Toxicity and efficacy of Prosopis juliflora seed pods extract on induced osteoarthritis in Wistar rats

Introduction

Due to advancement in biological research, average life span of human being incr eases with decrease in death rates. However longer lifespan predisposed humans to v arious noncommunicable diseases like heart attacks, cancer, asthma, diabetes as well as musculoskeletal disorders. When taking into both death and disability, all musculoskeletal disorders combined accounted for 6.7% of the total global disability-adjusted life years, which was the fourth greatest burden on the health of the world's population (third in the developed countries) (Woolf, 2015) [40]. Osteoarthritis (OA), rheumatoid arthritis, gout, low back pain, and neck pain are important musculoskeletal disorders responsible for aforesaid conditions. Osteoarthritis (OA) is the most common joint disorder involving joint cartilage, underling bone, synovial membrane and characterized by progressive cartilage destruction, hypertrophy of bone at the margins, subchondral sclerosis, and range of biochemical a nd morphological alterations of the synovial membrane and joint capsule, pain, and loss of function (Pal et al ., 2016) [21]. In USA, approximately 10% in men and 13% in women are suffering from symptomatic knee OA and numbers are likely to increase due to the aging an d obesity (Zhang & Jordan, 2010) [41]. According to one epidemiological study (Pal et al ., 2016) [21] , knee OA was found to be 28 .7% in India. Gender (female are more prone to OA than male), obesity and sedentary works were associated factors for OA in India [2] . Till today, OA is considered be non -curable diseases with therapeutic strategies primarily aimed at analgesia and ameliorationjoint function . Intra-articular injections (hyaluronate, corticosteroid), arthroplasty or rarely autologous chondrocyte implantation into the dam aged area are some therapeuti c strategies of OA (Bennell et al ., 2012; Hunter & Felson, 2006) [3,8] .

International Journal of Advanced Biochemistry Rese arch 2025; SP-9 (7): 731-741 In one survey, almost 70,000 joint replacement surgeries were performed in India in the year 2011 (Pachore et al ., 2013) [20] . Thereare many animal models that reproduce key aspects of human OAin terms of natural history, mechanisms, signs, and symptoms (Poole et al ., 2010) [22] . Dunkin Hartley guinea pigs develop spontaneous OA at age of 18 months onwards, which is similar to those of human OA (Jimenez et al ., 1997) [9] , however much longer period is required. For rapid development of OA in laboratory animal surgery or chemical methods is used. Among surgical method, medial meniscal tear, partial medial meniscectomy, destabilization of the medial meniscus, and anterior cruciate ligament transection are frequently used method for induction of knee OA (Takahashi et al ., 2018) [33]. Among chemical method, monosodium iodoacetate (MIA) rat model is well established, and the induced OA resembles human degenerative OA in terms of the histological and painrelated behaviours (Takahashi et al ., 2018) [33] . From ancient era to till days, ayurveda is one of the leading systems of medicine used for treatment of many geriatricdiseases in India. In this traditional medicine system, most of the plants prevalent in India have been identified and their used in different diseases have been documented. However, some plants which were introduced in last one or two centuries in India are not well studied and their medicinal property yet to be documented. Prosopis juliflora is one of the most widespread hyper accumulating plants introduced in India nearly 130 years ago to prevent advancement of desert (Tewari et al ., 2001) [35] . Because of Prosopis juliflorais not ancient Indian plant, their medicinal property are not documented in Indian ancient literature. In some recent research, medicinal and other properties of Prosopis juliflorahave been reported viz., as a part of layer diets (Khobondo et al ., 2019) [11] , hepato-protectant activity (Hassan et al ., 2019) [7] , antibacterial activity (Shah et [27,32,36] and, immunomodulation (Shah et al ., 2018) [27] . In some unpublished research, Prosopis julifloraseed pods are effective in amelioration of clinical OA in humans (personal communication with Ayurvedacharya), however no preclinical studies have been carried out to support such hypothesis. Looking to the paucity of information present study was planned with following objectives. 1) To estimate LD50 of Prosopis juliflora seed pods extract by acute oral toxicity study (up-and-down procedure) in Wistar rats;2) To study the repeated dose 28-daysoral toxicity and efficacy of Prosopis juliflora seed pods extracton induced osteoarthritisin Wistar rats.

