In vitro and In vivo Anti-in lammatory Activity of the Extracts of Whole Plant Argyreia imbricata (Roth) Sant. & Patel

Sebastin V*1, Gopalakrishnan G2, Sreejith M3, Anoob Kumar K I4 1Department of Pharmaceutical Chemistry, Malik Deenar College of Pharmacy, Kerala, India 2Department of Pharmacy, Annamalai University, Annamalai Nagar, Chidambaram, Tamil Nadu, India 3Department of Pharmaceutical Chemistry, Nazareth College of Pharmacy, Thiruvalla, Kerala, India 4Department of Pharmaceutical Chemistry, KVM College of Pharmacy, Alappuzha, Kerala, India


INTRODUCTION
Plants used in traditional medicinal systems are the source of several drugs commonly in usage for the treatment of a variety of ailments today. These drugs were initially discovered and developed by research on plants (Jose and Kumar, 2016). A medicinal plant can be suitably termed as chemical factory as it contains numerous unique bio active molecules such as glycosides, alkaloids, saponins, resins, oleoresins, sesquiterpene lactones, essential and ixed oils. India has the oldest, richest and most diverse cultural traditions associated with the use of an enormous variety of herbs (Suman et al., 2013). Plants of genus Argyreia belongs to the family Convolvulaceae are one among them. Many plants of this genus, mainly, A. speciosa was analyzed well in the aspects of phytochemistry, pharmacognosy, and pharmacological properties (Joseph et al., 2011;Modi et al., 2010;Jaiswal and Tailang, 2018). Argyreia imbricata, another one plant of this genus is commonly found in south India at an altitude up to 300m mean sea level which has not been previously explored scienti ically to our knowledge was selected for our research. In our previous effort, a preliminary phytochemical screening and the spectral characterization of active constituents present in the different dried extracts of whole plant material of Argyreia imbricata was done successfully (Sebastin et al., 2019). As a part of an investigation of different pharmacological activities, previously, the anti diabetic potential of the extracts was evaluated in vitro and in vivo. Now, it was focused on the evaluation of anti-in lammatory activity by in vitro and in vivo methods.

Plant materials -Collection and extraction
The whole plant of Argyreia imbricata was collected from Mekkarai, the village located close to the hillocks of the Western Ghats in the Tirunelveli District of Tamil Nadu, India. After proper identi ication and authentication, the collected material was dried, powdered and extracted by soxhlet apparatus assembly by using the solvents of increasing polarity viz., petroleum ether, chloroform, ethyl acetate, and methanol (Sebastin et al., 2019). The dried extracts obtained were used for the experiments.

Anti-in lammatory activity -In vitro
It was done by stabilization of human red blood cell (HRBC) membrane with different approaches such as heat-induced and hypo-tonicity induced membrane-lysis. All the prepared extracts were subjected to in vitro assays in triplicate, and the results were expressed as mean ± standard deviation.

HRBC membrane stabilization method
The study was designed about the procedure of (Seema et al., 2011;Anosike et al., 2012;Chowdhury et al., 2014;Patel and Desai, 2016). Whole human blood collected freshly from the healthy volunteers was mixed with an equal volume of sterile Alsever solution (2% dextrose, 0.8% sodium citrate, 0.05% citric acid, 0.42% sodium chloride, and 100ml distilled water) and centrifuged at 3000rpm for 10min., and the packed cells obtained were washed and reconstituted with sterile isosaline (0.85% sodium chloride in water, sterilized by autoclaving) as 10%v/v suspension. HRBC suspension (1ml) and each test extracts (1ml) in different concentration (100, 200, 400, 600, 800, 1000µg/ml) was taken in individual tubes. Normal control tube contains HRBC suspension and Alsever solution only. Standard control tube contains diclofenac sodium 200µg/ml instead of test extracts. All the tubes were subjected to incubation (37 • C for 30min) and then centrifugation. The supernatant was collected, and its haemoglobin content was estimated spectrophotometrically (560nm), and the percentage haemolysis and protection were calculated by

Heat-induced haemolysis
Test extracts were dissolved in isotonic phosphate buffer solution. The reaction mixture contains test extracts (5ml) in different concentration (100, 200, 400, 600, 800, 1000µg/ml) and 10%v/v HRBC suspension (0.1ml). Normal control tube contains saline, and the standard control tube contains diclofenac sodium 200µg/ml instead of test extracts. A batch of prepared tubes was kept at 54 • C for 20min, in a regulated water bath. Another batch tube was kept at −10 • C for 20min in a freezer. Then, all were centrifuged at 3000rpm for 3min, the supernatant was collected, and the haemoglobin content was estimated spectrophotometrically (540nm), and from this, the percentage inhibition of haemolysis by the tests was calculated by

