1. INTRODUCTION:

Kidney stone production corresponds to one of the earliest and most prevalent human disorders. Urolithiasis typically results in crystal aggregation, the formation of insoluble particles, and may be linked to metabolic and systemic diseases. Approximately 2 to 3% of instances of end-stage renal disease are caused by nephrolithiasis and nephrocalcinosis.

The complicated underlying pharmacological mechanism, as well as the phytochemical components of dietary plants that are causing their defence against the stone formation, explain the inherent function of nutritional herbs in avoiding the development of kidney stones¹. The victim's behaviour is altered by kidney stones because of the victim's tremendous anxiety of pain and the possibility of kidney failure. No satisfactory, efficient medication to dissolve or stop the occurrence of urinary stones is currently available in modern medicine^2,3. There are just three medicines that work: allopurinol, D-penicillamine, and acetazolamide. Even though many medications and therapies, including thiazides, cellulose phosphate, magnesium oxide, and pyridoxine, among others, have been attempted⁴. Many common plants have medicinal properties because they contain natural antioxidants, particularly phenolic compounds⁵. Through its diuretic properties, consuming Corn Silk Tea promotes urine production and facilitates kidney stone removal. Additionally, these aids improve issues with blood pressure and diabetes. Before consuming. The unripe fruit of the bitter orange, Citrus aurantium (Rutaceae), is used in traditional treatments to treat urolithiasis. Prior research has demonstrated that Citrus aurantium contains a variety of bioactive substances, including as polyethoxylated flavones (PMFs), flavonoid glycosides, and alkaloids. These active ingredients have pharmacological activity, antioxidant properties, and antibacterial properties⁶. Oranges, which are grapefruits, and lemons, as well as the juices made from these fruits, are essential parts of the modern diet and are becoming more and more well-liked due to current health fads. The use of these liquids may have a variety of effects on how kidney stones develop. On the one hand, juice consumption's preventive advantages include the amount of fluid intake it requires, its elevated citrate material, and the alkalinizing impact it has on the pH of the urine⁷.

2. MATERIALS AND METHOD:

2.1 Plant material- Samples of baby corn silk were gathered in Maharashtra and grown naturally in the Ahmednagar area. Fresh sour oranges were obtained from a farm in the Ahmednagar district's vicinity, in accordance with letter number PVPC/Bot/2022-23-/57 dated August 29, 2022, authenticated by Dr. S. P. Giri, Head, Department of Botany, PVP College of Arts, Science, and Commerce, Pravaranagar, Maharashtra.

2.2 Animals and Ethical Considerations:

The research project utilized male Wistar rats that averaged 250–300 g which were obtained and collected by Lacsmi Biofarms Pvt. Ltd. in Pune, India. Animals were nourished on regular feed and provided to accommodations that complied with the usual light and dark (12:12h) cycles and environmental temperature (22 2°C). The IAEC committee gave its blessing to the research's methodology. 1942/PO/RE/S/17/CPCSEA /2022/01/07

2.3 Chemicals and Reagents:

Ethylene glycol, gelatin solution, iodine, magnesium Solvents including water, ethanol, methanol, chloroform, glacial acetic acid, and phytochemical reagent. Potassium iodide, panisaldehyde, Dragendorff's reagent, acetate buffer, and Potassium bromide purchased from LOBA Chem, Mumbai⁸.

2.4 Extraction and Fractionation:

The water-based extraction part's solvent was corn hair, and reflux extraction was used in the procedure. The maize silk was extracted using 100g of the medication in 500 milliliters of water, and it was then macerated for 24 hours at ambient temperature after being evaporated⁹. The extracts were then subjected to an initial phytochemical screening as described in conventional procedures. A test was conducted using water-dilution of sour orange juice¹⁰. Column chromatography was performed using gradient elution method.

3. PHARMACOGNOSTIC EVALUATION:

3.1 Organoleptic evaluation:

According to WHO Quality Control Techniques for herbal medicine, an organoleptic examination of corn silk and sour orange was conducted with respect to their color, dimension, odor, shape, taste, surface, and fracture¹¹.

