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Discussion Chapter 7
Shodhana of Kupeelu Page 147
DISCUSSION
There are many poisonous plants reported in ancient scriptures of Ayurveda which
are being still practiced widely in a number of diseases after proper Shodhana
(purification/detoxification). It is reported that aconite (Vatsanabha) purified by
cow’s urine is converted to cardiac stimulant, whereas raw aconite is cardiac
depressant1. Likewise the plant Kupeelu (Strychnos nux-vomica Linn.) known as nux-
vomica, is described under the ‘Upavisa Vargas’(poisonous group)2, it’s seeds have
been used successfully in many diseases3 after proper Samaskar, known as
Shodhana. Nux-vomica was introduced in Europe in the sixteenth century, but was
not much used in medicine, being chiefly employed to poison dogs, cats, crows, etc4.
It is cited in the treatises of Ayurveda that the ‘Visha’(poison) becomes
‘Amrita’(nectar) after logical administration5 and the ancient physicians of Ayurveda
successfully used this drug to combat number of diseases after proper purification in
some specific media. Various Shodhana (purification) procedures have been adopted
for purification of nux-vomica seeds and these methods are either mentioned in the
classics of Ayurveda or practiced traditionally6,7,8,9. The role of these purificatory
procedures on the major chemical constituents of Kupeelu is yet to be evaluated.
By taking these facts as background, the present study entitled “A Comparative
Pharmacognostical and Phyto- pharmacological Evaluation of Raw and Shodhita
Kupeelu (Strychnos nux-vomica linn.) Seeds” has been taken and carried out in the
following phases:
1. Conceptual Study
2. Pharmacognostical Study
3. Pharmaceutical Study
4. Analytical Study, and
5. Pharmacological Study.
CONCEPTUAL STUDY
Concept of Visha & Upavisha:
Visha means sadness, grief, sorrow, disappointments, etc. Hence, it is a substance
which creates “Vishada” by producing harmful effects on mind and body10. It
prevades the whole body immediately after ingestion or it’s entry into the body and
causes vitiation of the healthy dhatus11. The use of Visha dravya in Ayurvedic System
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of Medicine is available since Samhita period. Brihat Trayee texts describe the types
of poisons and their medicinal use in details. The toxic effects of the poisonous plants
are also described. The therapeutic or the pharmacological doses of these plants are
clearly elaborated. As per Charaka, poisonous dravya when used in its proper dosage
and in proper manner and in proper stage of the disease can prove as a medicine.
According to origin, Visha dravyas are classified into two types i.e. Sthavara Visha
(plant poison), Jangama Visha12 (animal poison). Among the above two types of
poisons, plant poison is most frequently used in medicine. When poisonous plants are
used in the medicines they act very fast in the body in low dose according to the
intensity of action. Depending upon the severity of toxic effects, Visha drugs have
been classified as Visha and Upavisha. Upavishas are the substances which exert
toxic effects on the body but do not cause instant morbidity. Usually they have
delayed onset of action which starts within 15-45 minutes after consumption58.
Kupeelu is included under Upavisha category which is having specific action on
spinal cord. If Kupeelu is used in crude and unpurified form, it exerts deadly
poisonous and even fatal effects. It develops restlessness, suffocation, tremors and
convulsion gradually and ultimately leading to death due to respiratory arrest within
2-4hrs13. However, after the purification, the Kupeelu turned to be a very effective
medicine to treat a number of diseases. Even the purified Kupeelu is claimed to be a
potent drug in countering old age problems and specially recommended during
senility as Rasayana (antioxidant) 14.
While going through the history of modern toxicology it is revealed that by 1500
BC, hemlock, opium, arrow poisons, and certain metals were used to poison enemies
or for executions. Notable poisoning victims include Socrates, Cleopatra, and
Claudius. Socrates was given a potent infusion of hemlock (Conium maculatum)
seeds15. These seeds contain an alkaloid coniine which is a neurotoxic agent like
strychnine and causes death by blocking the neuromuscular junction. By the time
certain fundamental concepts of toxicology began to take shape especially by the
studies of two renowned scientists Paracelsus (~1500AD) and Orfila (~1800 AD).
Paracelsus (1493 – 1541) was one of the first to distinguish between the therapeutic
and toxic properties of substances in modern era. His famous words were:
"All substances are poisons; there is none which is not a poison. The right dose
differentiates a poison and a remedy."
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Paracelsus determined that specific chemicals were actually responsible for the
toxicity of a plant or animal poison. He also documented that the body's response to
those chemicals depended on the dose received16.
The similar concept was also explained by Charaka 2000 years before. Charaka
opined that a deadly poison can become a very good medicine if it is administered
properly i.e. used in proper dosage, in proper manner and in the proper stage of the
diseases17.
