INTRODUCTION:

No detailed Phytochemical and Pharmacological have been done on the plant Bryonia laciniosa, but however some phytochemical studies were carried out on fruits of the plant. It has been reported that the extraction of dry ripe fruit of Bryonia laciniosa with petroleum ether gave 10.2% dark viscous oil. Scientific Name: Bryonia laciniosa Linn. Family: Cucurbitaceae, Hindi Name: Shivlingi.

Botanical Description: Annual slender herbs; Leaves palmately 5-lobed, scabrous above, smooth beneath, margin denticulate; Peduncle (in male flowers); Calyx tube 2-4x3-6 mm, lobes spreading; Corolla greenish-yellow, shortly papillose, lobes ovate, acute; Female flowers fasciculate; Fruits spherical, yellowish-green, six striped; seeds grey, belted, attenuate with raised projections on both faces. Flowering and fruiting April to December in Indian conditions. Useful Parts: Whole plant and leaves. Traditional Medicinal Uses: According to Ayurveda, the plant is hot, pungent, and alternative; Leaves good for inflammations. Chemical Constituents: The plant possess bryonin (Bentley. R et.al. 1880).

1. Anti-Asthmatic Activity:

Asthma is characterized by an increase in air way resistance which is due to three main factors

1) Contraction of smooth muscles in the bronchioles

2) Oedema of the bronchiolar mucosa

3) Increased bronchiolar secretion

Asthma can be generally classified as either ‘Extrinsic’ or ‘Intrinsic’.

Intrinsic asthma: Attack of asthma not attributed to allergic reaction.

Extrinsic asthma: Asthmatic attack due to an allergic reaction

In extrinsic asthma, individuals are exposed to some specific allergens, such as dust, fumes, cotton fibres, etc; it leads to the development of specific antibodies (types IgE). Some of these antibodies circulate in blood, but most become attached to the surface of cells, particularly the mast cells. The IgE antibody can bind specifically to the receptor on the surface of the mast cells and combine with antigen or anti IgE. Re exposure to the original sensitizing antigen results in hyper sensitivity reaction causing disruption of mast cells and release of the contents of their granules into nearby tissues leading to synthesis and release of other mediators which are responsible for bronchial and vascular effect and inflammatory events. For evaluating anti – asthmatic drug, both invivo and invitro models are available. For the in vitro model ‘Guinea pig tracheal chain method’ is employed. Here the blockade of constriction of trachea (i.e., Dilation) is taken as parameter. Guinea pig is taken as experimental animal. In the invivo model protection against histamine Aerosol is employed. In the present work, the invivo model employed is mast cells degranulation rate studies by Atopic allergy.

The effect of 70% alcoholic extract of dried aerial parts of Bryonia laciniosa was based on the degranulation rate of sensitized peritoneal mast cells of albino rats when challenged with antigen. Triple antigen was used as adjuvant and prednisolone was used for comparison as standard (Bentley.R et.al. 1880).

2. Analgesic Activity:

Pain is an unpleasant sensory and emotional experience associated with actual or potential tissue damage.

Different procedures have been used for testing analgesics and are summarized below:

a) Thermal Method: A popular method which is applicable to mice uses an electrically heated plate at 55degree C+/-0.5degree C which is placed in an open cylinder. The mouse is placed in the cylinder and after a latent period it begins to show signs of discomfort, it may rise its hind feet, lick its fore paws or jump out of the cylinder. The last two named signs determine the end points. The reaction time is noted in seconds at different time intervals after drug administration

b) Mechanical Method: In this method the pressure is applied on the tail of a rat. Insensitiveness to the applied pressure is the criteria of testing analgesics by pressure methods.

c) Electrical Method: Electrodes are implanted or introduced to the skin, scrotum, tail or ear of the experimental animal. The voltage that has to be applied across the electrode (terminals) in order to cause the animals to squeak or struggle is recorded before and after administration of the substances under test.

d) Chemical Method: Injection of Phenyl quinine in mice through intra– peritoneal route, Benzoquinone, Bradykinin or Acetic acid can also be used. The response to the irritant solution is a characteristic writhing. It is usual to count the number of the total writhes in group of six mice. (Bentley.R et.al. 1880).

3. Anti-Convulsant Activity:

Drugs acting on the CNS may be broadly grouped as 1.CNS stimulants 2.CNS depressants The CNS depressants are most useful class of therapeutic agents.

For including convulsion commonly used techniques are:

1. Electrical stimulation of brain by maximal electro shock induced method (MES) in rats.

2. Chemical stimulation: Chemical commonly used is pentylene tetrazol.

The analgesic activity of 70% alcoholic extract of dried aerial part of Bryonia Laciniosa was screened by maximal electro shock induced convulsion in rats (Bentley. R et.al. 1880).