3.2 Microscopic evaluation:

3.3 Physicochemical analysis:

Based on the methodology outlined in WHO quality assurance techniques for medicinal materials^{12, 13} physicochemical characteristics such as ash value, moisture content, and absorption values had been established^{14, 15}. (Fig 1)

3.4 Phytochemical investigation:

Each of the extracts was reveal to phytochemical analysis for of chemical components using procedures as report valuable. All tests were finished in triplicate¹⁶. (Table 1)

3.5 Thin layer chromatography (TLC) investigation.:

Aqueous extract, of Corn silk Stationary phase: Silica Gel G, Mobile phase: chloroform: methanol Aqueous extract of Sour orange Stationary phase silica Gel G Mobile phase: chloroform: methanol^{17,
18, 19}.

3.6 Acute Toxicity:

In accordance with recommendation 423 of the OECD (Organization for Economic Co-operation and Development²⁰.

3.7 Experimental design:²¹

Animals were fed with 0.75% ethylene glycol in drinking water to rats for a period of 28 days to induce urolithiasis. After 28 days, rats were housed in metabolic cages for collection of urine for the detection of kidney stones. X rays and urine microscopy were used for identification of kidney stones. Urolithiasis induced rats were used for the further study. They were divided into seven groups: Normal Control (NC), Urolithic control (UC), Standard drug (SD), Corn silk drug (CS), Sour orange drug (SO), combined drug (CS):(SO), Fraction extract drug (FE), The test group of Urolithiatic rats received aqueous suspension of corn silk and sour orange, combined, fraction by oral administration 28 days for evaluation of efficacy and toxicity. The rats were divided into seven groups of six animals as given below

Table 1- Experimental grouping of animals

Group	Group Type	Experimental design
I	Normal Control (NC)	Received normal water.
II	Urolithic Control (UC)	Urolithiasis was induced by administrating 0.75% of ethylene glycol
III	Standard Drug (SD)	Urolithiatic rats received cystone by oral administration for 28 days
IV	Test (500mg/kg) (CS)	Urolithiatic rats received corn silk extract by oral administration for 28 days
V	Test (500mg/kg) (SO)	Urolithiatic rats received sour orange extract by oral administration for 28 days
VI	Test (500mg/kg) (50:50) (CS.SO)	Urolithiatic rats received corn silk and sour orange extract by oral administration for 28 days
VII	Test (FE) (125 mg/kg)	Urolithiatic rats received fraction of column

3.8 Statistical Analysis:

Data of independent observations are shown as Mean ± S.E.M. Statistical analysis was done using one way analysis of variance (ANOVA) followed by Dunnett’s t-test. p<0.05 was considered statistically insignificant.

4. RESULTS:

4.1: Extraction and Fractionation:

The Corn silk has been extracted with aqueous water 100g of drug in 500ml of water extracted for 3 hrs, maceration at room temperature for 24 hours and evaporated. Percentage yield of extract was found to be 73%. Further the extract was used to confirm the mobile phase for the column chromatography, by performing TLC with different mobile phases. Final selected MP chloroform: methanol (2.5:2.5) RF value 0.66. Fresh Sour orange juice diluted with water and test was performed. Fresh juice was used for TLC using mobile phase of chloroform: methanol RF value 0.81.

4.2: Microscopic evaluation:

Corn silk was observed under microscope slide with drop of water and corn hair fibres Corn silk hair with drop of sudden red oil globules present. Sour orange peel was observed oil glands, epidermal cell sour orange pulp observed globular chromoplast. Shown in (Fig 1)


CS fibers	CS Oil globules

SO oil Glands	SO Globular Chromoplast

Fig 1: Microscopy of corn silk and sour orange

4.3 Preliminary phytochemical analysis:

The results of qualitative phytochemical analysis of corn silk and sour orange

Table 2: Preliminary phytochemical analysis

Sr no.	Phytochemical	Test performed	Corn silk	Sour orange
1.	Carbohydrates	Molisch’s test	+	+
2.	Alkaloids	Magner’s dragendorff	+	-
3.	Tannins	Gelatin lead acetate	+	-
4.	Reducing sugar	Felling’s test	+	+
5.	Protein	Biuret test	-	-
6.	Cardiac glycoside	Ring test	+	+
7.	Steroids	Ring test	-	+
8.	Phytosterols	Liebermann Burchard	-	-
9.	Phenols	FC method	-	+
10.	Flavonoids	Aluminum chloride	+	-
11.	Amino acid	Million tests	+	+
12	Fixed oils	Spot test	-	+
13.	Terpenoids	Liebermann Burchard	+	_-

+ = Present; – = Absent.

