INTRODUCTION:

Urolithiasis is one of the painful urological disorders. It has high incidence throughout the world. In India many traditional medicines have been used in treatment of urolithiasis from ancient times. Herbal medicines have been used as alternative medicine because of reduced recurrence rate, mortality and morbidity compared to surgical and medical therapies. Currently use herbal drugs for primary health due to poverty, limited availability and high cost of modern medicine. Phytotherapy is potent in treatment of stones with fewer side effects with good efficiency in preventing recurrence of renal stones¹.

Herbal medicines have been used in treatment of urolithiasis since long times, in Ayurveda, Naturopathy, Unani, Siddha and Homeopathy². All medicines generally they are derived synthetically or from plant sources must satisfy the basic requirements like safety and effectiveness³.

In ancient times, medicine is prepared to each individual based upon symptoms and health condition of patient, yet now situation is replaced, herbal medicines are being prepared on large scale due to its wider acceptance throughout the world. In large scale production of herbal drugs, maintenance of quality is the main task. Hence Standardization is the essential step for the development and production of herbal drugs to confirm its identity, establish quality, purity and detects possibility of adulteration by various parameters. So herbal formulations must be standardized to validate the entire manufacturing operation. Standardization plays a major role in the quality control procedures. Hence, better standardization methods are needed to conform the quality of products. To regulate quality, safety and efficacy of herbal drugs, quality control methods need to be improved at each and every stage of production⁴. Standardized herbal products of reliable quality and unambiguous phytoconstituents are required for clinical trials to produce efficient therapeutic results.

It is very important to detect these phytochemicals present in various plants, alternative system formulations such as Ayurvedic, Siddha, Unani and Homeopathic formulations. The therapeutic activity of herbal drug depends on phytochemical concentration so it is essential to develop analytical methods.

Analytical methods such as various chromatographic and spectroscopic techniques are commonly used for quality evaluation of herbal drugs. Sometimes, the data generated from a single method is not enough to ascertain the quality of a herbal formulation. Hence hyphenated analytical techniques such as GC-MS, LC-PDA, LC-MS, LC-MS/MS and LC-NMR are used in analysis of active constituents of herbal formulations.

The present review encompasses the phytotherapy of urolithiasis, analytical methods for estimation of phytochemical markers in marketed Homeopathic formulation such as Clear stone drops formulation. Various analytical methods were reported such as UV, TLC, HPTLC, HPLC, LC-MS and LC-MS/MS, GC-MS methods for concurrent estimation of these phytochemicals of importance in urolithiasis.

Importance of Traditional systems of medicines:

The treatment of urolithiasis involves dissolution and passage of existing stones and prevention of stones recurrence. The stone recurrence rate is more than 50% after 5-10 years of incidence⁵. There is no efficient medicine accessible for treatment of renal stones, especially for prevention and stone recurrence in Allopathy. Currently, conservative measures like taking proper diet, increased consumption of water to ensure adequate urinary dilution and follows standard pharmaceutical medicines like alkali therapy, thiazide diuretics, allopurinol and surgical procedures like extra corporeal shock wave lithotripsy (ESWL), percutaneous nephrolithotomy, ureteroscopy, are used⁶.These are effective to some extent for several types of renal stones, but none of them are 100% effective because of high cost, risk of long-term fertility problems, potential side effects and recurrence of stones. Therefore, complementary treatment is an alternative approach for renal stone treatment.

Herbal medicines used in various traditional systems of Medicine:

Ayurveda:

In Ayurveda ‘Pashanabheda’ category plants protested to be useful in the treatment of renal stones. The term ‘Pashanabheda’ (Pashana=stone, Bheda=break) is a Sanskrit word used to group of plants with activities like diuretic and anti-urolithiasis⁷.

Siddha:

Siddha system of medicine not only curing a disease but also gives us an enormous perception and accompanies to healthy life. In this traditional system of medicine diseases are classified into 4448 numbers. “Kalladaippu” is one among them⁸. The clinical characteristics of Kalladaippu can be coincided with Urolithiasis. “Kalladaippu”, “KALPIRAMIYAM” are synonyms means renal stones in Siddha medicine⁹.

Unani:

Renal stones include Nephrolithiasis, Ureterolithiasis and Cystolithiasis. In Unani Nephrolithiasis called as Hisat-e-Kulyah, Ureterolithiasis as Hisat-e-Halib and Cystolithiasis as Hisat-e-Masanah¹⁰.In Unani System of Medicine, treatment to control urolithiasis is to expel out the destroyed stones by consumption of plenty of fluid, by taking of Unani medicines containing activitieslike tafteet-e-hisat (litholytic/lithotriptic), idraar-e-baul(diuresis), tahleel-e-waram (resolution) dafey-e-tashannuj (anti-spasmodic), alongwith taqwiyat-e-kul’yah¹¹ (Nephroprotective).