(up-anddown procedure) of Prosopis juliflora seed pods extract in Test compound Preparation: Prosopis juliflora seedpods extract (PJSPE) was used as a test compound. Fully matured seedpods of PF were collected around SDAU campus in the month of June 2019. The seedpods of PF were thoroughly washed with distilled water, and dry under shade in the laboratory. The dried PF seedpods were powdered by mixture grinder machine. Finally, powdered PF seedpods sieved through the kitchen strainer and fine powder was collected for extraction. Fifty gram of powdered PF seedpods was kept in 1000 mL conical flask and added 1000 mL distilled water. The closed conical flask kept in a shaker with shaking during initial six hours and no shaking for remaining eighteen hours. The mixture was filtered with filter paper. The water from the mixture was removed by heat in water bath temperature with 50 C temperature. Finally, experiments were conducted using the residue collected from the previously mentioned process. For maintaining the constant volume of throughout the study, rats were dosed 1 ml/100g of body weight. The dose formulations were prepared by suspending the PJSPE in distilled water. For acute study, dose was prepared on daily basis, and for 28 days studies, dose formulations were prepared on weekly basis.

Animal Procurement

A total 48 female Wistar rats were procured from laboratory animal facility of Torrent Research Centre, GIDC Bhat, Ahmedabad, Gujarat 382424, India,for these two studies. All the procured female rats were kept under acclimatization for a period of 15 days enrolling in the studies.

Housing and environmental conditions

Animal management and treatment procedures complied with the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), New Delhi, India. All the rats were housed in polypropylene cages at laboratory animal house facility in an environmentally controlled room with 22-3 "C temperature and 30-70% humidity with continues monitoring. Light/dark cycles of 12/12 hours were provided throughout the acclimatization and study period. Corncob was used as bedding material. Rats had ad libitum access to standard pellet diet (VRK Nutritional Solution, Sangli, Maharashtra, India) and RO drinking water throughout the study period. The composition of pellet diet is presented in Table 3.2. Rats were identified by tail markings. All essential management procedures were adopted to keep the rats free from stress.

Acute oral toxicity study (up-and-down procedure) of PJSPE in Wistar rats This experiment was design as per OECD Guidelines for the Testing of Chemicals, Section 4, Test No. 425 (OECD, 2008). A total five Wistar rat were dosed sequentiallyat 48 hours intervals. All five rats were fasted prior to dosing. Feed but not water was withheld from 5:00 p.m. on the day preceding dosing. Following the period of fasting, the rat was weighed, dose was calculated, and the test substance was administered in a single dose by gavage using a stomach tube. After dosing, feed was withheld for a further 3 hours. In first 30 minutes after dosing, rats were observed individually at least once, thereafter daily for a total of 14 days.

Repeated dose 28-days oral toxicity and efficacy of PJSPE on induced osteoarthritis in Wistar rats Induction of osteoarthritis (OA) Osteoarthritis was induced by intra-articular injection of monosodium iodoacetate (MIA; cat. #I2512; Sigma-Aldrich, St. Louis, MO, USA). MIA dissolved in physiologic saline

gauge, 0.5-inch needle (1 mg/joint). Anesthetized rats were given single intra-articular injection of MIA through the infrapatellar ligament of the femorotibial joint. Care was taken to ensure that the needle was not advanced too far into the cruciate ligament. OA was induced in all 48 rats on Day 1 of study.

