A SURVEY AND ANALYSIS OF POISONOUS PLANTS
OF FORENSIC INTEREST FROM HARYANA
A
SYNOPSIS
For the Registration of
DOCTOR OF PHILOSOPHY IN FORENSIC SCIENCE
DEPARTMENT OF GENETICS
Supervisor: Submitted by:
Dr. Rajvinder Singh Kiran Kumari
Assistant Professor Research Scholar
Deptt. of Genetics Deptt. of Genetics
M.D. University, Rohtak (Haryana) M.D. University, Rohtak (Haryana)
MAHARSHI DAYANAND UNIVERSITY, ROHTAK (HARYANA)
(NAAC ‘A’ grade accredited University)
(2015)
1
INTRODUCTION
Plants can be found all around us as part of interiorscapes in homes as well as in landscape
plantings outdoors. Plants which are poisonous have been known since early times. Few
poisonous species have a world-wide distribution but each geographical area can have its
own flora of poisonous plants. Several spontaneous plants are potentially toxic for human
beings and livestock under special circumstances. The plant exposures are one of the most
frequent poisonings reported to poison control centres (Vetter, 2000; Tinggi, 2003; Bruni et
al. 2010). Poisonous plants comprise the third largest category of poisons known around the
world (Bhatia et al. 2014). History also revealed that plants involved over 90% of all
poisonings in the nineteenth century and earlier. It is also estimated that more than 50,000
people die every year worldwide from toxic exposure. The incidence of poisoning in India is
among the highest in the world.
In India there are around 700 poisonous plant species belonging to encountered in India
include over 90 botanical families Common poisonous plants encountered in India includes
irritants plant (Castor, Glory lily and Croton), cardio toxic plants (Aconites and Yellow
oleander), Neotoxic plant (Colotropis, Datura and Shychos) and many more including
miscellaneous toxic plants (Shah and Vadlamudi, 2004; Kulkarni et al. 2015).
The accidental ingestion of toxic plant portions (for instance seed, fruit, root, etc.) can cause
severe poisoning or even death (Mclntire et al., 1990; Vetter, 2000). Animals, children even
adults may be accidentally injured by exposure to poisonous plants (Adhikari, 2012).
Poisonous plants such as Datura, Strychnos, Calotropis, Croton, Thevetia and Ricinus etc.
appear to be the commonest poisoning agents in India (Qureshi et al. 2001). Many of
poisonous plants are also responsible for suicide poisoning (Khajja et al. 2011).
Forensic toxicology deals with the chemistry and analysis of various types of poisons
including plants. A sound knowledge about poisonous plants can lead to a good scrutiny of a
medico-legal case. In India, there are so many cases have been reported where the criminals
had used food material mixed with stupefying plant agents such as Dhatura etc to loot the
passengers in the buses and trains. Detection of plant poisons in trace amount is a difficult
part of analytical toxicology process, as the nature of plant poison is seldom or hardly known.
Sometimes the forensic case files submitted to the laboratory request the screening for a
specific plant or plant-derived component, which might or might not be present.
The diagnosis of intoxicated patients is usually based on the morphological analysis of
ingested plant portions; this procedure requires experience in systematic botany (Bruni et al.
2
2010). Means, the identification of poisonous plants may be difficult as these plants do not
appear distinctly different from their nontoxic relatives (Douglas, 2008). The diagnosis of
intoxicated patients is usually based on the morphological analysis of ingested plant portions;
this procedure requires experience in systematic botany (Bruni et al. 2010). Means, the
identification of poisonous plants may be difficult as these plants do not appear distinctly
different from their nontoxic relatives (Douglas, 2008).
The problem of plant poisons may be aggravated by absence of knowledge of poisonous
plants. It makes crucial to have knowledge about surrounding poisonous plants. Information
gathered by generating database of poisonous materials from any particular region may be
life saving.