Anti-in lammatory activity-In vivo
Based on the results of in vitro anti-in lammatory evaluation, the extracts selected were subjected to in vivo evaluation in paw oedema of the experimental animals induced by carrageenan. The evaluation was designed about the procedure of (Mohan et al., 2013;Arul and Smith, 2017;Gupta and Gupta, 2017   to the animals of the entire group by intraperitoneal injection except for normal and in lammatory control. The paw volume of the injected animal was started to measure from zero hours by plethysmometrically and continued at the interval of one hour for ive hours. The percentage inhibition of in lammation was calculated by Percentage inhibition of inflammation = Vc−Vt Vc × 100 Where, Vc-mean an increase in paw volume in a control group of rats; Vt-mean an increase in paw volume in rats treated with test extracts. The results were expressed in mean ± SEM (Standard Error Mean) 6 experimental animals in each group. ANOVA and Dunnett's test assessed statistical signif-icance. P-values < 0.05 were considered signi icant.

RESULTS AND DISCUSSION
Results of HRBC membrane stabilization assay are presented in Table 1. In this study, all the four tested extracts showed a concentration based activity; notably, the ethyl acetate extract (1000 µg/ml) showed signi icant activity (71. 33 ± 0.60) comparing with other tested extracts and standard drug. Of course, the methanol extract also showed a signi icant concentration-dependent cell protection activity. Still, it was observed that its highest concentration (1000 µg/ml) showed a less percentage (63.16 ± 0.79) comparing with the ethyl acetate extract in  Table 3 which revealed that under isotonic condition, the ethyl acetate extract (1000 µg/ml) showed signi icant activity (70.10±0.85) comparing with other tested extracts in both hypotonic an isotonic condition. The results indicated that the activity of extracts was in luenced by its concentration and the changes in the tonicity of the medium. Generally, it is well-known that the integrity of the cell membrane plays an essential role in the viability of the cell. Exposure of RBC to different adverse factors such as hypotonicity, heat and cold activates free radical-induced lipid per oxidation that makes the cell highly susceptible to secondary damage and the in lammatory mediators' released increase the permeability of cell membrane that causes leakage of serum protein and luids into the cells. But the membrane stabilization prevents this. In this study, the tested extracts particularly, the ethyl acetate and methanol extracts perhaps stabilized the RBC membrane by blocking the release of lytic enzymes and in lammation mediators. The previous literature indicated that some saponins and lavonoids pos-sess a signi icant stabilizing effect on a lysosomal membrane in vitro and in vivo evaluation (Oyedapo et al., 2010). In our previous study, the ethyl acetate and methanol extract showed positive results in the test for lavonoids and saponins (Sebastin et al., 2019) that may be responsible for their membrane stabilizing effect. Based on this result, the ethyl acetate and methanol extracts of Argyreia imbricata were selected for the in vivo evaluation in the paw oedema induced by carrageenan. It is an acute test model used for assessing the anti in lammatory activity of tests. Oedema induced by carrageenan is a biphasic event in which the irst phase is concerned with histamine and serotonin release, and the next one is due to prostaglandins, proteases and lysosome release. The third one is the granuloma formation (Arul and Smith, 2017). In the in vivo evaluation, there is no signi icant difference between the initial paw volume and the paw volume in each hour of observation in the group I (normal control) animals. But, in the in lammatory control animals (group II), the elevation of paw volume is observed in each hour of observation, and it reached its maximum in the 5 th hour of observation. In case of standard control (group III), a slight increase of paw volume from its initial level was observed in the 1 st hour of observation. But in the next three observations, it was continuously decreased and reached the minimum in the last (5 th hour) observation. A similar observation is found in the animals of group IV-VII which are treated with ethyl acetate and methanol extracts in the dose of 200 mg.kg −1 and 400 mg.kg −1 respectively. However, the results indicated that the test extracts in the dose of 400 mg.kg −1 showed signi icant results comparing with the standard control, particularly, the methanol extract Tables 4 and 5.

CONCLUSIONS
In the present study, the whole plant Argyreia imbricata was collected, identi ied and authenti-cated, dried, powdered and extracted by soxhelation with solvents of ascending order of polarity viz., petroleum ether, chloroform, ethyl acetate, and methanol. Dried extracts obtained were subjected to anti-in lammatory evaluation by in vitro and in vivo methods. Among the four tested, two, the ethyl acetate and methanol extracts showed signi icant activity in the in vitro evaluation. Based on this result, these two extracts were selected for the in vivo evaluation. In this evaluation also, the selected methanol and ethyl acetate extracts exhibited a signi icant activity, particularly, the methanol extract which suggested that some compounds from these extracts appeared to be promising for the treatment of in lammation. Our future studies in the direction of toxicity evaluation, identi ication and isolation of the active constituents from these extracts may give signi icant results valuable for further researches.