INTRODUCTION:

A. Precatorius (Fabaceae), commonly known as Gunja, is a woody twinning herb, found almost in all tropical countries, ascending to altitudes up to 1 200 m.¹ with distinctive toxic Rakta seeds through black mark at the base. Leaves of the plant look like tamarind leaves having 20-40 leaflets. The plant is much branched, perennial, woody, slender, spiky twining. The stems of the plant are wrinkled, cylindrical with smooth surface and brown in color.

Leaves of the plant are differentiated by 7-20 pairs of leaflets, stipulate, turgid, obtuse, oblong, and truncate at both ends. Flowers are pinkish white, auxiliary racemes but shorter than leaves. Pods are oblong, hairy, turgid, 3-5 seed are found with a black mark at the base.² The seeds of Shodhita Gunja are used to treat alopecia (Indralupta)³,⁴, itching (Kandu)⁵, edema (Shotha)⁶, urinary disorders (Prameha)⁷ etc. Indian tribes are used Gunja seeds as purgative⁸, nervous disorders⁹, pneumonia, skin diseases¹⁰, abortifacient, antifertility¹¹ and as antimicrobial agents¹². It is good for eyes and Shukrajanan¹³. The root of Gunja is emetic, alexiteric and also used in nervous disorders, sore throat, dry cough, rheumatism, and leucoderma^14,15. According to Ayurvedic literature, the seeds of Gunja are poisonous and it should be given to the patients after Shodhana¹⁶. Shodhana is an Ayurvedic technique which can remove impurities, make less toxic, minimize the side effect and improve the therapeutic efficacy of a drug¹⁷. Though some research work has been carried out on the effect of Shodhana on Gunja kernels¹⁸ there is very least research work on pericarps of Gunja Shodhana. So the aim of the present study was to establish the effect of Shodhana on the percentage of various physicochemical values and acute toxicity of Ashodhita and Shodhita red Gunja pericarps.

MATERIAL AND METHODS:

Separation of pericarps:

All the three varieties of seeds were coarsely grounded and pericarps were separated.

Shodhana of Gunja in cow’s milk:

The red Gunja pericarps (250 g) were wrapped in a cotton cloth to form a Potali. The Potali was hanging in an iron pot holding cow’s milk in such a way that the Poali was totally immersed in milk but did not contact the bottom of the vessel. Heating (90°C) was given to the vessel by hot plate constantly for 6 h (2 Prahara) to aid the Swedana process. During the Swedana process, an adequate amount of fresh cow milk was added at regular gaps to uphold the level of milk in the vessel. After 6 h, the Potali was removed from the milk and pericarps were washed with hot water thrice and kept it for shade-dried ¹⁹.

Shodhana of Gunja in Kanji (sour gruel):

The process of Shodhana in Kanji was similar to that of milk except that the boiling time was 3 hours instead of 6 hours¹⁹.

Table 1: Ingredients of Kanji²⁰

Drug	Botanical name	Part used	Quantity
Rice	Oryze sativa L.	Seeds	2.5 kg.
Kulthi	Dolichos uniflorus Lamk	Seeds	2.5 kg.
Saindhav Lavana	Sodium Chloride		2.5 kg.
Urad	Vigna radiate L.	Dough	625 gm.
Haldi	Curcuma longa L.	Rhizome	625 gm.
Rai	Brassica nigra L.	Seed	625 gm.
Hingu	Ferula assa-foetida L.	Resin	625 gm.
Sarson	Mustard	Seed Oil	625 mL.
Vansha patra	Bambusa arundinaceae Retz.	Leaves	625 gm.
Jeerak	Cuminum cyminum L.	Seeds	375 gm.
Shunthi	Zingiber officinale Rosc.	Rhizome	375 gm.
Distilled water			80 Lt.

Preparation of Kanji (Sour gruel):

Rice and Kulthi were taken in stainless steel pots along with 40 liters of distilled water separately. The pots were placed over a mild fire, boiled and reduced to one fourth and filtered through cotton cloth. Both the prepared Kwath of rice and Kulthi were mixed and kept in an earthen pot layered inside with mustard oil. Hingu was fired in oil and rest of the ingredients (3-8) were mentioned above were also put in the pot. The mouth of the pot was tied by cloth and kept for 15 days and filtered before use. Thus 20 liters of Kanji was prepared (Table 1).

