Pharmacological Evaluation of Different Extracts of Asparagus officinalis (Asparagaceae) as an Analgesic, Anti-Inflammatory and Anti-arthritic Agent in Rats

Background: To treat the joint pain arial part of Asparagus officinalis (asparagus) has historically been used. However, its efficacy for rheumatoid arthritis has not been pharmaceutically evaluated. We explore the phytochemical analysis anti-inflammatory, analgesic and anti-arthritic activity of petroleum ether, ethanol and aqueous extracts of Asparagus officinalis aerial part. Materials and Methods: Tail-flick method was used to evaluate the analgesic activity anti-inflammatory activity was carried out using paw oedema induced with carrageenan and CFA induced arthritic model was used to evaluate the potential in anti-arthritic activity in rats. The Petroleum ether, ethanolic and aqueous extracts were dosed orally in three divided doses (75, 150 and 300 mg/ kg). For anti-inflammatory and analgesic activity diclofenac sodium at 10 mg/kg was used as standard, whereas in anti-arthritic model prednisolone at 5 mg/kg and methotrexate at 0.5 mg/ kg were used as standard. One-way ANOVA followed by Dunnett’s multiple range test were used to analyse statistical significance between means. Results: The results revealed a dose-controlled anti-inflammatory, anti-arthritic effect with different extracts whereas at some extent analgesic activity was observed. Four compounds were present and confirmed by LCMS/MS. The CFA model's findings showed improved defence against arthritic lesions and changes in body weight. Additionally, Asparagus officinalis significantly improved rheumatoid factor, changed WBCs, and favourably altered radiographic and histological alterations. Conclusion: The findings indicate that Asparagus officinalis is a strong anti-arthritic and anti-inflammatory compound that may be suggested for the treatment of both chronic and acute inflammation.


INTRODUCTION
Pain and Inflammation are both prominent symptoms of Rheumatoid arthritis (RA) which is a significant worry marked by non-specific joint inflammation, articular tissue loss, and joint abnormalities.As the condition advances, the likelihood of bone degradation and cartilage breakdown increases, resulting in significant impairment. [1]An immune system response known as inflammation may be brought on by a variety of causes, such as infections, damaged cells, and toxins. [2]The creation of prostaglandins is triggered by infections (germs) such as viruses, fungi, bacteria, and illnesses, medical disorders, and external traumas such scratches or injury from foreign objects, chemicals, or radiation, [3] interleukins (IL-1 and IL-6) and tumour necrosis factor (TNF-α) are examples of inflammatory cytokines that have been demonstrated to have a significant role in inflammation and joint destruction that occurs throughout the progression of arthritis. [4,5]Articular cartilage and the subchondral bones are degraded as a result inflammation induce in synovial membranes.Despite substantial research, the specific cause of RA is unknown.Recent research suggests that increased oxidative stress due to free radical production may be a key factor in the progression of RA. [6] Cyclooxygenase isoenzyme (COX) plays a major role in development of arthritis apart from cytokines (IL-1, IL-6, TNF-α, etc.) [7,8] Excessive cytokines (pro-inflammatory) can form a positive bridge between RA fibroblasts and macrophage-like synovial cells.Inflammatory cytokine inhibition is the most common molecular target in the treatment of RA. [9] Non-steroidal anti-inflammatory drugs (NSAIDs) and glucocorticoids have been widely employed in the treatment of RA. [10] These analgesic medicines, are reported to relieve only 50% of the pain particularly NSAIDs, that too roughly 30% of people in most cases. [11]However, due to serious adverse effects shows in heart, gastrointestinal tract and kidney function, this drug is now lost their major role as first-line treatment in response to current findings. [12]In recent decades, new categorized drugs are available for this treatment such as glucocorticoids and immunosuppressant are effective anti-inflammatory medications, but they have their serious adverse effects that make long-term use difficult.As a result, we are now depending upon herbal treatment which may show lesser adverse effect and may better safety profile as compared to existing one. [13]Scientists are always in search of better alternate treatment.In that case Ayurveda is always preferred choice.
Through literature survey and endangered species existing in India especially in Southern Region we observed many plants which are still unexplored for various pharmacological activities. [14]They are traditionally use in sub local rural areas but scientific validation is not yet reported.The primary motivation for researching traditional plants is because many of the existing allopathic medications are made from plant material, and many of the structures employed in allopathy are comparable to plant constituents.
According to a recent WHO (World Health Organization) estimate, 80% of people globally rely on herbal remedies for some of their basic medical requirements.Approx 485 plant species belonging to 100 families are used for arthritis as traditional drug. [15,16]The Asparagaceae family includes Asparagus as a part.Mentioned in one of the earliest treatises of human knowledge, the Rig-Veda, was written between 4500 and 1600 BC. [17] Added to this, very less work is available on Asparagus officinalis whereas other members of same class are reported for many pharmacological activities such as Asparagus racemosus, Asparagus sprengeri and Asparagus acutifolius are all members of the same genus. [18]Asparagus officinalis contains steroids, amino acids, saponins, fructans (Asparagosine and Asparagose), flavonoids and ferulic acid.Ethanolic and aqueous extracts of this plant have been use in Southern Interior region for treatment of many diseases but traditionally it is used to treat kidney and bladder stones, asthma, and cough. [19]Asparagus officinalis was found to have inhibitory effects on both cyclooxygenases-1 and -2, indicating that it possesses anti-inflammatory properties.Linoleic acid was one of the compound present in this plant and found active in various pharmacological activities [19] includes liver toxicity treatment, [20] anti-microbial, [21] anti-arthritic, [15] anti-oxytoxic, antiulcer, hypertensive and anticoagulant effects, [22] Ethanolic extract of Asparagus was reported anti-diabetic at dose of 50 mg/kg and 100 mg/kg, [23,24] Another study was also shown that 400 mg/kg of ethanolic extracts act as potential protective agent against oxidative stress in liver and kidney organs. [25]Based on our survey, we designed this study to access the pharmacological evaluation of different extracts of Asparagus officinalis (Asparagaceae) as an analgesic, anti-inflammatory and anti-arthritic activity in experimental rats.