Naturopathy and Yoga¹²:

It is a system of therapy that uses natural agents like air, water, herbs and physical forces for the treatment of diseases without use of any drugs. Naturopathic treatment for kidney stones as follows:

· Hydropathy:

It includes application of hot, cold, ice or mud pack, hot fomentation across renal region to relieve colic pain.

· Diet therapy:

Should take Plantain pith juice or Ash gourd juice, with empty stomach, one glass in early morning and late evening. Diet contains low protein diet and consumption of more amount of water.

· Herbal remedies:

· Yoga:

Yoga has been used in medicine and health care practices to improve health. Pavanmuktasana, Uttanpadasana, Bhujangasana, Dhanurasana improves renal function¹³.

· Accupuncture

Homeopathy:

Homeopathic medicine can be prescribed based on symptoms of the patient, uses substances produces similar symptoms. Homeopathic preparations are much diluted preparations made by use of either water or alcohol.

In Indian traditional system of medicine various marketed formulations have been used for treatment of urolithiasis, commonly used marketed formulations listed in the table-1.

Table-1: Marketed Formulations of Traditional Systems of Medicines used for Urolithiasis

Ayurveda	Unani	Siddha	Homeopathy
Gokshurada churna¹⁴	Hajral Yahud Pishti²¹	Amukkara Chooranam²⁷	Lycopodium clavatum³⁴ 200c
Chandraprabhavati¹⁵	Kushta Hajrul-Yahood powder²²	Megarajanga Chooranam²⁸	Thlaspi Bursa Pastoris³⁵
Neeri tablet¹⁶	Safoof‑e‑Pathar phori²³	Nandukkal Parpam²⁹	Stonil³⁶
stonevil capsule¹⁷	Sharbat buzoori motadil Syrup²⁴	Nerunjiver Kudineer³⁰	Clear stone drops³⁷
Cystone tablet¹⁸	Majoon hajrul yahood²⁵	SeenakaraParpam³¹	Phosphorus³⁸
Calcury tablet¹⁹	Renax²⁶	Sirupeellai Samoola³²	Berberis Vulgaris ³⁷
Trinapanchamool²⁰	MajunSang-e-Sarmahi Paste²⁴	Venkara chunn³³	Thuja occidentalis³⁹

DISCUSSION:

Literature review suggested that various in vitro, quality evaluation, comparison studies are carried out on listed marketed formulations to evaluate their antiurolithiatic activity.

In all these formulations Tribulus terrestris, Moringa oleifera, Dolichos biflorus, Raphanus sativus, Aerva lanata, Berberis species, Lycopodium clavatum and Pareira brava herbs appeared frequently in many marketed formulations. Almost all Unani physicians recommended Hajral yahud (Lapis judaicus)-Jews stone for urolithiasis as a single drug or in combination with other drugs in various dosage forms. Mostly homeopathic medicines are diluted alcoholic preparation of single herb (mother tinctures) or in combination with other in various dosage forms. The preparations available are mostly mother tinctures or pills. Berberis is found as the common ingredient in most of the Homeopathic formulations.

From the analysis of labeled data of marketed formulations in Ayurveda, Siddha, Unani and homeopathic formulations, commonly used plants containing major constituents are chemically unique.

Phytoconstituents:

The Phenomenon of stone formation is a complex process, there are several types of kidney stones, calcium oxalate stones are most common one⁴⁰.

Conventional medicines target only one feature, unlike allopathy, herbal medicines have various phytochemicals, exerts their anti-urolithiasis effect by multiple mechanisms ⁴¹explained in figure1.

Table -2: Mechanism of action of markers

Marker	Class	Mechanism of action
Berberine, Curare	Isoquinoline alkaloid	Anti oxidant, anti inflammatory, antibacterial, diuretic, urinary alkalizing, Inhibits calcium oxalate crystals deposition in renal tubule, Antispasmodic, skeletal muscle relaxant
Chlorogenic acid, Protocatechuic acid	Polyphenols	Angiotensin converting enzyme inhibitor, diuretic, Anti oxidant, anti inflammatory, antibacterial, phospholipase inhibition
Rutin, Quercetin, Kaempferol	Flavanoids	Anti oxidant, anti inflammatory and antilitholytic property
Oleanolic acid, Rosmarinic acid, Ursolic acid, Lupeol	Pentacyclic triterpenoid	Antioxidant, anti-inflammatory, diuretic, and angiotensin-converting enzyme inhibition
Otosenine, Flourosenine, Floridanine Eremorphilane sesqui terpenes	Pyrrolizidine alkaloids	Anti-hemorrhagic, diaphoretic, diuretic, an emmenagogue, and oxytocic.

