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Discussion Chapter 7

Shodhana of Kupeelu 47

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


Shodhana of Kupeelu Page 148

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."



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



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,



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



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



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),



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



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 _ + + _ + +



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.



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



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.



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



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



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


Shodhana of Kupeelu 2

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.



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



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



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



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



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.



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