1. INTRODUCTION:

Lithiasis or stony secretions in the urinary system has been identified together as a major disease in every human and animal. Calculus (stone) may harm the excretory hollow epithelial tissue of the organ, resulting in the impaired excretory activity of the organ. The most important of human excretory organ stones is the metallic element salt, which relates to the metallic element and the metabolism of salt in the tissue and tract system^1-4. Ammonium-magnate-phosphate (struvite stone) is the most prevalent form of phosphate stone, accounting for 5-15 percent of all stones. This stone is huge, has a coral shape, and is in contrast. Stones are formed as a result of a bacterial infection, especially proteus bacteria in the urinary system. This bacterium releases the urease enzyme, which breaks down urea into ammonia, making urine alkaline, resulting in a reduction in struvite solubility which facilitates stone formation. Stones occur as a result of a bacterial infection, particularly in the urinary system with proteus bacteria^5-13.

Nutrients have been linked to improvements in human health. In today's civilization, antinutrients, on the other hand, are significantly less common.

Antinutrient overload can result in nausea, bloating, headaches, rashes, and nutritional deficiencies. Toxic amino acids, phytic acid, gossypol, oxalates, goitrogens, lectins (phytohaemagglutinins), protease inhibitors, chlorogenic acid, and amylase inhibitors are only a few examples of antinutrients found in plant sources. In this view, the present study aimed to evaluate the nutritive phytochemicals, mineral elements, antinutrients, and invitro litholytic activity of B. rubra leaves and stem pod by calcium phosphate inhibition potential of this leafy vegetable and traditional medicinal plant which have enormous therapeutic properties including antiurolithiatic activity¹⁴.

2. MATERIALS AND METHODS:

Plant materials collection, authentication, screening of phytochemicals, semipermeable membrane preparation, invitro calcium phosphate synthesis, and compression into tablets, calcium phosphate inhibition by the decalcified and detannated concoction of B.rubra were already described in our earlier studies¹⁵. In the present study, invitro litholytic activity by calcium phosphate inhibition potential of leaf and stem pod of B. rubra in addition to the evaluation of mineral elements and antinutrients were carried out.

2.1 Extraction of plant material:

A hydroalcoholic extract of the decalcified leaf was prepared using 70% ethanol by maceration for five days. The concentrated extract was obtained by evaporating the solvent using a rotary evaporator. The extract obtained was dried completely to constant weight at room temperature. The same procedure was followed for preparing an extract from the stem pod.

2.2 Estimation of Nutritive phytochemicals:

2.2.1 Estimation of flavonoid content:

The aluminium chloride colorimetric test was used to quantify the total flavonoid content. A reaction mixture containing 1mg of sample and 4ml of distilled water was produced in a 10ml volumetric flask. After 5 minutes, 0.30mL sodium nitrite (5%) and 0.3mL aluminium chloride (10%) were added to the flask. After 5 minutes, 2ml of 1M sodium hydroxide was treated and diluted to 10ml with distilled water. A set of quercetin reference standard solutions (20, 40, 60, 80, and 100g/ml) was generated in the same way. The absorbance of the test and standard solutions against the reagent blank at 510 nm was measured using a UV/visible spectrophotometer. The total flavonoid concentration was calculated in milligrams of QE per gram of extract¹⁶.

2.2.2 Determination of total polyphenols content:

A spectrophotometric approach was used to determine the quantity of phenolics in the extracts¹⁷. The total phenol content was determined using the Folin-Ciocalteu assay. The amount of phenolics in the extracts was determined using a spectrophotometric method. The Folin-Ciocalteu test was used to assess the total phenol content. For the reaction, a volumetric flask was filled with 1mL of extract and 9mL of distilled water (25ml). The mixture was completely mixed with 1mL Folin-Ciocalteu phenol reagent. 10mL sodium carbonate solution (7percent ) was added to the mixture after 5 minutes. The liquid volume has been raised to 25 millilitres. A set of standard gallic acid solutions (20, 40, 40, 60, 80, and 100g/ml) was made using the same process.An ultraviolet (UV)/visible spectrophotometer was used to measure the absorbance of the test and standard solutions against the reagent blank at 550nm after 90 minutes at room temperature.

