Anti-Arthritic Activity of Annona Reticulata Leaves Methanolic Extract on Adjuvant Induced Arthritis in Rats

Research Article

Austin Med Sci. 2023; 8(1): 1074.

Anti-Arthritic Activity of Annona Reticulata Leaves Methanolic Extract on Adjuvant Induced Arthritis in Rats

Suneel Kumar Akunuru¹*; Venkatarathanamma Vakita¹; Varsha Kommajosyula²

¹Acharya Nagarjuna University, Andhra Pradesh, India

²SRM Institute of Science and Technology, Tamil Nadu, India

*Corresponding author: Suneel Kumar Akunuru Acharya Nagarjuna University, Andhra Pradesh, India. Email: [email protected]

Received: March 22, 2023 Accepted: May 06, 2023 Published: May 13, 2023


The research is expected to assess the effect of Annona Reticulata Methanolic leaves Extract (ARME) on progression of adjuvant induced arthritis in Wistar rats. ARME was obtained through bioactivity guided fraction by 5-Lipooxygenase inhibitory activity. ARME was administrated at 100 and 200mg/kg body weight via oral route for a period of 35 days to the experimental models. On day 8, via a single intra-plantar injection of 0.1mL suspension of heat killed Mycobacterium tuberculosis (100μg/animal) Arthritis was induced, in incomplete Freund’s adjuvant of left foot pads of Wistar rats (female). The paw volume was observed by recording the hematological and biochemical parameters, hind limb paw volume, pathological and radiography changes to evaluate the effect of the extract. On day 35, levels of pro-inflammatory cytokines (TNF-a, IL-1Β) and inflammatory mediators (PGE2, LTB4) was measured in serum samples. On the day of sacrifice, the liver was collected to record the stabilizing ability of lipid peroxide, activities of enzymatic antioxidants catalase, Superoxide Dismutase (SOD) and non-enzymatic antioxidants reduced glutathione (GSH) levels. ARME treated groups; 100 and 200mg/kg and prednisolone 10mg/kg significantly decreased hind paw volume, reduced serum levels of TNF-a, IL-1Β, PGE2 and LTB4, reduced MDA levels and increased levels of catalase, SOD and GSH in the liver. The promising results of serum biochemistry, haematology, histopathology and radiographic changes suggest that the administration of ARME was able to effectively modulate the inflammatory response and conquer the advancement of arthritis in the experimental animal model. Results obtained from the acute oral toxicity limit test establish that ARME (LD50>2000mg/kg) was safe in accordance to the OECD guideline 423. These findings may help to overcome some hurdles in the treatment of rheumatoid arthritis.

Keywords: Rheumatoid arthritis; Annona reticulata leaves; Methanolic extract (ARME); Inflammation; 5-Lipooxygenase; TNF-a; IL-1Β; PGE2; LTB4


Annona reticulata, most commonly known as ramphal in the Asian subcontinent in Southern Asia belongs to the family of Annonaceae. Most species of this family are known to contain medicinal properties which play a key role in metabolism. The extracts obtained from each part of the plant range through various medical potential. Annonaceous acetogenins have been known to have potential as anticancer agents. The extracts were obtained from the stems as well as the barks of Annona reticulata [1-4]. The role of herbal alternatives to manufactured medicines is gaining momentum in today’s world more specifically, the function in the treatment of arthritis.

The human body has various mechanisms in place to prevent infections/disease. One such protective mechanism is inflammation; which is a defense mechanism against toxins, foreign substances and microbes invading the human vessel, disrupting homeostasis [5]. Rheumatoid Arthritis (RA) is an autoimmune disorder where in, deregulated white blood cell i.e., neutrophil activation leads to RA [6]. One of many mechanisms involved in RA is the release of ROS and other proteolytic enzymes which involves both; the innate and acquired immune systems [2]. RA is an inflammatory condition which affects ~1% of adults, which may lead to premature death or physical disability if not addressed in time [7]. RA has a higher occurrence in females as compared to males at an age group above 50. Various pathways within the human body are involved in the inflammatory process. Arachidonic Acid (AA) metabolism plays a major role in the same. Most manufactured drugs block the Cyclooxygenase (COX) and Lipoxygenase (LOX) pathways. As any other drug, the anti-inflammatory drugs display many side-effects which range from gastrointestinal irritation, ulcers to hypertension and cardiac abnormalities [8, 9].

Many intermediary mediators such as PGE2, LTB4 play a role in the COX and LOX pathways and thus can be the potential target to prevent inflammation [10]. Hence, herbal sourced products are more reliable and cause fewer side effects to those of manufactured drugs. They were used as 5-LOX inhibitors to treat inflammatory disorders such as arthritis [11].

