INTRODUCTION:

Rheumatoid arthritis is a chronic autoimmune multisystem disease that develops when the immune system unintentionally attacks body tissues and joints. An inflammatory synovitis is the end outcome, which typically progresses to joint ankylosis and articular cartilage loss.^1-4A prolonged inflammatory reaction in the joints, a feature of rheumatoid arthritis, may eventually cause joint degeneration and deformity.

Joints are more likely to feel pain, swelling, and stiffness after extended periods of inactivity. The joints of the fingers and hands, as well as the wrists, feet, and knees, are frequently affected, and the symptoms frequently follow a symmetrical pattern (both body sides being affected). The effects of this may be felt throughout the body, and some examples include a reduction in red blood cell count, inflammatory conditions of the heart and lungs, and inflammation of the lungs.^5-8There is a 1 in 150 chance that a woman in her 35s to 60s will acquire this illness. In addition to the symptoms (such as pain, edoema, and joint stiffness), multiple organs are also impacted. Joint degeneration quickly leads to irreversible dysfunction and deformity in the affected joints, and it is often irreversible. As a result, early in the course of the illness, proper diagnosis and treatment are essential.^9-13An immune-mediated inflammation is what distinguishes RA from other chronic inflammatory diseases. This disorder affects only 1% to 2% of the world's population. Each of these symptoms is detrimental to health and may contribute to the onset of depression in a person.^14-16Additionally, synovial cells produce lubricants like fibronectin, collagen, and hyaluronic acid, which form the synovial interstitium's structural foundation, to make joint mobility easier.^17-20Inflammation is caused by the synthesis of specific soluble pro-inflammatory mediators such prostaglandins and it may be possible to manage inflammation by regulating these mediators' production.^21-24

The traditional Korean medicinal Thuja orientalis (L) (Cupressaceae) has been used to treat haemorrhages, dermatitis, gout, and chronic tracheitis.²⁵ This plant's diterpenoids, flavonoids, and essential oils shown anti-inflammatory²⁶, cytotoxicity against cancer cells²⁷, as well as neuroprotective²⁸, anti-diabetic²⁹, and fungitoxic properties.³⁰The TNF-a-induced vascular inflammation was reduced by the Thuja orientalis extract.³¹Among many other illnesses, it is used as a anticancer, analgesic³², and anti-allergic³³, antimicrobial³⁴, antiviral.³⁵The leaves of Thuja orientalis have long been used in traditional medicine to treat rheumatoid arthritis.³⁶

MATERIALS AND METHODS:

Materials

According to previously published reports, plant materials, extract preparation, acute toxicity assessment, and phytochemical contents were used.

Plant Material:

The leaves of Thuja orientalis were gathered in November. The leaves were physically cleaned with a cotton cloth. In the shade, the leaves were allowed to dry.

Preparation of extract:

To improve extraction by reducing particle size and increasing surface area, the material from dry leaves was treated in a mechanical grinder.The powdered, pulverised material was soaked in 70% Methanol for three days before being passed through a muslin cloth. Filter paper was used to separate the filtrate that was produced after three sequential macerations. In a rotary evaporator, the filtrate was dried to a semisolid bulk at low pressure. The yield was determined using after the semisolid bulk had been weighed. For later usage, the extract was kept in storage at a temperature below 8°C.

Animal:

Albino Wistar rats (220–250g, male) were employed for the experiment after clearance from the institutional animal ethical committee with protocol reference number RAP/7002A/2022. The animals were housed in identical lab environments with relative humidity levels between 55 and 65percent and temperatures between 25 and 30°C. There were no restrictions on the animals' access to food or water.

Chemicals and Drug:

The chemicals used were methanol, diclofenac sodium, chloroform, Freund's Complete Adjuvant Injection (Sigma Chemicals, USA), Rankem, New Delhi, and carboxymethyl cellulose (Loba Chemie, Mumbai) (Akums Drugs and Pharmaceuticals, India). Only drugs of the pharmaceutical grade were applied in this experiment.

Induction of arthritis:

This method and a mercury plethysmometer were used to measure the initial volumes of the rear foot of the experimental animals (shown on the left). After anaesthetised to the rat, 0.05mL of FCA (heat-killed Mycobacterium tuberculosis in sterile paraffin oil) was injected into the subplantar region of the left hind paw. The adjuvant arthritis had done its job on days 7 and 8, when inflammatory edoema in the hind paw was at its greatest.

Experimental design:

For scientists looking to learn more about how the immune system affects arthritis, FCA is a good model. The animals were split up into 5 groups with an average of 6 animals per group (n=6) one week before the experiment's start.