MATERIALS AND METHODS:

Collection: The entire plant with fruits was collected from Bangalore university campus. The plant was washed to remove adhering materials and dried under shades. It was then powdered and sieved through a fine mesh and the fine powder was used for phytochemical and pharmacological investigations (Dhingra, O.D. et.al., 1995).

1. Mesenteric Mast Cell Count by Atopic Allergy Method for estimating Anti Asthmatic Activity:

Principle:

The mechanism behind this model (Atopic allergy) to degranulate mast cell is as follows – In this classical immunological model, asthma is mediated by reaginie antibodies (Horse serum) bound to mast cells present in the system (Airway mucosa, mysentry etc). On exposure to antigen (Triple antigen), antigen – antibody reaction takes place on the surface of mast cells, subsequent exposure to the allergen (Horse serum) results in hyper sensitivity reaction, leading to degranulation (disruption) of mast cells and release of pharmacologically active agents like Histamine, prostaglandins and other potent chemical mediators are responsible for precipitation of asthma (Katzung B.G. 2007)^.

Material:

1. 1.Horse serum (obtained from Veterinary hospital, Bangalore, Turf Club, Bangalore)

2. Triple antigen–containing 20,000 million B. pertusis organism

3. Prednisolone–in a dose of 10mg/kg body weight 4.70% alcoholic extract of Bryonia laciniosa–300 mg/kg body weight.

Procedure The anti asthmatic activity of 70% alcoholic extract of Bryonia laciniosa was done by mesenteric mast cell count by Atopic allergy method in rat.

1. Albino rats of either sex weighing between 150- 200 gms were selected, divided into three groups, each containing six rats.

2. The rats were sensitized by injecting 0.5 ml of horse serum and 0.5ml of triple antigen subcutaneously.

3. Group I was kept as control without any drug treatment i.e., only distilled water was given during treatment days.

4. Group II was kept as standard and was administered with predisolone (10mg/kg body weight orally). Group III was treated with 70% alcoholic extract of Bryonia laciniosa in a dose of 300mg/kg body weight orally.

5. On the 5th day after sensitization of rats, they were treated with standard and test drug respectively till 12th day.

6. On the 13th day sensitization, all the animals were sacrificed and the intestinal mesentry was taken for further studies on the mast cells.

7. The mesentries of the sacrificed animals along with pieces of intestine were kept in ringer solution.

8. The mesentry pieces were challenged with horse serum (5%) for 10 minutes.

9. Later pieces of mesentry were stained superficially with toludine blue. The tissue was first immersed in 0.1% toludine blue (in 4% aqueous saline) for 10 minutes [12].

10. The tissue was then treated with xylene for 5- 10 minutes.

11. Finally it was rinsed with acetone thrice and place on a microscopic slide and stretched with the help of a needle. The intestinal pieces were cut and removed.

12. The tissue was examined under 200X magnification.

13. The number of intact and disrupted mast cells, in ten randomly selected fields for each tissue was counted. Three slides per each animal were studied.

2. Eddy’s hot plate method for estimating analgesic activity:

Principle In this method heat is used as source of pain. Animals are individually placed on a hot plate maintained at constant temperature (55 degree C) and the reaction of the animal such as paw licking or jump response is taken as end point. The method was first described by Eddy and Leimbach (Kiritikar K.R et al., 1987).

Materials Eddy’s hot plate analgesiometer maintained at 55degreeC +/- 0.5degreeC

1. Albino mice of either sex weighing between 20- 30 gms

2. Standard drug Ibuprofen in a dose of 100mg/kg body weight

3. Bryonia laciniosa (70% alcoholic extract) in a dose of 500mg/kg body weight. The analgesic activity of the 70% alcoholic extract of dried aerial parts of the plant Bryonia Laciniosa was carried out in mice using Eddy’s hot plate analgesiometer .

Procedure:

1. Albino mice of either sex weighing between 20- 30 gms were taken and divided into three groups of six animals each and marked.

2. The animals were fasted for 18 hours prior to the experiment with water ad libitum

3. Group I was kept as control and was administered with distilled water orally. Group II was administered with the standard drug Ibuprofen at a dose of 100mg/kg body weight and group III was administered with the test drug at a dose of 500mg/kg body weight.

4. After administration of test and standard drug, the test for analgesia was carried out by placing the mice on electrically heated plate at 55degreeC +/- 0.5 degree C and noting the signs of discomfort, i.e., it may lick its fore paws or jump out of the plate. The time was noted in seconds. Test was carried out similarly for animals of control group.

