INTRODUCTION:

Urolithiasis or kidney stone is a process of super saturation of urinary calcium oxalate or calcium phosphate crystals over its solubility. Factors that accelerate the formation of calcium oxalate or phosphate stone include supersaturation, dehydration, hypercalciuria, hyperoxaluria, hypernatremia and hyperuricosuria. Uric acid stone is less common than oxalate or phosphate stone. The current treatment includes the alkalization of urine that helps to maintain the pH of the urine and decrease the process of calcium oxalate supersaturation¹.

The Embelia ribes commonly known as vidanga, belonging to family Myrsinaceae. It is traditionally used as appetizer, carminative, laxative, anthelmintic and in liver disease². The main chemical constituent of Embelia ribes is “Embelin” and other chemical constituents are volatile oil, fixed oil, rasin, tannin, christembine (alkaloid), phenolic acids like caffeic acid, vanillic acid, chrorogenic acid, cinnamic acid, O-cumaric acid³. Various pharmacological activities such as antifungal⁴, antiviral⁵, antimicrobial⁶, anti-acne⁷, anthelmintic⁸, insecticidal⁹, anti-inflammatory¹⁰, anti-prolifetaion^11-13, anti-pyretic¹⁴, anti-cancer¹⁵,anti-hyperhomocysteinemic, anti-alzehimer, anti-hyperlipidemic¹⁶, anti-histaminic and bronchodialator¹⁷,anti-oxidant¹⁸ and were reported.

Ipomea hederacea is commonly known as kaladana or ivy leaf morning glory belonging to family Convolvulaceae. Traditionally it is used as purgative, fever, headache and bronchitis. Plant contains steroidal glycosides such as methylpentoside, and other glycoside of jalapinolic acid and its methyl esters (orizabins), sitosterol¹⁹. A number of pharmacological activities like anti-oxidant and hepatoprotective²⁰, antimicrobial²¹, antifungal²² were reported.

MATERIALS AND METHODS:

Plants Material:

The seeds of Embelia ribes and Ipomea hederacea were purchased from the local market, washed and then dry to remove any extraneous material from the crude drugs. The crude drugs were approved from the department of pharmacognosy, Faculty of Pharmacy, Raja Balant Singh Engineering Technical Campus Bichpuri Agra, Uttar Pradesh (Reg. No. 202010 and 202011). The crude drugs were subjected to pulverization.

Chemicals:

Various solvents such as ethanol (Changshu Yangyuan Chemical), chloroform (Thermo fisher scientific), diethyl ether (Thermo fisher scientific), n-hexane (Thermo fisher scientific), Dragendorff’s reagent (CDH), acetone (Thermo fisher scientific), Mayer’s reagent (CDH), sodium hydroxide (Qualigen fine chemicals thermoelectron LLS), lead acetate (Thermo fisher scientific), ferric chloride (Qualigen fine chemicals thermoelectron LLS), nitric acid (Thermo fisher scientific), Ninhydrine (Merck specialities), Biuret reagent (CDH), ruthenium red (CDH), sulphuric acid (Thermo fisher scientific), Fehling reagent (Qualigen fine chemicals thermoelectron LLS), Molish reagent (CDH), Benedict reagent (Thermo fisher scientific), Millon’s reagent (CDH), Cystone (Himalaya herbal healthcare) were purchased by the institute for the research work.

Determination of Extractive value of Embelia ribes and Ipomea hederacea²³:

The seeds of both the plants were powdered to desired size and then macerated with ethanol, chloroform, diethyl ether, n-hexane, acetone and water. Two gram of finally powdered crude drug was placed in a conical flask and mix with the solvents (30mL) ethanol, chloroform, diethyl ether, n-hexane, acetone and water separately. The flasks were corked and kept aside for 24 hrs at room temperature with shaking frequently. The mixtures were filtered through Whatmann filter paper no. 1. The obtained filtrates of various solvents were then transferred to watch glass, concentrated to dryness by keeping filtrate for complete evaporation of solvents.

