tannic acid and it also contained mimosine compound which is a toxic alkaloid.18Mimosa pudica plants extract was effective in neutralizing the main toxic effects of Russell’s viper and Saw scaled viper venoms.20
Concerning with acute toxicity study, LD50 of the ethanolic extract of Stachytarpheta indica L. (aerial parts) was more than 5000 mg/kgin the present study. The minimal lethal dose of ethanolic extract of Stachytarpheta indicaL. was 3000 mg/kg when Silambujanakiet al. evaluated the acute oral toxicity by Litchfield and Wilcoxon method.26 The other study showed LD50 for ethanolic extract of that plantwas 3000 mg/kg.27 In the study of Joshi et al., the maximum non-lethal dose of the ethanolic extract of Stachytarpheta indicaL. leaves was found to be 2000 mg/kg on albino mice by Staircase method.28
Many toxicity studies had been done for Amaranthus spinosus L. The methanolic extract and 50% ethanolic extract of Amaranthus spinosus L. were observed to be safe at 2000 mg/kg on Swiss albino mice by OECD 423 guideline.29, 30 Another study showed the methanolic extract of Amaranthus spinosus L. had no behavioural change nor mortality at dose of 2000 mg/kg.31 This study revealed LD50 of 95% ethanolic extract of Amaranthus spinosus L. was more than 5000 mg/kg in Wistar albino rats by OECD 425 guideline.
Acute toxicity study of the ethanolic extract of Mimosa pudica L.was carried out using acute toxic class-limit test dose guidelines of OECD 425 on Swiss albino mice and >2000mg/kg of LD50 was taken as cut-off value32, 33 In other study, the oral acute toxicity of the ethanolic extract of leaves was determined in albino mice by OECD 420 and no mortality was observed at a dose of 4000mg/kg.34 There was also no mortality up to 3200 mg/kg of 50% alcoholic extract in acute toxicity study on Swiss albino mice.35
Conclusion
All the data were expressed as oral administration of crude extracts at the highest dose of 5000mg/kg resulted in no mortalities or evidence of adverse effects, implying that three selected plants were non-toxic. However, further investigation is needed to confirm and prove on its chronic toxicity effect.
The authors are thankful to Department of Medical Research (PyinOoLwin Branch) for permitting to conduct this research and Laboratory Animal Services Division for supporting laboratory animals.
The authors declare that they have no competing interests.
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- ၀င်းအောင်၊ ဆေးသုတေသနဦးစီးဌာန၊ ၂၀၁၆ မြွေကိုက်ရောဂါကျန်းမာရေး အသိပညာပေး သုတေသန ဆောင်းပါးများ။ကျန်းမာရေးနှင့် အားကစား၀န်ကြီး ဌာန၊ စာမျက်နှာ - (၂၂) ။
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- လှမင်းထွန်း၊တိုင်းရင်းဆေးနှင့်အဆိပ်ဗေဒ။တိုင်းရင်းဆေးပညာဒီပလိုမာအတွက်တင်သွင်းသောစာတမ်း၊မန္တလေး၊ တိုင်းရင်းဆေးသိပ္ပံ၊ ၂၀၀၀ (စာမျက်နှာ - ၃၆ - ၆၃ / ၈၈ -၉၉ / ၁၀၀)
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