1. INTRODUCTION:

The complicated immune response towards inflammation takes place when the body is exposed to various causative agents, like microbial contamination, immunological reactions, physical trauma, or foreign substances. It manifests as redness, wheal, flare, myalgia and malfunction at the influenced area, and can also lead to systemic symptoms like fever and increased white blood cell count¹.

As a result, infection is usually considered as a natural defense procedure which boosts up the body's potential to beat off infection². Regardless of how, it involves a cascade of cellular and molecular processes, including the discharge of pro-inflammatory substances and also yields in manufacturing of oxygen radical. When inflammation is not properly controlled, it can disturb the usual balance amongst cellular responses and molecular actions. Then, continuous activation of immune cells by circulating cytokines can gradually change the acute inflammation to chronic. Chronic inflammation is lined up with an elevated hazardof developing many diseases, including malignant tumors, rheumatoid arthritis, obesity, CVS disorders, neurodegenerative diseases and diabetes mellitus³.

Pain is an unpleasant sensation triggered by real or perceived injury, eliciting various responses such as avoidance behaviors and changes in social interactions. While rooted in sensory experiences, pain is also influenced by psychological factors including emotions like depression, anger, and anxiety, as well as cognitive processes such as expectation and appraisal⁴. These emotional and cognitive components play a crucial role in shaping the perception of pain. Because pain can disrupt daily functioning and contribute to mood disorders like anxiety and depression, it can greatly diminish the quality of life for individuals⁵.

Although many drugs are available for managing pain and inflammation, but they often accompanied by significant adverse effects⁶. Mostly used non-steroidal anti-inflammatory drugs can cause gastric lesions, cardiovascular disorders, liver and kidney damage⁷. Corticosteroids can lead to complications such as osteoporosis, glucose intolerance, adrenal suppression, and peptic ulcers, while opiates may induce tolerance and dependence. Moreover, these drugs may not always provide adequate relief for some patients. This underscores the importance of developing alternative anti-inflammatory and analgesic agents with fewer or different adverse effects, allowing clinicians to tailor treatment based on individual patient needs and co-morbidities⁸. Therefore, further there is a growing need for invulnerable, and efficacious plant-based NSAIDs and analgesic drugs that offer minimal side effects. Several studies have reported that medicinal plants have therapeutic potential useful in management and treatment of various diseases^9-11.

The leaves of P.orientalis (Linnaeus) Franco, also called Biota orientalis or Thuja orientalis (Linnaeus), family Cupressaceae, have been used extensively as antitussive and expectorant in Chinese traditional medicine for more than two thousand years¹². It was first documented in the "Shen Nong Ben Cao Jing" (Shen Nong’s Herbal), where it was classified as a high-quality Chinese medicine for its purported benefits in relieving tremor, alleviating numbness, and potentially extending lifespan. Similarly, the "Compendium of Materia Medica" records its properties as promoting longevity, nourishing the heart, and calming the mind. In clinical use, it is employed alone or combined with other herbal drugs to treat chronic bronchitis, coughs, bronchiectasis, insomnia and asthma¹³.

Modern scientific studies concluded that the abstraction or portion ratio of the P.orientalis leaves show Non-Steroidal Anti-inflammatory activity in various cell-proliferation assays^14,15 or animal models^16,17. However, from what I know and understand from the information that I have, no study has evaluated the P.orientalis leaves collected from India, yet. Therefore, the unbiased study was to evaluate the NSAIDs and analgesic effects of P.orientalis leaves ethanol extract¹⁸.

2. MATERIAL AND METHODS:

2.1. Chemicals

All the chemicals used were of maximum purity (>99%). Ferric chloride, HCl, Molisch's solution, acetic acid, formalin, dextran, Dragendorff's reagent, magnesium metal strips and sulphuric acid were obtained from Loba Chemie Ltd., Mumbai. Ethanol was obtained from Jai Chemicals Works, Jaipur. Carageenum (Pharma Grade) was obtained from Sigma Chemical Co., USA.

2.2. Plant material collection and extract preparation

The plant leaves (Figure 1) were collected from local area of Jaipur, Rajasthan, India and authenticated from Botanical Survey of India, Jodhpur. A voucher specimen (JCP/PHCOG/2019/22) of the plant was deposited in the laboratory. The leaves were washed under tap water, and distilled water, and then dried in shade. The dried leaves were then powdered to prepare the coarse powder and stored carefully in moisture proof container until use. The leaves powder was extracted by maceration with ethanol (in ratio 1:10) for three days at room temperature as previously described¹⁹. The above preparation was drained out with Whatman filter paper No 1, and made to rest on water bath for being viscous and finally dried under reduced pressure and then stored until used for further experiments.