The extractive values (EV) were calculated by using following formula:

Weight of residue (g)

EV = ------------------------------------------------------------- × 100

Weight of Embelia ribes / Ipomea hederacea (g)

Phytochemical screening^24,25:

The phytochemical screening was evaluated by different methods according to their classification like tests for alkaloids was performed through Dragendorff’s test and Mayer’s test by adding the Dragendorff’s and Mayer’s reagent in diluted HCl in all extract and filtered. The test for flavonoids was performed by adding the few drops of lead acetate solution in the extracts. The test for tannins and phenolic compounds were investigated by ferric chloride and diluted HNO₃ solution. Furthermore the amino acid and protein were screened by Ninhydrine and biuret test respectively. Moreover the steroids, mucilage, glycosides and carbohydrates were screened through the Salkowaski, Ruthenium red solution, Borntrager’s test, Molish and Fehling test respectively. All the observation was measured accordingly.

In-vitro anti-urolithiatic activity¹:

Preparation of Calcium Oxalate (CaOx) Crystals:

Solution of calcium chloride and sodium oxalate were prepared by dissolving 1.47g of calcium chloride fused in 100mL distilled water and 1.34g of sodium oxalate in 100mL of 2N H₂SO₄. Separately both the solutions were mixed in the ratio of 1:1 with continuous stirring to precipitate out calcium oxalate. The resultant calcium oxalate crystal was free from traces of sulfuric acid by treatment of ammonia solution, washed with distilled water and dried at a temperature 60°C for 2 h. Calcium oxalate (CaOx) crystals were confirmed by microscope view (Niddle shaped).

Crystallization occurs according to the following reaction:

CaCl₂+Na₂C₂O₄--------->CaC₂O₄ + 2NaCl

Preparation of Semi-permeable membrane:

The semi permeable membrane of egg behaves like a biological membrane for in-vitro model. Apex of eggs was punctured in order to squeeze out the entire content. Empty eggs were washed thoroughly with distilled water and placed in a beaker consisting 2N-HCl solution for overnight, which lead to the complete decalcification of semi permeable membrane. Then semi permeable membrane was removed carefully from egg shells; washed thoroughly with distilled water and placed it in ammonia solution for neutralization of acid traces, and then rinsed with distilled water. It was stored in refrigerator at a pH of 7-7.4 under the moistened condition.

In-Vitro anti-urolithiasis by dissolution method:

Artificial calcium oxalate crystals were prepared by standard method described above with some modification. One of synthetically prepared calcium oxalate crystals were weigh and mixed with 2mL of each extract and standard drug Cystone packed in separate egg based semi permeable membranes by suturing. Each treatment was repeated three times. After completion of treatment the semi permeable membrane was allowed to open in petri dish and dry at 50ºC the remaining calcium oxalate crystals were weighed and the percentage dissolution was calculated, by the following formula.

Initial weight of CaOx – Final weight of CaOx

% dissolution =------------------------------------------------------------× 100

Initial weight of CaOx

Anti-urolithiasis by Turbidity method¹:

The precipitation of calcium oxalate crystals at 37ºC was measured with the help of Nephelometer (Table Model C) at 200 NTU. Artificial synthesized kidney stone of calcium oxalate was prepared and kept in a semi-permeable membrane with 2mL of each extract and standard drug Cystone. The semi-permeable membrane was hanged in buffer solution (pH- 8.4) and whole assembly was placed in an incubator (37ºC) for 24 h. The turbidity of solution was measured every 24h with refilling of extract and standard for 6 days. The result was analyzed statistically.

RESULT AND DISCUSSION:

In the present study the extractive value of Embelica ribes and Ipomea hederacea in chloroform, ethanol, diethyl ether, n-hexane, acetone and water extract were determined (Figure-1). Extractive value provides the yield percentage of phytocostitutents in the solvent. The result was shown in Table-1. The extractive value of ethanol and diethyl ether extract of Embelia ribes was found to be maximum (2.5%). The chloroform (2%), n-hexane (2%), acetone (1.5%) and water (1%) extract of Embelia ribes showed slight less extractive value. The extractive value of n-hexane and water extract of Ipomea hederacea was found to be maximum (14%w/w and 6%w/w). The diethyl ether (2.5%), acetone (2%), ethanol (2%), and chloroform (1.5%) extract of Ipomea hederacea showed very less extractive value.

