SNake Plants: anti venin...

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SNake Plants: anti venin...

Postby asnhs_archimedes » Sat Sep 13, 2008 4:52 am



hi experts!!!

Good day! I have problems with my project about snake plants as antivenin... we dont know the proper proceedures in doing our ecoeriments. here's our paper and pls do some corrections about it..

Snake Plant (Sansevieria zeylanica)
Leaf Extract: Venom Deactivator



A Research Proposal
Draft 4


Jan Christopher A. Go
Kristian R. Tesoro
Angelie M. Ruiz
Student researchers




Julie Ann M. Bonsubre
Research adviser






AGUSAN DEL SUR NATIONAL HIGH SCHOOL
San Francisco, Agusan del Sur
CHAPTER 1
INTRODUCTION

Background of the Study

Snake bite, specifically cobra bite, is a major health hazard that leads to high mortality and great suffering in victims. Conservative sources estimate that the number of accidents globally reach one million, resulting in 600,000 envenomations and more than 20,000 deaths annually. Other sources place annual incidences globally at 5 million with about 40,000 or more deaths – close to 10% mortality attributed to malaria. In India alone more than 200,000 cases are reported and an estimated 35,000 to 50,000 people die each year. A community-based retrospective survey in Kenya estimated that only 19% of the annual 151 snake bites per 100,000 people were potentially of venomous snakes. (Owuor & Kisangau, 2006)
Researches for antivenin drugs have been in demand in the society particularly, snakebites. It is the most common cases that need immediate actions. Due to the fact that venom is the cause of the fatality rate of such cases, antivenin drugs are really needed to be discovered. In the local place of San Francisco, Agusan del Sur, Philippines, most of the albularyos (local herbalist) uses herbs for such snakebites. Among these herbs is the snake plant.

This study is designed to test the potential use of antivenin content in common available herbs like snake plant. It is also designed to determine the feasibility of using snake plant leaf extract as an antivenin agent. This could be a great help in the production of more accessible, inexpensive, but effective antivenin drugs.








Statement of the Problem

This study aims to discover the antivenin effect of snake plant leaf extract. Specifically it inquires about the following question:

1. Is there a significant difference between the antivenin effects of snake plant leaf extract compared to the other laboratory antivenin drugs that already proven?

2. Is there a significant difference on the antivenin effect of snake plant leaf extract exposed to different concentration of extract?
a. 10, 000 ppm
b. 1, 000 ppm
c. 100 ppm

3. Is there any significant difference on the antivenin effect of snake plant leaf extract if we will consider different exposure of time it takes after the extract is injected into the frog?
a. 15 min.
b. 30 min.
c. 45 min.

Objectives

This study seeks to discover the antivenin effect of snake plant leaf extract. Definitely it intend to:

1. Compare the significant difference between the antivenin effects of snake plant leaf extract and to the other laboratory antivenin drugs that already proven.

2. Discover the significant difference on the antivenin effect of snake plant leaf extract exposed to different concentration of extract.
a. 10, 000 ppm
b. 1, 000 ppm
c. 100 ppm

3. Analyze the significant difference on the antivenin effect of snake plant leaf extract if we will consider different exposure of time it takes after the extract is injected into the frog.
a. 15 min.
b. 30 min.
c. 45 min.

Hypotheses

Based on the foregoing research problems, the researchers derived on the following hypotheses (Ho):

1. There is no important difference between the antivenin effects of snake plant leaf extract and to the other laboratory antivenin drugs that already proven.

2. There is no significant difference on the antivenin effect of leaf extract exposed to different concentration of extract.
a. 10, 000 ppm
b. 1, 000 ppm
c. 100 ppm

3. There is no significant difference on the antivenin effect of snake plant leaf extract if we will consider different exposure of time it takes after the extract is injected into the frog.
a. 15 min.
b. 30 min.
c. 45 min.

Assumptions

It is assumed that snake plant leaf extract contains bioactive components that can be used as antidote for snake bites. It is also assumed that snake plant is more effective antivenin agent compared to commercial antivenin.


Significance of the Study

The need of antivenin drugs in all venom producing animals specifically snake venom here in the Philippines is to be acted upon. The rainy season is fast approaching. Inline to that, snake comes out more often. Let us stick to the fact that snakebites are very widespread this time. Since snakebites need an immediate action availability of antivenin medicines are badly needed.

Furthermore, venom could already resist the commercial antivenin drugs available in the market. Research for a more available antivenin drug is also a major concern since it causes danger to mankind.

This study is found to be timely and relevant to test the potential use of antivenin content in common available herbs like bowstring hemp. The feasibility of using snake plant leaf extract as antivenin agent can be a great help in the production of more accessible, inexpensive but effective antivenin drugs.