Plant material
Arial part of Asparagus officinalis was procured form botanical Garden of Southern region.The specimen plant was identified and authenticated by NISCAIR, New Delhi, India with reference number NISCAIR/RHMD/consult/-2021/3827-28.Fresh plant material was cleansed with tap water to remove dirt and dried for 10 days in the sun and shade before being crushed into a powder in an electric grinder.

Preparation of extracts
Powder of dried aerial plant part (2.5 kg) was added in the soxhlet's apparatus using Petroleum ether, Ethanol and water for successive extraction.The final traces of all three solvent extracts were removed using the dried vacuum method.All extracts were kept at temperature between 2-4°C for further use.

Chemical and drugs
We bought carrageenan and Complete Freund's adjuvant from Sigma Chemicals (St Louis, USA).Ind. Swift Laboratories (Baddi, India) provided pure samples of prednisolone and diclofenac sodium, while MacLeod's Laboratory provided a gift sample of methotrexate (Mumbai, India).The study's other reagents and compounds were all of analytical grade.

Animals
Sprague dawley rats (130-150 gm) were utilised with the institutional animal ethics committee's previous consent (1125/ PO/Rc/S/07/CPCSEA) with protocol no B-098.The animals were kept in normal housing with a 12:12 light-dark cycle and temperatures between 24 and 28°C.Standard feed pellet diet (Safe Diet procured from Samitek Instruments) were provided to animals and water was allowed ad libitum.

Drugs and dosage
75 mg/kg, 150 mg/kg, and 300 mg/kg of the extracts were administered orally as a suspension in 5 mL/kg of 1% carboxy methyl cellulose and 1.0% v/v tween 80.As an oral suspension, methotrexate (0.5 mg/kg p.o.) and prednisolone (5 mg/kg p.o.), were utilised as standards for anti-arthritic activity.Whereas diclofenac sodium (10 mg/kg p.o.) was used as standard in analgesic and anti-inflammatory activities.

