Phytochemical & Pharmacological Investigation of Stems of Passiflora foetida

Aims: To carry out extraction, preliminary phytochemical analysis and invivo analgesic screening of extract of the stem of Passiflora foetida L. Methodology: Passiflora foetida L; Family: Passifloraceae, is an exotic fast-growing perennial and medicinal vine occurring in Germany, France and other European countries and USA and grown in different parts of India. Dried stems of Passiflora foetida L was coarsely powdered and maceration was done using Soxhlet apparatus. The ethanolic extract of stems of Passiflora foetida L was subjected to preliminary phytochemical tests. Then subjected to in vivo analgesic activity. Results: Phytochemical investigation of the stem of Passiflora foetida L preliminary test showed the presence of carbohydrates, glycosides, flavonoids and steroids. Acute toxicity study of ethanolic extract of stems of Passiflora foetida L was carried out and extracts were found to be safe up to 2000 mg/kg body weight. Pharmacological activities of stems of Passiflora foetida L was carried out from ethanolic extract. Conclusion: Phytochemical investigation of ethanolic extract of stems were carried out and Analgesic activity by tail flick method in rats and acetic acid induced writhing method in mice, showed statistically significant activity (P=.05) when compared to control. The ethanolic stem extract of Passiflora foetida L proved to have significant pain relieving action in a dose dependent manner. Original Research Article Antony et al.; JPRI, 33(58B): 408-414, 2021; Article no.JPRI.79723 409


INTRODUCTION
Medicinal plants which form the strength of traditional medicine is being explored extensively for the treatment of various health issues by subjecting to intense pharmacological studies. Number of traditional plants have been used in the treatment of various ailments which has been acknowledged and has made known the value of medicinal plants as potential sources of new compounds of therapeutic value and as sources of lead entities in the drug development. In developing countries and now in developed countries, it is estimated that about majority of the population rely on/or turning to traditional medicine for their primary healthcare. There arises therefore a need to screen medicinal plants for bioactive compounds as a basis for further pharmacological studies [1]. Passiflora foetida is a biennial herb, commonly referred to as red fruit passion flower of the class Dicotyledonea. It is a evergreen climber with twisted slender structures on the stems that help in climbing. Stems sometimes angular to 15 cm height. The stems have a disagreeable odour [2].
A review article on Passiflora reported that different parts of the plant either in fresh or dried form can be extracted for medicinal use in European countries as anxiolytics. A decoction prepared from the leaves and fruits is used to treat psychological disorders and asthmatic conditions. Paste prepared from the leaves of Passiflora foetida L is useful for the treatment of skin infections and headaches.It is believed to have antispasmodic, sedative and hypotensive activity [3] as well as effective antidepressant properties [4]. Three polyketides, alpha-pyrones named Passifloricins from Passiflora foetida resin were isolated [5], several natural and hemisynthetic passifloricins showed Leishmanicidal activity [6]. Passiflora edulis commonly known as passion fruit expressed free radical scavenging properties [7], antibacterial and cytotoxic properties [8], anti-helmenthic activity of Passiflora edulis Linn, using ethanol and water extracts of leaves [9], roots of Passiflora foetida Linn showed significant anti bacterial properties against different bacterial strains [10].
It was learnt that no substantial work on the stems of Passiflora foetida L was carried out both in the chemical investigation & screening for analgesic activity, hence the aim of present work is to carry out screening for analgesic activity.

Experimental Procedure for Preparation of Ethanolic Extract
The stems of Passiflora foetida L (3 kg) were cleaned, shade dried and broken down into pieces and powdered into a coarse powder by a mechanical grinder. The powder was then passed through sieve no. 40. 750 gms of powder was extracted with ethanol in soxhlet extractor for 72 hrs. A dark brown residue of yield of 12.3% was obtained after concentrating the extract which was done under reduced pressure. Then placed in desiccator for further use.

Selection of animals:
Wistar albino rats of either sex weighing between 100-150g and albino mice of either sex weighing between 20-25g were obtained from KSHEMA, Deralakatte Mangaluru. These animals were used for the acute toxicity and analgesic studies. The animals were stabilized for 1 week; they were maintained in standard condition at room temperature, 60 ± 5% relative humidity and 12 h light and dark cycle. They had been given standard pellet diet supplied by Hindustan Lever Co, Bombay and water ad libitum throughout the course of the study. The animals were handled gently to avoid giving them too much stress, which could result in an increased adrenal output.
Acute toxicity studies: Acute toxicity study was conducted to determine the median lethal dose (LD50) of the ethanolic extract of stems of Passiflora foetida L [11]. The acute toxicity study was carried out in adult female albino rats by "up and down" [12] method and OECD guidelines 425 [13].
The animals were divided into 5 groups (n=6). The first group was treated after fasting overnight with a oral dose of 100mg/kg of body weight with the ethanolic extract of the stems of Passiflora foetida L suspended in 0.5% sodium carboxy methyl cellulose (NaCMC). The animals were observed continuously for 2-3 hours for general behavioral, neurological, autonomic profiles and finally death after 24 hour [14]. There was no mortality and no signs of toxicity at this dose level. Similarly, the other groups were treated with an oral dose of 2000 mg/kg body weight of ethanolic extract of stems of Passiflora foetida L and there was no mortality and no signs of toxicity and the extract were found to be safe at this dose level. The observations were tabulated according to Irwin's table [15].

Selection of dose:
From preliminary toxicity studies of ethanolic extract of stems of Passiflora foetida L, it was observed that none of the treated animals died and thus it was concluded that these fractions were nontoxic up to 2000 mg/kg body weight were chosen for the study for analgesic activity, three dose levels were chosen in such a way that, middle dose was approximately one tenth of the maximum dose during acute toxicity studies, and a low dose, which was 50% of the one tenth dose, and a high dose, which was twice that of one tenth dose (200 mg/kg, 100 mg/kg, 400 mg/kg).
Preparation of different doses: The ethanolic extract was prepared in 0.5% Na CMC of dose 100, 200, 400 mg/kg body weight, given to rats and mice by oral route.