Figure 1: Platycladus orientalis

2.3. Animals:

The adult Wister rats(150-180g) of both sexes were employed for up to 12 hours light and 12hours dark cycles. During the experimental period Standard pellets obtained from Hafed, Rohtak, India was utilized as a basal diet. The food and drinking water ad libitum were given to control and experimental animals. Later onshuffled into various groups, the rats adopt to a new temperature, altitude, climate and environment for one week under standard conditions of humidity, temperature, and light/dark cycle.

2.4. Preliminary phytochemical screening of the extract:

Preliminary phytochemical screening of the extracts was performed to identify the presence of alkaloids, flavonoids, sterols, tannins, phenolic compounds, saponins, carbohydrates, proteins and amino acids by using method described by Harborne, 1998^20-22.

2.5. Drug dose:

For the rat experiment, the doses of an anti-inflammatory and analgesic were 100mg/kg body weight and 200mg/kg body weight, respectively.

2.6. Animal grouping:

For analgesic and anti-inflammatory research, four groups of animals were separated. Each gathering comprised of six creatures of one or the other sex. The groups were:

Group I: Negative control - Distilled water;

Group II: Test drug- alcoholic portion of P.orientalis -POE-100;

Group III: Test drug- alcoholic portion of P.orientalis-POE-200;

Group IV: Positive control- Standard drug ASA-10/ Diclofenac (10mg/kg). Here all results for edema and drug treatment in animals represented as: n for number of animal = 6, ns stands for = non-significant, *p<0.05 for significant, **p<0.01- means more significant, POE 100: 100mg/kg; POE 200: 200mg/kg; ASA-10: Acetyl salicylic acid-10mg/kg.

2.7. Evaluation of anti-inflammatory activity :

2.7.1. Carrageenan induced rat paw edema:

Acute inflammation was developed by sub plantar injection of 0.1ml of 1% 0.1ml of 1% carrageenan in normal saline in the hind paw of rats 1 h after the introduction of the test drug in body as well as positive and negative controls. The paw volume was evaluated at 1h, 2h and 3h after carrageenan mixture, using plethysmograph^23-24.

2.7.2. Dextran induced paw edema:

Just like in the carrageenan-induced paw edema model, the rats were treated with Dextran. In this research study, 1% dextran in 0.1 milliliters was used. At 1, 2, and 3hours, the carrageenan-induced paw edema model's volume was calculated^25-26.

2.7.3. Formalin induced paw edema:

One dose of the test drug was given to all groups for a week. On last day, edema volume already introduced was checked before drug treatment. After the treatment drug administration, paw edema of the mice was prompted by sub-plantar infusion of 0.1ml of 3% formalin arrangement in saline after 1h. Paw volumes were checked at 3h, 24h and 48h after formalin infusion as described before in carrageenan model^27-28.

2.7.4. Rat paw edema caused by histamine:

In this model, mice's paws became edematous after administration of an SC injection of 0.1 milliliters of freshly prepared histamine containing one percent histamine in normal saline. The paw edema was measured in the same manner as in the carrageenan-induced paw edema model. At 0, 1, 2, and 3hours, the paw volume was measured^29-30.

2.7.5. Plant Fiber actuated neoplasm in rats:

Plant fiber give rise in neoplasm development in rodents was carried out for chronicanti-provocative review. This design addresses the elimination and multiplication periods of irritation. Moving plant fiber pieces and disinfecting them in an autoclave prompted the creation of the 100mg-tall cotton pellets. The rodents were anesthetized with chloroform; the back and posterior sides were clean shaved and swabbed with 70% alcohol (v/v). A cut of 1cm was made in the intra-scapular district. Midline cut of 1cm was made in the intrascapular district Sterile cotton pellet (100mg) was introduced in each tunnel. Air was taken out from the passage and afterward entry point was shut with stitches. Starting from the day of implantation the test drug will be introduced for consecutive seven days. On the eighth day, the rats were killed. Plant fibers were taken out, cleaned of any extra tissue, dried in an 800C hot air oven for an overnight, and the rats were weighed. The granuloma tissue weight was determined by comparing the pellet's initial and final weights following drying. The amounts of granulation tissue produced per 100 grams of body weight were used to represent the outcomes^31-34.