4.4 Thin layer chromatography (TLC) investigation.

TLC of sour orange RF value - 0.81

TLC of corn silk RF value - 0.06

5. RESULTS OF ANIMAL ACTIVITY:

5.1 Acute Toxicity:

There were no behavioral changes; signs of toxicity and mortality observed after the administration of 2000 mg/kg, 1000 mg/kg of dose. Hence drug was said to be safe when given orally.

5.2: Calcium oxalate crystals study :

The values for Ca⁺⁺oxalate crystals reported in figure showed that oxalate was significantly higher in urolithic induced rats when compared with the normal control rats especially in week 2. Urine was collected in metabolic cage (Fig 2) and (Table 2).


Normal control	Urolithic induced

Test drug (CS+SO)	Test drug (FE)

Fig 2. Calcium oxalate crystals

5.3:X- ray study:

42 animals were distributed into 7 groups. The following (Table 3) and (Fig 3) indicates the number of animals showing efficacy after treating them for 28 days. Hence, we can conclude that drug has shown significant efficacy. In disease control group all 6 animals showed presence of stone after treatment period. In standard drug group 2 animals showed presence of kidney stones. In test drug group 1 animal showed presence of kidney stones.


Before Treatment	After Treatment

Fig 3: X rays

Effect of test drug on kidney stones found in animals after treatment

Table 3 Effect of test drug on kidney stones found in animals after treatment

Group	No. of Animals	Stone found in no. of animals after treatment
Normal Control	6	0
Urolithic Control	6	6
Standard	6	3
Test (500mg/kg) (CS)	6	2
Test (500mg/kg) (SO)	6	2
Test (500mg/kg) (50:50) (CS.SO)	6	1
Test (FD)	6	1

5.4 Conc. of phosphorus, uric acid and protein:

Phosphorous, uric acid and protein excretion was reversed to normal level which was elevated after the induction, as shown in (Table 4, Fig 4). Phosphorous, uric acid and protein were significantly elevated in 24hr urine of Urolithic rats as compared to normal control rats. Fraction drug treatment reduced the phosphorous, uric acid and protein to near normal.

Table 4: Effect of test drug on concentration of phosphorus, uric acid and Protein

Group	Phosphorus	Uric acid	Protein
C	5.50±0.5	1.04±0.04	2.41±0.12
EG	8.10±0.20	1.39±0.02	3.29±0.14
S	5.41±0.31**	1.12±0.04^***	2.47±0.15
EG + CS	6.15±0.27	1.14±0.03	2.28±0.20
EG + SO	6.13±0.29	1.16±0.03	2.30±0.15**
EG+CS+SO	5.13±0.10***	1.21±0.02	2.29±0.12
EG + FR	4.10±0.9***	1.19±0.02**	2.40±0.11***

Fig 4. Effect of test drug on concentration of phosphorous, uric acid, protein.

5.5 Weight of Organs:

Animals were sacrificed and the weights of the liver and kidney were taken after 28 days of receiving the test medication. As shown in Table No. 5, the weight of the animals' kidneys was found to be higher than that of the control group due to the presence of calcium oxalate crystals.

Table 5: Wt. of organs after sacrificing animals

Groups	Left Kidney (gm)	Right Kidney (gm)	Liver (gm)
Normal Control	0.130±0.02	0.129±0.012	1.385 ± 0.6
Urolithic Control	0.197±0.042	0.194±0.032	10308 ± 0.4
Test Drug (CS.SO)	0.1590±0.05	0.1627±0.04	10224± 0.3
Test Drug (FE)	0.1470±0.04	0.1519±0.03	10119± 0.2

5.6 Histopathology result:

Photomicrograph of kidney sections showing:

Control group structures of kidney with preserved nephron structures, stroma and renal pelvis. EG group showing cystically dilated renal tubules (star) with degenerative changes of tubular epithelium, epithelial cells. Std (stars) degenerative changes absence of ca oxalate crystals, EG+CS (curved arrow) in tubular epithelium, shrinkage, EG+SO degenerative changes in some renal tubular epithelium EG+FD absence of ca oxalate crystals (arrows). shown in (fig no 5)


Control Group (x100)	EG Group (x400)	Std Group (x400)

EG + CS Group (x400)	EG + SO Group (x400)	EG + FD Group (x400)

Fig no 5: Histopathological observation in kidney of animal.