Tail flick method (Analgesic activity)
Analgesiometer was used to measure the analgesic effect in rats.A nichrome wire within the instrument is heated to the necessary temperature and kept there by heat regulators.The current through the bare nichrome wire was maintained at a consistent strength (4 Amps).The rat was housed in a rat cage with only its tail sticking out.The centre of the tail was positioned on the platform such that it was barely above the hot wire but not touching it.The animal's sudden, recognisable flick or tail raising in response was used to measure the animal's latency (reaction time).To prevent any tissue injury to the animal, a cut-off duration of 10 sec was planned.On the seventh day following a one-week oral medication treatment, the reaction times for each group were tested at 30, 60, 90, and 120 min. [26]

Carrageenan induced paw oedema in rats (Acute-inflammation)
After one week of oral medication treatment, all groups except the control group received 0.1 ml of 1% w/v carrageenan injections into the rat paw.After a gap of one hour, two hours, three hours, four hours, and six hours, the paw oedema volume was measured using a digital plethysmometer (Model 7140, UGO Basile, Italy).Anti-inflammatory activity was determined by calculating the proportion of oedema that was inhibited compared to the control.The formula used to get the percentage inhibition of oedema was: oedema (% inhibition) = (A-B)/A X 100.where A denotes the volume of the paws in the control group and B denotes the volume of the paws in the group that received the test medication. [27]

Complete Freunds Adjuvant induced arthritis (Chronic inflammation)
By injecting 0.1ml (0.1% w/v) of deceased Mycobacterium TB bacteria homogenised in liquid paraffin into the left hind paw, arthritis was developed.On the fifth day, a second CFA booster was administered, while the control group received an equal volume of normal saline with minor modifications as per Dar et al. (2019).The drug treatment programme began on day one and lasted for 21 days.The paw volume was assessed using a digital plethysmometer on days 1, 7, 14 and 21.(Model 7140, UGO Basile, Italy).The proportion of oedema that was inhibited in comparison to the control was interpreted as an anti-inflammatory response.Methotrexate (0.5 mg/kg twice weekly) and prednisolone (5 mg/kg once daily) were employed as benchmark medications for comparison.The verneir calliper device was used to measure the ankle joint diameter on the 1 st , 7 th , 10 th , 15 th , and 20 th days. [28]

Sample Collection
For the analysis of rheumatoid factor, blood samples were taken on the 21 st day from the retro orbital plexus, and serum was obtained by centrifuging at 3500 rpm for 30 min at 4°C and stored at -80°C.Then, animals from each group were euthanized for an ankle joint histology study and an x-ray of the femur bone.

Preparation of quality control and calibration samples
Main stock solutions of Stigmasterol, β-sitosterol, Rutin, Telmisartan (Internal Standard for Rutin and Quercetin) and Metoprolol (Internal Standard for Stigmasterol, β-sitosterol) were made at 1 mg/mL concentration in methanol.

Sample preparation
Extracts were dissolved in methanol by looking at the solubility of Stigmasterol, β-sitosterol, and Rutin.And for Quercetin extracts were dissolved in DMSO.Prepared samples were diluted in acetonitrile containing internal standard + methanol + water.For LC-MS/MS (Liquid chromatography-tandem mass spectrometry) analysis, 10 μL were injected.