Various properties of Visha have been described in the table no. 2.1. Due to its
subtleness (Sukshma), the poison vitiates Rakta (blood) because the latter has the
property of moving through the subtle channels in the body. Anirdeshya rasa or
Avyakta rasa (indistinct taste) is the characteristic feature of both the poison and
water. The latter dominates the composition of Kapha. Therefore, the Avyakta rasa
attribute to poison causes augmentation or aggravation of Kapha. Marmas or vital
organs are soumya in nature. Therefore, the poison which has opposite properties like
tikshna (sharpness) adversely afflicts these vital organs. The term ‘Vikasi’ is derived
from the root ‘Kas’= ‘hims’ (meaning violence) with the preposition ‘Vi’. By
implication, this term means excessive violence or the suppression of the ‘Ojas’
resulting in looseness of the joints. The term ‘laghu’ (lightness) implies instability for
which therapeutic measures do not produce the desired results. The term ‘Visada’
means freedom from sliminess as a result of which the dosas do not remain adhered to
the tissue elements, and therefore, constantly circulate all over the body. These
attributes of the poison together exhibit specific actions like aggravation of Vata18.
As stated earlier the Seers of Ayurveda have classified poisons into two types
depending on their origin. Apart from the Sthavara and Jangama, there is a third type
of poison called Gara visha which is prepared by the combination of various types of
substances. ‘Gara’ is a technical term specifically used in the texts for this type of
artificial poison which produces it effects after a long time to cause diseases and
death19.
Susruta has also described three categories of poisons including this artificial
(Kritrima) prepared variety20. When the poison is produced by the combination of two
non-poisonous substances, then it is called as ‘Gara’. However, when the poison is
produced by the combination of two poisonous materials, then it is called Kritrima.
Another type of poison, known as ‘Dusi visha’ has also been described by the ancient
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Acharyas. According to the Charaka, ‘Dusi visha’ vitiates blood (Rakta) and
produces symptoms like Aru (eczema in the head), Kitima (psoriasis) and Kotha
(urticaria). Depending upon the location of the dosas and the constitution of the
patients, poison produces several other complications. This type of poison afflicts all
the three dosas and may cause death of the patient21.
Susruta in this context also opined that a constant use of some particular time (i.e.
cloudy/windy day, rainy season), particular place (i.e. marshy country) and particular
diet (e.g. wine, seasamum, Kulattha etc.) sexual intercourse, excessive exercise as
well as regular day sleep tends slowly to poison the fundamental Dhatus and this slow
poison (Dusi visha) ultimately manifests some specific symptoms in the body22.
Table 2.3 shows various Upavishas as mentioned in Rasatarangini with their
fatal dose & fatal period. It is evident from the table that only one raw crushed seed of
Kupeelu is capable of producing death within 4 hrs. Therefore, Shodhana as well as
administration in proper dose should be strictly followed prior to its use in medicine.
Role of Media in Shodhana
The concept of Shodhana is reported for the first time in the Charaka Samhita in
the context of Danti Dravanti Kalpadhyaya. To reduce the ‘Vikasi’ property of Danti
root, Charaka mentioned a specific Samaskara (processing) by Agni23. Acharya
Vagbhata also mentioned the Shodhana of plant drugs in detail in the context of
Bhallataka Rasayana and Amrita Bhallataka24.
In the present study the Shodhana of Kupeelu seed was performed by following
the principles of Nimajjana, Swedana & Bharjana in various media.
The media employed in the Shodhana process has very significant role for
eradicating the toxic chemical components partially or by transforming them to non-
toxic substances. Sometimes media act like solvent to dissolve the material thereby
separating them from the insoluble impurities, like Godugdha, used for Guggulu
Shodhana or Churnodaka used for the Shodhana of Manashila. Media may also
provide some new organic or inorganic principles to the drug which may be
responsible for enhancing their therapeutic efficacy. Previous study reveals that
Gomutra Shodhita Vatsanabha is turned to be cardiac stimulant whereas the crude
one is claimed to be as cardiac depressant1.
In this study, an attempt has been made to validate the different Shodhana
procedures of Kupeelu with various media like,
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Shodhana of Kupeelu Page 151
Gomutra (cow urine),
Godugdha (cow milk),
Goghrita (cow ghee) ,
Kanji (sour gruel),
Eranda taila (castor oil),
Ardraka swarasa (fresh ginger juice) and
Water.
Though Gomutra and Kupeelu possess similar properties like Katu, Tikta, Rasa;
Katu Vipaka and Ushna Veerya, yet it is used frequently for the Shodhana of Kupeelu
seeds. It may be due to its Lekhana property which ultimately leads to expel out the
poisonous Strychnine and Brucine from the seeds. As the cow urine is an alkali media
it may also hydrolyze the toxic alkaloids Strychnine and Brucine to their less toxic
derivatives.
Godugdha possess properties like Madhura, Snigdha, Guru, Sheeta etc., which are
just opposite to that of Kupeelu. This may counteract the adverse effect of the drug.