Group 1 - Normal Control (1% CMC)

Group 2 - Negative Control (0.05ml of FCA)

Group 3 - Standard Drug (Diclofenac sodium 10mg per kg orally)

Group 4 - Methanolic extract of Thuja orientalis leaves (200mg/kg body weight orally)

Group 5- Methanolic extract of Thuja orientalis leaves (400mg/kg body weight orally)

By injecting 0.05ml of FCA toxicant into the left hind paw of animals, the immunogenic arthritis was triggered (sub planter region).

A mercury plethysmometer was used to accurately record the swelling in the paw volume on a regular basis, i.e., on the 1,7,14 and 21 days. A digital balance was used to record the changes in body weight, which were, beginning on the day of injecting the FCA toxicant about 30 minutes before the FCA injection.

Parameter assesment:

Paw volume: The initial paw volume was measured using a plethysmometer on day 0 (before FCA injection). On the days 1, 7, 14, and 21, the dimensions of each paw were also measured. The difference between the end and starting paw volumes was calculated using the change in paw volume.

Body weight: Each animal was given the chance to report its body weight before the experiment and at various points after receiving the proper treatment. Each animal was weighed using a digital balance that was zeroed out and calibrated for animals. Each animal's weight was then noted.

Locomotor activity: We monitored the locomotor activity of each individual rat. Body mobility was determined by timing how long each animal took to travel a certain distance. It was noted on day 21 of the trial.

Arthritic score: Grades ranging from 0 to 4 were then issued in accordance with the physical signs of arthritis in an animal's paw that were seen after therapy.

Grade 0: Absence of edema

Grade 1: A slight degree of edema

Grade 2: The swelling of several paw digits

Grade 3: Ankle and wrist swelling

Grade 4: Severe edema in the wrist and fingers

Blood and serum analysis: Blood was collected from each animal on day 21 and placed into a test tube containing an anticoagulant through the retroorbital plexus (EDTA). Two portions of the obtained blood were separated. The quantity of Red Blood Cells, White Blood Cells, Hemoglobin (Hb), and Erythrocyte Sedimentation Rate, among other haematological parameters, were measured using conventional methods in the first phase (ESR).

X-ray radiography: The experimental rats' individual arthritis-induced legs were x-rayed before to sacrifice in order to assess soft tissue swelling, the kind of bone degeneration, and the presence of any constricted spaces in joints.

Statical analysis:

ANOVA was used to statistical analyses all of the data, and Dunnett's multiple comparison tests were used to identify differences between groups using GraphPad Prism 5.0. P values of 0.05 or higher were regarded as significant values. For six rats in each group, the findings were presented as mean SEM.

RESULT:

Assessment of parameters:

Paw volume:

The results given in table 1 the paw volume was significantly reduced by the methanolic extract of Thuja orientalis (400mg per kg) 0.36±0.02 to 1.35±0.02 at 21^st day, when compared to the negative control. Thuja orientalis (200mg per kg) also lessens the size 0.38±0.03 to 1.40±0.04 of the paws at 21^st day, when compare to the negative control, though not as significantly as the 400 mg dose.Standard group (diclofenac sodium 10mg/kg) was significantly reduced the paws size 0.34±0.02 to 1.03±0.07 at 21^st day, when compare to negative control.

Table 1: Thuja orientalis leaf extracts effect on paw volume in Wistar rat arthritis produced by Freund's complete adjuvant. Every value is shown as the mean SEM.

Groups

Days (ml)

Day 1^st

Day 7^th

Day 14^th

Day 21^st

Group-1Normal control (vehicle)

0.27±

0.03

0.27±

0.03

0.27±

0.03

0.27±

0.03

Group-2 Negative control

(FCA 0.5 ml/kg)

0.40±

0.03^a

0.82±

0.03^a

1.10±

0.02^a

1.51±

0.07^a

Group-3 Standard (Diclofenac sodium 10 mg/kg)

0.34±

0.02^ab

0.74±

0.02^ab

0.81±

0.03^ab

1.03±

0.07^ab

Group-4MTO

(200 mg/kg)

0.38±

0.03^abc

0.78±

0.07^abc

0.98±

0.03^abc

1.40±

0.04^abc

Group-5MTO

(400 mg/kg)

0.36±

0.02^abd

0.75±

0.07^abd

0.91±

0.07^abd

1.35±

0.02^abd

Changes in Body weight:

Thuja orientalis (400mg/kg) when compared to the negative control, the body weight was showed a notable increases in body weight in initial 7^th day161±2.94 thereafter the weight substantially increases 170±4.88.Thuja orientalis (200mg per kg) also increasementin body weight, the body weight was showed a notable increasement in body weight 166±1.16to 175±4.16. The increasement of body weight in standard group 194±4.41 as compare to negative control. (table 2)

Table 2: Effect of a Thuja orientalis leaf extract on body weight in Wistar rats with arthritis caused by Freund's complete adjuvant The mean SEM is used to illustrate each value. A one-way ANOVA statistical analysis is followed by Dunnett's multiple comparisons.

