Evaluation of Anti-asthmatic and Antioxidant potentials of seed extract of Strychnos nux vomica L.

 

Sheetal Singh*¹, G. S Chakaraborthy²

¹Noida Institute of Engineering and Technology (Pharmacy Institute), Gr. Noida 201306, U.P, India

²Parul Institute of Pharmacy and Research, Parul University, Vadodara, Gujarat

 

ABSTRACT:

The main aim of this study is to clarify the benefits of poisonous plant Nux Vomica L. in asthma. Asthma is one of the major public health problems for the developed and developing countries. Worldwide, it is estimated that 300 million people are affected with bronchial asthma. India has an estimated 15-20 million asthmatics with a prevalence of about 10% and 15% in 5-11 year old children. Asthma is a allergy which cause inflammation so we need the drug which poses anti allergic and anti-inflammatory action. The objectives of the study are screening the strychnos nux-vomica for its in vivo anti-asthmatic and antioxidant activity. The activity will be correlated with the various phytoconstituents present in the plant which might be responsible for the activity. Three different in vivo screening models are used these are clonidine-induced catalepsy in mice, clonidine-induced mast cell degranulation in rats, milk-induced leukocytosis and Eosinophilia in mice to get the best result of the prepared extract. All three models work on different receptor and also have different mechanism of acting on asthma or inflammation. Nux vomica is the plant which shows its action on both allergy and inflammation so it is chosen for this study and asthma is diseases which also have allergy and inflammation. The findings will be justified with the traditional claims of the plant. Strychnos nux vomica seed extracts showed the positive result of in vivo activity. From this study it was identified that hydro alcoholic seeds extract of this crude drug could be used to control the asthma.

 

 

INTRODUCTION:

Plants has served the human race as well as all the living creatures since the inception of civilization for maintain a good and better health with their aided values either with respect to activity or with their desired effects due to various active constituents. Some of the flora are also being used apart from their poisonous nature but given in appropriate conditions and symptoms proves to be a better healing touch. Among them one of the plant is Nux-vomica (Strychnos nux-vomica). Strychnos nux-vomica, (S. nux-vomica) in hindi commonly known as kuchla, and in english also known as Crow-fig. Nux vomica consist of dried ripe seeds of Strychnos Nux vomica Linn. It occurring naturally to East India and mainly collected from forest in Sri Lanka, North Australia and India.

 

In southern parts of the continent its also has seen its presence. It is also available in the forest of Bihar, Orissa, Konkan, Gorakhpur and Mysore.¹ In sixteenth centuary this drug is mainly use to kill animals only because of its poisonous nature.

 

The word Strynous nux vomica is drive from Greek word Strynos meaning poisous and nux means a nut possessing with vomiting or nauseating effect. It was introduced in medical practice from 1640 onwards². Its scientific classification is described below:

 

Scientific Classification:

Kingdom	:	Plantae
Division	:	Magnoliophyta
Class	:	Magnoliopsida
Order	:	Gentianales
Family	:	Loganiaceae
Genus	:	Strychnos
Species	:	Nux-vomica
Synonyms	:	poison nut, Quaker buttons, Kuchla, Crow-fig

 

 

Chemical Constituents:

The dried seeds of S. nux-vomica contain 2.6%–3% total alkaloids, out of which 1.25%–1.5% is strychnine, 1.7% is brucine, and the rest are vomicine and igasurine. Some other minor alkaloids are α-colubrine, β-colubrine, 3-methoxyicajine, protostrychnine, novacine, n-oxystrychnine, pseudostrychnine, isostrychnine, and chlorogenic acid, and glycoside, loganin³. Crude drug kuchla is used in the treatment of anemia, lumbago, asthma, bronchitis, constipation, diabetes, malarial fever, skin disease, paralysis, muscle weakness, and appetite loss. Suppressive effect of S. nux-vomica was observed on induction of ovalbumin-specific IgE antibody response in mice. Strychnine was identified and analyzed in detoxified kuchla seeds using liquid chromatography–electrospray mass spectrometry. Strychnine and brucine were separated and quantified in S. nux-vomica by nonaqueous capillary electrophoresis.⁴

 

EXPERIMENTAL SECTION:

Plant material:

The dried seeds were obtained from the authentic sources and it was authenticated. Further they were placed in a beaker containing water to remove the debris and foreign substances adhering to it. The seed which floats on the surface of water, broken and, black in colour were rejected from the study. The seeds which settled at the bottom of the beaker were selected for the study, washed and finally dried in hot air oven at a temperature not exceeding 25°C for purification process.⁵

 

Detoxification (Sodhana) process:

Sodhana also known as detoxification process was performed for of nux-vomica seeds. As described in Ayurvedic Rasashastra. The Rasashastra describes stepwise procedure for detoxification (sodhana) of nux-vomica and it was adopted in the present study which is depicted below.⁶

 

Step 1: The clean and dried seeds (500gm) were kept in cow’s urine for 7 nights. The urine was regularly changed every day.

