|Year : 2013 | Volume
| Issue : 3 | Page : 156-160
Anti-scorpion venom activity of Andrographis paniculata: A combined and comparative study with anti-scorpion serum in mice
Ranjana S Kale1, Satish E Bahekar1, Shailesh R Nagpure1, Kartik J Salwe2
1 Department of Pharmacology, Mahatma Gandhi Institute of Medical Sciences, Sewagram, Wardha, Maharshtra, India
2 Department of Pharmacology, Mahatma Gandhi Medical College and Research Institute, Pillaiyarkuppam, Puducherry, India
|Date of Web Publication||17-Dec-2013|
Ranjana S Kale
Department of Pharmacology, Mahatma Gandhi Institute of Medical Sciences, Sewagram, Wardha - 442 102, Maharashtra
Source of Support: None, Conflict of Interest: None
Objectives: The objective of this study is to evaluate the anti-scorpion venom (ASV) property of Andrographis paniculata in comparison with anti-redscorpion venom serum and this study aimed to determine its combined effect with anti-redscorpion venom serum.
Materials and Methods: Ethanolic extract of the plant AP was obtained using soxhlet apparatus. Swiss albino mice weighing 20-30g were used. Lyophilized venom sample of Mesobuthus tamulus and Lyophilized monovalent enzyme refined immunoglobulin anti-scorpion venom serum (ASV) was used. Using lethal dose of scorpion venom (25.12μg/g), the venom neutralizing ability of plant extract (1 g/kg) and ASV individually as well as in combination was studied using in vivo and in vitro methods. Mean survival time, protection fold and percentage survival of animals over the period of 24 h were the parameters used.
Statistical Analysis: Results were analyzed using Student's t-test.
Results: Ethanolic extract of AP (1 g/kg) showed some protective effect against scorpion venom. ASV was found more effective than plant extract. But, when plant extract and ASV were used in combination, potency of ASV was found to be increased both in vivo and in vitro.
Conclusions: Present study demonstrates that, both plant extract and ASV have their own scorpion venom neutralising ability in vivo and in vitro, but their combination is most effective in venom neutralizing ability.
Keywords: Andrographis paniculata , anti-scorpion venom, lethal dose, Mesobuthus tamulus
|How to cite this article:|
Kale RS, Bahekar SE, Nagpure SR, Salwe KJ. Anti-scorpion venom activity of Andrographis paniculata: A combined and comparative study with anti-scorpion serum in mice. Ancient Sci Life 2013;32:156-60
|How to cite this URL:|
Kale RS, Bahekar SE, Nagpure SR, Salwe KJ. Anti-scorpion venom activity of Andrographis paniculata: A combined and comparative study with anti-scorpion serum in mice. Ancient Sci Life [serial online] 2013 [cited 2020 Apr 9];32:156-60. Available from: http://www.ancientscienceoflife.org/text.asp?2013/32/3/156/122999
| Introduction|| |
Scorpion sting is one of the common life threatening acute medical emergencies and also can be considered as a neglected public health problem in tropical and sub-tropical countries including India.  In Maharashtra, particularly in Vidarbha and Konkan region, scorpion sting is a major health hazard especially, during paddy harvesting season immediately after rainy season leading to high mortality and morbidity rates. Indian red scorpion (Mesobuthus tamulus) belonging to Buthidae family is the most lethal amongst all the poisonous species of scorpions in India. 
Red scorpion venom causes massive release of neurotransmitters, which result in various clinical features resulting from envenomation.  Scorpion venom is a potent sodium channel activator.  It causes, delay in closing of neuronal sodium channels, which results in "autonomic storm" leading to sudden pouring of endogenous catecholamines into circulation leading to transient sympathetic and parasympathetic stimulation. , Symptoms such as profuse sweating, vomiting, increased salivation, bradycardia, priapism, cold extremities or transient hypertension followed by development of severe cardiovascular manifestations such as hypotension, tachycardia, tachypnea, pulmonary edema, tingling numbness and restlessness. ,, Morbidity and mortality are result due to acute refractory pulmonary edema, cardiogenic shock and multi-organ failure.  There are different opinions regarding to the treatment of scorpion sting. In the past years, various regimens have been tried including lytic cocktail, insulin, atropine, nifedipine, betablockers and captopril,  but all these have failed to reduce mortality and morbidity substantially. Even serotherapy has been a matter of debate and controversy in this matter.  However, α1 -blocker prazosin had shown to reduce fatality rate to 1%. 