Groups

Body weight (gm)

Day 1^st

Day 7^th

Day 14^th

Day 21^th

Group-1Normal control (vehicle)

160±

0.56

165±

2.98

168±

3.88

177±

5.02

Group-2 Negative control (FCA 0.5 ml/kg)

170±

0.43^a

165±

3.43^a

161±

3.98^a

148±

5.66^a

Group-3 Standard (Diclofenac 10mg per kg)

176±

1.12^ab

181±

3.19^ab

185±

3.97^ab

194±

4.41^ab

Group-4 MTO (200 mg per kg)

166±

1.16^abc

169±

2.89^abc

173±

3.49^abc

175±

4.16^abc

Group-5 MTO (400 mg per kg)

158±

2.12^abd

161±

2.94^abd

163±

3.45^abd

170±

4.88^abd

Locomotor activity: Slab (animal to cover a distance of two meters)

Rats given Thuja orientalis methanolic extract (400mg per kg, p.o.) had considerably better locomotor activity on day 21(123±0.73 to 114.6±0.38) when compare to rats given the negative control. Standard group had considerable better locomotor activity 121.0±0.10 to 113.6±0.23 as compare to negative control. (table 3).

Table 3: Thuja orientalis leaf extracts effect on paw volume in Wistar rat arthritis produced by Freund's complete adjuvant. Every value is shown as the mean SEM. One-way ANOVA statistical analysis is followed by Dunnett's multiple comparisons.

Groups	Locomotor activity (sec)
Groups	Day 0^th	Day 21^th
Group-1 Normal control (vehicle)	130.5± 0.01	121.67± 0.41
Group-2 Negative control (FCA 0.5ml/kg)	121.5±0.01^a	100.0± 0.55^a
Group-3 Standard (Diclofenac 10mg per kg)	127 ± 0.19^ab	113.6±0.23^ab
Group-4 MTO (200mg per kg)	121.0 ± 0.10^abc	110 ± 0.15^abc
Group-5 MTO (400mg per kg)	123 ±0.73^abd	114.6 ± 0.38^abd

Arthritic score:

When FCA was injected in the subplantar area, the arthritic score was considerably elevated (p<0.05). The arthritic score was lower from days 7 to 14 of the experiment, but this was not very noteworthy. The rat was treated with both the precise Thuja orientalis in comparison to the normal control rats. When compared to the normal control group, the arthritis score in the negative control group was higher until the 21st day of the experiment. However, treatment with a methanolic extract of Thuja orientalis significantly decreased the scale of arthritic score from the 14th to the 21st day (p<0.05) at the end. (table 4)

Table 4: Thuja orientalis leaf extract's anti-arthritic properties in Wistar rats given Freund's complete adjuvant treatment. The data is presented as the average standard deviation of the mean.

Groups

Mean of arthritic score in Freund induced arthritis in rats

Day 1^st

Day 7^th

Day 14^th

Day 21^th

Group-1Normal control (vehicle)

Group-2 Negative control (FCA 0.5 ml/kg)

1.66±

0.43

2.83±

0.16^a

3±

0.12^a

3.50±

0.66^a

Group-3Standard (Diclofenac 10mg per kg)

1.76±

0.21^ab

2.16±

0.166^ab

1.66±

0.33^ab

0.66±

0.41^ab

Group-4MTO

(200 mg per kg)

1.66±

0.42^abc

2.50±

0.8^abc

2.16±

0.49^abc

2.33±

0.46^abc

Group-5MTO

(400 mg per kg)

1.58±

0.12^abd

2.33±

0.94^abd

2.63±

0.45^abd

2.18±

0.88^abd

Haematological parameters:

When compared to the negative control group, Thuja orientalis (400mg per kg) methanolic extracts significantly (p<0.05) improved blood parameters. The RBC numberswere increased from 2.62±0.32 to 4.12±0.33 per microliter, but the WBC number and ESR have decreased from 8754±8.26 to 7068±6.45 and 4.04±0.07 to 3.62±0.09 respectively. Hemoglobin levels have risen from 7.40.86 to 12.650.91gm/dl. ( table 5).

X-ray radiography:

The radiography of FCA-induced arthritis revealed that the joints' gaps had enlarged, the arthritic rats had developed soft tissue edoema, and there was some bone damage. In the group that got 10mg of the reference drug per kg of body weight, this destruction was lessened. The 400mg per kg P.O. methanolic Thuja orientalis leaf extracts decreased soft tissue edoema and also reduced joint gaps when compared to the negative control group. (Shown in fig. 1)

DISCUSSION:

Rats' paw volume, as well as a number of arthritic parameters, haematological and biochemical values, and other factors, were shown to be diminished by the plant's extract in the current study, which sought to evaluate the potential of the methanolic extract of Thuja orientalis leaves.Any operator's influence on RBC adjustment is likely to have an impact on lysosomal layer adjustment because the RBC film follows the lysosomal layer. Additionally, RBC interaction with a Hypotensive medium results in film lysis, and RBC quality is dependent on the layer's integrity.