2.8. Evaluation of analgesic activity:

2.8.1. Acetic acid induced:

The extract will be administered orally one hour prior, and the standard medication diclofenac (10 mg/kg) will be administered i.p. 12 hours prior to acetic acid administration. On intra-peritoneal infusion of acidic corrosive 1%, 10 ml/kg a trademark reaction called the squirming condition will be noticed. This disorder is described by a progression of stomach muscles withdrawal, expansion of rear appendages, wandering aimlessly developments of the body, opisthosomas. This conduct will be seen after 5min of organization of acidic corrosive and counted for all out 10min when the organization of the medication. The results will be expressed as % protection of writhes comparing with the control^35-37.

2.8.2. Paw licking response in rats induced due to formalin:

The impact of alcoholic concentrate of Platycladus orientalis on formalin induced paw licking reaction was assessed by same method as followed for formalin prompted rear paw edema. The animals were observed for half an hour following formalin injections. It was determined that the duration of time spent licking the injected paw was a sign of pain. The time taken for the beginning of paw licking was at first estimated. The neurogenic and inflammatory pain are represented by the second phase of the nociceptive responses, which typically peak between 15 and 30 minutes after formalin injection. Subsequently, the reappearance of paw licking was estimated at 0-5min., 6-10min., 11-15min., 16-20 min. and 21-30minutes^38-39.

2.9. Data Analysis:

The study's mean and standard error of the mean (SEM) are used to represent its entire findings. The SPSS rendition 26 programming was utilized to investigate the information. Trial configuration was totally randomized plan. ANOVA was used to examine group differences. Subgroup examination was finished by Tukey's numerous correlation tests. When P 0.05 and 0.01, differences were considered significant.

3. RESULT AND DISCUSSION:

3.1. Extraction yield:

The ethanol extract of leaves powder was observed as a dark brown sticky mass which was dried and the extraction yield was found to be 7.31% w/w which was calculated as a percentage of the weight/weight (%w/w) yield of the dried extract obtained.

3.2. Phytochemical Screening:

The phytochemical analysis of P. orientalis leaves extract revealed the presence of phytochemicals of different categories shown in Table 1 which are well-known to support therapeutic activities in medicinal plants.

Table 1: Phytochemical constituents of the P. orientalis extract based on screening

Sr. No.	Phytochemical constituents	Presence
1	Alkaloids	+
2	Tannins	++
3	Glycosides	++
4	Carbohydrates	++
5	Flavonoids	+++
6	Saponins	-
7	Triterpenoids	-
8	Steroid	+

+++ = show +ve within 5 min; ++ = show +ve after 5 min. but within 10 min; + = show +ve after 10 min. but within 15 min.; - = show negative even after 15 min.

3.3. Anti-inflammatory activity of extract:

3.3.1. Anti-inflammatory effects in carrageenan induced paw edema: The anti-inflammatory activity of P.orientalis in alcoholic extract in carrageenan-induced paw edema reported in Table 2. POE-100 groups showed its anti-inflammatory activity after 1 h with 5.34%, 2h 8.1% and 3h 36.70%. POE-200 dose group represent major decrease in paw edema volume at 1 h 36.44%, 2h 46.53% and 3 h 48.44%, while POE-200 group showed higher anti-inflammatory activity to standard acetyl salicylic acid at all time intervals. Thus, it can be concluded that P.orientalis extract has potential for anti-inflammatory activity in early as well as in later phase.

3.3.2. Anti-inflammatory effects in dextran induced paw edema: The consequences of calming movement of alcoholic concentrate of P.orientalis in dextran actuated paw edema are given in Table 3. When compared to the control group, the POE-100 and POE-200 groups significantly inhibited inflammation in the dextran-induced paw edema model. At 1h (30.80%), 2h (50.68%), and 3h (46.86%), the dose group 200 showed significant smaller paw volumes. In a dose-dependent manner, alcoholic extract of P.orientalis demonstrated anti-inflammatory activity.

Table 2: Anti-inflammatory potential of P.orientals in carrageenan induced paw edema.

Treatments	Dose (mg/Kg)	After 1h		After 2h		After 3h
Treatments	Dose (mg/Kg)	Edema volume (ml)	% Inhibition	Edema volume (ml)	% Inhibition	Edema volume (ml)	% Inhibition
Control	Saline	0.71±0.03	-	0.87±0.02	-	0.96±0.01	-
ASA	10	0.50±0.03**	29.33	0.42±0.03 **	49.33	0.46±0.03**	52.23
POE	100	0.68±0.04 ns	5.34	0.78±0.02ns	8.1	0.60±0.03**	36.70
POE	200	0.45±0.03**	36.44	0.45±0.01**	46.53	0.50±0.04**	48.44