Step 2: The seeds after 1st step were collected and subjected to swedana. Swedana is a process in which seeds obtained after 1^st step are placed in a muslin cloth with banana leaf and tried. Further they are completely dipped in cows milk and boiled in a low flame for 3 H.

Step 3: Then the seeds of Strychnos nux-vomica were collected and washed with water. The seed coat and embryo were removed. The seeds were roasted with cow’s ghee in low flame on pan. Till a colour change from light brown to dark brown was observed with a crispy nature. Then the seeds were powdered. The nux vomica which is in unprocessed form (UNV) and the seeds detoxified by sodhana process (PNV) were subjected to phytochemical and acute toxicity studies.⁷

Selection of plant:

The dried seeds of plant Strychnos nux-vomica were collected from campus of NIET, Greater Noida. The detoxified seeds of Strychnos nux-vomica were then subjected to size reduction to get powder of desired particle size. The powder was then stored in a clean dry air tight container. They were mixed in equal ratio (1:1) and extraction was carried out by maceration process with hydro-alcoholic solvent for 7 days with occasionally shaking every 4 h and placing them in dark condition so as to minimize the light entrapment and maintaining a proper temperature 35°C. After 7 days filtration was carried out and the marc was re-macerated with solvent for another day. The filtrate was concentrated over water bath by maintaining a temperature not more than 35°C with continuous stirring. The extract was concentrated to a syrupy mass and placed in desicator for removal of moisture.⁸

 

Experimental animals:

Wistar albino rats weighing 150-200gm of either sex and Swiss albino mice 18-20gm of either sex were utilized for antiallergic activity. They were obtained from Central Animal House of Pharmacy Institute NIET, Greater Noida. The exploratory creatures were housed in standard polypropylene confines at 25±2°C with 12 hrs light and 12 h dim cycles. Every one of the creatures was bolstered with standard rodent feed and water not obligatory was given. The litter in the enclosures was restored thrice seven days to guarantee hygenicity and most extreme solace for creatures.

 

ANTIASTHMATIC ACTIVITY:

Clonidine-induced Catalepsy in Mice:

Catalepsy is a situation in which mice maintain imposed posture for long time before regaining the normal posture. The bar test was used to know the effect of Nux vomica extracts on clonidine-induced catalepstic mice. Firstly Clonidine (1mg/kg, s.c.) was administrated to mice (n=6) which was pretreated 60 min before with distilled water (vehicle), extracts of Strychnos nux vomica and chlorpheniramine maleate which is standard drug (10mg/kg, i.p). The time required to remove the paws from bar will be noted for each animal and the duration of catalepsy was measured at 0, 15, 30, 60, 90, 120, 150, and 180 min. Decrease in the duration of catalepsy was calculated versus control. Standard and test group was compared with control.⁹

 

Clonidine-induced Mast Cell Degranulation in Rats:

The histamine concentration has been estimated to be about 0.3 m. in rat mast cell granules The clonidine and compound 48/80 act through the dynamic expulsion of granules without causing any damage to the cell wall. Clonidine releases histamine from mast cells in a similar style to a selective liberator like compound 48/80. This model is used to check mast cell stabilization potential of test drug.¹⁰

Adult healthy Albino Wistar rat were devided into 4 groups. Each group contain six rats.

·       Group-I received vehicles 5 ml/kg, (p.o.)

·       Group-II received sodium cromoglycate 50mg/kg, (i.p.)

·       group-III and IV received extract of Strychnos nux vomica L. in different dose

·       The treatment will be continued for seven days. On day 7^th day, 2h after the assigned treatment mast cells will be collected from the (i.p.). 10ml of normal saline solution will be injected into (i.p.) and abdomen will be gently massaged for 90 s.

·       The peritoneal cavity will be carefully opened and the fluid containing mast cells will be aspirated and collected in siliconised test tube containing 7 to 10 ml of RPMI-1640 Medium (pH 7.2-7.4).