Scope and Limitation

This study, Snake Plant (Sansevieria Zeylanica) Leaf Extract: Venom Deactivator focuses on the extraction of antivenin substances from the leaves of snake plant.

This study is found to be timely and relevant to test the potential use antivenin content in common available herbs like snake plant (Sansevieria zeylanica). The feasibility of using snake plant leaf extract as a venom deactivator can be a great help in the production of more accessible, inexpensive but effective antivenin drugs.

Snake Plant is a very common plant found almost in every community. This study requires only 500 grams of fresh, young, green leaves of snake plants which can be found anywhere in the Philippines.

The study could be a big step for us to establish new approach in our warfare against fatal and serious poisonous diseases.

Definition of Key Terms

Snake plant common name for members of a genus of plants, and for the hemp-like fiber produced by them. Formerly used for bowstrings, this fiber is long, silky, elastic, resistant to rotting in water, and about equal in strength to hemp. The individual strands of fiber are obtained from the elongated leaves of the plant. This is also known in the native land as “la suerte”.
Extract is a substance made by extracting a part of a raw material, often by using a solvent such as ethanol or water. Extracts may be sold as tinctures or in powder form. Extract is obtain from the plant.
Deactivator to cause to be inactive; remove the effectiveness of
Ether more specifically ethyl ether or diethyl ether, colorless, liquid compound, (C2H5) 2O, that boils at 34.6° C (94.3° F). Extremely volatile and flammable, with a powerful, characteristic odor and a burning, sweetish taste, ether is only slightly soluble in water (about 6 per cent), but mixes in all proportions with most organic liquids, such as alcohol and carbon disulphide. Ether itself is one of the most important organic solvents and is widely used in the laboratory as a solvent for fats, oils, resins, and alkaloids.
Rotary Evaporation the processes of Rotary Evaporation (ROTAVAP) which will produce concentrated, sticky crude extract.
Serum liquid part of blood: the fluid that separates from clotted blood, similar to plasma but without clotting agents.
TLC thin layer chromatograph is a chromatographic technique that is useful for separating organic substances. Because of the simplicity and rapidity, it is often used to monitor the progress of organic reactions and to check the purity of products and separate spots that are unresolved by one solvent.
Venom a poisonous fluid produced by an animal and injected by a bite or sting in order to immobilize prey or defend itself. Venoms are produced by a wide range of animals, including snakes, scorpions, spiders, and fish.

Vertical Kymograph An instrument used to record the temporal variations of any physiological or muscular process; it consists essentially of a revolving drum, bearing a record sheet (usually of smoked paper) on which a stylus or pen point travels to and fro at right angles to the motion of the cylinder; the drum is rotated by a mechanism at a presumably uniform rate, or the rate is indicated by a time marker which registers on the sheet. In some types the record sheet surrounds the drum, which rotates spirally, to allow a continuous record at different levels of the sheet; in other cases the record sheet is a long roll




Snake Plant: Venom Deactivator - 1 -






Running head: Snake Plant: Venom Deactivator







Snake Plant (Sanseviera zeylanica) Leaf Extract:
Venom Deactivator







Jan Christopher A. Go
Kristian R. Tesoro
Angelie M. Ruiz













AGUSAN DEL SUR NATIONAL HIGH SCHOOL
San Francisco, Agusan del Sur


Snake Plant: Venom Deactivator - 2 -


Snake Plant (Sanseviera zeylanica) Extract:
Venom Deactivator


Snake bite is a major health hazard that leads to high mortality and great suffering in victims. Conservative sources estimate that the number of accidents globally reach one million, resulting in 600,000 envenomations and more than 20,000 deaths annually. Other sources place annual incidences globally at 5 million with about 40,000 or more deaths – close to 10% mortality attributed to malaria. In India alone more than 200,000 cases are reported and an estimated 35,000 to 50,000 people die each year. A community-based retrospective survey in Kenya estimated that only 19% of the annual 151 snake bites per 100,000 people were potentially of venomous snakes. (Owuor& Kisangau, 2006)

The purpose of this study is to make a venom deactivator against cobra’s venom using the leaf extract of the snake plant.