Annona reticulata leaf extracts were tested for its various effects on the human body such as: antioxidant, anti-hypersensitive, anti-diabetic, anti-microbial, anti-inflammatory activities. The role of the leaf leaf extract was not explored in inflammatory and arthritis models to test its efficacy. Hence, the properties of the methanolic extract from the leaves of Annona reticulata were tested for 5-lipoxygenase inhibition and its role in treatment of arthritis.

Materials and Methods

Plant Material and Preparation of Extract

Annona reticulata plants’ leaves were obtained from the forest area of Kondapalli, Andhra Pradesh, India, in January. The authentication of the plant was done in Department of Botany from Acharya Nagarjuna University by Prof. Dr. S. M. Khasim.

The initiation of the experiment was done by drying the leaves in the absence of sunlight followed by severing the leaves into small pieces. The pieces were ground into a coarse powder. Using the Soxhlet extraction method, the powdered material was extracted using: Hexane, Ethyl Acetate, Methanol and water. The raw material of the leaves was then extracted using the non-polar to polar grade above solvents (hexane, ethyl acetate, methanol, aqueous methanol, and water) using soxhlet apparatus.The extracts were concentrated and later dried using a rotary evaporator under limited pressure. 5-LOX; potent inhibitory fraction was obtained through bioactivity fractionation was often used for in vivo efficacy and toxicity studies. The most potent 5-LOX inhibitor was that of the methanolic extract (ARME).

Experimental Animals

Strain of Female Wistar rats weighing from a range of 180g to 220g were housed in a group of 3 per cage. Cages were made of polypropylene and stainless steel with the bedding of paddy husk and standard rodent diet of Nutrilab manufactured in Provimi Pvt. Ltd. Water used was UV sterlised supplied ad libitum to all cages. The temperature was maintained at 22±3°C, humidity of 30% - 70% and light (12h light and 12h dark). All experiments involving animals were performed in line with the standard protocol and guidelines of the Animal Ethical Committee, Govt. of India Reg.No. 1629/PO/a/12/CPCSEA, after proper approval.

In vitro 5-Lipoxygenase (5-LOX) enzyme inhibitory assay 5-LOX enzyme inhibitory activity of AMME was measured using the method of [12,13]. The assay mixture contained 80M linoleic acid and 10 μL potato 5-lipoxygenase enzyme in 50 mM phosphate buffer (pH 6.3). The reaction was initiated by addition of the enzyme buffer mix to linoleic acid (substrate) and its activity was observed as increase in absorbance at 234 nm.

The reaction was observed in UV-Kinetic mode on Varian Cary-50 UV- Vis spectrophotometer for 2 minutes. Various test substances were used to observe the inhibitory potential at different concentrations of the test substances, for 120 seconds before addition of the substrate. Assays were done in triplicates and an average was used for calculation. Inhibition percentage was calculated by comparing increase of absorbance to control of enzyme activity. The activity of plantextracts was compared with the standard positive control Nordihydroguaiaretic acid (NDGA).

Acute Toxicity Study

The acute toxicity of ARME was determined as per the OECD 423 guideline (Acute oral toxicity class method) [14]. Six animals (Step I and II) were used for screening of acute oral toxicity. The animals were observed for clinical signs for a period of 2 weeks/14 days after a single oral dose of ARME was administered at 2000mg/kg body weight.

Adjuvant Induced Arthritis

In vitro screened potent bioactive fraction was used to test its efficacy in vivo for chronic inflammation using Freund’s Complete Adjuvant [FCA] Induced Arthritis in Wistar rats as the experimental model. Wistar rats were divided into 4 groups of 6 animals per group. Group I animals served as arthritic control and received 0.5% CMC. The required quantity of test substance was weighed using an analytical balance and transferred into a mortar and pestle for further breakdown. The required volume of CMC was added and triturated to obtain a final concentration of 10 and 20mg/mL of homogenized test substance. For positive control, standard drug Prednisolone 10mg/kg in 0.5% CMC was used. From day 0 to day 35, animals were administered their respective dose (dose volume calculated based on measured body weights recorded on a weekly basis) based on the group, via the oral route. After dose administration, the rats were immediately transferred to the respective cages. The treatment regimen was as follows; Group I- arthritic animals without drug treatment, Group II- arthritic animals treated with ARME (100mg/kg), Group III- arthritic animals treated with ARME (200mg/kg) and Group IV was arthritic animals treated with Prednisolone (10mg/kg).

