INTRODUCTION:

The plant, Lagenaria siceraria (Family: Cucurbitaceae), known as bottle gourd, is a common fruit vegetable used throughout India.¹ It is a large pubescent, climbing or trailing herb, either wild or cultivated.Since time immemorial the fruit is used as nutritive agent.²It has been used as medicine traditionally in India, Brazil, European countries, China, Hawaiian island, etc for common ailments.³ It is reported to have antihyperglycemic,⁴ antihyperlipidemic,⁵ antioxidant,⁶ cardioprotective,⁷ immunomodulatory,⁸ antiinflammatory,⁹ diuretic activities.

The analgesic and anti-inflammatory activity of Lagenaria siceraria has not yet been documented. Current drugs for inflammation such as NSAIDs and opiates are not beneficial in all cases, due to their side effects and potency. Hence search for other alternatives seems necessary and beneficial. This study aimed to investigate in vivo anti- inflammatory and analgesic potential of extracts from Lagenaria siceraria.

MATERIALS AND METHODS:

Animals:

Healthy adult albino rats of Wistar strain weighing 180-250g & albino mice were used for this study. The animals were obtained from Lakshmi biofarms alephata, Pune India. On arrival, the animals were placed randomly and allocated to treatment groups in polypropylene cages with paddy husk as bedding. Animals were housed at temperature of 24±2 ºC and relative humidity of 30-70%. A12:12 light: day cycle was followed. All the animals were allowed to free access to water and fed with standard commercial pelleted rat chaw (M/S Hindustan Lever Ltd. Mumbai). All the experimental procedures and protocols used in this study were reviewed by the Institutional Animal Ethics committee (and were in accordance with the guidelines of the CPCSEA.Approval was obtained from the CPCSEA/IACE for animal studies in this project by proposal no: VIPER/IEAC/UG/2017-16

Preparation of extract:

Dried and powdered Fruit & leaves of Lagenaria siceraria were extracted by using ethanol in soxhlet apparatus. The total extract obtained was dried at 60 °C on steam bath Followed by a vacuum oven (50 °C) to obtain dried extracts. The extractive Value was calculated as % w/w yield and was found to be 6.78%.

Acute toxicity study:

Acute oral toxicity was performed by following OECD-423 guidelines (acute toxic class method), albino rats (n=6) of either sex selected by random sampling were used for acute toxicity study (OECD, 2002). The animals were kept fasting for overnight and provided only with water, after which the extracts were administrated orally at 5mg/kg body weight by gastric intubations and observed for 14 days. If mortality was observed in two out of three animals, then the dose administrated was assigned as toxic dose. If mortality was observed in one animal, then the same dose repeated again to confirm the toxic dose. If mortality was not observed, the procedure was repeated for higher doses such as 50,100 and 2000 mg/kg body weight.

Pharmacological Evaluation:

Analgesic activity:

1. Tail immersion test¹⁰:

The rodent tail withdrawal reflex can be elicited by immersion of tail in hot water at 55⁰CThis test is specific for opioid like central analgesics and is used to differentiate them from peripheral analgesics.

Methodology:

Young female mice weighing in between 20 to 60g are used.They are placed into individual cylindrical holder leaving the tail hanging out freely.The animals are allowed to get acclimatized the lower to holder for 30 min. before testing .The lower 5 cm of portion is marked.This part of tail is immersed in freshly filled water at exactly 55⁰c temperature .The reation time is recorded using a stop-watch of 0.5 s accuracy.After each determination the tail is carefully dried.The reaction time is determined before and periodically 0.5,1,2,3,4 and 6 h after the oral and s.c. administration of the the substance. The cut off time of immersion is 15 s. The withdrawal time of control mice usually lies between 1 and 5.5 s. A withdrawal time more than 6 s is regarded as a positive analgesic response. ED50 values can be calculated for each compound and time response curve (onset, peak and duration of action) can be plotted.