The following literatures were reviewed to support this hypothesis/assumption.
The traditional healers are the first line defense against illnesses. The success of these healers is vaguely understood, partly due to their unknown materia medica and occult-mystical nature of their practice, but direct testimony from victims confirms success of their treatments. Biomedicine ignores their practice but they serve more snake bite accident victims than modern practitioners. The healers, especially the elderly and spirit inspired, are reputed to have an inherent expertise to handle these cases. In both societies they are recognized as healthcare providers with a profound socio-cultural understanding (Owuor& Kisangau, 2006)
Antiserum is the only therapeutic agent available throughout the world. A major drawback of serum therapy is its prohibitive cost and chance that victims are often some distance away from medical care when bitten. Serum sickness is a possible side effect of serum therapy that results in inflammation of certain tissues, and other symptoms. Generally anti-venom serum is a scarce commodity and in the world market – sometimes even governments with money to purchase large quantities cannot obtain it. There is a crisis in the quality and supply of antivenin serum in the sub-Saharan Africa due to fallen production and business pressures resulting from privatization of production plants. (Owuor& Kisangau, 2006)
The aim of this paper is to evaluate the ethnoveterinary remedies used by certain hunters in Trinidad. Plants are used to treat snakebites and scorpion stings and for hunting success. During the research some hunters claimed that their dogs either started hunting or hunted better after they had treated them in various ways with medicinal plants. This study has evolved out of

Snake Plant: Venom Deactivator - 3 -

an interest in a non-experimental evaluation of Trinidad and Tobago's ethnopharmacopoeia. This evaluation establishes whether the plant use is based on empirically verifiable principles or whether symbolic aspects of healing are more important. Hunters are principally interested in the following game animals: agouti (Dasyprocta agouti), matte (Tupinambis negropunctatus), tatou (Dasypus novemcinctus), deer (Mazama americana trinitatis), lappe (Agouti paca), manicou (Didelphis marsupialis insularis), wild hog/quenk (Tayassu tajacu). The hunting season lasts from October 1st to February 28/29, then there is a closed season for the rest of the year. There is no comprehensive published information available on the number of hunting dogs in the country. Hunting dogs are typically foxhounds, 13 inch and 9 inch beagles, coonhounds (all original stock imported) and mixed breeds. These dogs are usually scent and not sight hounds. (Lans, 2001)
Snake bite remedies are of interest since they may have recognizable therapeutic or toxic effects and are steeped in cultural beliefs that invariably conflict with formal health care practices. Ethnobotany, the study of the interaction between plants and people, is invaluable in discovering new herbal medicines and plant-derived drugs. However, glossing antiophidian plant reports for premier species in new antivenom development is a challenge in drug discovery. Studies exploring pharmacopoeia of unrelated cultures for plants treating specific medical conditions (snake bites in this study) present one way of validating anecdotal field reports, corroborating and selecting promising lead plants. This paper presents antivenin therapies from the Luo and Kamba ethnic groups of Kenya. The fact that the two communities have a fragile biomedical health delivery system, are highly exposed to snake bites yet documentation on plant remedies used as antivenin in Kenya is far less than the practice set the basis for this study. In addition the current study lays a basis for similar studies in other Kenyan communities. (Owuor& Kisangau, 2006)
Comparative studies in the two ethnic groups are made to reflect the degree of consensus of herbal remedy usage in these areas that have different agroecological zones, socio-cultural and socio-economic diversities; consensus is an indicator of the likely efficacy of the remedies in question. The immediate short-term goals of the study were aimed at conserving largely oral ethnomedical knowledge and availing to the scientific world plant therapies used as antivenin in the two communities. The long-term goal is to actualize conventional snake bite therapy options with effective, cheap, accessible and less iatrogenic (allergic) plant compounds. (Owuor& Kisangau, 2006)





Chapter 2
Methodology

This chapter presents the research methods and research design


I. Material and Equipment

Gloves Cotton Swabs Tuberculin syringe
Mask Thread Amphibian ringer’s solution
Wax paper Dissecting pad Vertical Kymograph
Scalpel Dissecting set Varnish
Forceps Scissors Ether
Blade Burner Millimeter Flask
Match Vials Paper
Ballpen Double folded Gauze Volumetric flask
Filter Paper ROTAVAP Methanol


III. Procedure


A. ACQUISITION OF TEST ORGANISM

The frogs were collected at Brgy. Karaos, San Francisco, Agusan del Sur, at the land and swampy area near the house of the researcher. 50 frogs were gathered. 30 frogs are subjected with the leaf extract with 3 different concentration and remaining 15 were grouped as the control setup. The remaining frogs were used as extra for emergency purposes. The 50 frogs were placed in a big aquarium.

B. ACQUISITION OF PLANT SAMPLE

The snake plants were collected at barangay 5, San Francisco, Agusan del Sur. Two hundred gram of leaf was gathered. One hundred twenty-five grams are subjected in thirty frogs with three different concentrations. And fifty grams of leaf was group as the Control Setup. The remaining twenty-five gram of plant was used for emergency purposes. The collected plant samples were placed in a basket.

C. EXTRACTION
Snake plant (Sanseviera zeylanica) was collected at Brgy 4, San Francisco, Agusan del Sur. This study focuses and uses its parts: leaf as antivenin agent. The leaves of snake plant were air-dried for 3 days and were pounded after through mortar and pestle. One hundred grams of the extracted leaf was weighed by an analytical balance and soak to 200 ml acidified methanol in a volumetric flask. After 24 hrs, extracts were filtered using double folded gauze and a filter paper. This was subjected to leaf extraction using the Rotary Evaporation Apparatus. The obtained leaf extract was secured in sterile vials.