Experimental design

The study was carried out on 48 MIA induced OA female Wistar rats. All the 48 rats were randomly divided into eight different groups of which Group I, II, III and IV served as a progressive phase while Group V, VI, VII and VIII served as reversal phase. Each group consisted of six rats. Group I and V served as control and received only normal saline as vehicle. Group II, III, IV of progressive phase and Group VI, VII, VIII of regressive phase received PJSPE dissolved in normal saline at doses of 250 mg/kg (low dose), 500 mg/kg (mid dose) and 1000 mg/kg bodyweight (high dose) respectively, by oral gavage daily for period of 28 days. The dosing was started from same day of induction of OA in progressive phase while in reversal phase dosing was initiated from day 29 of the study. The detail of experimental design is depicted in Table 1. The rats were fasted overnight prior to blood collection and necropsy. On study day 29 (Progressive phase) and 57 (Regressive phase), form all rats of particular phase, blood was collected from the retro-orbital plexus with the help of a heparinised capillary tube in clot activator for clinical chemistry and in EDTA for haematology. Haemoglobins (Hb), haematocrit (HCT), total erythrocytes count (TEC), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobins concentration (MCHC), red cell distribution width (RDW%), total leucocytes count (TLC), neutrophils (Absolute and%), lymphocytes (Absolute and%), monocytes (Absolute and%), eosinophils (Absolute and%), total platelet count,, and mean platelet volume (MPV) were analysed on the day of collection using an Exigo Eos Vet, Boule Diagnostics AB, Sweden. Blood smears were prepared within 3 hours of collection and stained with Giemsa for evaluation of platelet, erythrocyte morphology and basophils count. Basophils percent were counted in blood smear and converted to absolute count. Alanine Aminotransferase (ALT), Aspartate Aminotransferase (AST), Alkaline Phosphates (ALP), gamma-glutamyl transferase (GGT), total protein, albumin, blood urea nitrogen (BUN), uric acid, creatinine, cholesterol, triglyceride, calcium, phosphorus, magnesium, and glucose were analysed on the day of collection using a Randox Monaco fully automatic clinical chemistry analyser, Randox Laboratories Ltd. UK. All the rats of progressive and regressive phase were euthanized, on 29 th and 57 th day of the study respectively. The rats were fasted overnight prior to necropsy. Terminal body weights were collected prior to necropsy. All animals in the study were subjected to a full detailed necropsy. Both left and right femorotibial joint, peripheral nerve, skeletal muscle, brain, stomach, intestines, liver, kidneys, adrenals, spleen, heart, thymus, trachea, lungs, ovary, uterus, cervix, vagina, urinary bladder, and lymph nodes. The liver, kidneys, adrenals, thymus, spleen, brain, and heart of all animals were trimmed of any adherent tissue, and wet weight were be taken as soon as possible after dissection. Tissues for microscopic examination were fixed and preserved in 10% neutral buffered formalin. Fixed tissues except bone with joints were trimmed, labelled, and washed under running tape water for 2 hours. Dehydration was done using ascending grade (30%, 70%, 90%, and 100%) of isopropyl alcohol. The dehydrated tissues were cleared by two changes of xylene and impregnated in melted paraffin wax. The entire tissue processing was performed in automatic tissue processor (Leica TP1020). The paraffin-impregnated tissues were embedded using Leica EG1160 paraffin embedding station and cooled on Leica EG1150 C Cold Plate. The 4 to 5 thick sections were cut using Leica RM2255 fully automated rotary microtome. The sections were taken on poly-L-lysine-coated (0.1% w/v in H2O) slides and stained with Harris Haematoxylin and Eosin (HE). The sections were deparaffinised by xylene, rehydrated through descending grade of isopropyl alcohol and water. The hydrated tissue sections were stained with haematoxylin, differentiated in acid alcohol and blueing was carried out by ammonia water. Then tissue sections were stained with eosin, dehydrated with absolute isopropyl alcohol, followed by xylene wash, and mounted with DPX (Suvarna et al ., 2013) [29] . The entire staining was performed in Gemini AS Automated Slide Stainer, Thermo Scientific. After fixation in 10% neutral buffered formalin for 6 days, all the joints were decalcified with formic acid-sodium citrate method (Luna, 1968) [14] for a period of 12 days. After decalcification, as per Gerwin et al . (2010) (Gerwin et al ., 2010) [5] , frontal sectioning of jointswas performed by cutting into two approximately equal halves, an anterior, and a posterior one, along themedial collateral ligament in the frontalplane. Joints were processes routinely as describe earlier. Anterior and posterior half of the joint were embedded in a single paraffin block with the cut planes facingdown. Three 8 sections were cut from each paraffin block atapproximately 200 mmsteps to obtain three sections from each half (anterior and posterior) of the joint. The first section was stained with HIM as describe earlier, while remaining two sections were stained with Toluidine blue to detect areas of proteoglycans loss in cartilage. Toluidine blue staining method was adopted from Gerwin et al .