Some important botanical survey of poisonous plants from countries like Brazil (Agra et al.,
2007); Nigeria (Agaie et al.,2007, Adediwura and Kola, 2012); Zordan (Al Quran, 2005);
China (Huai et al., 2010) and South Africa (Botha and Penrith, 2008) had already been
conducted. In India, the studies on poisonous plants are available from Meerut district, Uttar
Pradesh (Tomar and Singh, 2007); Rajasthan (Katewa et al., 2008) Sabarkantha district,
Gujrat (Jangid and Sharma,2011); Sagar district, Madhya Pradesh (Choubey and Khare,
2011); Cachar district, Assam (Choudhury et al., 2011); Mehsana, North Gujrat (Desai and
Patel, 2012); Goa (Dias, 2012) and Rourkela, Odisha (Pasayat et al., 2013) and Udhampur
district, J&K (Bhatia et al., 2014).
Haryana is basically an agriculture state and large percentage of population is engaged in
agriculture and related activities. Among the major flora of Haryana, variety of poisonous
plants also grows. A lot of work has been reported on the vegetation of Haryana. Some
important botanical survey of plants from district like Kurukshetra (Lal and Yadav, 1983);
Mewat, Gurgaon (Sharma et al. 1992; Sharma and Ahmad 1995); Khetawas, Jhajjar
(Panghal et al. 2010); Mahendergarh (Yadav et al. 2010); Tosham block, Bhiwani (yadav et
al. 2010); Aravalli Hill Range in Khanak, Haryana (Kumar et al. 2013); Jind (Singh and
Kumar, 2013); Karnal (Kumar and Singh, 2013); Ambala (Vashisht and Kaur 2013);
Tosham block of district Bhiwani (Yadav et al. 2014); Karnal (Kaur and Vashisht, 2014)
Jhajjar (Arya et al. 2015); Yamuna Nagar (Vashisht, 2015).
No exclusive and forensically oriented research work on poisonous plants from Haryana has
been reported from Haryana. In recent pilot survey from Rohtak city has reported occurrence
of some poisonous plants (Singh et al., 2015).
3
Phytochemical profiling is as important as identification of poisonous plants. Forensic
toxicological analysis requires a deep knowledge of phytochemicals in poisonous plants.
Standardization for chemical profiling of poisonous plants is a tedious process. In a major
forensic contribution the plant poisons were forensically analysed using some chemical and
instrumental methods of analysis (Goutam and Goutam, 2006). Literature has also revealed
use of Thin Layer Chromatography (TLC) for the poisonous plant extracts (Singh et al.
2006). Since there are hundreds of species of poisonous plants therefore, the extraction of
active components of poisonous plant from viscera has also remained a challenge for the
experts. Although general extraction and screening procedures exist in the literature but an
exclusive and ideal forensic extraction procedure would be able to extract all components of
interest of a particular poisonous plant. This will prove the efficacy of the methods to ideal
with matrix effect and concentrations.
The understanding of the chemical profiles of poisonous plants is also crucial for the
development and implementation of effective forensic methodologies to solve cases in future.
For imparting better understanding, knowledge and research aptitude about plant poisons in
the students there is urgent need to enhance this field and conduct some field survey in the
native area.
