Comprehensive phytochemical characterization of Persea americana Mill. fruit via UPLC/HR-ESI–MS/MS and anti-arthritic evaluation using adjuvant-induced arthritis model

Persea americana Mill. (avocado fruit) has many health benefits when added to our diet due to various pharmacological activities, such as preventing bone loss and inflammation, modulating immune response and acting as an antioxidant. In the current study, the total ethanol extract (TEE) of the fruit was investigated for in vitro antioxidant and anti-inflammatory activity via DPPH and cyclooxygenase enzyme inhibition. Biological evaluation of the antiarthritic effect of the fruit extract was further investigated in vivo using Complete Freund’s Adjuvant (CFA) arthritis model, where the average percentages of body weight change, inhibition of paw edema, basal paw diameter/weight and spleen index were estimated for all animal groups. Inflammatory mediators such as serum IL-6 and TNF-α were also determined, in addition to histopathological examination of the dissected limbs isolated from all experimental animals. Eighty-one metabolites belonging to different chemical classes were detected in the TEE of P. americana fruit via UPLC/HR-ESI–MS/MS. Two classes of lyso-glycerophospholipids; lyso-glycerophosphoethanolamines and lysoglycerophosphocholines were detected for the first time in avocado fruit in the positive mode. The TEE of fruit exhibited significant antioxidant and anti-inflammatory activity in vitro. In vivo anti-arthritic activity of the fruit TEE improved paw parameters, inflammatory mediators and spleen index. Histopathological findings showed marked improvements in the arthritic condition of the excised limbs. Therefore, avocado fruit could be proposed to be a powerful antioxidant and antiarthritic natural product.


Introduction
Rheumatoid arthritis (RA) is an autoimmune disorder characterized by systemic inflammation that damages joints, impairing mobility and perhaps decreasing lifespan (Smolen and Aletaha 2015).According to Prescha et al. (2018), the clinical condition of RA was significantly correlated with the availability of foods and nutrients that regulate antioxidant and anti-inflammatory defense mechanisms.Food quality assessment and its relationship to anti-inflammatory status in RA patients may help to discover dietary practices and nutrient intake that are crucial for enhancing antioxidant defenses, thus delaying RA symptoms (Jalili et al. 2014).Due to its high polyphenolic flavonoid content, which has been associated with antioxidant, anti-inflammatory and analgesic properties, adding fruit like avocado to RA patients' diet may help to reduce inflammatory symptoms.
Alligator pear (Persea Americana Mill., Lauraceae), is an edible fruit with creamy smooth flesh and bumpy skin, (Bhuyan et al. 2019).It is a very nutritious fruit as it contains fat-soluble vitamins which are less common in other fruits and valuable constituents including A, C, E, K1, folate, B-6, niacin, pantothenic acid, riboflavin choline, carotenoids, phytosterols and high-monounsaturated fatty acids (Dreher and Davenport 2013).The seeds and peels are rich sources of bioactive metabolites, such as phytosterols, triterpenes, catechins, hydroxycinnamic acids, pro-anthocyanidins, and glycosylated abscisic acid derivatives.The bark, fruit and leaf are used in traditional medicine in South and Central America.They are used for the treatment of various ailments such as menorrhagia, hypertension, stomach problems, bronchitis, diarrhea and diabetes (Adeyemi et al. 2002).Several health benefits were reported including antioxidant actions (Rodríguez-Carpena et al. 2011).The aqueous leaf extract of avocado produced a significant inhibition of carrageenaninduced swelling with a similar effect to that produced by indomethacin in the same duration.(Adeyemi et al. 2002).Natural products and their constituents possess advantages over synthetic alternatives with respect to potency, accessibility, low cost, lower side effects, superior safety and efficacy.These therapeutic natural phytoconstituents include steroids, glycosides, phenolics, flavonoids, fatty acids that have anti-inflammatory properties in addition to their ability to halt inflammatory processes (Yatoo et al. 2018).