(Gerwin et al ., 2010) [5] and Schmitz et al . (Schmitz et al ., 2010) [26] . Microscopic scoring of OA was performed as recommendation of OARSI histopathology assessment system Pritzker et al . (Pritzker et al ., 2006) [23] and modified Mankin score. The modified Mankin score was adopted fromTakahashi et al . (Takahashi et al ., 2019) [33] with little modification as we used toluidine blue rather than Safranin O staining. On basis of cartilage structure, cellularity, toluidine blue staining intensity, and tidemark integrity, a total score 0-15 was given. In this system, higher values indicating more severe cartilage degeneration. We also used Pritzker et al . (Pritzker et al ., 2006) [23] scoring system. In this system on basis of cartilage histopathology grade and stage assessment, the score ranges from 0 to 24, with higher values indicating more advanced cartilage degeneration was calculated.

The statistical analysis of data generated on various parameters was subjected to statistical analysis using 2-way analysis of variance (ANOVA). Pairwise comparisons with control, for each phase separately, was made using

Results and Discussion

Acute oral toxicity study (up-and-down procedure) of PJSPE in Wistar rats The five rats were dosed with 175, 550, 2000, 2000, 2000 mg/kg of PJSE respectively with oral gavage. All rats were survived up to 14 days period after dosing. There were not any variations in behavioural pattern or any clinical signs noted in rats during the 14-day observation period. On basis of acute oral toxicity of the PJSE utilizing the OECD 425

Main test, LD 50 was calculated, and it was greater than P J N J . Plant products and herbal medicine are used to treat various pathological conditions of humans and animals since ancient era, due to its ameliorative effect or due to cultural belief. World Health Organisation (WHO) advocating the use of herbal medicine along with routine treatment and they also recommend performing scientific trials on herbal medicine to evaluate the efficacy and toxicity of various herbal medicine (WHO, 1993) [39] . Very few studies have been planned to estimate LD 50 of various herbal medicine, as most of these products are considered as safe in humans as well as in animals in short term as well as long-term exposure. TraditionalLD 50 study was conducted with different protocols however, most of new LD 50 studies were conducted on basis of OECD 425 guideline with main test or limit test to derived LD 50 of a different herbal compounds (Saleem et al ., 2017; Shi et al ., 2019) [24,28]. Hassan et al . (Hassan et al ., 2019) [7] conducted acute study to find out the LD50 of methanol extract of PJ leaves in rat. In their study, intraperitoneal PJ leaves extract administration gave LD 50 value of 44. P J . J

. In this study LD 50 is more than 2000 mg/kg. Probably the route of exposure (oral vs. intraperitoneal) as well as part of PJ (seedpods vs. leaves) have significant effects on LD 50 in rats. In acute oral gavage toxicity study of Prosopis farcta extract in rat, LD 50 was 540 mg/kg (Moayeri et al ., 2018) [17]. In experiment conducted by Vasile et al . (Vasile et al ., 2019) [38] , LD50 of Prosopis Alba exudate gum was found to be >1000 mg/kg. This difference is probably due to different species Prosopis used in the studies . In one cattle study, PJ seedpods induced neuronal vacuolation, loss of neurons, degeneration of nerves and atrophy of masseter and other masticatory muscles after 200 days oral exposure (Tabosa et al ., 2006) [30]. In goats, after 270 days oral exposure, neuronal vacuolation of trigeminal nuclei, Wallerian degeneration in mandibular and trigeminal nerves and atrophy of associatedmuscles were observed (Tabosa et al ., 2000) [31]. Findings of these studies suggest that long term exposure (about 200 days or more) PJ have toxic effect in animals while short term exposure may be unable to produced appreciable gross or microscopic changes.