4
LITERATURE REVIEW
In humans, the Cerbera odollam was observed to be responsible for about 50% of the plant
poisoning cases in Kerala. Hence a survey was conducted among veterinarians of Thrissur
District to find out the prevalence of various plants poisoning as well as the management
practices adopted to control it (Gaillard et al. 2004). In an unusual case of poisoning
involving the white seed variety of Abrus precatorius causing serious manifestations in a
middle-aged male who had consumed the seeds on the advice of a folk medicine practitioner
was reported (Pillay et al. 2005). A publication from Mangalore reported that people from
rural areas who are less accessible to medical health care facilities or those who are ignorant
of the facilities, sometimes prefer local hakims for treatment of common ailments. But due to
their ignorance of the fact that such practitioners lack scientific expertise, they suffer dire
consequences from such primitive therapies. This person who availed therapeutic services
from a hakim, ended fatally as a result of overdoses of a root preparation obtained from Glory
Lily plant (Samanta and Uday, 2005). In another paper poisoning by Glory Lily was
reported from Karnataka. Some plant poisons were covered and phytochemically analysed
(Goutam and Goutam, 2006). An unusual case reported poisoning involving the white seed
variety of Abrus precatorius that caused serious manifestations in a middle-aged male who
had consumed the seeds on the advice of a folk medicine practitioner. Abrus precatorius
(Jequirity; Rosary pea), a plant that grows wild in most parts of India has been rarely reported
in Indian medical literature, even though cases do get reported periodically from around the
world (Pillay et al. 2005). Report on Dhatura poisoning was also published (Binev, 2006;
Amini, 2012). Poisoning due to accidental ingestion of poisonous plant aconite in Europe and
U.S.A has also mentioned in literature (Fujita et al. 2007). Each year over 100,000 exposures
to toxic plants were reported to poison centers throughout the United States (Froberg et al.
2007).
An account of 32 poisonous plants occurring on the Aravalli hills of Rajasthan was presented.
The information on the poisonous plant species was gathered from the tribals during
ethnobotanical field survey (Katewa et al. 2008). In a fatal poisoning due to an accidental
consumption of Adenia dgitata was reported where a 17-year old boy died of rapid
progressive multiple organ failure 96 hours after ingestion Adenia digitata fruits. The victim
had accidentally ingested the fruits of Adenia digitata, mistaking it for the edible passion
fruit, Passiflora edulis (Shetty et al. 2008). Jatropha curcas was reported responsible for
5
death of five family members, who presented within a few minutes with complaints of
vomiting and diarrhea (Shah and Sanmukhani, 2010). There are some plants like Cerbera
odollum, Cleistanthus collinus, Strychnos nux –vomica, Abrus precatorius, Colchicum
autumnale and Datura which are toxic and used for homicidal and suicidal purposes. Those
plants which are allergens or less poisonous, are used in burglary purpose in buses/trains.
Mucuna prurita is most useful plants for burglary purpose. Sometimes accidental poisoning
by poisonous plants has also been observed in tribal area of south Rajasthan (Khajja et al.
2011). Poisoning due to accidental ingestion of Dieffenbachia plant (Dumb Cane) has also
been mentioned in the literature (Adhikari, 2012). Some plants are also used in bioterrorism
to produce harmful effects or even mass destruction also mentioned in literature (Duncan
and Smith, 2012). Literature has been observed toxicological effects of extract from stem
bark, leaf and seed kernel of yellow oleander in albino rats was carried out (Khan, 2010).
The neurobehavioral, biochemical and ultrastructural alterations of Datura stramonium L
leaves in mice model has been reported (Devi et al. 2012). The acute toxicity of Ricinus
communis on Swiss mice has been discussed in literature (Pingale, 2011). In developing
countries, traditional medicine is widely used to treat many of non-infectious and infectious
ailments and it is estimated that approximately a total of 80% of the world’s population use
traditional medicine (Karthishwaran and Mirunalini, 2010). According to one estimate,
25% of the commonly used medicines contain compounds isolated from plants (Mukhtar et
al. 2008). High mortality has been reported due to consumption of Argemone mexicana and
Cassia occidentalis seeds in northern part of India (Verma et al., 2001, Panigrahi et al.,
2014). An important study was made to collect and systematically document the traditional
knowledge of poisonous plants of Udhampur District for the benefit of humanity before it is
entombed forever. A total of 90 toxic plants were listed from the study site. Most dominant
toxic families were Fabaceae, Asteraceae, Solanaceae, Apocynaceae and Euphorbiaceae.
Most of the poisonous plants were herbs (57.1%) and the whole plant toxicity was reported to
be the highest followed by leaves (Bhatia et al. 2014; Karthika et al. 2014).