The various activities of Godugdha like Preenana, Jeevana, Vajikarana,
Vayasthapana etc., may convert the poisonous Kupeelu seed to therapeutically more
potent one. A previous study also reported that boiling in milk converted the
strychnine into less toxic isostrychnine (Cai et al., 1990)27.
Ghee is viewed as a superior cooking fat because it doesn’t burn during cooking,
unlike butter and fats which are liquid at room temperature. When the seeds are fried
with Goghrita, it may takes on the properties of the seeds due to its “Samaskarasya
Anubartanat” action. Ghee also provides a soothing and cooling effect, helping to
offset the irritant effect of the seeds. By frying with Ghee, seeds became swollen, soft
and easily made into powder form.
Kanji is having the property to pacify Vata & Kapha and it is also used as
Shoolaghna28. Hence, Shodhana in Kanji may enhance the same properties of the
Kupeelu seeds.
Eranda Taila is specifically used in different painful conditions like Amavata,
Pristha & Guhya shula, Kati shula etc., as it is claimed as Vatahara29. When the
seeds are fried in Eranda taila, it penetrates readily into the seeds due to its Sukshma
property and may potentiate the Vatahara & Shothara activities of the seeds. Previous
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study shows that Ricinoleic acid is the main component of castor oil and it exerts anti-
inflammatory effects30.
Ardraka swarasa is Madhura in Vipaka and it pacifies Vata & Kapha. For this
reason to obtain better therapeutic efficacy, the traditional Unani practioners use the
Kupeelu seeds in acute rheumatic conditions after purifying it in Ardraka Swarasa31.
Water (Jala) is used as an intermediate media for Kupeelu Shodhana in Unani
System of Medicine32. The main aim of Shodhana of Kupeelu seeds is to remove the
toxic alkaloids Strychnine & Brucine. It is reported that these two alkaloids are
slightly soluble in cold water as well as boiling water33, 34. This may be the reason that
the Unani practitioners have selected the most common and easily available media for
Shodhana of Kupeelu seeds. Moreover the method of applying heat in Swedana
process may break these toxic alkaloids, converts and removes them from the seeds to
the water and helps in converting the toxic drug into a nontoxic.
The Drug Kupeelu
The drug Kupeelu was not described in the ‘Brihat Trayee’ texts of Ayurveda. A
drug described as Vishamustika is found in the Surasadi gana of Sushruta
Samhita35 but it is not botanically identified as S.nux-vomica36. Dalhana also
mentioned it as Raja Nimba37. Its uses in Ayurveda were recorded from the period
of “Brinda Madhava” (9th A.D.). The drug Visamusti was mentioned in the English
translation of Brinda’s Siddha Yoga edited by P. V. Tiwari, while describing
‘Vatavyadhi chikitsa’. Later it was mentioned by different authors with number of
synonyms. (Table 2.6) Synonyms like Visatinduka, Kupeelu, Visamusti etc., indicate
toxic nature of this tree. While searching through various Ayurvedic literatures
different pharmacological actions of seeds such as Shothahara, Putihara,
Vedanasthapana, Uttejaka, Nadibalya, Deepana, Pachana, Grahi,
Shoolaprashamana, Hridayottejaka, Kaphaghna, Kasahara, Vajikarana, Balva,
Kushthaghna, Kandughna, Swedapanayana etc., are available38,39. The other
parts of Kupeelu like root, bark, leaves, and fruits are also used as medicine in
various diseased conditions. Purified Kupeelu seeds are also claimed to be potent
drug in countering old age problems and specially recommended during senility
as Rasayana 40 (antioxidant). The plant is also found to have analgesic & anti-
inflammatory41, anti-oxidant42, anti-tumor43, anti-snake venom44, anti-diarrhoeal45 and
hepatoprotective46 activities when studied in animal models. Classical text books of
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Ayurveda suggest certain compound formulations containing purified seeds of
Kupeelu as one of the ingredient for the management in various diseases. (Table 2.7)
In a previous study, 16 alkaloids have been seperated and identified from the
crude nux vomica and 80% of them are strychnine and brucine, as well as their
derivatives such as isostrychnine and brucine N-oxide47. Strychnine (C21H22O2N2 ;
m.p. 286 to 2880 C) and brucine (C23H26O4N2 ; m.p. 1780 C) have been reported as
the most important and strongly toxic alkaloids present in this, besides other minor
alkaloidal constituents48. It is also reported that nux vomica in large doses,
producing tetanic convulsions and eventually death and in lesser doses it may
manifest mental derangement49. So it is mandatory to purify or properly processed
nux vomica seeds prior to its administration in therapeutics. There are also few
reports of previous research works advocating a variety of methods of
purification of nuxvomica seeds as per Chinese50, Unani51 and Ayurveda52
system of medicine.
Many of the strychnine poisoning resembles the clinical features of tetanus and
hence strychnine poisoning has to be diagnosed from tetanus. The Table 2.8 indicates
some of the distinguishing points between strychnine poisoning and tetanus.
Strychnine toxicity (LD50
value) is also varying from one animal to another and it
is reported in Table 2.9.