Aim of the current study
The main aim of the study is to perform a complete phytochemical characterization of Avocado fruit extract, investigate the in vitro antioxidant, anti-inflammatory and in vivo antiarthritic activity, in an attempt to use the fruit as a protective and curative natural plant material for arthritis.Possible beneficial effect of two doses of fruit extract was compared to methotrexate, which belongs to a class known as disease-modifying antirheumatic drugs (DMARDs) and is considered the first line of treatment for rheumatoid arthritis but possesses many adverse events reported in the literature.The effect of avocado consumption on CFA-induced arthritis in rats was studied in addition to histopathological examination of excised hindlimb.

Plant material
The fruits of avocado (Persea americana Mill., Lauraceae) were collected during May-June 2017 from local markets and were kindly identified by the Fruit Department, Institute of Agriculture, the National Research Centre.

Preparation of the extract
One kilogram of the deseeded fruits was extracted with 80% ethanol till exhaustion.The combined filtered extract was evaporated to dryness under vacuum to yield 80 g of total ethanol extract (TEE).

Evaluation of radical scavenging potential
DPPH radical scavenging activity of TEE of avocado fruit samples at 62.5, 125, 250, 500, and 1000 µg/ml concentrations was assessed according to the method adopted by Ibrahim et al. (2021).The absorbance was measured spectrophotometrically at 517 nm against ascorbic acid as the reference standard.The % inhibition of DPPH was calculated as follows: Where A c = absorbance of the control, A t = the absorbance of the sample.

Evaluation of anti-inflammatory activity
The cyclooxygenase inhibition efficacy of TEE of avocado fruit was performed.Procedures were followed as per manufacturer's instructions (Blobaum and Marnett 2007).Indomethacin and celecoxib were used as standard antiinflammatory compounds against COX1 and COX2 activity.

Acute toxicity study
The acute toxicity test for TEE of avocado fruit was carried out to evaluate any possible toxicological signs using 'The Limit Test' with a subjected dose of 2 and 5 g/kg orally according to Economic Cooperation and Development (OECD) 425 guidelines (OECD 2008).

Complete Freund's adjuvant (CFA) arthritis
CFA arthritic model is sensitive to anti-inflammatory and immune-inhibiting medicines for the study of pathophysiological and pharmacological control of the inflammatory process (Bevaart et al. 2010).Arthritis was induced by intraplantar injection of 0.1 ml of adjuvant into the surface of right hind paw of rats (Lao et al. 2001).The left hind paw was injected with an equivalent volume of saline (Noh et al. 2021).

Experimental design
Rats were randomly assigned to five groups (n = 8) and received their respective treatments as follows: Group I: served as the negative control group which received oral and intraplanar injections of normal saline.Group II served as positive control, receiving an intraplanar injection of 0.1 ml CFA.Group III: received CFA followed by daily oral methotrexate (MTX 0.25 mg/kg) for 28 consecutive days as the reference standard drug.Groups IV and V received CFA followed by a daily oral dose of 500 and 1000 (mg/kg) of TEE of avocado fruit for 28 consecutive days, respectively according to results observed from the acute toxicity study.

Collection of data
Paw edema and joint thickness were monitored on the 7th, 14th, 21st, and 28th days by using a plethysmometer and vernier calipers, respectively to confirm the development of arthritis.The mean percent change of injected paw diameter with respect to basal values and percent inhibition of paw edema was calculated at the end of the study p e r i o d u s i n g t h e f o l l o w i n g f o r m u l a : %Inhibtion in paw edema = 100 × 1 − VT

VC
Where VC = mean paw edema volume in the control group and VT = mean paw edema volume in the drugtreated group.
At the end of the experimental period (28 days), animals were euthanized and sacrificed by decapitation.Blood was collected and centrifuged at a speed of 4000 rpm at 4 °C for separation of serum which was deep frozen at (− 80 °C) till the time of biochemical estimations.Both left and right hind paws were excised then weighed.for determination of mean % paw weight of the basal value of the right hind paw.Finally, hindlimbs from each group were immersed in buffered formalin (10%) for histopathological investigations.
The relative organ weight of the spleen is indicative of immunological function.Therefore, the spleen of all animals was isolated, washed with cold saline then blotted dry between two filter papers and weighed to estimate spleen index % calculated as follows:

Biochemical analysis
TNF-α and IL-6 were estimated by using a solid-phase sandwich ELISA test kit for rats, according to the manufacturer's instructions (Elabscience, USA, Elabscience Biotechnology Inc, USA).