The extract of Thuja orientalis leaves, administered in a range of dosages, significantly altered the RBC layer in the current investigation. The layer counteracting the characteristic of Thuja orientalis can be seen as the interference activity of the plant with the entry of neutrophil lysosomes.According to the study's final conclusions, the fact that Thuja orientalis leaves extract at an oral dose of 400 mg per kg suppressed (controlled/inhibited) the proliferative edematous reaction (paw swelling) in FCA models when compared to the reference dose of 10 mg/kg/p.o. is due to the presence of antioxidants in plants.

In the current investigation, the first lesionsarthritic lesionsoften manifested 3 to 5 days after FCA injection, but the second lesionsinflammation of the non-injected hind pawpresented 7–21 days later. In contrast to the negative control, the Thuja orientalis leaf extracts at doses of 400 mg per kg. showed an improvement in locomotor activity. This could be as a result of plant extracts possibly reducing rat genic or musculoskeletal discomfort.

Muscular strength and mass are reduced as a result of the weight loss brought on by rheumatoid arthritis (rheumatoid cachexia), which may be related to tissue deterioration due to the decreased ability of the rat gut to absorb 14C-glucose and 14C-leucine and the lysosomal proteases to further enhance the proteolysis of muscle proteins. Additionally, rats in the current study who received 400 mg/kg/p.o. of the Thuja orientalis leaf extract showed a noticeable recovery of their body weight. It is a result of reduced PGE2 levels and an increase in the intestine's earlier diminished capacity for nutrition absorption. The release of IL-1 and TNF in the ligament condition also results in an increase in WBCs and platelet count. ESR respect is also raised by proteins that change in response to annoyance. In addition to this, rheumatoid factor, an auto-immune response directed against the Fc component of IgG, is a better serologic marker for joint discomfort. Research has shown that extracts from Thuja orientalis groups at 400 mg/kg/p.o. individually altered modifications in haematological parameters, while extracts at levels above reduced negative effects on RBC, WBC, and Platelets.

In addition, treatment with Thuja orientalis leaf extract at doses of 400 mg per kg considerably filled up the widening joint spaces, or you might say they were successful in narrowing the joints and noticeably constricting the joint design in models.The results of the 400 mg per kg b.w. methanolic extract of Thuja orientalis leaves indicated and noted (p<.05) raised in haematological parameters such RBC and Hb, in contrast to the negative control group, and more noted (p<0.1) in decrement in the WBC and ESR.The main contributors to its hematopoietic effect may be alkaloids, glycosides, flavonoids, and phenolic compounds, which have been documented to have bioactivity.Additional research is necessary because active biochemical substances may be the cause of elevated biochemical and haematological markers.

CONCLUSION:

Thuja orientalis leaf methanolic extract contains glycosides, alkaloids, tannins, and flavonoids. According to the results, it was discovered thattreatment with Thuja orientalis leaf extract at doses of 400 mg per kg considerably filled up the widening joint spaces, and we can say that they were successful in narrowing the joints and noticeably constricting the joint design in models which concludes that this pharmacological research supports the folk remedy and the traditional ancient use of Thuja orientalis leaves in the supportive treatment and management of rheumatoid arthritis.

ACKNOWLEDGEMENTS:

The lab equipment and resources provided by Rajiv Academy for Pharmacy allowed the writers to conduct and effectively conclude their research. The authors are extremely grateful.

INTERESTS IN CONFLICT:

No conflict of interest exists, according to the authors.

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Received on 02.11.2022 Modified on 21.04.2023

Research J. Pharm. and Tech 2024; 17(3):1055-1060.

DOI: 10.52711/0974-360X.2024.00164

Hematological parameter	RBC (× 10⁶ /μl)	WBC (×10³ /μl)	ESR (mm/1st h)	Hb (g/dl)
Group-1 Normal control (vehicle)	5.43±0.33	7014±4.2	3.32±0.07	13.65±0.31
Group-2 Negative control (FCA 0.5 ml/kg)	2.62±0.32^a	8754±8.26^a	4.04±0.07^a	7.4±0.86^aa
Group-3 Standard (Diclofenac 10mg per kg)	4.83±0.33^ab	7036±6.6^ab	3.58±0.04^ab	12.87±0.37^ab
Group-4MTO (200 mg per kg)	3.82±0.29^abc	7018±7.16^abc	3.72±0.21^abc	9.96±0.86^abc
Group-5MTO(400 mg per kg)	4.12±0.33^abd	7068±6.45^abd	3.62±0.09^abd	12.65±0.91^abd