2.2.3 Estimation of saponin:

An anisaldehyde reagent was used to determine total saponin. Water was used to make the sample solution. 10mg diosgenin was dissolved in 16mL methanol and 4 mL clean water for the standard saponin solution. Standard diosgenin solutions (20, 40, 60, 80, and 100 g/ml) were made using 80percent aqueous methanol. After that, pipetted as soon as possible after thoroughly mixing 500g of sample and 500μl of 0.5% anisaldehyde reagent were combined and set aside for 10minutes to determine total saponins. The tubes were then filled with 2mL of 50percent sulphuric acid reagent and swirled together. After that, the tubes were held at a constant temperature of 60ºC in a water bath. Tubes were chilled for 10minutes before being measured at 435nm for absorbance. A standard was created using the same way. From the calibration curve of the standard, the number of saponins was determined as saponin equivalent¹⁸.

2.2.4 Estimation of tannin content:

The Folin-Ciocalteu technique was used to determine the tannins¹⁹. The sample extract was mixed with 7.5ml distilled water, 0.5ml Folin-Ciocalteu phenol reagent, 1 ml 35percent sodium carbonatesolution, and 0.1ml sample extract in a volumetricflask (10ml), which was then diluted to 10 l with distilled water. Before being left at room temperature for 30minutes, the liquid was properly mixed. Tanic acid reference standard solutions (20, 40, 60, 80, and 100g/ml) were prepared in the same way as before. The absorbance of the test and standard solutions against the blank at 725nm was measured using a UV/Visible specrophotometer. Tannic acid content was measured in milligrams of tannic acid per gram of extract.

2.3 Elemental analysis:

Calcium was determined by titration against the sample solution with 0.01M EDTA as per the procedure given by AOAC/BIS/FSSAI. Iron in the plant samples was determined by an Atomic absorption spectrophotometer with air acetylene flame using Cathode Lamp-Fe – 248.3 nm and measuring the absorbance of the iron at 248.3nm. Magnesium was determined by titration against the sample solution with 0.01M EDTA as per the procedure given by AOAC/BIS/FSSAI. Phosphorous in the plant samples were determined by the calibration curve method and measuring the absorbance at 460 nm. Potassium was estimated by Flame Photometer. A flame photometer measures the photoelectric intensity of color imparted to the flame of a merker type burner under well-controlled settings²⁰.

2.4 Estimation of antinutrients:

2.4.1 Estimation of oxalate content:

The material is boiled in dilute sulphuric acid to remove the oxalate ions (0.5N). Then, by titrating the extract with a standard KMnO4 solution, the oxalate content was calculated volumetrically. 1g of material was weighed in an electronic weighing scale, then transferred to 30ml of 0.5 N sulphuric acid and placed in a water bath to heat for 15minutes. To remove the oxalate ions, the material is cooked in dilute sulphuric acid (0.5N). The oxalate content was then estimated volumetrically by titrating the extract with a standard potassium permanganate solution¹⁹.

2.4.2 Estimation of phytate:

The method of Haug and Lantzech (1983) was used to analyze phytate²⁰. According to this approach, a suitable amount (0.8-1.0gram) of the sample was extracted with 0.2N HCl by filling the conical flask with 25ml of 0.2N HCl and shaking it for 1hour at 30ºC and 80 revolutions per minute on a shaker. A test tube with a ground glass stopper was filled with 0.5ml of extract. A known iron content acidic ammonium iron-III sulfate solution was added, and the tubes were closed with a stopper and secured with a clip. After 15 minutes of cooling in ice water, tubes were heated for 30minutes in a boiling water bath before being allowed to cool to room temperature. The contents of the tube were combined and centrifuged at 3000 revolutions per minute for 30 minutes.When 1ml of the supernatant was moved to another test tube and 1.5ml of 2,2-bipyridine solution was added, the light pinkish tint appeared. The absorbance was measured at 519nm in distilled water. The supernatant was tested for phytate levels and iron reduction. At 519nm, the absorbance of standards with known phytate concentrations was determined. Phytate concentration in micrograms was displayed on the X-axis, while Optical Density (O.D) was plotted on the Y-axis.