5. The observations were made at 30’ and 60’.

6. The results are tabulated (Lewis G. P. et al., 1977).

3. Electroshock method for estimating anti convulsion activity:

Principle For inducing convulsion by electro shock, a rectangular pulse current of high voltage (150 mA) is employed. The electro shock was given to each rat for 0.2 seconds with the help of convulsion meter through pinna electrodes. Drugs likely to be effective in Grandmal epilepsy usually confer protection against electrically induced convulsion in animals. The MES convulsions are divided into five phases:

(1) Tonic flexion

(2) Tonic extension

(3) Clonic convulsion

(4) Stupor

(5) Recovery or death

A substance is known to posses anticonvulsant property, if it reduces or abolishes the extensor or recovery phase of MES convulsion.

Materials:

1. Electro convulsion meter

2. Standard drug–Carbamazepine in 1% Tween 80 solution in a dose of 40mg/kg bodyweight.

3. 70% alcoholic extract of Bryonia laciniosa in 1% Tween 80 solution in a dose of 500mg/kg body weight.

Procedure:

1. Albino rats of either sex weighing between 150- 200 gms were weighed, marked and divided into three groups containing 6 rats each.

2. The animals were fasted for 18 hours prior to the experiment with water ad libitum.

3. Group I received carbamazepine (40mg/kg body weight) and Group II received 0.2ml of 1% Tween 80 solution and served as standard and control respectively. Similarly Group III received 500mg/kg body weight of 70% alcoholic extract of Bryonia laciniosa.

4. The electro shock was given to each rat for 0.2 seconds with the help of convulsion meter through pinna electrode and the effects were observed.

5. The results are tabulated (Satoskar R.S. et al., 1991).

RESULTS AND DISCUSSION:

1. Anti Asthmatic Activity:

The anti asthmatic activity of 70% alcoholic extract of Bryonia laciniosa was done by mesenteric mast cell count by Atopic allergy method in rats. The number of intact and disrupted mast cells, in ten randomly selected fields for each tissue was counted. Three slides per each animal were studied. The results of the test and standard in mesenteric studies are given in Table-1. Increase in % granulation was recorded in Byonia laciniosa treated samples compared to control. 56.27% was recorded in Bryonia laciniosa treatment and 81.26% granulation was recorded in predinisolone (figure1). Based on graphical picture Percentage protection of Bryonia laciniosa treated samples over control increased significantly and the value was 43.88 (figure-2). Based on statistical analysis ‘t’ value for Bryonia laciniosa treatment was 4.857 (Table-2).

Fig.1- Percentage degranulation and granulation of mast cells

Fig.2- Percentage protection of prednisolone and Byonia laciniosa against mast cell degranulation

2. Analgesic Activity:

After administration of test and standard drug, the test for analgesia was carried out by placing the mice on electrically heated plate at 55degreeC +/- 0.5 degree C and noting the signs of discomfort, i.e., it may lick its fore paws or jump out of the plate. The time was noted in seconds. Test was carried out similarly for animals of control group. The observations were made at 30’ and 60’.The results were tabulated in Table-3. It was found that Bryonia laciniosa showed fairly good analgesic activity at 30 and 60 minutes when compared with standard drug.

Bryonia laciniosa treated group showed an increase in response time to pain stimuli when compared to the control group (Table-3). The increase in response time was from 5.83 to 8.50 seconds at 30 minutes and from 5.67 to 10.50 seconds after 1 hour of treatment. Response time to pain stimuli shown by Morphine sulphate was 15.33 and 18.17 seconds respectively after 30 and 60 minutes of treatment. Statistical test revealed that analgestic activity was significantly increased in Bryonia laciniosa treated group (Table-4).

3. Anti Convulsant Activity:

For inducing convulsion by electro shock, a rectangular pulse current of high voltage (150 mA) is employed The electro shock was given to each rat for 0.2 seconds with the help of convulsionmeter through pinna electrode and the effects were observed. The results are tabulated (Table- 5).

In Bryonia laciniosa treated group % reduction of extensor phase was 39.27. In Carbemazepine treated group % reduction of extensor phase was 95.58. The statistical analysis revealed that there was significant increase in anticonvulsant activity in the case of Bryonia laciniosa treated group (Table-6).