Table -3: Analytical methods of markers

Marker

Structure

Analytical method

Berberine

Class: Isoquinoline alkaloid

Estimation of Berberine by

1) UV⁴²:

UV-1800, Shimadzu

Wave length-428nm

Beers range-10-50 μg/ml.

2) HPTLC⁴³:

Plates: silica gel 60F254 (20x10 cm)

Solvent: n-Propanol: Formic acid: Water (9: 0.1: 0.9 v/v/v).

Detection: at 350nm in Fluorescence mode

3) HPLC⁴⁴:

Column: Sunfire RPC18, (250 × 4.6 mm ID, 5 μm).

M.P:1%ammunium chloride: Acetonitrile.

Flow rate: 0.8ml/min

Wavelength: 254nm.

4) LC-MS⁴⁵:

Column: Acquity UPLC C18 (100 ×1.0 mm, 1.8μm)

M.P: water (containing0.1% formic acid): acetonitrile (30:70v/v)

Flow rate: 0.45mL/min

Detection: from m/z 50 to 3000 using quadrupole time-of-flight mass spectrometer

Chlorogenic acid

Class: Polyphenol

Rutin

Class: Flavanol glycoside

Quantitative analysis of chlorogenic acid, berberine and rutin by LC-UV/MS⁴⁶:

Column: Pinnacle II (25 cm × 4 mm, 5-μm)

Elution: Gradient elution by using formic acid: water acetonitrile

Detection: UV-350nm

Rt10.39, 18.58, and 33.81

Carrier gas-Nitrogen

Ionization source: 70 eV (Electron impact ionization source)

Sarsapogenin

Neotigogenin

Class: Steroidal saponins

Isolation of steroidal saponins by NMR⁴⁷:

Steroidal saponins are identified by Proton and C₁₃ NMR.

High performance liquid chromatography (HPLC) analysis of phenolic profiles⁴⁸:

Column: Zorbax SB-C18, 250 x 4.6 mm,5um

M.P: Gradient elution

10 mM phosphoric: methanol

flow rate of 1 mL/min at room

Wave length:306 nm and 333 nm

Protocatechuic acid(PC)

Class: Polyphenol

Chlorogenic acid (CA)

Class: Polyphenol

Kaempferol-3-O-rutinoside (K-O-R) ,Rutin

Class: Flavonoid glycoside

PC, CA, and K-O-R by HPLC⁴⁹:

Standard and sample solutions are prepared with methanol

Column: Agilent -C18 (150 × 4.6 mm,5um

M:P: Trifluoro aceticacid: methanol (gradient elution)

Flow rate: 0.7ml/min.

Detection: UV at 254nm

R_t: 5.36, 8.22 and 17.04 min

Flavanol glycosides by HPLC⁵⁰:

Column:C_{8 (}25cm x 7 mm,7um)

Mobile phase: Propanol: Water (14:86)

Flow rate:1.8 ml/min

Detection: PDA at 254nm

This method is better method when compared with gradient elution mode.

Phenolic compounds by HPLC⁵¹:

Column: Agilent C18(250 × 4.6 mm ,5 μm)

Elution: Gradient Elution using methanol

Flow rate :0.8ml/min

Detection: Diode array detector at 278 nm

Eugenol:

Class: Phenylpropanoid

Eugenol by UV⁵²:

Eugenol by UV, HPTC, HPLC⁵³:

λ max of eugenol:281 nm.

HPTLC: silica gel F254

Solvent for HPTLC: methanol, chloroform (95:5)

Solvent for UV, HPLC: MeOH: H2O: ACN (50:25:75)

Rt 12.79.

Rosmarinic acid:

Class: Phenol

Ursolic acid:

Class: Pentacyclic triterpenoid

Rosmarinic acid by HPLC⁵⁴:

Column: Inertsil ODS-3 C-18 column (25cmx 4.6 mm,5um)

M.P: methanol: 0.01Mphosphoric acid (Gradient elution)

Detection: at 285nm

R_t:30.07.