2.5 In vitro litholytic activity:

The extracts' litholytic activity in vitro was assessed by weight variation and dissolution of prepared calcium phosphate tablets²¹. Exactly weighed calcium phosphate tablets were placed with 3ml of extract prepared in DMSO solution (50mg/ml and 100mg/ml concentrations of each) and packed in the semipermeable bag prepared from egg separately and sutured. Calculax was also prepared in DMSO (50mg/ml) and used as standard. The semi-permeable bags were allowed to suspend in 100ml tris buffer (0.1 M) separately in conical flasks.

1(a)

1(b)

1(c)

Figures 1(a) and 1(b) shows the presence of egg in Hcl overnight and prepared semipermeable membrane preserved in Tris buffer until use.

The tablets prepared from synthesized calcium phosphate were shown in figure 1(c). Similarly, negative control (one exactly weighed calcium phosphate tablet) has been placed in the semi-permeable bag with 10ml of water. Each sample treatment was carried out twice and an average was taken.All the flasks were subjected to incubation at 37±10º C for 5 weeks. For each bag, the weight loss of calcium phosphate tablets at an interval of two weeks has been evaluated after incubation and complete drying in an oven for 5hours at 40ºC. The calcium phosphate dissolution was estimated by calculating the initial weight and final weight of the tablets with the help of a formula

% Dissolution = (W initial – W final) × 100/W initial

W – weights of calcium phosphate tablets before and after the incubation with the extract.

Statistical analysis:

The mean and standard deviation are used to express the data. One-way analysis of variation (ANOVA) was used in the statistical analysis, followed by Dunnnet's test.

3. RESULTS AND DISCUSSION:

In our earlier studies, phytochemical screening of leaves, stems, and pods of B. rubra detannated concoction showed many phytoconstituents including polyphenols, flavonoids, and saponins, and steroids were found to be absent. The concoction showed a considerable reduction in the weight of calcium phosphate confirming the litholytic activity of non-tannin molecules of the plant.In traditional medicinal practices, the whole plant of B. rubra is given as an infusion till the expulsion of stone. In the present study, nutritive and antinutritive phytochemicals and inhibition of calcium phosphate by stem pod and leaf were determined to confirm the exact plant organ responsible for the observed activity.

The findings of mineral elements screening of B.rubra leaves, stems, and pods were presented in table 1. Magnesium can bind with oxalate due to which both diminishing and absorption of oxalate takes place thereby acting as a stone preventive. Potassium will improve hyperoxaluria and also it is a good source in the control of diuretic and hypertensive implications. Potassium citrate along with thiazides is prescribed for kidney stone treatment at the same time it is impaired by poor long-term compliance, gastrointestinal upset, and unpalatable taste. Because higher calcium consumption binds oxalate in the gut, a higher calcium diet (1200 mg/day) has been linked to a lower incidence of kidney stone development. Calcium can bind to dietary oxalate as a result, preventing it from being absorbed^22,23. The plant selected contains all the nutrients in considerable quantities required for preventing and managing many diseases.

Table 1: Elemental analysis of B. rubra organs/100gms

S. No	Parameter	Dried Pod	Dried Stem	Dried Leaf
1	Phosphorous	17.5mg	182mg	46.3mg
2	Potasssium	203mg	865mg	345mg
3	Magnesium	74mg	148mg	102mg
4	Calcium	262mg	561mg	317mg
5	Iron	9.6mg	19.2mg	8.0mg

The results of nutritive phytochemicals are shown in table 2. Tannins have shown potential antiviral, antibacterial,and antiparasitic effects. It was reported that certain tannins can inhibit HIV replication selectivity and are also used as a diuretic^24-27. Tannins are sometimes known as anti-nutrients because they inhibit the absorption of certain elements into the body. Tea and coffee, for example, contain tannins, and drinking too much of these beverages without milk can cause calcium and iron deficiencies, which can lead to osteoporosis and anaemia²⁸. Tannins also precipitate proteins. Furthermore, ingesting significant amounts of tannins is not recommended because they may have carcinogenic and antinutritional properties, posing a risk of negative health impacts. Our plant contains an average amount of tannins that may not have such harmful effects and also consuming B. rubrawith milk is most suitable for patients with any type of urinary stones. Saponins have been discovered to have anticancer, immunostimulatory, and hypocholesterolemic properties^29,30. It also affects protein digestibility^31,32by inhibiting various digestive enzymes such as trypsin and chymotrypsin which is one of the advantages for people consuming B. rubra where protein consumption in people with kidney stones is harmful. The presence of these nutritive phytochemicals may show the pharmacological activities of the plant.