Statistical analysis
Data collected from the animal study were analyzed using GraphPad Prism software (San Diego, CA, USA) by performing ANOVA (1-way) followed by Tukey's post hoc analysis with a probability level set at p < 0.05 for significance.Farid et al. (2022).Each injected sample (10 μl) was passed through a Phenomenex In-Line filter disk (3.0 mm, 0.5 μm).Waters X select HSS T3 (2.1 × 150 mm, 2.5 μm) column is used, temperature (40 °C).The mobile phase A containing 5 mM NH 4 HCO 2 buffer at pH 3 with 1% methanol was used for the +ve ionization mode while 5 mM NH 4 HCO 2 buffer at pH 8 containing 1% methanol for the −ve mode.Acetonitrile100% was used as a mobile phase B for both ion modes.The flow rate was 0.3 ml/min, a linear gradient for a run duration for 28 min was used: (90% A and 10% B) for 20 min, (10% A and 90% B) for 5 min then (90% A and 10% B) for the remaining 3 min.For both ion modes, a capillary voltage of 4500 V, 500 °C is a source temperature, and the MS acquisition is ranged from 50 to 1000 m/z.Further, the peaks and spectra were processed using the Analyst TF 1.7.1 and PeakView ® 1.2 Softwares (SCIEX, Framingham, MA, USA).The tentative identification of the compounds was carried out by comparison of their masses, fragmentation pattern, and molecular formulae with those in the literature.
The antioxidant activity may be due to the presence of phenolic compounds in the fruit where phenolics are composed of one or more aromatic rings bearing one or more hydroxyl groups and are therefore potentially able to quench free radicals by forming resonance-stabilized phenoxy radicals (Rice-Evans et al. 1996;Bors and Michel 2002).
Cyclooxygenases involved in inflammation exist in two isoforms, COX-1 and COX-2.COX-1, which is present in most tissues, has a beneficial effect maintaining the normal lining of the stomach and intestines by protecting them from digestive juices (Vane and Botting 1998;Hawkey 2001).Contrarily, COX-2 is effectively induced in pathological conditions only such as inflammation, where proinflammtory mediators, cytokines and endotoxins activate its production (Mitchell et al. 1994).Therefore, COX-2 has been considered as the most appropriate target for assessment of anti-inflammatory drugs rather than COX-1, since its inhibition leads to adverse side effects such as reduced mucosal blood flow, retardation of mucous secretion that aids in ulcer healing and decrease in renal blood flow (Wallace and Chin 1997).

Acute toxicity studies
There was no mortality at the end of the observation period, hence the LD 50 was estimated to be larger than 5000 mg/kg and was considered safe.Avocado TEE extract was given at two dose levels (5 and 10%).

