Urinary system is a collection of organs (kidneys, bladder, urethra and ureters) in the body associated with filtering out exceeding amount of fluid from the blood helping to preserve homeostasis and kidneys are the prime organs, since they control the acid base equilibrium and the blood balance of the water and salt. The cycle of excretion is the prime function with the control of concentrations of varying electrolytes in bodily liquids and the preservation of regular blood potential of hydrogen ions (pH) levels^1,2. Management of ion composition of plasma ions, its osmolarity and concentration of plasma hydrogen ions (pH) constitute the majority parts of kidney function³.

The filtering membrane consists of the triple framework offering protection and specific characters needed to create the primary glomerular filtrate, i.e. the ultra-filtrate fenestrated mucosal lining of the glomeruli capillaries, the basement membrane and the podocytes filtering blood, that are transformed into urine by the tubular network⁴. Over period and aging there is a decrease of both glomerular filtration rate (GFR) and renal flow of renal plasma which starts at around 30 years of age⁵. Hyperuricemia is a clinical disorder marked by excessive production or under-secretion of uric acid, generally defined as serum urate (SU) > 7 mg/dl, may occur in up to eighteen per cent of certain populations where its most prominent diagnostic symptoms are characterized by crystallization and accumulation of uric acid in joints and underlying tissues but the exact mechanism for uric acid mediated tissue trauma remains uncertain. Presently, chronic hyperuricemia therapy agents can be classified into dual major classes: uricostatic drugs (e.g. allopurinol) by competitively inhibiting xanthine oxidase and uricosuric drugs (e.g. probenecid) that disable urate reuptake in the renal tubular system⁶. The strength of latest research supports the notion that hyperuricemia is a significant trigger for ischemic heart disease while insulin resistance can be indirect and influenced by elevated rates of plasma triglycerides; also urate inhibits the release of insulin in differentiated pancreatic islets of rats and prevents the insulin release stimulated by glucose⁷. Approximately twenty out of hundred people on allopurinol which is popularly used in this indication have recorded adverse effects due to mainly hypersensitive allopurinol syndrome (e.g., rash, cough, hepatitis, eosinophilia, renal failure)⁸. Further most noted side effects entail gastrointestinal aversion, alopecia, suppression of the bone marrow and even can cause death. Many side effects occur like nausea, vomiting, neuro-muscular diarrhea also due to colchicine therapy⁹. Capparis moonii Wight, in Marathi Waghati, Rudanti in Sanskrit, adonda in telugu is widely acknowledged as Indian caper or large capers, also is a member of the Capparidaceae family (Capparadaceae), typically occurring in the Konkan area, and is seen to be growing vigorously under hot semi-dry conditions¹⁰. Its fruits contain beta-sitosterol, l-stachyhydine and rutin^11-14. On the basis of the data that flavonoids show great action in hyperuricemia, and also considering the amount of side effects involved with regular therapy, the present study aimed at evaluation of anti-hyperuricemic activity of these caper fruits alcohol extract which was not yet ventured.

MATERIALS AND METHODS:

Collection and authentication of plant:

Dried fruits of Capparis moonii W. from Hamidiya Dawakhana, Pydhony, Bhuleshwar, Mumbai were collected and authenticated by Dr. (Mrs.) Bindu Gopalkrishnan, Asst. Professor, Department of Botany at Mithibai College of Arts, Chauhan Institute of Science and Amrutben Jivanlal College of Commerce and Economics, Mumbai and samples were sent to the Pharmacology Department, Oriental College of Pharmacy, Sanpada, Navi Mumbai- 400705.