Repeated dose 28-days oral toxicity and efficacy of PJSPE on induced osteoarthritis in Wistar rats

All 48 rats of the study were survived up to terminal scarifies. Rats in the treatment group of progressive and reversal group did not show any in-life observations and were comparable to control of their respective phase. The body weight summary rats of all groups are presented in Table 2 and3. Body weight of rats in the treatment group of progressive and reversal group did not show any significant changes and were comparable to control of their respective phase. The haematology summary of rats of all groups is presented in Table 4 and5. Haematology of rats in the treatment group of progressive and reversal group did not show any significant changes and were comparable to control of their respective phase. The clinical pathology summary of rats of all groups is presented in Table 6 and7. Clinical pathology of rats in the treatment group of progressive and reversal group did not show any significant changes and were comparable to control of their respective phase. During full detail necropsy on study day 29 th and 57 th , rats from all treated group did not show any test article related changes in any organs and were comparable to controls of their respective phase. Absolute and relative organ weight of rats in the treatment group of progressive and reversal group did not show any significant changes and were comparable to control of their respective phase. The absolute and relative organ weigh summary are presented in Table 8 and 9 and, Table 10 and 11 respectively. Osteophytes are characterised by formation of fibrocartilage like mass at the junction of synovium attached to cartilage or at the site of ligament or tendon insertions. In few cases, pannus formation was noted, characterized by formation of fibrovascular tissue that cover tibial plateau cartilage . In few rats, articular cartilage was markedly atrophied due to necrosis and sub -cartilage fibrosis was noted .

In progressive phase, on 29 th day of the study, rat suffered from OA showed fibrillation, fissure, erosion, loss of proteoglycans content on toluidine blue staining and minimal fibrosis. Summary of modified Mankin score and Pritzkerscore of joints of are presented in Table 13 and 14 respectively. Lesions were more severe in tibial plateau than femoral condyle in Group I and IV. No significant changes were observed in Pritzker score of the Group I and IV. In modified Mankin score, in control group, cartilage cellularity was slight decrease in four rats while moderately decrease in two rats. In Group IV, cartilage cellularity was slight decrease in four rats while moderately decrease in two rats. On toluidine blue staining, moderate loss of proteoglycans was evident in all six rats of Group I and IV (Figure 1). Superficial and transitional zone zones were more severely affected, and loss was staining was extended up to radial zone in few animals (Figure 2). Tidemark, a basophilic line between the cartilage and calcified cartilage was abnormal in three control rats and four rats of Group IV. The lesions in femoral condyle were less severe than tibial plateau and severity of modified Mankin score in Group IV was comparable to Group I. No significant changes were observed in modified Mankin score of the Group I and IV and findings of Group IV were comparable to Group I. Oral administration of PJSPE for 28 days did not show any effect on progression/advancement of histopathological lesions of OA in MIA induced OA at 1000 mg/kg dose. In reversal phase, on 57 th day of the study, rat suffered from OA showed all the lesions mentioned in 29