PHARMACOGNOSTICAL STUDY
Pharmacognostical study of a drug is the key for getting therapeutically potent
medicines prepared of genuine raw drug. Genuine raw drug can only be obtained if it
is collected from its natural habitat which grows in ideal soil. The soil which is devoid
of big stones, excessive water, without Valmika (snake nest) and nearer to water tanks
should be selected for this purpose. The soil which is unctuous, smooth, tight,
blackish-white or reddish in color and with grass is considered as the best soil for
collection of raw materials59.
Ayurveda also gives special importance on the characteristics of collected drugs.
The drug, collected for the medicinal usage should have some distinct characteristics
like, it should not be affected by smoke, rain, air or water and it should be collected in
the respective seasons60. The drug should be free from Krimi (pests), Visha (poison),
Shastra (weapon), Atapa (excessive sunlight), Pavana (strong wind), Dahana (fire),
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Toya (excessive moisture), Sambadha (diseases) and should not be grown on Marga
(road side)59.
The raw materials used in Ayurveda shall be collected according to part used,
season, potency etc., in a specific manner. Acharya Charaka recommended the
collection of fruits according to their season61. It is also advised not to collect unripen
or over-ripen fruit. Fruits which are well ripened shall only be collected from the
plants62.
In the present study, fully matured Kupeelu (Strychnos nuxvomica Linn.) fruits
were collected personally from the field of Bankura district, West Bengal in India
during the month of December and the seeds were then taken out from the fruit pulp.
The fresh seeds thus obtained, were used for pharmacognostical study both before and
after Shodhana.
On comparing macroscopically the organoleptic characters of the Shodhita
powdered samples with raw seeds, certain changes in color, odor and taste were
observed. It indicates the impact of various media during the Shodhana processes.
This might be due to the fact that during Shodhana processes, the constituents
responsible for color, odor & taste of the media might have been transported into the
Kupeelu seeds to a certain extent, which ultimately developed some new organoleptic
characters in the Shodhita samples. The organoleptic characters are presented here in
the below mentioned table, (Table 7.1)
Table 7.1: Comparative organoleptic characters of raw and Shodhita Kupeelu
Powdered
Samples
Organoleptic CharactersColor Odor Taste
KR Pale brown to yellowish gray Odorless Intensely bitter
KGM Whitish gray Characteristic ofurine
Salty bitter
KGD White Characteristic ofmilk
Sweetish bitter
KGG Golden brown Characteristic ofghee
Sweetish bitter
KGMD Grayish white Not Characteristic Bitter
KGMDG Grayish brown Characteristic ofghee
Sweetish-bitter
KET Reddish brown Pungent Moderately bitter
KKJ White Characteristic ofKanji
Moderately bitter
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Shodhana of Kupeelu Page 155
KAS Whitish grey Characteristic ofArdraka
Bitter with a little bitof pungent
KW Whitish Not Characteristic Bitter
KBW Whitish Not Characteristic Bitter
Histochemical studies reveal the presence of Strychnine, Brucine and
Hemicellulose in the raw seeds (Table 3.1). Strychnine was found to be present in the
middle portion of the endosperm whereas, Brucine in the outer part of endosperm.
Hemicellulose was found in the cell wall of seeds.
In Microscopic study of the powdered samples most of the characters of raw
Kupeelu seeds (Table 3.2) were observed in Shodhita Kupeelu samples. Only the
aleurone grains were found to be absent in the powder microscopy of all the Shodhita
samples. Other characters were similar to the powder drug of the raw samples.
Aleurone grains are membrane-bounded storage granules found in cells of the
aleurone layer in plants; contains either a protein matrix or protein-carbohydrate
bodies. As they are protein in nature they may be denaturized or hydrolyzed into some
other forms during the Shodhana process. Oil globules were found plenty in number
in the samples like KGD, KGMD, KGG, KGMDG, and KET etc., where oil based
media were used for Shodhana purpose. The powder microscopic characters of raw
and Shodhita Kupeelu are summarized in the below mentioned table. (Table 7.2)
Table 7.2: Comparative powder microscopic characters of raw and ShodhitaKupeelu
Powdered
Samples
Diagnostic CharactersLignifiedtrichomes
Oilglobules
Starchgrains
Aleuronegrains
Polygonalendospermiccells
Brownishcolourcontent
KR + + + + + +
KGM _ + + _ + +
KGD _ ++ + _ + +
KGG _ ++ + _ + +
KGMD _ ++ + _ + +
KGMDG _ ++ + _ + +
KET _ ++ + _ + +
KKJ _ + + _ + +
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Shodhana of Kupeelu Page 156
KAS _ + + _ + +
KW _ + + _ + +
KBW _ + + _ + +
PHARMACEUTICAL STUDY
Shodhana of Kupeelu seeds were carried out by ten different methods for the
purification of the drug. Each Shodhana procedure was repeated for three times to
establish the validation of the Pharmaceutical processing.
