Assessment of analgesic and anti-inflammatory potential of ethanolic extract of Gossypium arboreum leaves in experimental animals

Background and objectives: Gossypium arboreum commonly known as cotton plant, this variety of cotton plant available throughout India. Cotton plant was used traditionally for the treatment of infection, diarrhea and other inflammatory conditions. The aim of present study is to evaluate analgesic and anti-inflammatory activity of Gossypium arboreum leaves extract on experimental animals. Materials and Methods: The ethanolic extract of Gossypium arboreum leaves (EEGA) was subjected to assess its antioxidant potential using DPPH radical scavenging assay; further anti-inflammatory and analgesic activity was assessed by using carrageenan-induced rat paw edema and tail flick test respectively in experimental animals. Results: It was observed that free radicals were scavenged by the EEGA in a concentration dependent manner. The Ethanolic extract showed maximum 72% scavenging activities at 200 g/ml concentration. The ethanol extract exhibited significant analgesic activity in the tail-flick model (?<0.01) by increasing the reaction time of the mice to 8.9 sec at 180min after treatment in comparison to control (3.4 sec). The EEGA (100, 200 and 400 mg/kg, p.o.) showed dose-dependent, inhibition of carrageenan-induced rat paw edema from 30 min onwards (P<0.01), Conclusions: Present study revealed that the ethanolic extract of Gossypium arboreum displayed prominent analgesic and anti-inflammatory activity in experimental animals owing to its antioxidant property.


Introduction
In numerous diseases and disorders oxidative stress and inflammation play a vital role [1,2]. Free radicals particularly, the reactive oxygen species (ROS) creates oxidative stress within cells resulting in inflammatory and infectious condition. Phagocytic cells including polymorphonuclear leukocytes (neutrophils, eosinophils) and mononuclear cells (macrophage and lymphocytes) produce excessive amount of reactive oxygen species which play a significant role in the host defense mechanism [3,4].
Inflammation is a complex biological response of vascular tissues against aggressive agents such as pathogens, irritants, or damaged cells. The generation of oxygen free radicals is known to be involved in the development of the inflammatory response. In addition to their actions as noxious mediators generated by inflammatory cells, these molecules play also a crucial role for progression of inflammation in various organs [5]. Inflammation is the initial response and is characterized by the increased movement of plasma and innate immune system cells, such as neutrophils and macrophages, from the blood into the injured tissues. The standard signs of inflammation are expressed by increased blood flow, elevated cellular metabolism, vasodilatation, release of soluble mediators, extravasation of fluids and cellular [6].
Although non-steroidal anti-inflammatory drugs (NSAID) and opioids are widely used in the treatment of inflammatory diseases, these drugs have numerous side effects which include gastritis, peptic ulceration; gastrointestinal bleeding and tolerance may develops due to chronic administration of NSAIDs [7]. Therefore, it is necessary to identify analgesic and antiinflammatory agent with higher potency and minor side effects. diarrhea and other inflammatory conditions [11]. Gossypium arboreum leaf extracts are already reported for its antidiabetic, antihypertensive, antibacterial and antifungal activity [12][13][14].
Infusion of the leaf of Gossypium arboreum used to reduce respiratory complications like cough and cold as it reduce smooth muscle contractions [15,16]. The important active phytochemicals present in Gossypium arboreum are glycosides, monoterpenes, phenolic acids, triterpenoid, carbohydrates, flavonoids, alkaloids, fatty acids, and essential oils, some of these phytochemicals are reported for its antioxidant potential [11,17].
So, based on the literature review, reported pharmacological activities and chemical constituents found in cotton plant the present study is aimed to investigate analgesic and antiinflammatory potential of ethanolic extract of leaves of Gossypium arboretum.

Extraction
Leaves of Gossypium arboreum (Family Malvaceae) was collected in the month of January 2019 from the local region of Nanded district of Maharashtra state, and it was identified and authenticated from Taxonomy Research in Botany, N.E.S Science College, Nanded.
Leaves of Gossypium arboreum, were washed 2-3 times with tap water and then shade dried for 7 days then coarsely powdered using grinder. 100 g of dried powder was extracted with ethanol by using Soxhlet apparatus. The % yield of ethanolic extract of leaves of Gossypium arboreum (EEGA) was found to be 4.7% w/w.

Animals
Albino Wistar rats (180-220 g) and Swiss albino mice (20-30 g) of either sexes were used for the study. The animals were obtained from the animal house of Sudhakarrao Naik Institute of Pharmacy, Pusad, Maharashtra, India. The animals were housed in standard laboratory condition of dark,light cycle (12,12 h) and temperature (25 ± 2°C). Animals were given standard laboratory diet and water ad libitum. The experimental protocol was approved by the Institutional Animal Ethics Committee (Reg. No. 729/PO/Re/S/11/CPCSEA).