Fig. 1: Extractive value of Embelia ribes (ER) and Ipomea hederacea (IH)

Table 1: Result of determination of extractive value of Embelia ribes Burm.F. and Ipomea hederacea Jacq.

S. No.	Solvent	Weight of the crude drug (g)	Extractive value (ER) (%/w/w)	Extractive value (IH) (%w/w)
1	Chloroform	2	2	1.5
2	Ethanol	2	2.5	2
3	Di-ethyl Ether	2	2.5	2.5
4	n-Hexane	2	2	14
5	Acetone	2	1.5	2
6	Water	2	1	6

The preliminary phytochemical analysis was performed to analyze the primary phytoconstituents present in the Embelia ribes and Ipomea hederacea. The result of phytochemical analysis was shown in Table 2.

Table 2: Result of Phytochemical screening of Embelia ribes Burm.F. and Ipomea hederacea Jacq.

S.N	Phyto-constituents	*Embelia ribes*						*Ipomea hederacea*
S.N	Phyto-constituents	ERE	ERC	ERD	ERH	ERA	ERW	IHE	IHC	IHD	IHH	IHA	IHW
1	Alkaloids	+	+	+	-	-	+	-	-	-	+	-	+
2	Tannins	+	-	+	-	+	-	+	-	-	+	-	-
3	Amino acid	-	+	-	-	-	+	+	+	-	+	+	+
4	Protein	-	+	-	-	-	+	+	+	-	-	+	-
5	Mucilage	-	-	-	-	-	+	-	-	-	-	-	+
6	Glycosides	+	+	-	-	-	-	+	+	-	-	+	-
7	Carbohydrates	+	+	-	+	+	+	-	-	-	+	-	+
8	Acidic compounds	-	-	-	-	-	-	-	-	-	-	-	-

(+ve)= Present, (-ve)= Absent

Table 3: Result of in-vitro anti-urolithiatic activity by dissolution method of Ipomea hederacea Jacq.

S. No	Groups	Initial Weight of CaOx (g) at day-1	Final Weight of CaOx (g) at day-6	% Dissolution of CaOx
1	Control	1±0.00	0.99±0.003	1.33±0.33
2	IHC	1±0.00	0.32±0.014	68.33±1.45^**
3	IHE	1±0.00	0.44±0.020	56.00±2.08^**
4	IHD	1±0.00	0.46±0.008	54.33±0.88^**
5	IHH	1±0.00	0.23±0.008	76.67±0.88^**
6	IHA	1±0.00	0.38±0.014	62.33±1.45^**
7	IHW	1±0.00	0.28±0.017	72.00±1.73^**
8	Cystone	1±0.00	0.09±0.003	90.67±0.33^**

All values are Mean±SEM statistical comparison was performed by graph pad instat software using ANOVA followed by Dunnett’s Multiple Comparisons Test, ^**P<0.01, when all groups were compared with control group.

Table 4: Result of in-vitro anti-urolithiatic activity by dissolution method of Embelia ribes Burm. F.

S. No	Groups	Initial Weight of CaOx (g) at day-1	Final Weight of CaOx (g) at day-6	% Dissolution of CaOx
1	Control	1±0.00	0.99±0.003	1.33±0.33
2	ERC	1±0.00	0.34±0.006	66.00±0.58^**
3	ERE	1±0.00	0.16±0.007	84.33±0.67^**
4	ERD	1±0.00	0.23±0.012	77.33±1.20^**
5	ERH	1±0.00	0.30±0.006	70.00±0.58^**
6	ERA	1±0.00	0.33±0.012	67.33±1.20^**
7	ERW	1±0.00	0.30±0.003	70.33±0.33^**
8	Cystone	1±0.00	0.09±0.003	90.67±0.33^**

Table 5: In-Vitro anti-urolithiatic activity by Turbidity method of Ipomea hederacea Jacq.