LC-MS/MS analysis
For Stigmasterol and β-sitosterol Q TRAP 6500+ was run in positive ion mode for the analysis.The LC system utilised was the Sciex Exion LC, which included a vacuum degasser, two isocratic pumps, an AD Multiplate Autosampler with a temperature control set to 4°C, and a thermostatic column oven with a (40°C) temperature control (Sciex Exion, AD column oven).Kinetex EVO, C 18 , 5 μm particle size 50*4.6 mm stationary phase was utilised for the chromatography (Phenomenex, US).For the mobile phase, 0.1% formic acid in water served as the aqueous reservoir, while 0.1% formic acid in methanol served as the organic modifier.A common reverse phase gradient programme (time (min) / % B= 0.00/60, 3.0/99, 6.50/99, 7.00/99, 10.00/60 and 11.00/60) was used with run time of 11 min.Using  Analyst software version 1.6.3,data collecting and processing for quantification were carried out (AB SCIEX).By employing a Harvard infusion pump (Harvard Apparatus, Holliston, USA) linked directly to the mass spectrometer and infusing a 500 ng/ mL solution in water: methanol (50:50 v/v) at 10 μL/minute flow rate, the mass spectrometric settings were tuned for the substances.By using a 0.9 mL/minute flow rate of mobile phase without a column, flow injection analysis (FIA) was used to optimised the flow-dependent source parameters.The following optimised settings were used to run the Turbo V source with the ESI (Electro spray ionisation) probe: Positive polarity, 30 pressure for the curtain gas, 70 psi for the nebulizer gas, 55 psi for the heater gas, 5500 volts for the ion spray, and 550°C for the source temperature.The mass spectrometry was run in the MRM mode, which fixes both the parent ion and the fragment ion.The optimal declustering potential (DP) and collision energy (CE) for the Stigmasterol parent and fragment ions were 100 V and 5 V, respectively.For metoprolol, the parent and fragment ions' m/z values were 268.10 and 116.3, with DP and CE, respectively, of 110 V and 25 V (Figure 3).
For Rutin and Quercetin TRIPLE QUQD 5500+ was used.MRM for LC-MS/MS operated in positive ion mode for Quercetin and negative ion mode for rutin analysis.The LC system utilised was the Sciex Exion LC, which included a vacuum degasser, two isocratic pumps, an AD Autosampler multiplate with a temperature control at 4°C, and a temperature control thermostatic column oven with at 40°C (AD column oven.Exion, Sciex).The 50×4.6 mm Kinetex EVO, C 18 (Phenomenex, US) was used as stationary phase used for the chromatography with 5 μm particle size.The mobile phase consisted of 10mM Ammonium Acetae with 0.1% Formic acid in Milli Q as aqueous reservoir and (100%) methanol (organic modifier) at flow rate of 1 mL/minute was used.A programme with common reverse phase gradient (time (min) / % B= 0.00/5, 1/95, 2.50/95, 2.6/5 and 11.00/5) was used for Quercetin and % B= 0.00/60, 1/99, 2.50/99, 2.6/99 and 3/60) for Rutin with short run time of 3 min.Using Analyst software version 1.6.3,data collecting and processing for quantification were carried out (AB SCIEX).By employing a Harvard infusion pump (Harvard Apparatus, Holliston, USA) linked directly to the mass spectrometer and infusing a 500 ng/ mL solution in water: methanol (50:50, v/v) at 10 μL/minute flow rate, the mass spectrometric settings were tuned for the substances.By using flow injection analysis (FIA) and a 1 mL/ min flow rate of mobile phase without a column, flow dependant source characteristics were optimised.The Turbo V source with the ESI (Electro spray ionisation) probe was run for quercetin with   The Turbo V source with the ESI (Electro spray ionisation) probe was used for Rutin with the following optimised settings: Positive polarity, 40 psi curtain gas, 550°C source temperature, 55 psi heater gas and 50 psi nebulizer gas.Ion spray voltage is -4500V.
The mass spectrometry was run in the MRM mode, which fixes both the parent ion and the fragment ion.The Rutin parent and fragment ions employed had m/z values of 609.00 and 299,90, respectively, and collision energy (CE) and the ideal declustering

Statistically Analysis
Data were presented as mean standard deviation.Using a one-way ANOVA and Dunnett's multiple range test, the statistical analysis of the ankle joint diameter, paw oedema, % inhibition, and soft tissue thickness was conducted.a corresponding chance of error, a P<0.0001, b P<0.001, c P<0.05 was considered statistically significant.

Qualitative Phytochemical Screening
The presence of flavonoids, terpenoids, alkaloids, tannins, sterols, proteins and carbohydrates content in extracts was confirmed by different test analysis.

Analgesic Activity
In this study, highly significant (p<0.0001)effect was found at 120 min with dose of 300 mg/kg of petroleum ether extract (83.49%), followed by aqueous extract (80.5%) and ethanolic extract (78.22%).Moderate analgesic effect noted with ethanolic extract, aqueous extract and petroleum ether extracts at 75 mg/kg and 150 mg/kg.Response time was increased 124% to 204% with diclofenac sodium (Table 1).

Anti-inflammatory activity
The results (Table 2) were found extremely statistical (p<0.0001)significant difference with all extracts at a higher dose from 2hr  to 6 hr and less significant effect at lower doses also.Diclofenac sodium treated group showed 17.33% to 21.53% inhibition and petroleum ether extract was observed with the highest significant range of 13.14% to 22.95% inhibition followed by aqueous extract 9.90% to 20.74% inhibition and at last ethanolic extract was observed with a significant range of 11.17% to 20.46% inhibition.