In the context of the above discussion, it is expected that the real therapeutic agent for scorpion envenomation should be the one which should be able to neutralize venom or toxins following a bite.  Anti-scorpionvenom (ASV) is a Lyophilized monovalent enzyme refined immunoglobulin specific for scorpion M. tamulus.  ASV is an antidote and useful therapeutic agent capable of neutralizing circulating venom toxins if administered immediately and even at the late stages of envenomation. However, it is uncertain that whether ASV can reverse cardiac side-effects and prevent cardiovascular morbidity and mortality.  Usual mode of administration is either by intravenous (IV) or intramuscular route. Scorpion anti-venoms are rather species specific. 1ml of ASV can neutralize 1.0 mg of dried scorpion venom.  The certain lethal dose (LD 99 ) value of Indian red scorpion venom is 40g/0.5 ml of injected dose IV.  There is strong clinical correlation between clinical symptoms of envenomation and the level of scorpion venom antigens in the serum  hence, variable doses of ASVs are necessary for neutralization of changeable quantity of scorpion venom. ASV has been available for clinical use in India since 1997. ,
Numerous plant species are being used as folk medicines to treat scorpion sting. , Andrographis paniculata (AP) is one of these plants that has long been used in traditional medicine. It is widely found and cultivated in tropical Asian countries, northern parts of India, Java, Malaysia, Indonesia, West Indies, throughout South India. Sri Lanka is center of origin and diversity of species.  It is a herbaceous plant commonly known as "King of bitters"due to its extreme bitter taste. It is also known as "Kalmegh" and belongs to family Acanthaceae. The most common reported uses are for digestive problems, infections ranging from malaria, hepatitis, diarrhea, bronchitis, fever, common cold and snake bite. , Alcoholic extract of AP shows the maximum anti-venom activity by in vitro assay of human red blood cells membrane lysis.  Also, the ethanolic extracts of the AP have shown some protective effect against the red scorpion venom in mice by partial in vitro and in vivo neutralization activity. 
In this study we seek to compare the anti-scorpion venom property of AP extract with that of the anti-red scorpion venom and to study the effect of its combination of AP extract with anti-red scorpion serum.
| Materials and Methods|| |
Collection of plant materials
The plants were cultivated and collected from local garden of the institute during early rainy season (June and July). The plant was authenticated by a Botanist. The plants at flowering stage, i.e., after 3 rd to 4 th month of sowing, were cut at the base leaving behind about 10-15 cm of stem for plant regeneration.
Preparation of extract
Fresh aerial parts of plant were collected, cleaned by washing in tap water and shade dried. Fine powder was made and stored in an airtight container. The alcoholic extract was prepared according to procedure reported by Mahanta and Mukharjee.  40 g of dried powder was macerated in 95% ethanol overnight. It was then packed in the thimble of the soxhelet apparatus and was extracted using 95% ethanol refluxing at 60-80°C which yielded an extract dark green to brown in color. The stock extract thus, obtained was preserved in airtight glass container and kept inside a refrigerator at 4°C.
Scorpion venom sample
Lyophilized venom sample of M. tamulus was purchased from Haffkine Institute, Parel, Mumbai, Maharashtra and was stored at 2-8°C for future use, taking all necessary precautionary measures of handling and storage.
Anti-scorpion venom sample
Lyophilized monovalent enzyme refined immunoglobulin ASV of M. tamulus (SS100902; MFD September 2010 and EXP February 2015) was purchased from Haffkine Bio-pharmaceutical Corporation Ltd., Parel, Mumbai, Maharashtra and was stored at 2-8°C for future use, taking all necessary precautionary measures of handling and storage. Each 1 ml of reconstituted ASV neutralizes not less than 1.0 mg of dried red scorpion venom.