Table 2: Analysis of nutritive phytochemicals in B. rubra organs/100gms

S. No	Parameter	Dried Pod	Dried stem	Dried leaf
1	Total flavanoids	8.2mg/g	4.6mg/g	6.5mg/g
2	Saponins	1.9mg/g	1.2mg/g	1.4mg/g
3	Tannins	10.5mg/g	1.2mg/g	2.5mg/g
4	Total phenolics	12mg/g	1.5mg/g	3.2mg/g

The results of antinutrients are shown in table 3. The plant B. rubra was found to contain low amounts of various antinutrients such as oxalate and phytates. Antinutrients cause adverse effects by interfering with biological processes such as digestion, absorption, and utilization. Phytate has been found to reduce the absorption of minerals like iron, zinc, magnesium, and calcium from food. However, the plant's lower phytate content results in improved mineral absorption and consumption in humans³³. Oxalate can interfere with calcium absorption and utilization in the body, as well as irritate the gut. Oxalate can bind to calcium and form calcium oxalate, which is insoluble^34-37. Antinutrient levels can be reduced to acceptable levels as a result of processing. Antinutrients could be reduced to some degree by the use of soaking, boiling, and blanching techniques. Soaking reduces the amount of phytate, tannins, and oxalate in foods³⁸. Our plant B. rubra is considered healthy to eat since it contains a majority of antinutrients in low quantities and it is also decalcified to some extent. To make up for a nutritional deficit, B.rubra may be added to the diet.

Table 3: Analysis of antinutrients in B. rubra organs/100gms

S. No	Name of the test	Dried pod	Dried stem	Dried leaf
1	Phytates	2.7mg/g	1.8mg/g	2.0mg/g
2	Oxalate	0.8mg/g	0.6mg/g	0.5mg/g

Litholytic activity results revealed that both the extracts showed considerable reduction in weight of calcium phosphate tablet at a regular intervalof two weeks during five weeks with better activity by leaf. In the case of leaf, the result has shown considerable reduction after the first week at both the concentrations and the rate of reduction was more after two weeks thereafter. The rate of solubility of tablet was more in leaf extract followed by the standard at 50mg concentration after five weeks and at 100mg concentration, the rate was increased in case of standard. In the case of the stem pod, the result has shown a very slow reduction after every week intervals when compared to leaf at 50mg concentration but the rate of solubility was increased at 100mg concentration to an considerable extent. The rate of solubility of tablet was more in standard followed by the extract at both the concentrations after five weeks except for leaf at 50mg concentration (Tables 4-7). The in vitro model method showed that, both the extracts of B.rubra exhibited prominent Litholytic activity.

Table 4: Weight of calcium phosphate tablet before and after treatment at 50 mg

S. No	Extract	Conc.	Initial wt of CaP T (g)	Wt of CaP T (g) after one week	Wt of CaP T (g) after three weeks	Wt of CaP T (g) after five weeks	Reduction in wt after five weeks (g)
1	Control	50mg	0.1521±0.0001	0.1350±0.0002	0.1246±0.0003	0.1237±0.002*	0.1237
2	Standard calculax	50mg	0.1521±0.0001	0.1029±0.0002*	0.0910±0.0002*	0.0751±0.0001*	0.0751
3	Leaf	50mg	0.1522±0.0002	0.0977±0.0002*	0.0765±0.0001*	0.06017±0.001*	0.06017
4	Stem pod	50 mg	0.1528±0.0008	0.1002±0.0001*	0.0823±0.0002*	0.07897±0.0005*	0.07897

Values represent mean±SD, n=3. Data were analyzed by one-way analysis of variation (ANOVA) followed by Dunnett’s test. *P<0.05 and ns- non significant. Standard and test were compared with control group