2. Acetic acid writhing test¹¹

Methodology:

Swiss mice (24-30g) of either sex can be used. Acetic acid in a concentration of 0.02% is suspended in a 1% suspension of carboxy methyl cellulose. A liquor of 0.25 ml of this suspension is administered i.p. to mice in each group. Group of 6 mice are used for test and standard drugs. The mice are then placed individually into glass beaker and after 5 min. they are observed for a period of 10 min. and the numbered of writhes recorded for each animal. For the purpose of scoring, a writhes is indicated by stretching the abdomen with with simultaneous stretching of atleast one hind limb.

The percentage inhibition of writhing by an analgesic is calculated according to the following formula:

Average withers in control group - Average withers in treated group *100

%Inhibition = ----------------------------------------------------------------------------

Average withers in control group

Anti-inflammatory activity:

1. Carrageenan Induced Paw Edema¹²:

The animals were divided randomly into different groups with 6 rats per group as follows-

Group	Treatment	Dose
I	Control (Saline solution)	0.3 ml/kg
II	Diclofenac sodium	10 mg/kg
III	EELS	200 mg/kg
IV	EELS	400 mg/kg

Procedure:

The different extracts at the dose level of 200 and 400 mg/kg body weight were administered orally to the treated group and Diclofenac sodium at the dose level of 10 mg/kg body weight was administered intraperitonially to the standard group. After 30 min paw edema was induced by the injection of 0.1 ml of 1% freshly prepared suspension of carrageenan into the sub-planter region of the left hind paw of the each group of rat. The paw marked with ink at the level of lateral malleolus to facilitate subsequent reading. The paw edema volume was measured by mercury displacement with the help of plethysmometer at 0, 1, 3, and 5hr after injecting carrageenan. The difference between 0 h and Subsequent readings were considered as edema volume. The percentage inhibition of edema in various groups was calculated using the formula (Vogel and Scholliens, 2002).

% Edema Inhibition= (1 – Vt/Vc) X 100

Where,

Vt = Edema volume in the drug treated group.

Vc = Edema volume in the control group.

Statistical Analysis:

Difference in paw volume and their average, Percentage inhibition of edema and standard error mean (s.e.m.) were calculated for each experiment. Data was expressed as mean ± s.e.m. and statistical analysis was carried out by One Way ANOVA followed by Dunnet’s t-test at P<0.05 significance level using “Graphpad Instat” version 3.00 for Windows 95, GraphPad Software.

RESULTS:

Acute toxicity testing:

The plant extract did not exhibit any mortality upto the dose level of 2000mg/kg so, the extract safe for long term administration.

Phytochemical evaluation:

Phytochemical evaluation of Lagenaria siceraria fruit & leaf showed the presence of, flavonoids, carbohydrates, proteins, glycosides & saponins.

PHARMACOLOGICAL STUDIES:

Analgesic activity

1. Tail immersion method –

In tail immersion test the crude extract produced 32.42%(p<0.05) and 39.69% (p<0.05) elongation of tail flicking time 30 minutes after oral doses of 200 and 400 mg/kg body weight respectively (table1). After 60 minutes the extract showed 46.69% (p<0.05) and 54.52% (p<0.001) elongation of tail flicking time.

Table 1: Effect of Crude extract on Tail immersion method:

Group	Treatment	Dose	Basic reaction time (sec)	Reaction time in sec.
		Dose	Basic reaction time (sec)	15min	30 min	60 min	120min
I	0.5% CMC	1ml/kg	6.18±0.18	6.38±0.06	6.75±0.08	6.88±0.08	6.30±0.13
II	Ibuprofen	30mg/kg	6.42±0.12	10.17±0.10*	13.59±0.09**	17.61±0.11**	19.36±0.26**
III	L.siceraria	200mg/kg	5.48±0.06	6.11±0.06*	8.11±0.07*	10.28±0.10*	13.10±0.14**
IV	L.siceraria	400mg/kg	5.88±0.09	7.58±0.06*	9.75±0.06*	12.93±0.08**	15.11±0.13**

Result expressed as mean ± SEM from six observations; ** indicates P < 0.01 &

* indicates P < 0.05

2. Acetic acid induced writhing test:

In the acetic acid induced writhing test the extract of Lagenaria siceraria (200 and 400 mg/kg body weight) showed a significant (p<0.001) reduction in the number of writhes with 45.54%and 55.88% respectively (table-2)

Table 2: Effects of crude extract on acetic acid induced writhing response in mice.