 

The mast cells will be and centrifugation at low speed (400-500 rpm) and the pallet of mast cells will be taken in the medium. The mast cells suspension approximately (1 x 10 6 cells/ml) will be challenged with 0.5µg/ml of clonidine solution and stained with 1% toluidine blue and observed under high power microscope field.

 

Table: 1 Doses of Clonidine-induced Mast Cell Degranulation in Rats

Group	Treatment	Dose and route of administration	Parameters
Control Group	Distilled water	5 ml/kg p.o	Cell disruption count
Standard Group	Sodium cromoglycate	50 mg/kg i.p
Test Group	Test drug	Test dose p.o

 

                               T2-T1

% Protection = ––––––––––––– X 100.

                                   T2

T1 = Control group.                  T2 = Test group.

 

Total 100 cells will be counted from different visual areas and the number of intact and degranulated cells will be counted. Percent of decrease in clonidine induced mast cell degranulation will be calculated versus control. Standard and test group are compared with control group and control group will be compared with % of intact mast cells.¹¹

 

Milk-Induced Leukocytosis and Eosinophilia in Mice:

After parenteral injection of milk there is an increase in total leukocyte count (TLC) and this stressful condition can be normalized by administration of an antistress or adaptogenic drug. Furthermore leukocytes recruited during asthmatic inflammation, release the inflammatory mediators like cytokines, histamine, and major basic protein these increase the ongoing inflammation. This model is used to study the protective effect of test drug extract Nux vomica against milk-induced leukocytosis in mice¹². Albino mice will be divided six groups (n=6). All the mice were be anesthetized by using (i.p.) injection with pentobarbital sodium, and blood will be collected. They were further subjected for total cell and differential cell counts. Group I received orally distilled water (10ml/kg), group II, III, and IV will be given boiled and cooled milk (4ml/kg) s.c, group III, IV will be given test dose, by p.o 1 hour before milk injection. All mice were be anesthetized and blood will be removed from retro orbital site. Total leukocyte and eosinophil count will be done in each group before drug administration and 24 hours after milk injection.¹³

 

A blood is hallmark of both allergic and nonallergic asthma. Total 100 cells will be counted from different visual areas and the number of intact and degranulated cells will be counted. By noting the number of leucocytes and eosinophils before and after treatment the difference will be calculated. Standard group will be compared with control and test will be compared with standard¹⁴

 

 

Table 2: Doses of Milk-Induced Leukocytosis and Eosinophilia in Mice

Group	Treatment	Dose	Parameters
Control Group	Distilled water	10ml/kg p.o	Total Leucocyte Count and Eosinophils Count
Positive Control	Distilled water+ Boiled and Cooled milk	10ml/kg p.o+4ml/kg s.c
Test Group	Test Drug + boiled and Cooled milk	test dose p.o+ 4ml/kg s.c

 

RESULTS:

Clonidine-induced Catalepsy in Mice

Table 3: Result of Clonidine-induced Catalepsy in Mice

GROUP	Duration of catalepsy (sec) Mean ± SEM
	15 min	30 min	60 min	90 min	120 min	150 min	180 min
Control Group	62.8± 2.62	78.4± 2.82	78.6± 2.12	78.8± 3.80	82.4± 3.91	83.2± 2.88	121± 3.52
Standard Group	25.4± 2.72*	23.4± 2.08**	22.2± 1.67**	21.0± 2.84	29.6± 2.64**	29.9± 2.62**	54.0± 3.02**
Test Group (50 mg/kg)	48.4± 7.16*	56.8± 2.12**	67.6± 2.08*	61.0± 4.28**	61.8± 5.42**	62.5± 5.34*	98.4± 8.16*
Test Group (150 mg/kg)	44.6± 4.81**	45.2± 1.82**	47.6± 2.82	56.2± 2.16	55.0± 3.52**	48.6± 1.89**	91.2± 4.22**

Group-I = Vehicle (10ml/kg, i.p.)

Group-II = Chlorpheniramine maleate (10 mg/kg, i.p.)

Group-III = - treated with low dose of Strychnos nux vomica L. extract (50 mg/kg, p.o.)

Group-IV = treated with high dose of Strychnos nux vomica L extract (150 mg/kg, p.o.)