Phytochemical characterization: Some efforts have been made to identify chemical
characteristics of plant poisons in forensic context by using Thin Layer Chromatography
(Tiwari 1976; Goutam and Goutam 2006; Singh et al. 2006). Some useful data related to
TLC analysis of some plant alkaloids have also been reported earlier (Stahl 1969). Various
extraction methods and techniques i.e LC-MS, TMS, GC-MS and immunoassay for detection
of toxic alkaloids from blood were described (Beyer et al., 2009). Some useful data related to
6
Thin layer chromatography (TLC), high performance liquid chromatography (HPLC) and gas
chromatography- mass spectroscopy (GC-MS) techniques used for identification of plant
alkaloids have been reported earlier (Iranbaksh 2006, Singh et al., 2006). TLC
fingerprinting of the leaves of ricinus communis linn through High Performance Thin Layer
Chromatography (HPLC) was carried out for quantitative determination of ferulic acid and
geographical variation (Verma et al. 2011). Various qualitative and quantitative methods like
HPLC-ESI-MS, Spectrophotometric assay, TLC, HPLC, HPLC/MS, LC/MS/MS and digoxin
immunoassay has been employed for detection of various toxins of aconitine, cevadine,
colchicine, sparteine, strychnine from human blood and viscera matrices (Gaillard and
Pepin, 1999, Praveen et al., 2012). The application of GC/MS and FTIR was used for the
analyzed the chemical constituent of Extracts of Thevetia peruviana Seed has been reported
(Sangodare et al. 2012).
7
OBJECTIVES
Keeping in mind the significance of this research problem the following objectives have
been formed:
1. To generate epidemiological profiles of cases of plant poisoning in humans and
livestock occurred in last five years in Haryana.
2. To make a survey on the existence of poisonous plants in Panchkula, Rohtak,
Faridabad and Sirsa districts of Haryana.
3. Morphological and molecular identification of these plants.
4. To establishing a herbarium of the identified poisonous plants.
5. To perform Thin Layer Chromatography and Gas Chromatography-Mass
Spectroscopy of some forensically important poisonous plants for phytochemical
analysis.
6. To study toxicity and development of exclusive methods of extractions of toxic
constituents of these plants in animal model (mice).
8
MATERIALS AND METHODS
1. Generating epidemiological profiles: In order to generate database, record of cases of
plant poisoning happened in Haryana during last five years will be collected from Pt.
B.D. Sharma University of Health Sciences (Rohtak), Medical and veterinary
Colleges/Hospitals, and Forensic Science Laboratory, Madhuban. Database will be
comprised of various parameters like gender, age, mode and plant type responsible etc.
2. Selection of sampling sites and mode of survey: Survey for the existence of poisonous
plants would be carried out from Panchkula, Rohtak, Faridabad and Sirsa districts of
Haryana (Map-1). The sampling sites have been selected according to similar
geographical areas and insignificant literature on poisonous plants. Direct interviews will
be conducted of the informants such as herbal practitioners and local inhabitants for
knowing the distribution of poisonous plants in selected sites. These plants will then be
photographed and relevant parts (specimens) would be collected for further identification
up to species level. Some parts or whole plant bush would also be collected for herbarium
purpose.
Map-1 Study sites from Haryana (Panchkula, Rohtak, Faridabad and Sirsa)
9
3. Morphological and molecular identification: Morphological identification of plants
specimens would be made with help of available expertise and literature monographs
(Frohne and Pfander, 2005; Bhattacharjee and Bhattacharjee 2013). Isolation of
DNA from selected plants will be done by using methods available in the literature
(Saghai-Maroof et al. 1984; Deshmukh et al. 2007; Vural and Dageri, 2009; Ferri et
al. 2009; Bruni et al. 2010; Cook et al. 2015) and molecular identification of plant will
be done by using universal primers in PCR described as per literature (Linder et al. 2000;
Alvarez and Wendel, 2003; Fazekas et al. 2012; Zeng et al. 2015).