Preparation of extract:

Capparis moonii Wight dried fruits were grinded to produce a nearly smooth powder, and they were sieved for fine powder through the mesh. Capparis moonii W. powdered plant fruits (142gm) were placed in the Soxhlet extractor extraction compartment using cotton inserts to filter the solvent (ethanol 90 percent v/v) returning from crude powder to the round bottom flask for 72 hours. The siphon tube drained the compartment, with solvent flowing back into the bottom round flask. This loop was replicated until the powder was consumed, which was detected when the liquid in the siphon tube was clear. After the extracts have been evaporated to get the dried crude form, they were then placed in an appropriate container and stored in a refrigerator covered appropriately at 4°C till the time for their use. Alcoholic extract percentage yield was 7.88 per cent w/w. The entire study was then done on the basis of Capparis moonii W. alcoholic extract.

Qualitative phytochemical screening:

Preliminary chemical studies were performed on Capparis moonii W. alcoholic extract. The phytochemicals such as carbohydrates, flavonoids, glycosides, tannins, saponins, fixed oils/ fats, alkaloids, phenolic compounds, sterols/terpenoids and proteins were assessed to see if present.

Animals:

Animals procured were both male and female Albino Wistar Rats (140 to 200g) for carrying out acute toxicity which were obtained from Bombay Veterinary College, Parel, Maharashtra and male Albino Wistar Rats (140 to 200g) were acquired from Bharat Serums and Vaccines Limited, Thane West, Maharashtra for models, approved by the Institutional Animal Ethics Committee. The animals were kept in well ventilated, air-conditioned animal house at a constant temperature of 22±2°C, with a relative humidity of 55-60%. The animals were placed on bedding material, in spacious polypropylene cages with a paddy husk. The animals were held on normal diet with pellets and filtered water.

Acute oral toxicity:

The Acute oral toxicity test was performed with adherence to the Organisation for Economic Co-operation and Development (OECD) guidelines 423 where a total of nine animals in the weight range of 140 to 200g were first brought and acclimatized for about a week. They were then grouped in three groups as lower concentration (50mg/kg), intermediate (300mg/kg) and higher concentration (2000mg/kg) and fasted 2 hr before the administration of the fruit extract. After this they were observed for any abnormalities at 15, 30, 60, 120 and 180 minutes on 1^st day and then once a day till the 14^th day.

Drugs:

For standard Stamlo 2.5mg (Amlodipine) tablets (marketed by Dr. Reddy’s Laboratories Ltd., India) and Zyloric 100mg (Allopurinol) tablets (by Glaxosmithkline Pharmaceuticals Ltd., India) were acquired from a registered pharmacy shop. Phenol red (Kiran light Laboratories Pvt Ltd, Mumbai, India) was used. For inducing hyperuricemia, Potassium oxonate salt >98.0% (TCI Chemicals Pvt Ltd., India) was purchased. Uric acid and creatinine kits for bioanalysis were purchased from Transasia Bio-medicals Ltd., Mumbai, India.

Instruments:

Chem 7 Bioanalyzer by Erba Mannheim, London, United Kingdom, Micro centrifuge by Bio-lab, Mumbai, India and Ultraviolet Visible Spectrophotometer UV-1800, Shimadzu, Mumbai, India.

Preparation of Formulation:

Preparation of Standard: Tablets of Stamlo 2.5mg (Amlodipine) and Zyloric 100mg (Allopurinol) were taken and crushed to make a fine powder. The powder was then used to prepare the suspension with 0.5% w/v viscosity builder Sodium carboxy methyl cellulose (i.e. sodium CMC).

Preparation of negative control:

Phenol red was taken and added to saline solution to make a 3% saline solution. Potassium oxonate salt was used to prepare the suspension with 0.5% w/v sodium CMC.