Estimation of different physicochemical values:

For quantitative estimation of total ash and acid insoluble ash, all the samples of Ashodhita and Shodhita pericarps of Gunja were carried out as per Ayurvedic Pharmacopeia^20.The protein percentage was also calculated by the spectrophotometeric method described by Lowery et al ²¹ a standard curve at 700 nm absorbance was prepared with bovine serum albumin (B.S.A.) and 2% potassium sodium tartrate, 1% CuSO₄.5H₂O, 2% sodium carbonate in 0.1 NaOH were used as test solution²¹. Extraction with non-polar to polar solvents (alcohol and water) was made with the help of soxhlet apparatus and the percentage of each extractive was calculated after evaporating the respective solvents.

Experimental animals:

Acute toxicity test was performed as per Organization for Economic Co-operation and Development (OECD) guidelines 423 ²². Experiments were performed using Swiss strain albino mice of either sex weighing between 20-30 gms. Animals were acclimatized for one week before using them, before experimentation the animals were kept fasting overnight. The animals were randomly divided into 3 groups each containing six mice. The animals were housed in polypropylene cages, in a temperature controlled environment (23±2ºC). Lighting was controlled to supply 12 h of light and 12 h of dark for each 24-h period. The mice were fed with standard laboratory animal prescribed food with water ad libitum.

Preparation of Drug Sample:

The Ashodhita and Shodhita pericarp powder were taken the weighed quantity of powder were dissolved in 100 mL of distilled water and macerated in the cold water and kept overnight for extraction. The material was then centrifuged and supernatant obtained was diluted with water as required for per kg body weight of mice about three different doses (0.5, 1 and 2 mg/kg b.w.) level of the drug were administered intraperitoneal route of the animals.

Drug administration:

The test substance was administered in a single dose by intraperitoneal route. Animals were fasted 3 h prior to dosing (only food was withheld for 3 h but not water). Following the period of fasting, animals were weighed and test substance was administered intra-peritoneal at a dose of 0.5, 1 and 2 mg/kg b.w. After the administration of the drug, food for the mice was suspended for 2 h. The injected mice were observed after 2 hours intervals for 24 hours. The effect of the test drugs on the mice was scored with the use of nine degrees with a scale of 0-8. Scoring was performed at the time of peak effect. The base score below and above 4 was subnormal response and the base score for the abnormal sign was 0-8 and 4 for normal sign.²³

RESULTS:

Table 2: Shodhita red pericarp with milk

S.N	Parameters	Red pericarps
1.	Weight of Gunja pericarps	250 gm
2.	Volume of cow’s milk consumed	3 liter
3.	Total time consumed in the whole process of Shodhana of Gunja pericarps	7 hours
4.	Net volume of the total cow’s milk remained in the vessel of Dola-Yantra after the Shodhana process was over	3.3 liter
5.	Total consumed milk	1.0 liter
6.	Weight of Gunja pericarps obtained after the Shodhana process and dried in shade	220gm
7.	Colors after Shodhana process	Black

Table 3: Shodhita red pericarp with milk

S.N	Parameters	Red pericarps
1.	Weight of Gunja pericarps	250 gm
2.	Volume of Kanji consumed	2 liter
3.	Total time consumed in the whole process of Shodhana of Kanji pericarps	4 hours
4.	Net volume of the total Kanji remained in the vessel of Dola-Yantra after the Shodhana process was over	3.4 liter
5.	Total consumed Kanji	1.1 liter
6.	Weight of Gunja pericarps obtained after the Shodhana process and dried in shade	215 gm
7.	Colors after Shodhana process	Light yellow

Table 4: Physicochemical values Ashodhita and Shodhita red pericarps

SI. no	Parameters	Results
		Ashodhita	Milk	Kanji
1.	Total ash	2.1 (% w/w)	1.9 (% w/w)	1.6 (% w/w)
2.	Acid Insoluble ash	0.8 (% w/w)	0.6 (% w/w)	0.5 (% w/w)
3.	Protein	1.1	0.2	0.08
4.	Alcohol soluble extractive	7.8 (% w/w)	2.07 (% w/w)	1.11 (% w/w)
5.	Water soluble extractive	14.4 (% w/w)	12 (% w/w)	8 (% w/w)