Effect of daily oral treatment with two dose concentrations of avocado extract on % body weight change
At the end of the study period, adjuvant injection induced a significant depression in % body weight changes by 36.6% that of the normal rats.According to Challal et al. (2016), the adjuvant arthritis model is associated with catabolism which manifests in the depression of body weight gain.This was effectively improved by daily oral administration of MTX for 28 days, which significantly increased body weight change by 13.4% that of the diseased group.Likewise, daily oral intake of 10% extract significantly raised body weight change by 27.6%, contrary to the effect of 5% extract, which showed an insignificant change with respect to the control value at p < 0.05 (Fig. 2A).
Avocado fruit extract exerted an increase in the body weight of the experimental animals.This effect could be due to its curative anti-arthritic effect by increasing the absorption of glucose and leucine in rat intestine, which was lowered in arthritic conditions (Somasundaran et al. 1983;Rajendran and Krishnakumar 2010).In addition to its high fat content, the fruit is rich in unsaturated fatty acids (good fat).Polyunsaturated fatty acids are potentially helpful anti-inflammatory agents that might assist individuals at risk in several acute and chronic inflammatory conditions (Calder 2005).Moreover, a human study performed by Monnard and Dulloo (2021) declared that polyunsaturated fats modulate glucose metabolism and alterations in body composition in relation to central adiposity, hence improvement in lean body mass.Therefore, it could be proposed that extract may have improved metabolism and arthritis-induced cachexia (Challal et al. 2016).

Effect of daily oral treatment with two dose concentrations of avocado extract on % inhibition of paw edema (volume), change of basal paw diameter and basal paw weight
Adjuvant-injected rats given oral reference drug and extract at two dose levels daily for 28 days exhibited inhibition of paw edema to 20, 22.6 and 15% that of the untreated animals respectively, with superior effect observed in the higher extract concentration (10%) group at p < 0.05 (Fig. 2B).Untreated arthritic animals exhibited a highly significant increase in % change in basal paw diameter to 48% as compared to normal value.Daily oral administration of reference drug induced a significant reduction to 56.3% that of the control diseased group.In a similar manner, higher dose of extract (10%) revealed a significant decrease in % change of basal paw diameter to almost half the value of the control group with an equipotent effect to that of the reference value and a better effect than that of the lower dose extract group at p < 0.05 (Fig. 2C).After excision of the hind limb at the end of the study, adjuvant injection induced a significant elevation of % basal paw weight to 1.7 folds that of the normal value.Daily oral intake of reference and 10% extract for 28 days revealed a marked depression of basal paw weight to almost 78% that of the untreated control group, unlike the weaker effect observed from the low dose (5%) extract group, which showed a significant depression by 13.4% that of arthritic untreated animals at p < 0.05 (Fig. 2D).Results of the current investigation regarding arthritic paws revealed that avocado extract improved paw edema, diameter, and weight due to the presence of bioactive compounds in avocado extract; persenone A, a long chain fatty alcohol (Kim et al. 2000), in addition to persin compound (2-Hydroxy-4-oxohenicosa-12,15-dien-1-yl acetate) (Goudarzi et al. 2017), linoleic and oleic fatty acids (Chandel et al. 2018).Moreover, flavonoids such as quercetin and isorhamnetin have anti-inflammatory and antioxidant activities (Lesjak et al. 2018).

Effect of daily oral treatment with two dose concentrations of avocado extract on serum TNF-α
Pro-inflammatory cytokines TNF-α and IL-6 are key mediators in the pathogenesis of rheumatoid arthritis (Sharma et al. 2011;Wang and Zhong 2015) which enhance osteoclastogenesis and expression of matrix metalloproteinases, that culminates in cartilaginous degradation (Thummuri et al. 2015).In the current study, adjuvant injection induced a significant four fold rise in serum TNF-α level with respect to normal value.However, daily oral treatment with reference drug for 28 days normalized serum TNF-α.Oral administration of 5% extract significantly lowered TNF-α to 64% that of the arthritic untreated rats but was still higher than that of the normal and reference values.Likewise, daily intake of 10% extract significantly depressed serum TNF-α to 42.7% that of the untreated group, with an almost equipotent effect to reference standard at p < 0.05 (Fig. 2E).