Group	Treatment	Dose	Writhing	% Inhibition
I	Solvent control	-	21.42±1.34	-
II	Ibuprofen	30mg/kg	7.92±0.40**	64.72
III	L.siceraria	200mg/kg	11.62±0.70*	45.54
IV	L.siceraria	400mg/kg	9.45±0.60**	55.88

Values are mean ± SEM (n = 6); One-way ANOVA; **P<0.001, compared to control.

Anti-inflammatory activity

1. Carrageenan Induced Paw Edema:

In Carrageenan induced paw oedema test the L.siceraria extract produced 21.16% (p<0.05) and 31.66% (p<0.01) elongation of paw flicking time 1 hrs after oral doses of (200 and 400 mg/kg body weight) (table 3). After 5 hrs the L. siceraria extract showed 41.26% (p<0.05) and 49.20% (p<0.01) elongation of paw flicking time.

Table 3: Effects of crude extract on Carrageenan Induced Paw Edema in rat.

Group	Treatment	Oedema volume (ml)					% inhibition after 180 min
Group	Treatment	Dose	0 min	60 min	120 min	180 min	% inhibition after 180 min
I	Control (Saline solution)	0.3 ml/kg	27.56+1.93	52.55+1.63	91.56+2.54	105.56+2.53	--
II	Diclofenac sodium	10 mg/kg	18.33+1.84	32.93+1.66	38.63+1.26**	21.66+1.54**	85.04
III	L.siceraria	200 mg/kg	26.22+1.83	40.92+1.32	61.21+1.84	45.16+1.28**	62.77
IV	L.siceraria	400 mg/kg	22.33+1.84	36.93+1.66	45.63+1.26**	29.66+1.54**	77.46

Result expressed as mean ± SEM from six observations; ** indicates P < 0.01 &

* indicates P < 0.05

DISCUSSION:

In the acetic acid induced writhing test the extract of L.siceraria (200 and 400 mg/kg body weight) showed a significant (p<0.001) reduction in the number of writhes with 45.54%and 55.88% respectively (table 2). In Tail immersion test the L.siceraria extract produced 32.42% (p<0.05) and 39.69% (p<0.05) elongation of tail flicking time 30 minutes after oral doses of (200 and 400 mg/kg body weight) (table 3). After 60 minutes the L .siceraria extract showed 46.69% (p<0.05) and 54.52% (p<0.001) elongation of tail flicking time.

The constriction response of abdomen produced by acetic acid is a sensitive procedure for peripheral analgesic agents.¹³ This response is believed to be mediated by the prostaglandin pathways.¹⁴ The extract of L.siceraria produced antinociceptive activity and thus indicates the presence of analgesic components that might influence the prostaglandin pathways. In the Tail immersion test, the plant extract prolonged the stress tolerance capacity of the mice, indicating the possible involvement of a higher center.¹⁵

In the carrageenan induced paw oedema test the extract of L.siceraria (200 and 400 mg/kg body weight) showed. In Carrageenan induced paw oedema test the L.siceraria extract produced 21.16% (p<0.05) and 31.66% (p<0.01) elongation of paw flicking time 1 hrs after oral doses of (200 and 400 mg/kg body weight) (table 3). After 5 hrs the L.siceraria extract showed 41.26% (p<0.05) and 49.20% (p<0.01) elongation of paw flicking time. The constriction response of abdomen produced by carrageenan induced paw oedema is sensitive procedure for anti-inflammatory agents.

So from above discussion it is clear that L. siceraria having Analgesic and Anti-inflammtry potency.

CONCLUSION:

From the above investigation, it is quite apparent that ethanolic extract of L.siceraria fruit & leaves possesses potent analgesic and Anti-inflammatory effect. This is evidenced by significant increase in the reaction time by stimuli in different experimental models.

ACKNOWLEDGEMENT:

The authors acknowledge the facilities provided by Vishal institute of pharmaceutical education and research ale, pune.

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Received on 12.01.2019 Accepted on 16.03.2019

Asian J. Pharm. Tech. 2019; 9(2):75-78.

DOI: 10.5958/2231-5713.2019.00013.8