Dose Selection
The dose was selected as per literature survey, the dose 100mg/kg, 200mg/kg and 400 mg/kg was selected for this study. The data obtained were statistically analyzed using ANOVA followed by Dunnett's test to detect any significant difference among different means, with level of significance set at p< 0.05.
The results were expressed as mean ± S.E.M.

In-vitro antioxidant activity
Several concentrations (50, 100, 150 and 200 µg/ml) of the EEGA were tested for their antioxidant activity in DPPH (2, 2-dipheny 1, 1-picrylhydrazyl) radical scavenging activity. It was observed that free radicals were scavenged by the EEGA in a concentration dependent manner.
The EEGA showed maximum radical scavenging activities (72%) at 200 g/ml concentration, which is comparable to that of standard ascorbic acid (89%).

Discussion
Certain types of inflammatory injuries are mediated by reactive oxygen species. The most likely sources of these oxidizing agents are the phagocytic leukocytes (e.g., neutrophils,

Time in min
Diclofenac EEGA 100mg/kg EEGA 200mg/kg EEGA 400mg/kg monocytes, macrophages, and eosinophils) that invade the tissue [21,22]. These reactive radicals and oxidants may harm cell and tissues directly via oxidative degradation of cellular components [23]. Reactive oxygen species may also initiate and amplify inflammation via the release of several mediators involved in the inflammatory response [24].
Chronic health problems such as asthma, diabetes, hypertension, cancer, kidney and liver failure and certain inflammation, showed involvement of free radicals induced damage [25].
Antioxidants prevent free radical induced tissue damage by stabilizing the unstable free radical by donating electron or may reduce its formation or enhance their decomposition [26]. Free radical induced damage can be reduced by use of certain antioxidant agents derived from synthetic or natural source. The rich sources of natural compounds with antioxidants property are present in traditional Indian diet and medicinal plants [27,28]. In present study, the antioxidant activity of Gossypium arboreum leaves was performed by using DDPH radical scavenging assay. DPPH produce a stable free radical, and in present assay the measurement of electron donating ability of EEGA and that can be analysed by colour change in the reaction mixture. The changes in colour (from deep violet to light yellow) were measured at 517 nm on a UV-visible light spectrophotometer [25]. EEGA exhibited a significant DPPH radical scavenging activity and that was comparable to ascorbic acid. Radical scavenging ability of EEGA may be due to presence of certain phytochemicals such as lipid, proteins, flavonoid and glycoside responsible for antioxidant effect [17].
Analgesics act on peripheral or central nervous system and selectively relieve pain without significantly altering the consciousness. Central effect is due to raising the threshold for pain and inhibit the generation of impulses at chemoreceptor site, when act peripherally [29]. In present study, tail-flick method was used for screening of analgesic activity, it is mediated by spinal reflex to a nociceptive stimulus and reaction time to the stimuli is measured. EEGA at the dose of 400 mg/kg showed significant analgesic effect by increasing the reaction time for tail withdrawal when compared to that of control group animals. EEGA possesses flavonoids that may contribute to analgesic effect [30,31].
Carrageenan is the phlogistic agent; used to induce inflammation in the experimental animals to screen anti-inflammatory activity of compounds. When carrageenan injected locally into the rat paw, it produced inflammation, which was occur within 30 min [32,33]. Carrageenan showed biphasic response, the inflammatory symptoms observed due to release of histamine, serotonin and similar substances during first phase (2 nd hour) of administration; and in second phase (3 rd hour) activation of kinin-like substances, i.e., prostaglandins, proteases and lysosome responsible [34,35]. The EEGA inhibited the carrageenan induced rat paw edema, at both early and late phase.
Results revealed the inhibitory effect of EEGA on edema formation is probably due to the inhibition of the synthesis and release of the inflammatory mediators such as histamine, serotonin and prostaglandins. Anti-inflammatory effect of EEGA may be due to presence of flavonide, saponins are responsible for reducing the release of inflammatory mediators [30,36]. EEGA also reported for its membrane stabilizing activity which may contribute for its anti-inflammatory activity [37].

Conclusion
It can be concluded that the ethanolic extract of the leaves of Gossypium arboreum possess anti-inflammatory and analgesic activity thus validating the traditional claims. This knowledge could be tapped to formulate new agents to treat inflammatory ailments. Funding: This research received no external funding.