Effect of daily oral treatment with two dose concentrations of the avocado extract on serum IL-6
Similar to TNF-α, untreated arthritic rats showed a significant 2.6-fold elevation in serum IL-6 level.Oral treatment of diseased rats with the reference standard for 28 days significantly reduced levels by 56% with respect to untreated animals.Likewise, daily oral intake of 5 and 10% concentration of the extract significantly decreased serum IL 6 level to 67.6 and 54.4%, respectively as compared to the diseased animal group, however, values remained markedly higher than that of the normal group by 79 and 44%, respectively.Noteworthy, higher extract concentration showed a superior effect to that of the lower dose of extract at p < 0.05 (Fig. 2F).Avocado extract contains compounds such as apigenin (Karunaweera et al. 2015) procyanidins (Cui et al. 2018), monounsaturated fatty acids (Henrotin et al. 1998), chlorogenic and caffeic acids (Meng et al. 2014;Zhang et al. 2014) that could be responsible for reducing the proinflammatory mediator levels of TNF-α and IL-6 in rat serum.

Effect of daily oral treatment with two dose concentrations of the avocado extract on spleen index
Splenomegaly in rheumatoid arthritis is attributed to increased β cell proliferation and upregulation of immunoglobulin production (Nishiya et al. 2000;Lazaro and Morel 2015), which is indicated by splenic index.Untreated diseased animals exhibited a significant 2.3-fold increase in spleen index% with respect to that of the normal value.Oral administration of reference drug and 10% extract for 28 consecutive days significantly improved spleen index to near normal value, unlike the 5% extract group, whose spleen index was still significantly high as compared to normal, reference and 10% extract concentration values at p < 0.05 (Fig. 2G).It could be proposed that avocado fruit extract protected spleen from enlargement induced by adjuvant arthritis, showing comparable improvement to MTX.This can be explained by the presence of a chemical class identified in avocado fruit for the first time; Lysoglycerophospholipids that has an immunomodulatory effect.Phosphatidylcholine (PC) supplementation reduced the development of arthritis due to the inhibition of the neutrophil leukocyte-mediated inflammatory reaction (Hartmann et al. 2009).

Histopathological study
No histopathological changes were observed in the normal control group; articular surfaces, joint capsules and synovial membranes were intact (Fig. 3A).On the contrary, the +ve control group exhibited serious alterations characterized by severe synovitis, massive inflammatory cell infiltration, marked edema, fibroplasia and pannus formation (Fig. 3 B and C).Additionally, necrosis of cartilage was recorded in some examined sections (Fig. 3C).Meanwhile, treatment with reference induced an improved histological picture, and examined sections exhibited edema, few inflammatory cells infiltration (Fig. 3D) and pannus formation.Likewise, sections from arthritic rats treated with extract 5% showed moderate inflammatory cells infiltration, pannus formation (Fig. 3E), and edema.Furthermore, a more improved histological picture was noticed in sections from arthritic rats treated with extract 10%, as examined sections revealed congested blood vessel and mild inflammatory cells infiltration (Fig. 3F).

UPLC/HR-ESI-MS/MS metabolomic profiling of the TEE of avocado fruit
The TEE of P. americana fruit was analyzed using reversed-phase UPLC/HR-ESI-MS/MS in negative and positive ionization modes.Throughout the ca.30 min run time, metabolites were eluted according to their polarity, from the most polar to the least polar.According to available literature data, they were identified using retention time (Rt), MS data (molecular ion, fragmentation pattern, and predicted formula), compared to reported literature data (Di Stefano et al. 2017), MS databases (FooDB, HMDB and Massbank) (Wishart et al. 2018;Horai et al. 2010), in-house library and authentic sources.Figure 4A and B shows representative UPLC chromatograms of selected metabolites.Some of the chemicals discovered had previously been reported in Avocado.The occurrence of 81 metabolites in negative and positive modes belonging to various classes was shown by the high resolution of ESI-MS, including organic acids, phenolic acids, flavonoids, tannins, long chain fatty alcohols, fatty acids, lyso-glcerophospholipids, amino acids and coumarins (Table 1).