6. GC-HRMS RESULTS:

This isolated compound was subjected to gas chromatography high-resolution mass spectroscopy (GCHRMS) analysis to study the chemical composition of the extract. (Table no.6)

7. DISCUSSION:^22,23

The World Health Organization (WHO) states that the physiological and histological description of a medicinal plant is the first step in establishing its identity and purity and should be completed before any tests are conducted. Numerous studies have demonstrated the antioxidant and therapeutic benefits of corn silk and sour orange. It also has additional uses as an anti-fatigue medication, an antidepressant, and a diuretic that decreases hyperglycemia. Corn silk and sour orange are widely used in teas and nutritional supplements to address urinary problems. Corn silk and sour orange protein hydrolysate have been shown to have antiurolithiatic and hemato protective properties in experimental studies. Potential antiurolithiatic peptides from the hydrolysate have also been found. In conventional Persian medicine, the peel of a bitter orange and the hydrosol made from its bloom are utilized as neuroprotective and antidepressant medications. It is used orally as an appetizer, an aphrodisiac, a medical food that protects the liver, and a treatment for abdominal discomfort, dyspepsia, constipation, and polymenorrhea. Peels are also gastrotonic and helpful for gastrointestinal conditions. A traditional cough remedy calls for slicing the fruit, removing the seeds, and heating the seeds with a dusting of rock candy powder. Every morning, the warm juice should be slowly sipped after a light heating.

It is used to treat obesity and bedwetting as well as cystitis, edema, kidney stones, diuretics, prostate disorders, and urinary infections, reduces discomfort and increases urine output by relaxing and soothing the bladder's and urinary tubule lining. A renal stone is a solid item that is created from urine-based compounds. Kidney stones can be made of calcium oxalate, uric acid, in as well as cystine. Shockwave lithotripsy, uroscopy, percutaneous nephrolithomy, or nephrolithotripsy are all options for treating kidney stones. Severe lower back pain, blood in the urine, nausea, vomiting, a fever, chills, and bad-smelling or murky urine are all common symptoms. Diverse wastes are dissolved in urine.

8. CONCLUSION:

According to reports, it is used to treat ailments including hypercholesterolemia, urinary tract infections, and other related conditions. Sour oranges and corn silk are both employed as crucial ingredients in the creation of numerous medications. It has been shown to be non-toxic and safe for eating by people. Its possible anti-oxidant properties, medical uses as a diuretic, in lowering hyperglycemia, as an anti-depressant and an anti-fatigue drug, and its antiurolithiatic activity.

9. ACKNOWLEDGEMENT:

The Authors thanks for giving support and proper direction for making successful review article and also help to find new and innovative information for making review article. We would also like to thanks Pravara Rural College of Pharmacy Loni Pravaranagar, Maharashtra India for providing required facilities to carry out this Research work.

10. CONFLICT OF INTEREST:

We have No Conflict of interest.

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Received on 14.07.2023 Modified on 18.01.2024

Research J. Pharm. and Tech 2024; 17(10):4739-4744.

DOI: 10.52711/0974-360X.2024.00730

Sr no	Peak	Chemical name	Mol. Formula	Mol, weight	Time	Area
1	0.0394	2-Propanone, 1-hydroxy-	C3H6O2	74	4.35	1096209.66
2	0.0427	2-Furanmethanol	C5H6O2	98	4.76	678558.01
3	0.0427	1,3-Butadiene-1-carboxylic acid	C5H6O2	98	4.76	678558.01
4	0.0384	3,5,6-Trichloro-4-isopropylsulfanyl-pyridine-2-carbonitrile	C9H7Cl3N2S	280	5.01	176981.79
5	0.0384	Pyridine,1,2,3,6-tetrahydro-1-methyl	C6H11N	97	5.01	176981.79
6	0.1057	Cyclohexane,1Racetamido-2,3-cis-epoxy-4-trans-acetoxy	C10H15NO4	213	5.88	292104.95
7	0.0557	2,4-Dihydroxy-2,5-dimethyl-3(2H)-furan-3-one	C6H8O4	144	6.10	413655.20
8	10.21	4H-Pyran-4-one, 2,3-dihydro-3,5-dihydroxy-6-methyl	C6H8O4	144	10.21	4145091.00