4. Establishing Herbarium: The specimens would be deposited in herbarium to be
established in Department of Genetics, M.D. University, Rohtak, Haryana (India).
Specimens would be properly preserved in museum jars showing all relevant information
as per method described earlier (Jain and Rao, 1977).
5. Phytochemical analysis: Forensically important poisonous plants would be used for
phytochemical and toxicological analysis. The extracts of the selected parts of the plants
would be prepared in different solvents by using soxhlet apparatus. These extracts would
be taken for following chromatographic analysis:
i. Thin layer chromatography (TLC): Extracts will be spotted on pre-coated TLC
plates. A solvent system separating constituents of all the selected poisonous plants
would also be devised. After separation, the phytochemical constituents will be
visualized in non-destructive and destructive methods available in the literature
(Manual of Forensic Toxicology DFS, 2005, Goutam and Goutam, 2006). The
tables of hRf values would be generated.
ii. Gas Chromatography–Mass Spectroscopy (GC-MS): The extracts would also be
analyzed by using Gas Chromatograph Mass Spectrophotometer (Zibbu and Batra,
2011; Cock and Kalt, 2012; Shettima et al. 2013).
6. Toxicological analysis for determining toxicity levels: The toxicity of the extracts from
different plants would be determined with method available in the literature (Zohra and
Fawzia 2014; Zainuddin and Sul’ain, 2015).
7. Extraction of plant poisons from viscera sample: Chemistry of poisonous plant is very
complex subject. They may have a mixture of several hundred different chemical
compounds, and extraction and identification of toxic compound/s from viscera in most
of the cases is a tedious job and sometimes subjected to the reference material. Difficulty
10
arises mainly when many harmless compounds behave very much like the toxic
constituents in the same plant. For example, proteins and tannins under certain conditions
behave like alkaloids, and natural sugars mimic the reactions of glycoside. It means the
poisonous substances contained in the plants are diverse both in kind and in their effect
on the human body, and no exclusive and perfect method of extraction of plant poisons is
available. Some literature (FSL manual of Toxicology, 2005) is available for this
purpose but more elaborated study is required for achieving this objective. The
experimentation on the adult albino mice would be performed after taking necessary
Animal Ethical Clearance, in accordance with the guidelines in the care and use of
laboratory animals and after approved by the Institutional Animal Ethical Committee
(IAEC).
11
TENTATIVE PLAN OF WORK
Research work will be carried out as follows:
First Year:
1. Generating database of cases of plant poisoning.
2. Survey on existence of poisonous plants.
3. Collection of samples and their identification.
Second Year:
1. Establishing herbarium of poisonous plants.
2. Phytochemical analysis of poisonous plants of forensic interest.
Third Year
1. Determination of toxicity and development of exclusive extraction methods of
plant poisons from animal model (mice).
2. Compilation of thesis for submission.
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SIGNIFICANCE OF THE STUDY
It is an interdisciplinary type of study which will combine forensic science with following
related scientific fields:
Aid for Poison Control Information Centers: Information of this survey may be
helpful for Poison Control Information Centers to generally provide referral and
management advice for plant poisoning. The observations of the proposed study can be
directly helpful for the human beings while animal community can also be escaping
from the areas prone to poisonous plants.
Botanists & Ecologists: This study will generate a database of poisonous plants
which will provide a support to the botanists and ecologists to conduct future research
in universities and other related institutes.
Forensic Veterinary Toxicology: It would be very useful for veterinarians to
document plant poisoning cases through government reporting services.
Pharmacology: Pharmacologists can really be benefited with the findings of the
proposed research project. Plants contain a large number of biologically active
chemicals (e.g. digitoxin, colchicines and atropine) which may be extremely useful for
treating various human and animal diseases. There is also a need to explore the use of
plant extracts in the field of pharmacy especially studying antimicrobial activities.
13
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