Swiss albino mice of either sex weighing 20-30 g were used for the study. All the animals were kept in polypropylene cages and maintained temperature at 25 ± 2°C. They were kept in 12:12 h light: Dark cycle and fed on standard laboratory chow and water ad libitum. Animals were acclimatized to laboratory conditions for 10 days before the test.
Ethical clearance was taken from Institutional Animal Ethics Committee of institute where the research was conducted (MGIMS/IEAC/2/2012).
Calculation of LD 99 of red scorpion (M. tamulus) venom
Lethal dose (LD 99 is defined as the least amount of venom (dry/weight in grams) injected intraperitoneally to animals to result in death of 99% animals within 24 h. In this study, LD 99 of M. tamulus scorpion venom was calculated by probit analysis and the dose was calculated as 25.12 μg/g used for neutralization purpose. 
Acute toxicity of M. tamulus venom and its neutralization by plant extract and ASV
Animals were divided into four groups of six animals each (total 24). Each animal in all groups was dministered LD 99 dose of M. tamulus venom (25.12 μg/g) (i.p.) Group 1 received distilled water (DW) and was considered as control group. Group 2 received plant extract (1 g/kg), i.p. and was considered as test group. Group 3 received ASV (25.12 μg/g and was considered as standard group. Group 4 received plant extract and ASV, i.p. and considered as combination group. The plant extract as well as ASV was administered 5 min after the dose of venom. All groups received same volume of preparations. In all the groups, time of injection, duration of survival and number of animals which survived were recorded upto 24 h. All the experimental procedures were carried out at the same time of the day between 9.00 h and 12.00 h.
Neutralization of the lethal venom effect of M. tamulus venom by Alam and Gomes method
The neutralization test described by Alam and Gomes was followed.  Animals were divided into three groups of six animals each (total 18). LD 99 dose of M. tamulus venom was mixed in vitro with DW, plant extract and ASV separately for group 1 (control group); group 2 (test group) and group 3 (standard group). The mixtures were incubated for 1 h at 37°C and then centrifuged at 2000 rpm for 10 min. The supernatant was injected, i.p. into animals. Thus group 1 received DW incubated with LD 99 dose of M. tamulus venom, i.p. (control group).Group 2 received plant extract incubated with LD 99 dose of M. tamulus venom, i.p. (test group). Group 3 received ASV incubated with LD 99 dose of M. tamulus venom (standard group). All groups received same volume of admixture preparations. In all the groups, time of injection, duration of survival and number of animals which survived were recorded upto 24 h. All the experimental procedures were carried out at the same time of the day between 9.00 h and 12.00 h.
All the experiments were single blinded to prevent observational bias in which a post graduate student recorded survival time and animals which survived in each experiments. 
The statistical analysis was done using Student's t-test. P < 0.05 was considered statistically significant and <0.005 was considered highly significant.
| Results|| |
LD 99 of M. tamulus scorpion venom used was calculated by probit analysis and the dose was 25.12 μg/g. 
Acute toxicity of M. tamulus venom and its neutralization by plant extract and ASV
LD 99 of M. tamulus scorpion venom in first group caused 100% mortality. Ethanolic extract of the plant AP significantly increased mean survival time (0.60 h) and protection fold, but it could not completely protect the animals when used alone with scorpion venom. Administration of ASV alone with scorpion venom, not only increased mean survival time (15.04 h) and protection fold significantly but it also protected two animals (33.33%). Administration of ASV and plant extract with scorpion venom also increased mean survival time (19.30 h) and protection fold significantly, but it also protected three animals (50%). This means that administration of ASV and plant extract in combination is very much effective than used individually in all aspects, i.e., mean survival time, protection fold and complete survival [Table 1].
|Table 1: Mean survival time, protection fold, survived animals, and survival percentage against the LD99 of the Mesobuthus tumulus venom when challenged by the plant extract and anti-scorpion-venom immediately after venom administration|
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Neutralization of the lethal venom effect of M. tamulus venom by Alam and Gomes method
LD 99 of M. tamulus scorpion venom mixed with control in vitro caused 100% mortality. Ethanolic extract of the plant AP significantly increased mean survival time (2.24 h) and protection fold, but it could not completely protect the animals when used alone with scorpion venom. Administration of ASV alone with scorpion venom, not only increased mean survival time (22.29 h) and protection fold significantly, but it also protected three animals (50%). This means that, in vitro administration of ASV is very much effective than when used with plant extract in all aspects, i.e., mean survival time, protection fold and complete survival [Table 2].