The active constituents present in the drugs are also responsible for their toxic
effects. Therefore, it is not desirable to expel them out completely from the drugs.
The main aim of Shodhana process is to reduce the toxic constituents to some extent
or by potentiating their chemical transformation to non-toxic or relatively less toxic
substances. There may be some new principles added to the drugs which are
responsible for enhancing their biological efficacy. Hence, maximum beneficial effect
can be obtained by administering the Shodhita drugs within their therapeutic dosage
limit.
In this study, Shodhana of Kupeelu was performed by the process of Nimajjana
(dipping), Swedana (boiling), Bharjana (frying) etc., in various media.
In Nimajjana method, the Kupeelu seeds were kept immerse in some particular
media like Gomutra, Kanji, Ardraka swarasa , water etc., for a specific period of
time. In Swedana method the seeds were boiled under the bath of media in a Dola
yantra for a definite length of time.
Principles of Nimajjana & Swedana methods are similar to the common stages of
extraction process where the solvent enters through the pores into the cells resulting in
the swelling of the tissues and solution of the soluble constituents takes place within
the cells then there is escape of dissolved material through the solvent boundary layer
by the process of diffusion – finally separation of the solution from the drug occurs.
The rate of extraction depends mainly on the temperature gradient and concentration
gradient across the cell membrane. The rate of extraction and solubility is increased
by elevation of temperature. Rising temperature increases the concentration gradient
across the cell membrane thereby increasing mass transfer of active principles from
solid material to the solvent25.
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Shodhana of Kupeelu Page 157
The principles underlying the Swedana process is similar to the process of hot
continuous extraction where the continuous flow of liquid media occurs by the
temperature gradient due to heating26.
In Bharajana method, the seeds were fried by cow ghee in mild temperature for a
specific period of time. In Bharjana process, some physical & chemical changes like
reduction in hardness, increase brittleness, formation of new chemical compounds
take place which ultimately make the drug body friendly26.
During Shodhana of Kupeelu in various media, change in color of the media was
noticed and it might be due to the removal of color containing materials like tannin
from the seeds66. Taste of every media became bitter after Shodhana and it might be
due to the extraction of bitter principles like strychnine, brucine, etc. from the seeds.
Changes in organoleptic characters of Kupeelu seeds were also noticed and the final
weight obtained after each Shodhana procedure was noted accordingly. (Table 4.1-
4.20) The percentage of final weight obtained and the percentage of weight lost
during the each batch of Shodhana processes are duly calculated and presented in the
Table 7.3.
Table 7.3: Final quantity of Kupeelu seeds obtained after Shodhana procedures
Batch InitialQuantity(g)
FinalQuantityObtained(g)
Loss(g)
Loss % Average lossof 3 batches(%)
KGM-1 100 64.50 35.50 35.50 34.77
KGM-2 100 66.00 34.00 34.00
KGM-3 100 65.20 34.80 34.80
KGD-1 100 70.30 29.70 29.70 30.73
KGD-2 100 68.50 31.50 31.50
KGD-3 100 69.00 31.00 31.00
KGG-1 100 52.30 47.70 47.70 49.67
KGG-2 100 48.20 51.80 51.80
KGG-3 100 50.50 49.50 49.50
KGMD-1 100 70.30 29.70 29.70 30.40
KGMD-2 100 69.50 30.50 30.50
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Shodhana of Kupeelu Page 158
KGMD-3 100 69.00 31.00 31.00
KGMDG-1 100 63.20 36.80 36.80 36.10
KGMDG-2 100 61.80 38.20 38.20
KGMDG-3 100 66.70 33.30 33.30KET-1 100 72.30 27.70 27.70 25.27
KET-2 100 77.70 22.30 22.30
KET-3 100 74.20 25.80 25.80
KKJ-1 100 56.60 43.40 43.40 45.80
KKJ-2 100 54.70 45.30 45.30
KKJ-3 100 51.30 48.70 48.70
KAS-1 100 66.80 33.20 33.20 33.70
KAS-2 100 65.10 34.90 34.90
KAS-3 100 67.00 33.00 33.00
KW-1 100 68.30 31.70 31.70 30.93
KW-2 100 70.00 30.00 30.00
KW-3 100 68.90 31.10 31.10
KBW -1 100 88.60 11.40 11.40 13.53
KBW -2 100 86.30 13.70 13.70
KBW -3 100 84.50 15.50 15.50
After Shodhana with some specific media, all samples were peeled off with the
help of a knife, embryo were removed and then dried properly. The dried seeds were
made into powder form and the final weight of the each powdered sample was
recorded. Graph 7.1 shows that maximum loss in final yield (49.67%) was found
after Shodhana with Goghrita whereas minimum loss in final yield (13.53%) was
obtained after purification with boiling water. It might be due to the extraction of
more soluble mass from the seeds by Goghrita than boiling water.