Table 5: Weight of calcium phosphate tablet before and after treatment at 100mg

S. No	Extract	Conc.	Initial wt of CaP T (g)	Wt of CaP T (g) after one week	Wt of CaP T (g) after three weeks	Wt of CaP T (g) after five weeks	Reduction in wt after five weeks (g)
1	Control	100mg	0.1524±0.0002	0.1348±0.0007	0.1243±0.0001	0.1242±0.0002	0.1242
2	Standard calculax	100mg	0.1526±0.0009	0.1012±0.0001*	0.0844±0.0002*	0.0715±0.0002*	0.0715
3	Leaf	100mg	0.1526±0.001	0.1049±0.0008*	0.09413±0.0001*	0.0810±0.0003*	0.0810
4	Stem pod	100mg	0.1531±0.0009	0.1109±0.0001*	0.1020±0.0009*	0.09733±0.0005*	0.09733

Table 6: % Solubility of calcium phosphate tablet during treatment at 50mg concentration

S. No	Extract	Conc	% Solubility afterone week	% Solubility after three weeks	% Solubility after five weeks
1	Standard calculax	50mg	32.34714	40.1709	50.6245
2	Control	50mg	11.1768	18.0802	18.6719
3	Leaf	50mg	35.8081	49.7371	60.4664
4	Stem pod	50mg	34.4240	46.1387	48.3180

Table 7: % Solubility of calcium phosphate tablet during treatment at 100mg concentration

S. No	Extract	Concentration	% Solubility afterone week	% Solubility after three weeks	% Solubility after five weeks
1	Standard calculax	100mg	33.6828	44.6920	53.1454
2	Control	100mg	11.5485	18.4383	18.5039
3	Leaf	100mg	31.2581	38.3158	46.92005
4	Stem pod	100mg	27.5636	33.3768	36.4271

CONCLUSION:

The findings of this study show that leaf, stem, and pod extracts of the plant possess in vitro litholytic activity. The higher amount of phytochemicals, mineral elements, and lower amount of antinutrients may correspond to their greater litholytic activity. Further studies are required to identify the exact active principles of the leaf which were responsible for the determined activity is in progress.

ACKNOWLEDGMENT:

The authors would like to thank the JSS Academy of Higher Education and Research (JSS AHER), India for a research grant (REG/DIR(R)/ URG/54/ 2011-12) under the JSS AHER Research Grants / Financial assistance to one of the authors Mr. G Ramu, Faculty, Department of Pharmacognosy, JSS College of Pharmacy, Ooty, Nilgiris, Tamilnadu, India and for providing the necessary research facilities.

REFERENCES:

1. Khan SR. Calcium oxalate crystal interaction with renal tubular epithelium, mechanism of crystal adhesion, and its impact on stone development. Urol. Res. 1995; 23(2):71–79. https://doi.org/10.1007/bf00307936.

2. Korkmaz A, Kolankaya D. The protective effects of ascorbic acid against renal ischemia-reperfusion injury in male rats. Ren. Fail. 2009; 31(1): 36–43. https://doi.org/10.1080/08860220802546271.

3. Moreira MA, Nascimento MA, Bozzo TA, Cintra A, Dalboni MA, Mouro MG et al. Ascorbic acid reduces gentamicin-induced nephrotoxicity in rats through the control of reactive oxygen species. Clin. Nutr. 2014; 33(2): 296–301. https://doi.org/10.1016/j.clnu.2013.05.005.

4. Samir K. Shah, Kruti M. Patel, Pinal M. Vaviya. Evaluation of Antiurolithiatic activity of Betula utilis in Rats Using Ethylene Glycol Model. Asian J. Pharm. Res. 2017; 7(2):81-87. doi: 10.5958/2231-5691.2017.00014.4

5. Vrunda Zalavadiya, Vipul Shah, D.D. Santani. A Review on Urolithiasis and its Treatment using Plants. Research J. Pharmacognosy and Phytochemistry. 2013; 5(1): 1-8.

6. Dheepa Anand, Chandrasekar R, Sivagami B. A Critical Review on Antiurolithiatic Activity of Bioactive Phytoconstituents. Research Journal of Pharmacognosy and Phytochemistry. 2021; 13(2):95-0. doi: 10.52711/0975-4385.2021.00015

7. K Geetha, R Manavalan, D Venkappaya. Effects and Causes of Lithiasis. Research J. Pharmacology and Pharmacodynamics. 2010; 2(4): 261-267.

8. Archana R Dhole, Vikas R Dhole, Chandrakant S Magdum, Shreenivas Mohite. Herbal Therapy for Urolithiasis: A Brief Review. Research J. Pharmacology and Pharmacodynamics. 2013; 5(1): 06-12.