Anti-hyperuricemic study:

A) Phenol red excretion in rats

The rats were bifurcated into five groups with each group having 6 animals. The groups formed were Vehicle Control (0.5% NaCMC solution), standard (Amlodipine 1.8mg/kg), disease control ((phenol red 2.5 ml/kg), test groups of Capparis moonii Extract (CME) 100mg/kg and 200mg/kg. Wistar rats (male) having weight 140–200g were treated orally with the test compound (CME) of fruits or the standard (Amlodipine) suspended in 0.5 per cent (Na-CMC) 30 min prior to intravenous injection via the tail vein with 2.5ml/kg of a 3% Phenol Red in saline (Phenol sulfonphthalein). After the treatment, the Retro-orbital method was used to withdraw the blood samples after 30 and 180 min. Blood (0.2ml) was diluted with 2ml 0.9% sodium chloride (NaCl) solution and centrifuged. To 1ml of the supernatant, 1ml of 1% sodium carbonate solution and 8 ml of saline was added. Using spectrophotometer, extinction at 546nm was determined.

B) Potassium oxonate induced hyperuricemic model:

The rats were bifurcated into five groups with each group having 6 animals. The groups formed were Vehicle Control (0.5% NaCMC solution), standard (Allopurinol 10mg/kg), disease control (potassium oxonate in 0.5% NaCMC solution 250mg/kg), test groups (CME) 100mg/kg and 200mg/kg. Hyperuricemia was generated by injecting 250mg/kg intra-peritoneally with potassium oxonate injection suspended in 0.5 per cent (Na-CMC) twice, in two hrs fasted rats before the experiment began, to increase serum urate levels and these acted as hyperuricemic rats. The standard and test substances were suspended in 0.5 percent (Na-CMC) and given to the rats daily for at least five days in a row, one hr. post the potassium oxonate injection. Blood was obtained by retro-orbital plexus from rats and they were sacrificed after the collection of blood. The blood was permitted to clot for about one hour at room temperature and then centrifuged for 20 min at 1,000 x g to get the serum. The serum was deep freezed and preserved until it was assayed.

Statistical analysis:

The data were analyzed by means of GraphPad (version 3.05, 32 bit for Win 95/NT) with InStat Software. For each category the results are expressed as mean±SD. A one-way variance analysis (ANOVA) was used to analyze statistical differences followed by the Tukey-Kramer Multiple Comparisons test.

Tests at P<0.05 were considered statistically significant.

* * * * refers to p<0.001, * * refers to p < 0.01; # refers to p < 0.05.

RESULTS:

Qualitative phytochemical screening:

Capparis moonii W. qualitative phytochemical analysis of alcoholic extract indicated that carbohydrates, flavonoids, glycosides, tannins, saponins, fixed oils/fats, alkaloids, phenolic compounds, sterols were present while terpenoids and proteins were identified as absent. The presence of flavonoids and polyphenols and the other constituents contained in the extract, significantly correspond to the Capparis moonii Wight's anti-hyperuricemic activity in the alcoholic extract.

Acute oral toxicity studies:

Acute oral toxicity analysis was performed as recommended in OECD Guideline 423 and the findings showed that no toxic signs were found in clinical parameters during an acute toxicity test of up to 2000mg/kg. Therefore, it shows that the median lethal dose (LD₅₀) of Capparis moonii W. dried fruits alcoholic extract equals 2000mg/kg. Following detailed survey of different research papers and directed by the guide, doses were selected as 100mg/kg and 200mg/kg for the test groups.

Anti-hyperuricemic study:

A) Phenol red excretion in rats

Both the alcoholic, fruit extract test groups of Capparis moonii W. displayed dose-dependent activity behavior, revealed in the examination of extinction of phenol red wherein the higher dose retained more phenol red in blood plasma opposed to control and similar to amlodipine used as a standard. (More retention of phenol red in blood plasma implies better removal of uric acid from the blood).

Fig 1: Graphical representation of effect of alcoholic extract of fruits of Capparis mooni W. on concentration of Phenol red in blood plasma samples (μg/ml).

Note: Values are expressed as mean ± standard deviation (SD) (n=6). ***P<0.001 compared with toxicant control, **P<0.01 compared with toxicant control and #P<0.05 compared with Vehicle control. Data was analyzed using one-way ANOVA followed by Tukey-Kramer Multiple Comparisons test.