Arthritis was induced in all groups of Wistar rats on day 8. This was done using a single intra-plantar injection of 0.1mL suspension of heat killed M. tuberculosis (100μg/animal) incomplete Feund's adjuvant (Sigma-Aldrich, USA), into the sub plantar region of left hind limb. [FCA Preparation: The required quantity of heat killed M. tuberculosis H37Ra (Difco laboratories) was suspended in Freund (100μg/animal) in incomplete Feund's adjuvant (Sigma-Aldrich, USA), into the sub plantar region of left hind limb. [FCA Preparation: The required quantity of heat killed Mycobacterium tuberculosis H37Ra (Difco laboratories) was suspended in Freund’s Incomplete Adjuvant and triturated using a mortar and pestle to obtain a FCA suspension containing 1mg/mL of Mycobacterium tuberculosis H37Ra].

Animals were observed for clinical signs once day, everyday throughout the duration of the study. Before administration of dose body weights were recorded and weekly once there after during the course of the study and prior to the termination of the study. Paw volume was measured using a plethysmometer (UGO basile) on days 0 (prior to treatment), 7 (prior to AIA induction), 20, 24, 28 and 35. Oedema volume was calculated as the difference between the final and initial paw volume. The percent inhibition of oedema volume was calculated through the formula;

{(Vc-Vt / Vc)} X 100


V0 - Oedema volume of normal control group

Vc- Oedema volume of AIA control group

Vt - Oedema volume of treatment group

Blood Analysis

On day 35, blood was collect through a small puncture in the retro-orbital plexus, under isoflurane anesthesia. Using a Humstar 600 automated biochemistry analyser blood was centrifuged at a speed of 1500 rpm for a short span of 15 minutes total bilirubin, total protein, albumin, cholestrol, triglycerides, creatinine, urea and CRP AST/SGOT, ALT/SGPT and to separate serum from the sample. On day 35, the rats were sacrificed and blood was collected in 1% EDTA sample tubes for hematological analysis i.e., RBC, WBC, heamoglobin (Hb), Lymphocytes and PCV using an Automated Hematology Analyzer (Humacount 60ts, HUMAN and Germany). ESR (Erythrocyte Sedimentation Rate) is an assay used to screen for an active disease (presence or absence) and measured using modified Westgren Method in mm/hr.

Cytokines and Inflammatory Mediators

Blood was collected by retro-orbital plexus puncture on day 35 under isoflurane anaesthesia. The serum was separated by centrifugation at 1500rpm for 15 minutes and the samples were analysed for pro-inflammatory cytokines (TNF-a, IL-1Β inflammatory mediators PGE2 and LTB4) levels in serum was estimated using ELISA assay kit (R&D systems) according to manufacturer’s protocol. Lastly, the specific chromophore reactions were read at a wavelength of 540nm in a micro well plate ELISA reader (BioRad, USA).

Antioxidant Studies

All experimental rats were euthanized through an overdose of ketamine followed by necropsy after termination of the study. The liver was collected and washed with cold saline, preserved at a temperature of -20°C until antioxidant analysis. Bradford Assay was performed for protein estimation in liver samples (using 20% liver homogenate), for the different groups of rats.

The enzymatic antioxidant activities like catalase estimated by Sinha 1972, Superoxide Dismutase (SOD) estimated by Kakkar and the non-enzymatic antioxidant activities like reduced glutathione (GSH) estimated by Ellman.

Histopathology and Radiology of Knee Joint

The tibio-tarsal joint of all animals were dissected, fixed using 10 % buffered formalin and later decalcified in a 1:1 formic acid and distilled water for 7 days, after termination of the study. Dehydration of tissue samples at various concentrations of isopropyl alcohol i.e. 70, 80, 90 and 100 % was performed for a time period of two hours. It was then processed for paraffin embedding and sectioned into a thickness of 3-5 μm. The slides were examined under light microscope for histopathological lesions after staining using Hematoxylin and Eosin (H&E) and Toluidine Blue (TB). The ankle joints were fixed in 10% buffered formalin and was subjected to radiography on Siemens triodors (100 MA at 100cm focal distance, 40 KV and 2 mAs exposure). The lateral radiographs were retained and exposure was recorded.

Statistical Analysis

Analysis of data was performed using one-way ANOVA as the primary test followed by Dunnett’s test in graph pad prism software. All results were expressed in the form of Mean ± S.D and considered significant only if P>0.05.


5-lipoxygenase Inhibitory Activity

5-Lipoxygenase enzyme inhibitory activity of Annona reticulata extracts ARHEX, ARET, ARME, ARAM and ARW exhibited inhibitory activity 5-Lipoxygenase enzyme by showing IC50 values as 389.12, 191.10, 49.61, 111.37 and 135.71μg/mL respectively, Standard NDGA showed an IC50 value of 5.31 μg/mL.5-Lipoxygenase enzyme inhibitory action of Annona reticulata was depicted Figure 1.