9. R. Sathish, G. Jeyabalan. Anti- Lithiatic Effect of Ipomoea batatas (L) Leaves and Tuberous Roots on Ethylene Glycol Induced Urolithiasis in Rats. Res. J. Pharmacology and Pharmacodynamics. 2018; 10(1): 01-07. doi: 10.5958/2321-5836.2018.00001.0

10. D. Kayalvizhi, V. Sivakumar, M. Jayanthi. Phytochemical screening and antinephrolithiasis activity of ethanol extract of Aerva lanata on ethylene glycol induced renal stone in rats. Research J. Pharm. and Tech. 2015; 8(11): 1481-1486. doi: 10.5958/0974-360X.2015.00265.6

11. Spandana. K, Shivani. M, Himabindhu. J, Ramanjaneyulu. K. Evaluation of In Vitro Antiurolithiatic Activity of Vigna radiata. Research J. Pharm. and Tech. 2018; 11(12): 5455-5457. doi: 10.5958/0974-360X.2018.00994.0

12. Kushagra D, Rajiv S, Ruchi G, Neelesh M. In-Vitro Evaluation of Anti-Urolithic Activity of Leaves Extract of Costus igneus. Research J. Pharm. and Tech. 2020; 13(3):1289-1292. doi: 10.5958/0974-360X.2020.00237.1

13. Kamta Prasad Namdeo, Sumanta Naskar, Neeli Rose Beck. In vitro Antilithiatic study of Ethanolic extract of roots of Ipomoea digitata Linn. Research J. Pharm. and Tech. 2021; 14(1):369-372. doi: 10.5958/0974-360X.2021.00067.6

14. Popova A, Mihaylova D. Antinutrients in Plant-based Foods: A Review. Open Biotechnol. J. 2019; 13(1):68-76. http://dx.doi.org/10.2174/1874070701913010068

15. Ramu G, Navanita SK, Roan Ngoc AnhHuy, Duraiswamy B, Dhanabal SP. Solubility of calcium phosphate crystallization in vitro in presence of Basella rubra deproteinized concoction used in non-codified medicine for urinary stone. IJPSR. 2020; 32(11):74-83. https://doi.org/10.9734/jpri/2020/v32i1130547.

16. Omoruyi BE, Bradley G, Afolayan AJ. Antioxidant and phytochemical properties of Carpobrotus edulis (L.) bolus leaf used for the management of common infections in HIV/AIDS patients in Eastern Cape Province. BMC Complementary and Altern Med. 2012; 12(1):215-241. https://doi.org/10.1186/1472-6882-12-215.

17. Khanavi M, Hajimahmoodi M, Cheraghi-Niroomand M, Kargar Z, Ajani Y, Hadjiakhoondi A et al. Comparison of antioxidant activity and total phenolic contents in some Stachys species. Afr J Biotechnol. 2009; 8(6):1143-1147.

18. Inuwa HM, Aina VO, Gabi B, Aimola I, Toyin A. Comparative determination of antinutritional factors in groundnut oil and palm oil. Adv. J. Food Sci. Technol. 2011; 3(4): 275-279.

19. Akaneme FI, Igata D, Okafor H, Anyanebechi O. Breeding for nutritional quality for Corchorus olitorius, Annona muricata and Pantaclenthera macrophylla: a study of their nutritional contents. Afr. J. Agric. Res. 2014; 9(14): 1107-1112. http://dx.doi.org/10.5897/AJAR2014.8471

20. AOAC International. Guidelines for dietary supplements and botanicals (AOAC Official Method of Analysis, Appendix K). AOAC International, Rockville. 2013:1-32.

21. Yachi L, Bennis S, Aliat Z, Cheikh A, Idrissi MOB, Draoui M et al. Litholytic activity of some medicinal plants on urinary stones. AFJU, 2018; 24(3): 197-201. https://doi.org/10.1016/j.afju.2018.06.001.

22. Hedges LJ, Lister CE. Nutritional attributes of spinach, silverbeet, and eggplant. Crop and food research confidential report no 1928. Newzealand Institute for Crop and Food Research Limited. 2007; 1(15),1-29.

23. Kavitha V, SaradhaRamadas V. Nutritional composition of raw fresh and shade dried form of spinach leaf (Spinach oleracea). JPR Biomed Rx An International Journal. 2013; 1(8): 767-770.