B) Potassium oxonate induced hyperuricemic model:

It showed significant reduction in the uric acid and creatinine levels in dose dependent manner.

Uric acid:

Fig 2: Graphical representation of effect of alcoholic extract of fruits of Capparis mooni W. on concentration of uric acid in blood serum samples (mg/ml).

Note: Values are expressed as mean ± SD (n=6). ***P<0.001 compared with toxicant control, *P<0.05 compared with toxicant control and ###P<0.001 compared with Vehicle control. Data was analyzed using one-way ANOVA followed by Tukey-Kramer Multiple Comparisons test.

Creatinine:

Fig 3: Graphical representation of effect of alcoholic extract of fruits of Capparis mooni W. on concentration of creatinine in blood serum samples (mg/ml).

Note: Values are expressed as mean ± SD (n=6). ***P<0.001 compared with toxicant control, **P<0.01 compared with toxicant control and ###P<0.001 compared with Vehicle control. Data was analyzed using one-way ANOVA followed by Tukey-Kramer Multiple Comparisons test.

Histopathology studies:

Histological examination of liver and kidney tissues of rats induced with potassium oxonate to induce hyperuricemia was performed.

The liver tissues of the rats in all the groups i.e. vehicle control, standard group, potassium oxonate disease control group and test groups of 100mg/kg and 200mg/kg all showed no abnormalities in the observation.

Similarly, no abnormalities were detected in the tissues of kidney of the vehicle control group.

The group given only potassium oxonate as disease control showed major abnormalities of kidney tissues like moderately multifocal deposits of yellowish white crystals which were noted in renal tubules and occasionally in glomeruli. The affected areas showed degenerative changes of tubular epithelium, atrophy and/or cystic dilations of the tubules. In comparison with both control group and disease control group, the standard group kidney tissues displayed only occasional foci of yellowish white crystal deposits, which were noted in renal tubules and occasionally in glomeruli.

Simultaneously the test group of lower concentration (100mg/kg) showed abnormalities like moderately multifocal deposits of yellowish white crystals which were noted in the renal tubules and occasionally in the glomeruli. The affected areas showed atrophy and/or cystic dilations of tubules which resembled to that of disease control group, the test group of higher concentration (200mg/kg) showed better results with minimally multifocal deposits of yellowish white crystals which were noted in renal tubules and occasionally in glomeruli. Occasional foci of atrophy and/or cystic dilations of tubules were seen like that of the standard group.

On comparison, test group of lower concentration displayed moderate abnormalities while the test group with higher concentration showed minimal or occasional instances of abnormalities proving that the higher concentration of 200mg/kg showed better action in dose-dependent manner as compared to lower concentration of 100mg/kg and also it gave similar effect in protecting tissues from abnormalities caused by hyperuricemia like the standard group.

DISCUSSION:

Hyperuricemia constitutes as a significant factor of threat for gout growth. Hyperuricemia is caused by a spike in uric acid manufacturing; decrease in the instances of removing uric acid from the kidney or a combo of these processes. Gout production includes 3 steps: chronic hyperuricemia, the production of crystals of monosodium urate monohydrate (MSU), and connection of MSU crystals and the inflammatory system¹⁵. While hyperuricemia is spread across the globe, there are small numbers of agents for decreasing the uric acid in the blood and their usage is often restricted due to unwanted side effects¹⁶. A possible source of new anti-hyperuricemic substances can thus be obtained from the natural source¹⁷.