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Shodhana of Kupeelu Page 159
Graph 7.1: Average loss of weight after Shodhana of Kupeelu seeds
Regarding utilization of media, total volume of Ardraka swarasa was utilized
during the Shodhana process. It might be due to the several factors like rate of
evaporation and condensation of Ardraka swarasa regulated by the environmental
temperature, humidity, surface area etc., and also might be due to the increased
absorption of media by raw seeds. The percentages of average utilization of media are
presented here in the Graph 7.2.
Graph 7.2: Average utilization of media during Shodhana of Kupeelu seeds
100
34.77 30.73
49.67
30.4 36.125.27
45.833.7 30.93
13.53
0
20
40
60
80
100
120
Average % weight lossKR
KGM
KGD
KGG
KGMD
KGMDG
KET
KKJ
KAS
KW
KBW
0
20
40
60
80
100
11.28
38.33
68
4555
70
11.66
100
8.43 10.83
Average % utilization of mediaFGM
FGD
FGG
FGMD
FGMDG
FET
FKJ
FAS
FW
FBW
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Shodhana of Kupeelu Page 160
ANALYTICAL STUDY
The physicochemical analysis of different samples such as raw and Shodhita
Kupeelu seeds were carried out followed by quantitative estimation of their toxic
alkaloids viz. strychnine & brucine by HPTLC. Differences in all the physico-
chemical parameters were observed among the raw and Shodhita Kupeelu samples.
Qualitative tests reveal the absence of glycoside in Shodhita Kupeelu however, no
other difference regarding functional group was observed among raw and Shodhita
Kupeelu seeds.
In comparison to the raw seeds, moisture content was increased in all the Shodhita
samples except in KAS. It indicates more removal of intracellular water portion from
the raw seeds by the process of diffusion during the Shodhana process with Ardraka
Swarasa. The percentage of loss on drying of all the samples were recorded and
presented here in the Graph 7.3.
Graph 7.3: Loss on drying of raw & Shodhita Kupeelu seeds
The Graph 7.4 indicates that maximum ash content was found in the Gomutra
Shodhita sample and it might be due the addition of more organic and inorganic
contents like nitrogen, sulphur, phosphorus, hippuric acid, NaCl etc., from cow’s
urine to the raw seeds63.
3.39 3.67
5.03 5.276.28
4.13 4.03
5.28
2.78
7.21
9.17
012345678910
Loss on drying %KR
KGM
KGD
KGG
KGMD
KGMDG
KET
KKJ
KAS
KW
KBW
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Shodhana of Kupeelu Page 161
Graph 7.4: Ash value of raw & Shodhita Kupeelu seeds
Water soluble extractives of all the purified samples were found to be
decreased in comparison to the raw drug (KR). This might be due to the solubility of
the contents like strychnine, brucine, loganin etc., in water64, 65. These water soluble
contents dissolved and pulled out from the raw Kupeelu seeds during the Shodhana
process by diffusion. The water & alcohol soluble extractive values of Gomutra
purified sample were found to be lowest in comparison to all other samples. The
result indicates maximum removal of water & alcohol soluble compounds from the
raw seeds by Gomutra. The results are presented here in the Graph 7.5.
Graph 7.5: Water & Alcohol soluble extractive values of raw & ShodhitaKupeelu
00.20.40.60.811.21.41.6
1.11
1.58
1.26 1.221.13
1.01 1.07 1.06
1.42
0.98
1.43
Ash value %KR
KGM
KGD
KGG
KGMD
KGMDG
KET
KKJ
KAS
KW
KBW
0
5
10
15
20
25
30
35
40
Water (W.S.E) & Alcohol soluble extractIve (A.S.E) %
W.S.E
A.S.E
Discussion Chapter 7
Shodhana of Kupeelu Page 162
pH values of all the samples were found to be acidic. It was in the range between
4.51- 6.25 (KET-4.51; KGM-6.25). (Graph 7.6) Relatively higher pH was obtained
in the Gomutra purified sample which might be due to the alkalinity of cow’s urine
that neutralized the acidic pH of the raw drug to some extent.
Graph 7.6: pH values of raw & Shodhita Kupeelu
Alkaloids, oil, carbohydrate, protein and tannin are present in raw sample as
well as purified samples. Steroid, Flavonoid are absent in raw and the purified
samples. Glycoside was found to be absent in all the purified samples in comparison
to the raw drug. It might be due to the hydrolysis of loganin into an aglycone
loganetin, or converted into secologanin. Secologanin in turn is condensed with
tryptamine to produce strictosidine, the central precursor of many monoterpenoid
indole alkaloids53.
0
2
4
6
8
4.75
6.254.85 4.58
5.81 5.714.51 4.81
5.7 5.55 5.5
pH valuesKR
KGM
KGD
KGG
KGMD
KGMDG
KET
KKJ
KAS
KW
KBW
Picture a. Nature 468, 461–464 doi: 10.1038/nature09524
Picture a.