th day of the study with more severity. Summary of modified Mankin score and Pritzkerscore of joints are presented in Table 13 and 14 respectively. On 57 th day of the study, loss of cartilage (Figure 3), fibrosis (Figure 4) involved sub cartilage area and bone marrow space. Multinucleated giant cells numbers were increased in comparison to day 29 th of the study. No significant changes were observed in Pritzker score of the Group V and VIII. In modified Mankin score, in six rats of Group V and four rats of Group VIII, clefts/fissures were extended up to radial zone. Cartilage cellularity was moderately decrease in six rats of Group V and five rats of Group VIII. In one rat of Group VIII, cartilage cellularity was severely decrease. On toluidine blue staining, no staining was noted (severe loss of proteoglycans) in two rats of Group V and VIII, while three and four rats of Group V and VIII showed severe loss of toluidine blue staining. Tidemark loss, advancement, or duplication was noted in five rats of Group V and six rats of Group VIII. The lesions in femoral condyle were less severe than tibial plateau and severity of modified Mankin score in Group VIII was comparable to Group V. No significant changes were observed in modified Mankin score of the Group V and VIII. Oral administration of PJSPE for 28 days did not show any ameliorative effect on histopathological lesions of OA in MIA induced OA at 1000 mg/kg dose.

In the present study, oral administration of PJSPE at 1000 mg/kg for 28 days did not produce any pathological changes in organs. However, in past studies, dry ground pods of PF to cattle for 200 days or to goat for 270 days produced neuromuscular abnormality (Tabosa et al ., 2000, 2006) [30,31]. Study duration, species, and dose may be responsible for such discrepancy. In previous mentioned studies, cattle, or goat were fed ground pods of PF either 70% or 90% of dry matter basis for more than 200 days. Such high and long exposure of some unidentified toxicant present in PF may produce pathology in neutrons or muscles.

In the present study, monosodium iodoacetate at 1 mg/joint induced articular cartilage fibrillation, fissure, eburnation/ denudation, fibrosis, thickening of subchondral bone plate, pannus, and osteophyte formation. In the present study, lesions were milder in progressive phase (29 th day of OA induction) in comparison to regressive phase (57 th day of OA induction). Similar findings have been noted in previous studies (Guzman et al ., 2003; Takahashi et al ., 2018) [6,33]. In present study, lesions were more severe in medial tibial plateau than lateral tibial plateau and femoral condyle, which is in accordance with previous studies (Guzman et al., 2003; Takahashi et al ., 2018) [6,33]. This may be attributed to biomechanics of the rat stifle joint, in which medial compartment naturally load higher than the lateral compartment of the femorotibial joint. In 2010, OARSI published recommendations for histological assessments of OA in rat (Gerwin et al ., 2010) [5]. Even though this is probably more accurate histological assessments of OA in rat, due to complexity and applicability to MMT model only, this system does not become popular. Even after publication of this recommendations, modified Mankin score, or OARSI score proposed by Pritzker et al . (2006) [23] are more commonly used to for histological scoring of OA in rat (Shi et al ., 2019; Takahashi et al ., 2019) [28,34]. In present study, we used both the system to score OA. In our experience, OARSI score proposed by Pritzker et al . (Pritzker et al ., 2006) [23] is more accurate and easier to apply to evaluate OA model in rat than modified Mankin score. Forced ambulation, weight bearing test, tactile allodynia (Von Frey test), and radiographic examination are some tests used by research to access OA related pain, behaviour changes and cartilage abnormalities in past (Choi et [4,13,16,18]. Serum collagenases (MMP-3 and MMP-13), C-telopeptides of type II collagen (CTX-II), insulin-like growth factor-1 (IGF-1) and Osteopontin (OPN) are some biomarkers used by various researchers to evaluate the effect of various compound in induced OA rat model ( [2,10,12,15] . In the present study, we have not evaluated any pain related behaviouralteration or OA-related serum, urinary or synovial biomarkers. Researchers tested Prosopis juliflora extracts against some [1,25,37] . In some case, PF seed pods are effective in amelioration of clinical OA in humans (personal communication with Ayurvedacharya). In present study,

R protective or ameliorative effect on MIA induced OA. This is probably the first in which LD 50 of PJSPE was derived and in vivo toxicity and efficacy of PJSPE on experimentally induced osteoarthritis in Wistar rats was evaluated.

Acknowledgement

AcknowledgementAuthors are thankful to the Dean & Principal, College of Veterinary Science and Animal Husbandry, Kamdhenu University, Sardarkrushinagar, for providing necessary facilities and sanctions for conduct of research work.

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