Values are expressed as Mean ± SEM, n=6, ANOVA along with Dunnett’s test is use for statistical analysis. *p<0.05, **p<0.01, compared to control group

Clonidine (1mg/kg, s.c.) produced catalepsy in mice. The maximum duration of catalepsy was seen at 180 minute in vehicle treated group (121±3.52.) After the administration of clonidine. There was significant inhibition (p<0.05, p<0.01) of Clonidine induced catalepsy in the animal pretreated with extract (50, 150 mg/kg, p.o.) and the duration of catalepsy was found to be 98.4±8.16 and 91.2±4.22 seconds respectively. Chlorpheniramine maleate, (10 mg/kg, i.p.) treated group significantly reversed (p<0.01) the Clonidine induced catalepsy in mice.

 

Mast cell degradation induced by Clonidine in rats

Table 4: Result of Mast cell degradation induced by Clonidine in rats

Values are expressed as Mean ±

SEM, n=6, ANOVA along with Dunnett’s test is use for statistical analysis., **p<0.001, compared to control group

 

 

In this study the percentage protection of control group was found to be higher as compared with other groups. But the hydro alcoholic extract of seed of nux vomica showed significantly (p<0.001) decrease in mast cell degranulation. Treatment with standard Sodium Cromoglycate extract of seed of nux vomica (100mg/kg) and 150mg/kg p.o) was given for 7 days. The animal group which was pretreated with nux vomica extract showed the significant protection % in degranulation of mast cell at two dose form of nux vomica extract. These significantly decreased the percentage protection in rats. The protection in mast cell degranulation test drug (nux vomica extract) was comparable to standard control (Sodium Cromoglycate) (50mg/kg).

 

 

Milk-Induced Leukocytosis and Eosinophilia in Mice

Table 5: Milk-Induced Leukocytosis and Eosinophilia in Mice

Treatment	Dose	Different in number of leucocytes count	Difference in number of eosinophil count
		Before and after treatment	Before and after treatment
Normal		223.66±17.19	21.16±6.01
Negative control (milk)	4ml/kg (s.c)	4302.83±17.19###	134.50±9.9###
Positivecontrol (dexamethasone + milk)	1mg/ kg (s.c)	477.16±4.54***	26.16±7.25***
Hydroalcholic extract + milk	50mg/kg	2249.50±7.78***	69.00±14015***
Hydroalcholic extract + milk	150mg/kg	1557.66±26.48***	49.33±11.26***

# normal control group compare with milk treated group. * compare with milk treated grou

Values are expressed as Mean ±

 SEM, n=6, ANOVA along with Dunnett’s test is use for statistical analysis., **p<0.001, compared to control group

 

 

 

The results of present study revealed that Hydroalcholic extracts of Strychnos nux vomica L extract caused reduction in the count of inflammatory cells. Strychnos Nux Vomica L extract have a significant in antieosinophilic activity and have beneficial effect in asthma.

 

CONCLUSION:

Strychnos nux-vomica, in Hindi commonly known as kuchla, and in english also known as Crow-fig. Nux Vomica consist of dried ripe seeds of Strychnos Nux vomica Linn. It is also known as poisonous nut. The main constituent are strychnine and brucine. Crude drug kuchla is used in the treatment of anemia, lumbago, asthma, bronchitis, constipation, diabetes, malarial fever, skin disease, paralysis, muscle weakness, and appetite loss.

 

On administration of hydro-alcoholic extract in rats at a dose of 50 and 150mg/kg it was observed that in the clonidine induced catalepsy in mice the maximum duration was seen at 180 min in vehicle treatment after the test drug treatment the duration of catalepsy was decrease and there was significant inhibition (p<0.05, p<0.01) in duration of catalepsy. So we conclude that the cataleptic effect of clonidine in mice is due to histamine release, histamine is mediator and result shows the inhibition of catalepsy by hydroalcholic extract of S. Nux vomica.

 

Mast cell degradation induced by Clonidine in rats we conclude that administration of the hydroalcholic extract of S. Nux vomica reduce the degranulation of the cell. So the plant extract have antiasthmatic property.

 

From milk-induced leukocytosis and eosinophilia in mice model we get that the hydroalcholic extract of S. Nux vomica reduce the leukocytosis and eosinophilia count which was induced by the milk. Increase level of leukocytosis and eosinophilia is a indication of allergy. So we conclude that the extract have antiallergic activity.

Further this plant can be explored to its molecular level for gene expression and identifying the root cause of disease.

 

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Received on 11.06.2019           Modified on 04.07.2019

Accepted on 01.08.2019         © RJPT All right reserved