Discussion Chapter 7
Shodhana of Kupeelu Page 163
Schematic presentation of the chain reaction in Picture a. indicates that
Tryptophan (1) is decarboxylated by tryptophan decarboxylase to yield tryptamine
(2), which reacts with secologanin (3) to form strictosidine (4). After numerous
rearrangements, strictosidine (4) is converted into a variety of monoterpene indole
alkaloids, such as 19,20-dihydroakuammicine (5), ajmalicine (6), tabersonine (7) and
catharanthine (8).
HPTLC profiles of raw and Shodhita Kupeelu indicate that some peaks
disappeared and some new peaks appeared after Shodhana processes. In raw sample,
total 4 peaks were found whereas 3-8 peaks were observed in the purified samples
under 254 nm. This disappearance and newly appearance of peaks suggest the
extraction of some components like strychnine, brucine, loganin etc., and formation of
some new compound during Shodhana process. The Rf values of Strychnine and
Brucine standard were found as 0.54 & 0.34 respectively which were also present in
all the samples. (Table 5.3 and Graph 7.7)
Graph 7.7: Peaks observed under 254 nm in raw & Shodhita Kupeelu seeds
Decrease in Strychnine and Brucine content was found in all the Shodhita samples
when compared to the raw drug. Strychnine and Brucine contents were found to be
lowest in the sample purified by Gomutra-Godugdha (KGMD) when compared to the
other samples. (Table 5.4 and Graph 7.8) It might be due to the reason that during
Shodhana processes, some amount of Strychnine and Brucine were removed by
diffusion process into cow’s urine. Further boiling in cow’s milk also initiated more
4
8
5 5 56
34
3
5
3
1 1
0123456789
No. of peaks
Discussion Chapter 7
Shodhana of Kupeelu Page 164
diffusion of the alkaloids from the seeds as well as some amount of Strychnine and
Brucine might have been converted into their N-oxidative derivatives with lesser
toxicity67. Removal of some constituents from the raw seeds were also confirmed by
observing the changes in organoleptic characters like color, odor & taste occurred in
the media as well as in the samples during Shodhana processes.
Graph 7.8: % of Strychnine & Brucine in raw & Shodhita Kupeelu seeds
PHARMACOLOGICAL STUDY
Pharmacological study was carried out in two parts viz., acute toxicity study &
efficacy related study.
Part I: Toxicity study:
Kupeelu in crude and unpurified form exerts poisonous and even fatal effects.
It develops restlessness, suffocation, tremors and convulsion gradually and ultimately
leading to death due to respiratory arrest within 2-4hrs. The main toxic alkaloids
present in the seeds are Strychnine and Brucine which cause tetanic convulsion.
Normally inhibitory neurons in the spinal cord called Renshaw cells release glycine at
inhibitory synapses with motor neurons. This inhibitory input to motor neurons
prevents excessive muscular contractions. Strychnine binds to and blocks glycine
receptors which cause massive tetanic contractions. All skeletal muscles, including
the diaphragm contracts fully and remains contracted. Because the diaphragm cannot
relax, the person cannot breath. The normal delicate balance between excitation and
inhibition in the CNS is disturbed and motor neurons are firing without restraint68.
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
KR KGM KGD KGG KGMD KGMDG KET KKJ KAS KW KBW
% of Strychnine & Brucine
Strychnine Brucine
Discussion Chapter 7
Shodhana of Kupeelu Page 165
As expected raw Kupeelu sample (KR) treated animals showed severe convulsions
followed by death at the dose of 175mg/kg. Even at the dose of 100mg/kg also,
animals showed convulsions. This may be because of strychnine contents of seed
which blocked glycine receptors and lead to synchronized firing of neurons. Contrast
to this observation, between the two Shodhita samples, Kanji Shodhita Kupeelu (KKJ)
showed LD50 around 265mg dose level whereas, the sample purified through A. F. I
approved method (KGMDG) showed it at a dose level more than 1g/kg. (Table 6.2
and Graph 7.9) An increase in LD50 value indicates that subjecting to Shodhana
procedure is having the property of removing the toxic alkaloids strychnine & brucine
from the seeds. A. F. I method is found to be more effective than Kanji method in
reducing the toxicity of the drug. In quantitative HPTLC study it was revealed that
strychnine & brucine content were more reduced in KGMDG sample than KKJ
sample which also support the above results. (Table 5.4 and Graph 7.8)
Graph 7.9: Result of LD50 study in rats
Part II: Efficacy study:
It has been well established fact that Ayurvedic classics have emphasized various
methods of Shodhana (purificatory procedures) to overcome the undesired effects from
various poisonous drugs. The same is very true about Kupeelu seeds for which various
Shodhana methods are mentioned in Ayurveda. In the Pharmacological study, Kupeelu
seeds were subjected to two classical methods of Shodhana (Kanji and AFI methods)
and evaluated for analgesic and anti-inflammatory activities to know whether classical
purificatory procedure affect the efficacy or not i.e., whether efficacy will remain same
or it will enhance the efficacy or it will decrease the efficacy.