Organic acids
Three organic acids were identified in peaks 1, 2, and

Phenolic acids
Seven phenolic acids were identified in this study.Phenolic acids yielded deprotonated [M−H] − fragment ion

Flavonoids
Flavonoids undergo a fragmentation pathway within aglycone through the Retro-Diels-Alder (RDA) reaction yielding small neutral molecules and fragments, e.g., CO 2 , CO and H 2 O (Cuyckens and Claeys 2004).Fourteen different flavonol glucosides were characterized in the studied extract (quercetin, isorhamnetin, kaempferol, luteolin).) (Ding et al. 2007).Kaempferol has a potent antioxidant activity (Deng et al. 2019), which can be found in a wide variety of herbs and plant families with antiinflammatory activity (Periferakis et al. 2022).

Long-chain fatty alcohols
Avocado fruit is reported to be enriched with straight-chain fatty alcohols with different degrees of unsaturation, alkyl chain length, hydroxylation, and subsequent acetylation  (Bhuyan et al. 2019).These fatty alcohols are reported to exhibit antiviral, cytotoxic, antifungal, antioxidant and antimicrobial activities (Mukherjee et al. 2013).In our study, long-chain polyhydroxylated fatty alcohols were detected in avocado fruit crude extract in peaks 51, 53, 57, 59, 62, 73, 70, 72, and (Oelrichs et al. 1995).Persenone A is a promising agent that prevent inflammation-associated diseases (Kim et al. 2000).Persenone A along with persin were found to suppress superoxide (O 2 − ) and nitric oxide (NO) generation in cell culture (Domergue et al. 2000;Hashimura et al. 2001).

Long-chain fatty acids
Lauraceous acetogenins are a family of long-chain fatty acids with biologically active derivatives (Rodriguez-Saona and Trumble 2000).In this study, eleven long-chain fatty acids were detected in avocado fruit crude extract; 2 oxo-fatty acids (peaks 49 and 50) were detected and identified as oxohexadecenoic acid at  (Deas et al. 1974).Moreover, 6 straight-long chain, fatty acids in peaks (65, 67, 69, 76, 77, and (Farag et al. 2015), in addition to two saturated fatty acids detected (peaks 69 and 77); hexadecanoic acid (Yang et al. 2015) and heptadecanoic acid (Allen et al. 2015) (Ragheb et al. 2023).It was reported that PUFA (polyunsaturated fatty acids) are useful in alleviating several diseases such as cardiovascular, inflammatory heart diseases, atherosclerosis, autoimmune disorder and diabetes (Finley and Shahidi 2001).Siswadi and Saragih (2021) reported that hexadecanoic acid possesses important biological activities such as antioxidant, antimicrobial, hypocholesterolemic and antiinflammatory activities.In metabolic illness mouse models, hexadecenoic acid has anti-inflammatory effects on hepatic steatosis and insulin signaling.It has recently been described in human circulating monocytes and monocytederived macrophages as anti-inflammatory (Astudillo et al. 2018).Linolenic acid was shown to block the synthesis of prostaglandin which results in reduction of inflammation and prevention of certain chronic diseases.It was observed that the fatty acids and their amides can cover a wide range of therapeutic indications such as bacterial infections and inflammations (Tanvir et al. 2018).

Lyso glycerophospholipids (LGPLs)
The molecular structure of LGPLs consists of a hydrophobic acyl chain(s) attached to the positions 1 and 2 of the glycerol backbone and in position 3 hydrophilic phosphate head group was attached.Various kinds of LGPLs were discovered based on the type of hydrophilic head.
The detected LGPCs and LGPEs were identified with molecular formula CxHxNO8P, where the glycerol moiety acylated with one or two fatty acid(s), the phosphate group at position 3 was either connected to choline (phosphatidylcholine) or ethanolamine (phosphatidylethanolamine).The product ion at m/z 184 is a characteristic fragment ion to the phosphocholine head group.(Murphy and Axelsen 2011).Glycerophospholipids (GPLs)were also reported to decrease the pro-inflammatory cytokines as IL-1β, TNF-α and NO in LPS-activated microglia (Kim et al. 2020).Additionally, GPLs reduced the gastric mucosal lesions in rats after treatment with NSAIDs (Küllenberg et al. 2012).