Table-1 Data Showing Degranulation and Granulation of Mast Cells

Treatment	Control	Prednisolone	*Bryonia laciniosa*
Dose	0.2ML Saline	10mg/kg	300mg/kg
Wt. of animals in gm +/- sem	170.83 +/ - 5.85	165.17 +/- 3.25	165.005.45
Degranulated cells(x) +/- sem	16.26 +/- 0.5	4.29 +/- 0.38	9.9 +/- 0.35
Granulated cells (y) +/- sem	4.6 +/- 0.39	18.74 +/- 1.33	12.83 +/- 0.23
Total (x+y) +/- sem	20.87 +/- 0.85	23.02 +/- 1.47	22.83 +/- 0.35
%Degranulatin +/- sem	77.92 +/- 1.71	18.74 +/- 1.56	43.73 +/-1.12
%Granulatin +/- sem	22.08 +/- 1.70	81.26 +/- 1.56	56.27 +/-1.12
%Protection	--	75.95	43.88

Table-2 Statistical Data For Anti Asthmatic Activity

Group	Prednisolone treated group	*Bryonia laciniosa* treated group
Calculated ‘t’ value	12.9032	4.6999
Table ‘t’ value	4.587	4.587
Remarks	significant	significant

Table-3 The Analgesic Activity of 70% Alcoholic Extract of Bryonia laciniosa by Eddy’s Hot Plate Method

Treatment	Wt. of animals in gms +/- sem	Dose in mg/kg body weight	Response time in sec +/- sem
Treatment	Wt. of animals in gms +/- sem	Dose in mg/kg body weight	30 minutes	60 minutes
Control	20.83 +/- 0.83	--	05.83 +/- 0.31	05.67 +/- 0.42
Morphine Sulphate	26.33 +/- 0.95	100	15.33 +/- 0.49	18.17 +/- 0.75
Bryonia Laciniosa	27.00 +/- 1.13	500	08.50 +/- 0.43	10.50 +/- 0.50
Animals – Albino Mice (20-30 Gms)

Table-5 The Anti Convulsant Activity of 70% Alcoholic Extract of Bryonia laciniosa By MES Method.

Treatment	Control	Carbama zepine	*Bryonia laciniosa*
Wt. In gm +/- sem	166.33+/-6.4	144.67+/-14.63	171.17+/-6.23
Dose in mg/kg body wt	0.2ml	40	500
Flexion	3.00+/-0.365	13.33+/-0.333	2.33+/-0.211
Extensor	15.10+/-0.477	0.667+/-3.674	9.17+/-0.645
Clonic	1.83+/-0.167	0.33+/-0.211	1.17+/-0.307
Stupor	127.0+/-2.082	47.37+/-8.361	90.0+/-3.795
Recovery or death	Recovered	Recovered	Recovered
% Reduction of extensor phase	--	95.58	39.27
Animal – Rats (150-200 gms) Route - Oral

Table-6 Statistical Data For Anti-Convulsant Activity

Group	Carbamazepine treated group	*Bryonia laciniosa* treated group
Calculated ’t’ value	14.9867	5.7833
Table ’t’ value	4.587	4.587
Remarks	significant	significant

CONCLUSION:

Based on the results we concluded that 70% alcoholic extract of Bryonia laciniosa increased the anti-asthmatic activity, analgestic activity and also anti-convulsant activity in rat. The drug was found to be nontoxic. By doing further research in Bryonia laciniosa even we can

develop phytotherapeutics for Asthma, Epilepsy. Also we can use Bryonia laciniosa as pain reliever.

REFERENCES:

1) Bentley, R and H. Trimen, 1880. Medicinal Plants.Churchill,London,pp:3.

2) Dhingra, O.D. and J.B.Sinclair,1995.Basic plant Pathology Methods.2^nd Edn., CRC Press, Inc., Boca Raton,pp:359

3) Katzung B.G. (2007) Basic and Clinical Pharmacology. 10th Edn., McGraw Hill Companies Inc., New Delhi.

4) Kiritikar K.R. and Basu B.D. (1987) Indian Medicinal Plants, Vol. II, 2nd ed., Published by Lalit Mohan Basu, Allahabad, India, 1023-28.

5) Lewis G. P. and Whittle I’. J. I‘. (1977) Br. J. Pharmacol, 61,229-235.

6) Satoskar R.S., Bhandarkar S.D., Nayak V.K., Desai N.K., Kshirsagar N.A. (1991) J. Postgrad Med.,37(1):5-8.

Received on 11.08.2017 Accepted on 06.10.2017

Asian J. Pharm. Res. 2017; 7(4): 256-260.

DOI: 10.5958/2231-5691.2017.00040.5

	30 Minutes			60 Minutes
Group	Calculated ‘t’ value	Table ‘t’ value	Remark	Calculated ‘t’ value	Table ‘t’ value	Remark
Morphine Sulphate	16.3176	4.58	significant	14.5492	4.58	significant
Bryonia laciniosa	5.068	4.58	significant	7.3882	4.58	significant