S. No	Groups	Observation (200NTU)
S. No	Groups	Day1	Day2	Day3	Day4	Day5	Day6
1	Control	0.00±0.00	0.00±0.00	0.00±0.00	0.00±0.00	0.33±0.33	1.00±0.58
2	IHC	10.67±0.67	19.67±0.33	31.67±0.33	43.00±1.00	54.00±0.58	62.33±0.67
3	IHE	2.00±0.57	9.00±0.58	18.33±0.33	21.00±0.58	25.33±0.33	29.67±0.57
4	IHD	1.67±0.33	12.33±0.33	16.67±0.67	19.33±0.33	24.00±0.58	26.33±0.65
5	IHH	11.67±0.88	19.00±1.15	27.33±0.88	39.67±0.67	53.33±0.33	73.00±1.15
6	IHA	3.00±0.57	13.00±0.58	18.33±0.33	21.00±1.00	27.67±0.67	40.67±0.67
7	IHW	13.33±0.33	17.33±0.33	26.33±1.33	36.00±0.58	50.67±0.88	65.67±1.20
8	Cystone	20.67±0.67	35.00±1.15	68.00±1.15	70.33±1.45	75.00±0.58	85.67±1.76

All values are Mean±SEM statistical comparison was performed by graph pad instat software using ANOVA followed by Dunnett’s Multiple Comparisons Test, all values of Day 6 are significant P<0.01, when all groups were compared with control group.

Table 6: In-Vitro anti-urolithiatic activity by Turbidity method of Embelia ribes Burm. F.

S. No	Groups	Observation (200NTU)
S. No	Groups	Day1	Day2	Day3	Day4	Day5	Day6
1	Control	0.00±0.00	0.00±0.00	0.00±0.00	0.00±0.00	0.33±0.33	1.00±0.58
2	ERC	5.33±0.33	12.00±0.58	17.00±0.58	19.33±0.33	20.33±0.33	38.00±0.58
3	ERE	15.00±0.58	19.33±0.33	39.33±0.88	48.00±0.57	58.00±0.58	72.33±0.88
4	ERD	13.33±0.33	20.67±0.67	31.00±1.15	39.33±0.88	53.00±0.58	60.00±0.58
5	ERH	11.33±0.33	20.67±0.67	31.00±1.15	44.33±0.88	58.33±0.88	69.00±1.15
6	ERA	13.00±0.58	19.33±0.88	29.00±1.15	37.00±0.58	48.67±1.45	69.67±0.67
7	ERW	7.00±0.58	11.33±0.88	25.67±0.88	31.33±0.88	40.67±0.88	41.33±0.67
8	Cystone	20.67±0.67	35.00±1.15	68.00±1.15	70.33±1.45	75.00±0.58	85.67±1.76

CONCLUSION:

As per the results we concluded that extractive value gives the yield percentage of phytoconstituents, that showed the maximum dissolution and turbidity that confirmed the more phytoconstituents present in more yield percentage. As the urolithiasis is the process of supersaturation of calcium oxalate crystals, the result of the dissolution and turbidity leads to decrease this process. Further studies will be needed to reveal the active ingredient or lead molecule and mechanism that responsible for dissolving the renal calculi.

ABBREVIATION:

ERC- Embelia ribes Chloroform extract

ERE- Embelia ribes Ethanolic extract

ERD- Embelia ribes Diethyl ether extract

ERH- Embelia ribes n-hexane extract

ERA- Embelia ribes Acetone extract

ERW- Embelia ribes water extract

IHC- Ipomea hederacea Chloroform extract

IHE- Ipomea hederacea ethanol extract

IHD- Ipomea hederacea Diethyl ether extract

IHH- Ipomea hederacea n-Hexane extract

IHA- Ipomea hederacea Acetone extract

IHW- Ipomea hederacea water

CaOx- Calcium Oxalate crystals

DISCLOSURE OF INTEREST:

The authors declare that they have no conflicts of interest concerning this article.

ACKNOWLEDGEMENT:

The authors are thankful to Director, R.B.S. Engineering Technical Campus Bichpuri, Agra for providing necessary facilities for conducting this research work.

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Received on 12.06.2020 Modified on 16.08.2020

Research J. Pharm. and Tech. 2021; 14(7):3566-3570.

DOI: 10.52711/0974-360X.2021.00617