Experimental Procedure
Tail flick method: Acute nociception was assessed using a tail flick apparatus (Analgesiometer). Each rat was placed in a restrainer, before treatment the basal reaction time was measured by keeping distal one-third portion of the rats tail. Tail was placed over the nichrome wire without touching it. The extracts were immediately administered by oral route and reaction time was measured at 30 minute interval after the drug administration for 2 hrs. to prevent tissue damage 10 seconds cut off time kept. In the present study, adult male/female Albino rats (100 150 g, 5-6 weeks old) were selected. They were divided into 5 groups of 6 animals each. The first group served as control and received 2% NaCMC. The second group was treated with pentazocine (10 mg/kg body weight, p.o.). The third, fourth and fifth groups received 100, 200 and 400 mg/kg body weight of ethanolic extract of the stems of Passiflora foetida L as a suspension in 0.5%Na CMC. Reaction time was recorded after every 30 minutes for 120 minutes and the average values of reaction time after each time interval are calculated and compared with the pre test value by analysis of significance [16,17] Evaluation of the analgesic activity of the ethanolic extract of Stem of Passiflora foetida L by tail flick method and writhing method are represented in tables 1 & 2 respectively along with group treatment description.

RESULTS AND DISCUSSION
Acute toxicity studies: The ethanolic extract of stems of Passiflora foetida L did not exhibit any toxic effects up to 2000 mg /kg body weight when administered as single oral dose. Selection of dose The LD50 of the ethanol extract was more than 2000 mg/kg body weight in all the cases. Hence a dose of 100, 200 and 400 mg/kg body weight were chosen for the study.
The treatment of rats with the ethanolic stem extract of Passiflora foetida L at doses of 100, 200 and 400 mg/kg body weight respectively exhibited significant (P=.01) increase in reaction time while compared to control in case of tail flick method.
Pretreatment of mice with ethanolic stem extract of Passiflora foetida L at a dose 100, 200, 400 mg/kg body weight respectively produced a significant (P=.01) reduction in writhes induced by acetic acid while compared to control. The % inhibition of writhings or % protection were found to be 48.41%, 59.61% and 63.99% for ethanolic extract of stem at a dose 100, 200 and 400 mg/kg body weight respectively.
The ethanolic extract of the stem of Passiflora foetida L exhibited dose dependent analgesic activity in both methods experimented for analgesic activity.

Statistical Analysis
All the values reported are expressed as mean ±SEM. Analyzed by One way analysis of variance (ANOVA) followed by Dunnett's multiple comparison test.
The preliminary qualitative analysis was performed for testing the presence phytochemical constituents in ethanolic extract and results are shown in Table 3.
Results of analgesic activity by tail flick method and writhing method are reported in Tables 4 & 5 respectively.

Discussion
Analgesic action caused visceral pain in acetic acid induced writhing in mice, which may be due to the release of arachidonic acid from tissue phospholipids action that could trigger an inflammatory response via the cyclooxygenase pathway as well as prostaglandin synthesis. Increased levels of prostaglandins (PGE2 & PGF2α) in the peritoneal region increases pain  100 mg/kg body weight of extract of stem of Passiflora foetida L in 0.5% w/v NaCMC Group IV (Test) 200 mg/kg body weight of extract of stem of Passiflora foetida L in 0.5% w/v NaCMC Group V (Test) 400 mg/kg body weight of extract of stem of Passiflora foetida L in 0.5% w/v NaCMC   ** the mean difference is significant at the .05 level, when compared to the standard associated with inflammation by increase in capillary permeability. This method is useful for evaluating peripheral analgesics, as the less number of writhing shows inhibition of prostaglandin synthesis which is a peripheral mechanism of pain inhibition. The crude ethanolic stem extract of Passiflora foetida L proved to have significant pain relieving action in a dose dependent manner.
Phytochemical screening showed the presence of carbohydrates, glycosides, flavonoids & steroids which is a probability for its analgesic properties by qualitative tests. Flavonoids bring about their analgesic activity primarily by targeting prostaglandins [19] The tail flick method is used for study of centrally acting nociceptive action. The three groups showed significant increased pain threshold in a dose dependent gradient way [20,21]. The tail flick latency continued to ascend for all three doses till 90 min when compared to the control. There seemed to be no statistically significant difference in mean tail flick latency between the three drug groups at the prefixed time points of 0, 30, 60, and 90 minutes. Therefore, in the tail flick method, the three drug dosages were observed to be equally efficacious in analgesic effect during the experimental time period of 120 minute. This indicates that the stem extract of Passiflora foetida L can produce analgesic effect by peripheral central mechanism.

CONCLUSION
The Preliminary Phytochemical investigation of ethanolic extract of stems revealed the presence of carbohydrates, glycosides, flavonoids and steroids. But proteins, tannins, saponins, alkaloids, resins were absent. Analgesic activity of stem of Passiflora foetida L showed statistically significant and may be attributed to components like flavonoids and steroids. From studies carried on stem it is evident that Passiflora foetida L are endowed with significant analgesic activity against animal models, thereby justifying their use in traditional system of medicine.

CONSENT
It is not applicable.

ETHICAL APPROVAL
"All authors hereby declare that "Principles of laboratory animal care" (NIH publication No. 85-23, revised 1985) were followed, as well as specific national laws where applicable. All experiments have been examined and approved by the appropriate ethics committee"-, REF. NO. KSHEMA /AEC/13/2011)