24. Lin LU, Shu-wen LIU, Shi-bo JIANG, Shu-guang WU. Tannin inhibits HIV-1 entry by targeting gp41, Acta Pharmacol. Sin. 2004; 25(2): 213–218.

25. Akiyama H, Fujii K, Yamasaki O, Oono T, Iwatsuki K. Antibacterial action of several tannins against Staphylococcus aureus. J. Antimicrob. Chemother. 2001; 48(4): 487–491. https://doi.org/10.1093/jac/48.4.487.

26. Kolodziej H, Kiderlen AF. Antileishmanial activity and immune-modulatory effects of tannins and related compounds on Leishmania parasitized RAW 264.7 cells. Phytochem. 2005; 66(17): 2056–2071. http://doi.org/10.1016/j.phytochem.2005.01.011.

27. Katie EF, Thorington RW. Squirrels: the animal answer guide (Baltimore: Johns Hopkins University Press, 2006:p. 91).

28. Ricardo‐da‐Silva JM, Cheynier V, Souquet JM, Moutounet M, Cabanis JC, Bourzeix M. Interaction of grape seed procyanidins with various proteins about wine fining. J Sci Food Agric. 1991; 57(1): 111–125. https://doi.org/10.1002/jsfa.2740570113.

29. Banno N, Akihisa T, Tokuda H, Yasukawa K, Higashihara H, Ukiya M et al. Triterpene acids from the leaves of Perilla frutescenes and their anti-inflammatory and antitumor-promoting effects. Biosci. Biotechnol. Biochem. 2004; 68(1): 85–90. http://dx.doi.org/10.1271/bbb.68.85.

30. Kao TH, Huang SC, Inbaraj BS. Chen BH. Determination of flavonoids and saponins in Gynostemma pentaphyllum (Thunb) Makino by liquid chromatography-mass spectrophotometry. Anal. Chim. Acta. 2008; 626(2): 200–211. https://doi.org/10.1016/j.aca.2008.07.049.

31. Sen S, Makkar HPS, Becker K. Alfalfa saponins and their implication in animal nutrition, J. Agric. Food. Chem. 1998; 46(1): 131–140. http://dx.doi.org/10.1021/jf970389i.

32. Bureau DP, Harris a AM., Cho ab CY. The effects of a purified alcohol extract from soy products on feed intake and growth of Chinook salmon (Oncorhynchustshawytscha) and rainbow trout (Oncorhynchusmykiss), Aquaculture. 1998; 161(1-4): 27–43. https://doi.org/10.1016/S0044-8486(97)00254-8.

33. Morris ER. Phytate and dietary mineral bioavailability. In: Phytic acid: Chemistry and Applications (Pilatus Press: Minneapolis, 1986; 57–76).

34. Anitha R, Sandhiya T. Occurrence of calcium oxalate crystals in the leaves of Medicinal Plants. IJP. 2014; 1(6): 389-393. http://dx.doi.org/10.13040/IJPSR.0975-8232.1(6).389-93.

35. Nakata PA. Plant calcium oxalate crystal formation and its impact on human health. Front. Biol. 2012; 7(3): 254-266. https://doi.org/10.1007/s11515-012-1224-0.

36. Schlemmer U, Frolich W, Prieto RM, Grases F. Phytate in foods and significance for humans: food sources, intake, processing, bioavailability, protective role, and analysis. Mol Nutr Food Res. 2009; 53(2): S330–S375. https://doi.org/10.1002/mnfr.200900099.

37. Edet A, Eseyin O, Aniebiet E. Anti-nutritional composition and mineral analysis of Allium cepa (onion) bulbs. AJPP 2015; 9(13):456-459. http://dx.doi.org/10.5897/AJPP2015.4300.

38. Bishnoi S, Kheatrpaul N, Yadav RK. Effect of domestic processing and cooking methods on phytic acid and polyphenol contents of pea cultivars (Pisum sativum). Plant foods Hum Nutr. 1994; 45(4): 381–388. https://doi.org/10.1007/BF01088088.

Received on 28.05.2022 Modified on 20.12.2022

Research J. Pharm. and Tech 2023; 16(11):5155-5160.

DOI: 10.52711/0974-360X.2023.00835