Effect on body weight
Body weight of positive control group was decreased at different time intervals in comparison with normal control group.Higher dose of all three different extracts showed slightly changes in the body weights as compared to positive control group at 15 th day    and 21 st day but there was no statistical significant difference.Standard treated group (Methotrexate and Prednisolone) were able to keep the animal body weight in normal range.It was increased from 3.71% to 7.39% with petroleum ether extract and in aqueous extract treated group, it was noted from 0.79% to 14.76% (Table 3).

Effect on paw oedema
From 14 th day and 21 st day, the percentage of inflammation inhibition was found increase and statistically significant difference at all three doses of all three different extracts as compared with a positive control group.It was observed that    aqueous, ethanolic and petroleum ether extracts have statistically high significant anti-arthritic effects from day 14 th to 21 st day at all doses.Methotrexate showed 28.33% inhibition of oedema, Prednisolone showed 32.18% inhibition of oedema.Aqueous extract was observed with highest significant range of 16.71% to 29.66% inhibition followed by ethanolic extract 16.19% to 26.34% followed by petroleum ether extract 11.41 to 23.95% inhibition (Table 4).

Effect on Paw diameter (Using Vernier caliper)
It was observed that aqueous, ethanolic and petroleum ether extracts have statistically high significant anti-arthritic effects from day 14 th to 21 st day at all doses.Methotrexate showed 30.78% inhibition of oedema, Prednisolone showed 32.97% inhibition of oedema.The aqueous extract was observed with highest significant (p<0.0001)range of 24.45 to 34.21% inhibition followed by ethanolic extract 22.19 to 29.91% followed by petroleum ether extract 18.69 to 26.87% inhibition (Table 5).

Effect on arthritis symptoms (Using Visual Scoring)
It was observed that aqueous, alcoholic and petroleum ether extracts have statistically high significant anti-arthritic effect from day 14 to day 21 at all doses.Visual scorings were given based on walking, inflammation, joint deformities and pain assessment using Grimace scale (0= Normal to 5= Severe).Methotrexate showed 51.85% recovery, Prednisolone showed 81.48% recovery.Aqueous extract was observed with highest significant (p<0.0001)range of 18.52 to 66.67% recovery followed by ethanolic extract 14.81 to 55.56% recovery followed by petroleum ether extract22.22 to 48.15% recovery of arthritic symptoms (Table 6).

Effect on Spleen weight
It is very common during arthritis spleen weight increases.It was observed that aqueous, alcoholic and petroleum ether extracts were able to statistically normalise the spleen weight which shows the anti-arthritic effect.Methotrexate showed 18.28% recovery, Prednisolone showed 43.38% recovery.Ethanolic extract was observed with highest significant(p<0.0001)range of 22.09 to 32.05% recovery followed by aqueous extract 17.37 to 26.94% recovery followed by Petroleum ether extract10.30to 23.59% recovery of arthritic symptoms (Table 7).

Effect on RA Factor
An immune system protein called rheumatoid factor protein has the ability to assault the body's healthy tissues.The most typical association between elevated blood levels of rheumatoid factor and autoimmune illnesses.It was observed that aqueous, alcoholic and petroleum ether extracts were able to statistically decrease the RA factor value in arthritic animals.Methotrexate showed 38.43% recovery, Prednisolone showed 49.32% recovery.
Ethanolic extract was observed with highest significant (p<0.0001)range of 30.11 to 63.99% recovery followed by aqueous extract 23.60 to 57.34% recovery followed by Petroleum ether extract 36.31 to 55.98% recovery of RA factor (Table 8).

Effect on WBC's in Arthritic animals
As rheumatic arthritis is autoimmune diseases WBC's count increase is very common observation.The most common link between elevated WBC numbers is autoimmune disorders.It was observed that aqueous, alcohol and petroleum ether extracts were able to statistically decrease the WBCs value in arthritic animals.Methotrexate showed 43.32%, Prednisolone showed 65.21% decrease in WBC's count.The ethanol extract was observed with highest significant (p<0.0001)range of 52.07 to 77.19% decrease followed by petroleum ether extract41.94to 68.43% recovery followed by aqueous extract 43.09 to 64.98% decrease in WBC's count (Table 9).