Table 5: Effect of Ashodhita pericarps of red Gunja on acute toxicity test in mice

Awareness	Alertness	Slight CNS depression. This depression was directly related to the dose of test drug.
	Visual placing	No change in animal response when placed in different positions.
	Stereotypy	No effect was seen.
Mood	Grooming	unaffected
	Restlessness	Absent in the unaffected mice.
	Irritability	Aggressiveness
	Fearfulness	Absent
Motor activity	Spontaneous activity & reactivity	Sluggish, dose related
	Touch response	Present
	Pain response	Present
CNS excitation	Tremors	Absent
	Convulsions	Absent
Posture	Body posture	No effect
Muscle tone	Limb tone	Elongated
	Body tone	Tail both are dose-related
Reflexes	Pinna	Absent
	Corneal	Absent
Autonomic	Pupil size	No effect
	Salivation	No effect
	Body temperature	No change
	Skin color	Slight brown at site of injection
	Heart rate	Decreased
	Respiratory rate	Decreased

Table 6: Effect of milk Shodhita pericarps of red Gunja on acute toxicity test in mice

Awareness	Alertness	No change
	Visual placing	No effect
	Stereotypy	No effect
Mood	Grooming	No effect
	Restlessness	Absent
	Irritability	Slight excitation
	Fearfulness	Absent
Motor activity	Spontaneous activity & reactivity	No effect
	Touch response	Present
	Pain response	Present
CNS excitation	Tremors	Absent
	Convulsions	Absent
Posture	Body posture	No effect
Muscle tone	Limb tone	No effect
	Body tone	No effect
Reflexes	Pinna	No effect
	Corneal	No effect
Autonomic	Pupil size	No effect
	Salivation	No effect
	Body temperature	No change
	Skin color	Light brown at the site of injection
	Heart rate	Decreased
	Respiratory rate	Decreased

Table 7: Effect of Kanji Shodhita pericarps of red Gunja on acute toxicity test in mice

Awareness	Alertness	No change
	Visual placing	No effect
	Stereotypy	No effect
Mood	Grooming	No effect
	Restlessness	Absent
	Irritability	Slight excitation
	Fearfulness	Absent
Motor activity	Spontaneous activity & reactivity	No effect
	Touch response	Present
	Pain response	Present
CNS excitation	Tremors	Absent
	Convulsions	Absent
Posture	Body posture	No effect
Muscle tone	Limb tone	No effect
	Body tone	No effect
Reflexes	Pinna	No effect
	Corneal	No effect
Autonomic	Pupil size	No effect
	Salivation	No effect
	Body temperature	No change
	Skin color	light brown at the site of injection
	Heart rate	Decreased
	Respiratory rate	Decreased

Table 8: CNS activity and acute toxicity screening of Ashodhita pericarps of red Gunja

	Pericarps
	0.5 mg	1.0 mg	2.0 mg
Awareness
Alertness	4	4	4
Visual placing	4	4	3
Stereotypy	4	3	3
Mood
Grooming	4	4	4
Restlessness	4	4	4
Irritability	4	4	3
Fearfulness	4	4	3
Motor activity
Spontaneous activity & reactivity	4	4	4
Touch response	4	4	3
Pain response	4	3	3
CNS excitation
Tremors	3	3	3
Convulsions	4	4	3
Posture
Body posture	4	3	3
Muscle tone
Limb tone	4	4	3
Body tone	4	4	4
Reflexes
Pinna	4	4	4
Corneal	3	3	3
Autonomic
Pupil size	4	4	4
Salivation	4	4	4
Body temperature	4	3	3
Skin color	4	4	4
Heart rate	4	4	4
Respiratory rate	3	3	3

Table 9: CNS activity and acute toxicity screening of milk Shodhita pericarps of red Gunja pericarps

	Pericarps
	0.5 mg	1.0 mg	2.0 mg
Awareness
Alertness	4	4	4
Visual placing	4	4	4
Stereotypy	4	4	4
Mood
Grooming	4	4	4
Restlessness	4	4	4
Irritability	4	4	4
Fearfulness	4	4	4
Motor activity
Spontaneous activity & reactivity	4	4	4
Touch response	4	4	4
Pain response	4	4	4
CNS excitation
Tremors	4	4	4
Convulsions	4	4	4
Posture
Body posture	4	4	4
Muscle tone
Limb tone	4	4	4
Body tone	4	4	4
Reflexes
Pinna	4	4	4
Corneal	4	4	4
Autonomic
Pupil size	4	4	4
Salivation	4	4	4
Body temperature	4	4	4
Skin color	4	4	4
Heart rate	4	4	4
Respiratory rate	4	4	4