Uric acid is produced when hypoxanthine is converted to xanthine and then by Xanthine oxidase it gets converted to uric acid. Over manufacturing or removal of uric acid from the body leads to hyperuricemia that occurs in approx thirty per cent of the population is a growing global issue and substances that demonstrate the capability to increase uric acid removal from system or those that hamper uric acid synthesis have been approached for hyperuricemia therapy¹⁸. There was also a correlation between the elevated threat of hyperuricemia and the occurrence of hyperlipidemia, hypertension, diabetes, obesity and cancer¹⁹. According to literature survey, fruits of plant contain Beta-sitosterol, l- Stachyhydrin, Rutin, Gallotannins and Chebulinic Acid²⁰. Rutin has shown beneficial effect as a hypouricemic agent²¹. Commonly used extraction solvents for flavonoids like Rutin are alcohols²². Thus, the reason to perform this present study was to estimate the anti-hyperuricemic activity of alcoholic extract of Capparis moonii W. dried fruit which is a natural caper fruit rich in flavonoids.

Capparis moonii W. alcoholic extract was used to estimate the phenol red excretion in rats where it was observed that the extract showed dose-dependent type of action where the higher concentration of 200mg/kg showed higher amount of retention of phenol red in the blood suggesting that it has better ability to secrete urate out of the body of rats. In case of potassium oxonate induced hyperuricemia, similar results were obtained as 200mg/kg concentration showed significant reduction in serum uric acid levels as well as serum creatinine levels as compared to 100mg/kg concentration having near values to that of vehicle control and standard group. Therefore, it can be said that the test groups were significantly successful in lowering hyperuricemia.

Phenol red is a sulphonic acid dye also known as phenolsulphonphthalein²³. In models of phenol red excretion, uricosurics such as amlodipine cause a significant rise in the plasma concentration of phenol red in the blood which is used to calculate their ability to expel uric acid²⁴. Potassium oxonate is a salt variant of oxonic acid that serves as a selectively competitive uricase inhibitor and inhibits the action of hepatic uricase, which causes hyperuricemia in rodents²⁵.

One of the flavonoids present in Capparis moonii W. fruits is rutin. Earlier research verified that rutin decreased uric acid serum levels in hyperuricemic rodents. Urate transporter 1 (URAT1) and glucose transporter 9 (GLUT9) regulate the reuptake of renal urate while organic anion transporter 1 (OAT1) facilitates urate secretion and plays a significant role in the development of progressive kidney disease. It was capable of exhibiting anti-hyperuricemic impact by strikingly down-regulating kidney messenger ribonucleic acid (mRNA) and protein levels of messenger glucose transporter 9 (mGLUT9) and down-regulating kidney messenger urate transporter 1 (mURAT1) mRNA and protein levels resulting in lower availability of these respective sites for urate reabsorption while it increased renal messenger organic anion transporter 1 (mOAT1) expression which in turn enhanced urate secretion in rodents. The findings of the ongoing study indicate that Capparis moonii W. alcoholic extract can be beneficial in hyperuricemia as a result of its flavonoid content²¹.

CONCLUSION:

The findings of the current study indicate that Capparis moonii W. alcoholic extract of the fruits can be beneficial for hyperuricemia treatment. Additional studies are suggested to classify the active phytochemicals and illustrate the mode of action.

ACKNOWLEDGEMENT:

We would like to thank the Management for their support and the Pharmacology Dept. of Oriental College of Pharmacy for the work performed. We would also like to acknowledge Dr. Mrs. Mrinal Sanaye, I/c HOD, Dept. of Pharmacology, Prin. K.M. Kundnani College of Pharmacy for her support.

AUTHORS’ CONTRIBUTIONS:

Dr. (Mrs.) Vanita Kanase guided with designing the study, making of protocol and managed the work done. Shruti Shettigar performed the literature searches, performed the acute toxicity, models, phytochemical screening and completed the manuscript writing.

CONFLICTS OF INTEREST:

We announce we do not have conflicting interests.

AUTHORS’ FUNDING:

We thank Oriental College of Pharmacy for funding the project.

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Received on 13.05.2020 Modified on 05.08.2020

Research J. Pharm. and Tech. 2021; 14(6):3173-3178.

DOI: 10.52711/0974-360X.2021.00553