Effect on Joint Diameter in X-Ray
X-ray is good tool to see the deformities in bones and tissues around the same.It was observed that aqueous, alcohol and petroleum ether extracts were able to statistically cure the arthritis.Methotrexate showed 18.52%, Prednisolone showed 18.86% decrease joint diameter.Aqueous extract was observed with highest significant (p<0.0001)8.13 to 22.36% decrease followed by ethanolic extract 13.53 to 18.34% decrease followed by petroleum ether extract 9.87 to 17.15% decrease joint diameter (Table 10, Figure 1).

Histopathological Study
A histological investigation of the joints after CFA injection is shown in Figure 2. The joint architecture appears normal in the non-arthritic control group; The synovial membrane, a sizable joint gap, and articular cartilage as well as bone are all destroyed in the arthritic control group.On the other hand, treatments using aqueous, ethanol, and petroleum ether extracts of Asparagus officinalis stopped the degeneration of the articular architecture in the treated animals.The healthy control knee joint underwent a histological investigation and showed no signs of bone erosion, inflammation, cartilage degeneration, or cellular infiltration.The

Extracts quantification using LC-MS/MS
All three extracts were analysed using LC-MS/MS.Results depicted the presence of rutin, quercetin, β-sitosterol, stigmasterol (Table 11, Figure 3).Highest concentration of rutin was present in the ethanol extract i.e. 650.45 µg/gm whereas in other two extracts showed less than 100ug/gm.In case of quercetin, highest concentration was found in aqueous extract than other two extracts.The concentration of β-sitosterol and stigmasterol were present in higher quantity in pet ether extract and very less in ethanolic extract.Both compounds were absent in aqueous extracts.