Rosmarinic acid, ursolic acid byTLC, HPLC, MS⁵⁵:

TLC: silica gel GF₂₅₄(8x5 cm).

Solvent: Toluene: ethyl acetate: formic acid (7:2:1v/v)

HPLC: C18 column (250x4.6 mm, 5 μm)

M.P:methanol: tetrahydrofuran:0.1%orthophosphoricacid (55:5:40 v/v)

Flow rate: 0.7 ml/min

Detection: 210 and 330 nm

R_t: 5.45min (RA) at 330 nm 12.55 min (UA) at 210 nm

MASS: Q-Tof-Micromass

Ionization source: ESI in positive ion mode.

m/z of RA: 360

Oleanolic acid

Class: Triterpenoid saponin

HPTLC⁵⁶:

Standard and sample solutions were prepared by using methanol

Plates: silica gel 60 F₂₅₄ Solvent: toluene: ethyl acetate: formic acid (7:3:0.3, %v/v).

Detection: Densitometer

at 530 nm in UV, in visible mode used anisaldehyde sulphuric acid reagent

Rf: 0.56

HPLC⁵⁷:

C18 column (100 mm × 4.6 mm,1.8um)

M.P: Buffer: Methanol (gradient mode)

Flow rate:1.2 ml/min

Detection:210nm

R_t: 11.5 min

LC-MS/MS⁵⁸:

Column:(150mm×4.6mmi.d., 5.0um)

M.P: methanol–ammonium acetate (32.5 mM) (85:15, v/v)

Flow rate: 1ml/min

Detection: Electron spray ionization, negative ion mode.

Internal standard: glycyrrhetinic acid

Bebeerine

Class: Alkaloid

Cissampareine

Class: Alkaloid

Bebeerine and Cissampareine by UV, IR, NMR, MS^59,60:

Leaf and stem extract by column chromatography

21 fractions collected subjected to TLC, check for homogeneity of fractions, combined and concentrated and further analyzed by UV, IR, NMR, MS.

Bebeerine: 0.88, λ max: 265 nm, M–229

Cissampereine: Rf- 0.95, λ max: 265 nm, (M+1) ion peak at 607.

Analytical methods of markers:

The therapeutic activity of herbal drugs depends upon the active chemicals present in them. Standardization is necessary in order to establish quality of drugs, based on the concentration of their active phytochemicals. So, it is essential to develop analytical methods to detect these markers present in plants, different herbal formulations or alternative system formulations like Ayurveda, Siddha, Unani and Homeopathy. The efficacy of herbal medicines has a characteristic of a complex mixture of chemical compounds present in the herbs, only few constituents are clinically evaluated for therapeutic value. Analytical methods such as various chromatographic, spectroscopic methods are commonly used for quality evaluation of herbal drugs. Analytical methods of markers which are commonly present in all formulations explained in table no.3.

CONCLUSION:

There is no affordable treatment available for renal stones in allopathy without stone recurrence. Herbal drugs have gained remarkable acceptance being noninvasive, less cost, safe, and effective with less chances of recurrence. Hence people prefer to follow traditional systems like Ayurveda, Siddha, Unani, and Homeopathy for renal stone treatment. So, herbal formulations must be subjected to quality control tests to ascertain their quality, safety, purity and efficacy. Even though many types of analytical methods such as chromatographic and spectroscopic techniques are available for estimation of markers, methods should be carefully selected and improved extensively.

All commonly used herbs in existing formulations having major constituents are chemically therapeutic, so this review suggests the researchers to develop potent formulation for treating urolithiasis with enhanced efficacy and less side effects in best way as compared to existing treatment. Multidisciplinary research is needed to develop potent formulations as well as precise and sensitive research methods for analysis of markers. There is a demand for validated analytical methods for finding precise quality of herbal formulations used in renal stone diseases.

ABBREVIATIONS:

UV-Ultra violet visible spectroscopy, IR-Infra Red spectroscopy, NMR-Nuclear Magnetic Resonance spectroscopy, TLC-Thin Layer Chromatography, HPLC- High Performance Liquid Chromatography, HPTLC-High Performance Thin Layer Chromatography, LC-MS-Liquid Chromatography Mass Spectroscopy, GC-MS-Gas Chromatography Mass Spectroscopy, LC-NMR-Liquid Chromatography-Nuclear Magnetic Resonance Spectroscopy.

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Received on 14.01.2022 Modified on 23.03.2022

Asian J. Res. Pharm. Sci. 2022; 12(4):321-328.

DOI: 10.52711/2231-5659.2022.00055