Amino acids
Avocado contains 18 of the 22 amino acids required for consumption including all the eight essential amino acids.It contains more protein than an equivalent amount in cow's milk, which makes them a high-quality protein food with special value to vegetarian diet (Caballero et al. 2005).In this study, four amino acids were identified (peaks 8, 9, 11, and 19) ), the loss of H 2 O + CO from protonated tyrosine led to the formation of a fragment ion at m/z 136, Further loss of NH 3 resulted in the formation of the fragment ion at m/z 119 (Nishio et al. 2021);phenylalanine (peak19) [M+H] + 166 (C 9 H 11 NO 2 + ) the major fragmentation pathway of protonated phenylalanine started from the loss of H 2 O + CO to form a fragment ion at m/z 120.A fragment ion at m/z 103 was formed by the further loss of NH 3 (Farid et al. 2022).Muscle weakness, poor wound healing, weakened immunity, stunted growth, and dull skin and hair are all effects of deficiency of essential amino acids.Avocados are one of the few fruits that contain all essential amino acid (Caballero et al. 2005).

Conclusions
Avocados have become an increasingly popular food in recent years as they are rich in antioxidant content.Regular consumption of avocado fruits might improve antioxidant defenses of the body and could be considered an excellent source of anti-inflammatory compounds.
Screening of the in vitro antioxidant and anti-inflammatory activities of TEE of P. americana fruit showed a high antioxidant activity due to the presence phenolics, flavonoids, fatty alcohols and fatty acids.Moreover, the TEE of the fruit exerted a significant in vivo anti-arthritic activity by improving arthritic paw parameters of the diseased experimental animals and serum inflammatory biomarkers (IL-6 and TNF-α).This was further emphasized by the histopathological study of the rats' excised hindlimbs, which showed milder inflammatory cell infiltration and improvement in histological picture.A study of the chemical constituents of the TEE of the fruit by UPLC/HR-ESI-MS/MS spectrometry led to the identification of 81 metabolites belonging to various classes, most of which possess antioxidant and anti-inflammatory activities.
This study suggests recommending avocado fruit consumption by patients who are prone to arthritic problems.According to the findings of the current investigation and a review of traditional medicine literature, avocado fruit extract could be considered as a promising antioxidant and anti-arthritic natural product.

Fig
Fig. 1 DPPH free radical scavenging activity of TEE of avocado fruit in comparison with ascorbic acid

Fig. 2
Fig. 2 Effect of daily oral treatment with two dose concentrations of avocado extract on % body weight change (A) % inhibition of paw edema (B), change of basal paw diameter (C), and basal paw weight 4. Citric acid at [M−H] − 191 (C 6 H 7 O 7 ) − , malic acid at [M−H] − 133(C 4 H 5 O 5 ) − and homocitric acid at [M−H] − 205(C 10 H 17 O 4 ) − (Ragheb et al. 2023).While citric acid gave a fragment at m/z 111 corresponding to [M−H-CO 2 -2H 2 O] − .Organic acids have been shown to have numerous biological functions such as antioxidant and anti-inflammatory activities.Both citric acid and l-malic acid significantly decreased inflammatory cytokine TNFα activity and inhibited ADP-induced platelet aggregation (Tang et al. 2013).

Fig. 3
Fig. 3 Photomicrographs representative hematoxylin and eosin staining of paw sections of A control negative rat showing normal joint, articular surface and synovial membrane.B and C +ve control; showing massive inflammatory cells infiltration (black arrow), fibroplasia (blue arrow), pannus formation (yellow arrow) and necrosis of cartilage (red arrow).D Diseased rat treated with reference standard

Table 1
Metabolites tentatively detected via UPLC/HR-ESI-MS/MS analysis of avocado deseeded fruits ethanol extract in both negative and positive ionization modes