DISCUSSION
Arthritis is nothing but the presence of swelling, redness, pain or inflammation in a joint.The word "disease of the joints" in Greek is the origin of the word "arthritis."Acute or persistent joint inflammation is what's known as it, and discomfort and structural damage are frequently present alongside it.Arthralgia, or joint pain without inflammation, can be brought on by disease in the joint itself or in the nearby soft tissues, ligaments, and tendons.Subcutaneous nodules on the surfaces of the extensor muscles and soft tissue edoema around the affected joints are also observed. [29]levated WBC count represents the change in immunity.A minor to moderate rise in WBC counts is brought on by the production of the IL-Ib inflammatory response during arthritic circumstances.Granulocyte production and macrophage colony-stimulating factor both rise in response to IL-Ib. [1]Same was observed in our study arthritic control (14466 Cells/cumm) was observed with maximum count whereas it was significantly decreased in treated groups (4566.67 to 8400 Cells/cumm).
Rheumatoid factor (RF) was measured in serum collected on the terminal day indicates that as compare to arthritic control (22IU/ mL) significant decrease was observed in treatment groups.An immunoglobulin molecule known as serum rheumatoid factor (RF) is regarded as a "non-self " molecule that has the ability to activate the immune system.A major indication of RA in the RA aetiology may be abnormal variations in the blood levels of RF and CRP. [30]w oedema and joint swelling is an indicator of the anti-arthritis activity of various medicines.Determining foot swelling is a simple, sensitive, and rapid way to assess and assess the degree of inflammation. [1]With the help of plethysmometer and vernier calliper oedema and swelling was observed.Treatment groups were observed with significant decrease in swelling as compare to inflammation control group.
Same way X-ray and histopathology suggest the treatment as compare to arthritic group.Radiographic changes in the condition of arthritis are a useful diagnostic tool to get to know the severity of the arthritis.Soft tissue swelling is an early sign of radiology, but marked radiological changes such as bone erosion and narrowing of the joint space can only be observed during development. [31]mediately after the completion of experiment, the spleen was removed from the animal and the organ weight was measured.Animal organ weight was calculated.Spleen weight increase is very common symptom during arthritis. [30,32]Systemic autoimmune disorders are marked by an extremely high prevalence and destructiveness of splenic long-lived plasma cells.The research has shown that spleen-derived CD11b+Gr-1+ myeloid cells (SDMCs) play a pathogenic role in the deposition of splenic long-lived plasma cells in the sanroque lupus-prone mouse and that SDMCs can be accumulate. [33]rm qualitative phytochemical assays it is clear that Asparagus officinalis is reach in terpenoids and flavonoids.Various routes of action are already proven for mode of action against inflammation and arthritis by both flavonoids and terpenoids.Results of LC/ MS proved that the extracts contain higher concentration of rutin, quercetin, β-sitosterol and stigmasterol and the protective effect may be due to the molecular action of these compounds in obstacle of rheumatoid arthritis.
The central effects of the extract in inducing anti-nociception were assessed using tail flick experiments.The tests can also be characterised by their proclivity to respond to pain stimuli delivered via various neural pathways.The tail flick method is thought to be a supraspinally organised reflex and requires higher brain activity.It looks like that flavones extracted from diverse plants, such as quercetin and rutin, produce considerable anti-nociceptive reactions.It's possible that flavonoids are responsible for the extract's analgesic action. [30]The excessive expression of TRPV1 can be inhibited by quercetin, which indicates that quercetin might relieve thermal hyperalgesia. [34] the case of rheumatoid arthritis, several inflammatory cytokines, such as TNF, IL6, JAK, and the TNFR1 receptor, were discovered to be actively engaged in the signalling resulting in the inflammatory response that causes the destruction of bones and adjacent tissues of the synovial joints.The inflammatory response that is involved in the course of rheumatoid arthritis can be directly inhibited by disrupting the signalling pathways involved     The PI3K/Akt signalling pathway is thought to be a link between FLS cell proliferation and apoptosis.PI3K and Akt are highly expressed in RA-FLS cells and have an impact on FLS cell migration.Furthermore, many inflammatory cytokines, such as IL-17 and IL-21, can endorse FLS cell inflammatory proliferation by provoking and activating PI3K flavonoids, which can inhibit p-Akt expression in B cells. [35]Likewise, flavonoids can restrict the MAPK signalling pathway, thereby reducing RA symptoms. [36]In RA, NF-B and STAT are profoundly and sustainably activated, which flavonoids improve. [37]Triterpene decreases pro-inflammatory cytokines such IL-6, IL-1, TNF-α and increases the anti-inflammatory cytokine IL-10 in rats with arthritis caused by complete Freund's adjuvant (CFA). [38]cording to certain studies, the useful pharmacological effects of terpenes for arthritis are linked to the regulation of many intracellular signalling pathway proteins, including RANKL, the MAPK family, NFkB, PGE-2, COX-2, iNOS, matrix metalloproteinases, MPO, and c -FOS. [39]Angiogenesis is primarily mediated by VEGF/VEGFR2 signalling, which is a key target for anti-angiogenic therapies.When VEGF and VEGFR2 interact, VEGFR2 is phosphorylated on multiple tyrosine residues, triggering signalling cascades that promote EC proliferation, migration, survival, and permeability.Thus, VEGFR2 activation is a critical step in the angiogenesis process, and blocking VEGF/VEGFR2 signalling with VEGFR2 inhibitors can suppress angiogenic responses.β-Sitosterolis has already reported the improvement in RA by VEGF pathway. [40] lowering MMP-8 secretion and synthesis, Stigmasterol therapy is said to ameliorate joint-related pathology to levels close to normal.This drop in the levels of metalloproteinase MMP-8 and inflammatory mediators is most likely brought on by a decrease in the activity of the transcription factors MAPKs and NF-kBp65. [41]

CONCLUSION
According to the findings of this study, Asparagus officinalis is a plant that contains various chemical component groups that have anti-inflammatory, analgesic and anti-arthritic activities.In vivo tests have been done to assess these qualities.These findings support the plant's use in the conventional management of chronic inflammatory illnesses, and also suggest that it may be a candidate for the discovery of novel anti-inflammatory, analgesic and/or anti-arthritic compounds.We have identified the presence of rutin, quercitine, stigmasterol and β-sitosteroid.But during our analysis we observed many other molecular weight peaks which shows the presence of many more ingredient availability.
To determine the precise mechanism of action of Asparagus officinalis on rheumatoid arthritis, more research can be done.