Table 10: CNS activity and acute toxicity screening of Kanji Shodhita pericarps of red Gunja pericarps

	Pericarps
	0.5 mg	1.0 mg	2.0 mg
Awareness
Alertness	4	4	4
Visual placing	4	4	4
Stereotypy	4	4	4
Mood
Grooming	4	4	4
Restlessness	4	4	4
Irritability	4	4	4
Fearfulness	4	4	4
Motor activity
Spontaneous activity & reactivity	4	4	4
Touch response	4	4	4
Pain response	4	4	4
CNS excitation
Tremors	4	4	4
Convulsions	4	4	4
Posture
Body posture	4	4	4
Muscle tone
Limb tone	4	4	4
Body tone	4	4	4
Reflexes
Pinna	4	4	4
Corneal	4	4	4
Autonomic
Pupil size	4	4	4
Salivation	4	4	4
Body temperature	4	4	4
Skin color	4	4	4
Heart rate	4	4	4
Respiratory rate	4	4	4

DISCUSSION:

The objective of the present research work is to study the effect of Shodhana on the Gunja, which is attributable to pericarps of A. precatorius of red seed. In this perspective red pericarp of Gunja were processed in cow’s milk and Kanji and subjected to various physicochemical tests and acute toxicity study. The physicochemical evaluation is essential factors which facilitate and distinguishing the adulteration or inappropriate handling of the crude drug. The ash values are such type of quantitative standards which characterize the presence of different impurities like silica, oxalate and carbonate, this type of adulterant commonly occurring in crude drugs. As brought out by the present studies, the percentage of total ash is decreased in the Shodhita material suggested that leaching out of some amount of inorganic salts may have occurred during Shodhana while acid insoluble ash consists mainly silica and indicate contamination with earthy material²⁴. Similarly, the decrease in protein percentage in Shodhita sample is due to the dissolution of water-soluble protein in Shodhita material. Simultaneously the decrease was more prominent in Kanji as compared to milk, suggesting that Shodhana process by Kanji is much better than milk. The percentage of alcohol and water-soluble extractive value decreases, which is possibly due to the extraction of water and alcohol soluble substances like sugar, glycosides, in milk and Kanji (Table 4). This study proved showed that Shodhana in Kanji is more effective. These observations regarding organoleptic character showed that there was a change in the color of red pericarps (red to black), and the same time a minute decrease in weight was also observed in Shodhita pericarps but there is no change in shape except they become soft (2,3)^25,26. The aqueous extract of red pericarps of Gunja seed in three different doses was administered intra-peritoneal to the animals. The observations were taken on the neurological behavior autonomic studies. The injected mice were observed after 2 hours intervals for 24 hours. The effect of the test drugs on the mice was scored with the use of nine degrees with a scale of 0-8. Scoring was performed at the time of peak effect. The base score below and above 4 was the subnormal response and the base score for the abnormal sign was 0-8 and 4 for the normal sign. In the CNS activity including awareness (alertness), mood (irritability), motor activity (reactivity), CNS excitation (convulsion) and autonomic responses (skin color, heart, and respiratory rate) considerable variations were observed in Ashodhita and Shodhita red Gunja. Overall effects have been summarized in Table 5-10. On the basis of the present study, it was concluded that red Gunja is suitable for medicinal purposes with respect to their physicochemical and neurological behavior autonomic behavior. It is also interesting to note that the more reduction in toxicity was observed in Kanji Shodhita Gunja.

CONCLUSION:

The more reduction in toxicity was observed in Kanji Shodhita as compared to the milk Shodhita. The study showed that Shodhana in Kanji is more effective. Therefore, all the analytical tools used in this study can be used as a marker for Shodhana.

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Received on 08.01.2018 Modified on 12.02.2018

Research J. Pharm. and Tech 2018; 11(5):1935-1939.

DOI: 10.5958/0974-360X.2018.00359.1