D. CONCENTRATION

Using the leaf extract of Snake plant (Sansevieria zeylanica), 10 mL per extract was prepared. One mL per extract was mixed to 3mL methanol each and was labeled as 25% concentration. Two mL per extract was mixed to 2mL methanol each and was labeled as 50%. 3mL per extract was mixed to 1mL methanol each and was labeled as 75% concentration. 4ml of the stock solution was labeled as 100%. All the extracts were placed in closed and sterilized vials.


E. TREATMENT

Extraction method was conducted to test the effect of the Snake plant extract as antivenin deactivator.

Thirty frogs were gathered and subjected to One hundred twenty-five gram of snake plant leaf as the experimental setup. Fifteen frogs were gathered and subjected to Fifty gram of snake plant leaf as the control setup. Five frogs and Twenty-five leaves were used as extra for emergency purposes.

F. DATA GATHERING
The data were gathered by recording all the results and observations from the experiment conducted. In recording the muscle contraction of a frog, we need to look and observe the peak of a line in a vertical kymograph and record its corresponding value. List all the observations gathered in different time intervals, three replicates and different treatments. Record the volume of the venom and the volume of the extract from the plant samples used.


G. WASTE DISPOSAL

Collect the entire used sample and put it in the plastic bag/sack. Clean the place where the experiment was conducted. Put all the biodegradable wastes in a trash can separate from non-biodegradable. Chemical vials and excessing chemicals must be disposed properly by putting it on a thick plastic cellophane and burry it on the soil 30 m away from water sources.


Please help us.. thank you and God Bless..
asnhs_archimedes
 
Posts: 8
Joined: Sat Aug 23, 2008 7:03 am
Occupation: student
Project Question: Snake Plant: Anti-venum Agent
Project Due Date: August 25,2008
Project Status: I am just starting

Re: SNake Plants: anti venin...

Postby pinoy_escibidoo » Sat Sep 13, 2008 8:49 pm

Gretings and saluations...

Hi!!i never thought i could meet a student like me from the sam region!! I'm from ANHS, II-Darwin..

Your project is interesting...(How come i didn't think of that?) (char!) anyways i think the research paper is self is full of facts and findings..but ours have still many things to work on.. Just have fun in doing your project I think I can advice somethings but for now this is all what I can say.. try to send a private message to me!! I could help you out!!

PEACE OUT!!!
PRINCE OF TENNIS ROCKZZ!!!
pinoy_escibidoo
 
Posts: 4
Joined: Sat Sep 13, 2008 8:06 pm
Occupation: student
Project Question: magnetic levitation
Project Due Date: next year
Project Status: I am just starting

Re: SNake Plants: anti venin...

Postby barretttomlinson » Mon Sep 15, 2008 2:03 am

Hi,

You seem to have identified a very important problem, a great start for important research.

My best advice is that you need to have a serious conference with your research advisor very soon. I regret I have no knowledge of the rules governing Philippine science fairs. In the US you would have to submit your research plan to a Science Fair Research committee before starting any experimental work for two reasons: you are handling very dangerous materials (snake venom) and you are experimentling on live animals.

I am very curious about some questions:

1) Why do you think snake plant leaves have antivenin activity? A detailed discussion of the answer to this question would greatly improve your background of the study section. It is extremely important to be extremely careful to properly cite your sources and to quote any statements that you copy verbatim, with the proper attribution. (Failure to do this will get you expelled from the academic community.) You mention herbalists use these plants. A detailed discussion of this is very much in order - what part of the plant do they use, how do they prepare it, how do they advise using it, how do they know it is effective,etc.

2) If you do not have specific evidence that your leaf extract might be effective, how do you justify doing the test on live animals? Will the exposure to the extract hurt or kill the animals in the absence of envenomation? You don’t want the cure to be worse than the bite.

3) Do you know what is in the snake venom that causes toxicity? If so can you measure its activity by some kind of test without using the test animals? If so, you might be able to test the efficacy of your plant extract on inhibiting the toxin without using the test animal, at least initially.

4) Have you thought of how to obtain, handle and use snake venom safely, and what procedures you would take if you got some venom on or in you? Knowing the answers to these questions is key to getting permission to handle it.

5) I noticed you plan to dissolve your plant extract in methanol, and imply you will expose the frogs to the solution. Methanol is toxic to most animals. Will you poison them with your antivenin?

You are trying to address some very important issues, and I wish you every success with the project.

Best regards,

Barrett Tomlinson
barretttomlinson
Former Expert
 
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Joined: Wed Oct 03, 2007 12:24 am


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