Figure 1 :
Figure 1: Effect of Asparagus officinalis extracts on arthritic joints in X-ray.

Figure 2 :
Figure 2: Histopathology of rat joints after treated with different drugs.

Figure 3b :
Figure 3b: LC-MS picture of Rutin in Petroleum ether extract of Asparagus officinalis.

Figure 3c :
Figure 3c: LC-MS picture of Rutin in ethanolic extract of Asparagus officinalis.

Figure 3d :
Figure 3d: LC-MS picture of Rutin in Aqueous extract of Asparagus officinalis.

Figure 3f :
Figure 3f: LC-MS picture of Quercetin in Petroleum ether extract of Asparagus officinalis.

Figure 3g :
Figure 3g: LC-MS picture of Quercetin in Ethanolic extract of Asparagus officinalis.

Figure 3h :
Figure 3h: LC-MS picture of Quercetin in aqueous extract of Asparagus officinalis.

Figure 3j :
Figure 3j: LC-MS picture of β-sitosterol in pet ether extract of Asparagus officinalis.

Figure 3l :
Figure 3l: LC-MS picture of β-sitosterol in aqueous extract of Asparagus officinalis.
in the control of the inflammatory response, either by suppressing the aforementioned cytokines or by antagonisting the TNFR1 receptor.As a result, the TNFR1 receptor and the cytokines TNF, IL6, and JAK potentially serve as significant therapeutic targets for the therapy of rheumatoid arthritis.Flavonoids have anti-inflammatory, analgesic, and antioxidant properties.These effects are associated with the suppression of NF-B-dependent pro-inflammatory cytokines, VEGF, ICAM-1, and STAT3, as well as the activation of the antioxidant transcription factor Nrf2.

Table 2 : Effect of Asparagus officinalis extracts in Acute Inflammation (Using plethysmometer) activity on rats.
Statistical analysis of data was carried out by one-way ANOVA followed by Dunnett's multiple range test.The values are expressed as mean ± SD for each group (n=6), a P<0.0001, b P<0.001, c P<0.05 compared with negative control group.ns: non-significant.

Table 3 : Effect of Asparagus officinalis extracts on body weights of Arthritic animals.
Statistical analysis of data was carried out by one-way ANOVA followed by Dunnett's multiple range test.The values are expressed as mean ± SD for each group (n=6), a P<0.0001, b P<0.001, c P<0.05 compared with negative control group.ns: non-significant.

Table 4 : Effect of Asparagus officinalis extracts in Arthritis (Using plethysmometer) activity on rats. Groups (n = 6) 1 st Day 7 th Day 14 th Day 21 st Day
Statistical analysis of data was carried out by one-way ANOVA followed by Dunnett's multiple range test.The values are expressed as mean ± SD for each group (n=6), a P<0.0001, b P<0.001, c P<0.05 compared with negative control group.ns: non-significant.

Table 5 : Effect of Asparagus officinalis extracts in Arthritis (Using Vernier calliper) activity on rats.
Statistical analysis of data was carried out by one-way ANOVA followed by Dunnett's multiple range test.The values are expressed as mean ± SD for each group (n=6), a P<0.0001, b P<0.001, c P<0.05 compared with negative control group.ns: non-significant.

Table 7 : Effect of Asparagus officinalis extracts in Arthritis Animal Spleen Weight
c P<0.05 compared with negative control group.ns: non-significant.

Table 8 : Effect of Asparagus officinalis extracts on RA Factor in Arthritis.
b P<0.001, c P<0.05 compared with negative control group.ns: non-significant.

Table 9 : Effect of Asparagus officinalis extracts on WBC's in Arthritis.
Statistical analysis of data was carried out by one-way ANOVA followed by Dunnett's multiple range test.The values are expressed as mean ± SD for each group (n=6), a P<0.0001, b P<0.001, c P<0.05 compared with negative control group.ns: non-significant.

Table 10 : Effect of Asparagus officinalis extracts on Arthritic Joints in X-Ray.
Statistical analysis of data was carried out by one-way ANOVA followed by Dunnett's multiple range test.The values are expressed as mean ± SD for each group (n=6), a P<0.0001, b P<0.001, c P<0.05 compared with negative control group.ns: non-significant.