|Table 2: Mean survival time, protection fold, survived animals and survival percentage against the LD99 of the Mesobuthus tumulus venom when mixed with the plant extract and anti-scorpion venom in vitro|
Click here to view
| Discussion|| |
Neurotoxins of scorpion venom are highly lethal than those of snake venom.  LD 99 of M. tamulus venom is found to be 25.12 μg/g.  Ethanolic extract of AP in the dose of 1 g/kg was found to have some protective effect against red scorpion venom in mice.  Since 1997 ASV is available for clinical use in India. , Various studies conducted shows that 1ml of reconstituted ASV serum neutralized 1.2 mg of Indian red scorpion venomby IV route in an in vivo study in mice. 
In present study, when LD 99 of scorpion venom was injected in mice, it produced 100% deaths in both acute toxicity group and in vitro neutralization group (control groups), these results were comparable to previous study  [Table 1] and [Table 2].
The ethanolic extract of AP significantly increased mean survival time and protection fold, but could not protect the animals from death in both acute toxicity group and in vitro neutralization group (test groups). This confirmed some protective effect of plant AP [Table 1] and [Table 2].
In group 3 (standard groups), administration of ASV with lethal dose of scorpion venom, significantly increased mean survival time and protection fold as compared to control group and test groups in both acute toxicity and in vitro neutralization group. But, in acute toxicity group two animals survived and the percentage survival was 33.33%. In in vitro neutralization group, three animals were survived and the percentage survival was 50%. This shows that, ASV has more protective venom neutralizing action in vitro as compared to that of acute toxicity [Table 1] and [Table 2].
In group 4 (Combination group), in acute toxicity group, when combination of plant extract and ASV was administered along with LD 99 of scorpion venom, significantly increased mean survival time (19.038 h) and protection fold (71.51) ascompared to that of standard group. Three animals survived and percentage survival was 50%. This shows the potentiating action of plant when combined with ASV.
When the acute toxicity group was compared with in vitro neutralization group, the latter was more protective in terms of mean survival time, protection fold as well as percentage survival of animals in all aspects showing more in vitro neutralization effect. In other words in vivo, higher doses of ASV and plant extract of AP are necessary to have beneficial effect on animals.
Hence, it can be concluded that ASV as well as ethanolic extract of AP, has scorpion venom neutralization property. Administration of plant extract increased the potency of ASV also. This result is similar to previous studies.  This can be possible due to inactivation or precipitation of active venom components by plant extract, which can be attributed to certain bioactive components in the plant such as sitosterol, polyphenolic compounds, flavonoids, alkaloids, saponins, tannins, pentacyclicterpines, nitrocompounds, cinamic acid derivatives, which are the known compounds possessing protein binding and enzyme inhibiting properties.  Additionally, this plant contains andrographolide, major diterpenoids making up to 4%, 0.8-1.2%, 0.5-6% in dried whole plant, stem and leaf extract respectively.  This active constituent of plant can be possibly responsible for ASV action by modifying actions of proteins and enzymes. These also inhibit snake venom phospholipase A2 activities of Indian cobra venom. 
| Conclusion|| |
It can be concluded that both ASV and ethanolic extract of AP, have scorpion venom neutralization property in vivo and in vitro, when used individually, but their combination provides maximum benefit in all the aspects of anti-venom property. However, further studies are required for elaboration of potentiating action of plant with ASV in humans. Considering the scorpion sting as acute medical emergency, further studies to prepare injectable form of extract of AP will be helpful. Larger in vivo doses may be more beneficial.
| Acknowledgments|| |
We acknowledge with gratitude the cooperation and assistance we received from Dr. Sushil K. Varma, Head, Department of Pharmacology, MGIMS, Sewagram, our resident Dr. Arup Misra and Mr. Gopal Kulkarni, statistician IHMP, Pune without whose help the study could not have been completed.
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[Table 1], [Table 2]
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