0
200
400
600
800
1000
1200
KR KKJ KGMDG
175265
1098
LD50 in rats (mg/kg. body weight)
KR
KKJ
KGMDG
Discussion Chapter 7
Shodhana of Kupeelu Page 166
Anti-inflammatory activity: In carrageenan induced paw oedema test, the first
phase (up to 3 hours after injection of carrageenan) results from the concomitant
release of mediators: histamine, serotonin and kinins on the vascular permeability and
the second phase (up to 6 hours after injection of carrageenan) is correlated with
leukotrienes formation and release54. From the results, as depicted in the Table- 6.3,
both raw and possessed Kupeelu seeds at the given dose level, failed to exhibit any
significant response at the first phase. However, the test drugs KR, KGMDG and KKJ
non-significantly inhibited the second phase of carrageenan induced inflammatory
response indicates mild action of test drug on leukotrienes formation and release.
The formalin-induced inflammation in the rats foot may be conveniently divided
into two parts, the first involving 5-hydroxytryptamine as mediator and the second
mediator which is unrelated to 5-hydroxytryptamine55. In contrast to results obtained
in carrageenan induced paw oedema, both KR and KKJ significantly suppressed paw
oedema at both time intervals, which were found to be statistically highly significant.
The result shows that decrease in paw volume is nearly equal in both the samples after
24 h whereas the decrease is more in raw drug than the Kanji purified sample after
48h of Formaldehyde administration (Table- 6.4). Thus, the result indicates that these
two samples of Kupeelu (KR & KKJ) having significant anti-inflammatory activity
against formalin induced inflammation. However, the test drug KGMDG non-
significantly inhibited both the phases of formalin induced inflammatory response
indicates mild action of the test drug on proliferative phases of inflammation.
Analgesic activity: In analgesic activity study, the analgesic testing protocol was
selected such that both centrally and peripherally mediated effects could be
ascertained by adopting formalin induced pain and tail flick response methods.
Tail flick model, which is thermal induced nociception, indicates narcotic
involvement, which is sensitive to opioid μ receptors (Abbott and Young, 1988)56.
The ability of the drugs to prolong the reaction latency to thermally induced pain in
mice further suggests central analgesic activity. Table- 6.5 shows the data related to
the tail flick response in mice. In control group, the tail flick response showed
increase at various time intervals in comparison to the basal values. In-group
KGMDG elevation in pain threshold was observed at all the time intervals studied
except for the observation related to 60 min, at which a mild decrease was observed
when compared to the control group. Although the values are statistically
Discussion Chapter 7
Shodhana of Kupeelu Page 167
insignificant, yet it can be assumed that the drug KGMDG may has mild analgesic
activity against tail flick test. In the group treated with KR, a decrease in pain
threshold was observed at all the time intervals studied except for the observation
related to 60 min. at which a mild increase was observed when compared to the
control group. These values are statistically insignificant suggest that the drug KR has
failed to prove any sort of analgesic effect in tail flick test. Results observed in-group
KKJ shows elevation in pain threshold at 60 min., 120 min., and 180 min. of post
drug administration in contrast to the decrease in pain threshold at 30 min. and 180
min. interval. These values are not statistically significant but the percentage wise
increase in pain threshold suggests mild analgesic effect of the drug at particular time
intervals.
Formalin injection to plantar aponeurosis of rats shows pain response in two
phase’s viz., initial and late phase. The initial phase lasts for 0-10 min. of
formaldehyde injection; it is supposed to be mediated through modulation of
neuropeptides57. The second phase, which is observed 20-30 minutes of formaldehyde
injection, is supposed to be mediated through release of inflammatory mediators like
prostaglandin etc. Data related to the effect of test drugs on experimental pain
induction through Formalin is depicted in the Table- 6.6. In the first phase (0-10 min.)
all, the three-test drugs (KR, KGMDG and KKJ) exhibit a decrease of 19.59%,
36.29% and 16.65% respectively in the number of paw licking response. However,
the decrease in-group KGMDG was only found to be statistically significant when
compared to the control, which indicates the presence of marked analgesic activity in
the drug mediated through modulation of neuropeptides. Although, the percentage
wise decrease in other two groups i.e., KR and KKJ also indicates presence of mild
analgesic activity in these two drugs. While in the second phase of reading season, i.e.
20-30 min. after drug administration, a significant decrease of 34.01% was observed
in-group KR indicating its significant suppression effect on pain of inflammatory
origin. However, non-significant decrease i.e. 29.50% was observed in-group
KGMDG and an increase of 11.31% was found in-group KKJ that is also statistically
insignificant. Though the result found in-group KGMDG is not statistically significant
but the percentage wise decrease in number of paw licking is suggestive that the drug
KGMDG has mild inhibitory activity against inflammatory pain.
Discussion Chapter 7
Shodhana of Kupeelu Page 168
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