Introduction:

For basic healthcare, between 75 and 80 percent of the world's population still relies on herbal
medicine, particularly in underdeveloped nations.[1] This is mainly due to the widespread
perception that herbal medications are inexpensive and easily accessible in addition to having
no negative effects.[2] The World Health Organization (WHO) reports that the usage of herbal
treatments is two to three times more globally than the use of conventional medications.[3]
Many current medical practices have their roots in the ancient usage of plants for therapeutic
reasons, which predates human history. Use of natural ingredients is a key component of
traditional medicines (TMs). Natural products are used in many medical practices, including
traditional Chinese medicine (TCM), Ayurveda, Siddha and Unani. These practices have been
practiced for hundreds or even thousands of years worldwide and have developed into well-
organized, controlled medical systems.[4] Over millions of years, natural compounds have
developed chemical variety, which affects their biological activity and drug-like qualities.
These goods are now among the most crucial tools for creating novel scaffolds and lead
compounds.[4]
Regardless of regional, ethnic, or cultural borders, urolithiasis has been a major worldwide
health restriction for humans and animals over the last 20 years. It is the third most frequent
urinary tract issue, with a lifetime risk of 1–5% throughout Asia, 8–15% across America and
Europe, while 20% around the Middle East.[5] Today, there is a rise in the prevalence of
urolithiasis throughout India, while two "stone belts" have been scientifically detected here.[6]
Since the advent of extracorporeal shockwave lithotripsy (ESWL), that has developed into the
accepted method for removing kidney stones, open renal surgery for nephrolithiasis is now
uncommon and seldom carried out.[7] However, persistent remaining stone fragments, the
potential for infection, and the painful consequences of shock waves all point to the risk of
acute renal damage, decreased renal function, and increased stone recurrence as potential side
effects of ESWL.[8] Even though numerous randomized studies have shown the effectiveness
of pharmacological therapy, there are sometimes quite substantial adverse effects.[9] The main
issues in the treatment of urolithiasis are invasive surgical procedures and ineffective
medication therapies. Doctors continue to give thiazides and alkalizing medicines to treat
various kinds of stones. However, even after taking the medicine, the underlying risk factors
won't change, and recurrence will become very common.[10] It is thus advisable to explore for
an alternative via the use of phytotherapy or medicinal plants.
When kidney stones occur, inflammation may occur either upstream (as a causative reason) or
downstream (as a consequence).[11] It is a constituent of the intricate biological response of
vascular tissue to harmful stimuli, such as infections, injured cells, or irritants. The organism's
response to eliminate harmful stimuli and initiate the healing process is a defensive action.[12]
While modern medications are effective in treating inflammation and its related disorders, their
usage is generally limited owing to the accompanying adverse effects. In recent years, there
has been a growing recognition that medicinal plants, phytochemicals, as well as herbal
products have the potential to impact the progression of inflammatory diseases. Additionally,
they offer advantages in terms of safety, cost-effectiveness, and availability. These natural
substances can provide a combination of nutrients that assist in the repair and upkeep of
damaged tissues. Consequently, it is logical to conduct a scientific evaluation of traditional
medicines to determine their potential use in treating inflammatory conditions.[13]
Alternanthera dentata a member of the Amaranthaceae family has traditional claims of
possessing stone dissolving property according to ethnomedicinal evidence also there is claims
that the plant has anti-inflammatory and analgesic activity. Also, numerous plants in the
Aracaceae family have urolithiatic potential. Calamus floribundus Griff. belongs to the
Aracaceae family, and its urolithiatic potential has yet to be established. There are claims that
the paste of roots and shoots of Calamus floribundus is used to treat wounds. However, the
antiurolithiatic and anti-inflammatory potential of both the plants have not been scientifically
proven.
The Selected Plant

PLANT PROFILE
Plant 1: Alternanthera dentata Scheyground
Taxonomic classification of Alternanthera dentata
Scheyground
Scientific name: Alternanthera dentata
Order: Caryophyllales
Family: Amaranthaceae
Subfamily: Gomphrenoideae
Genus: Alternanthera
Species: A. dentata Scheyground
Common/English names: Little Ruby, Joseph's coat, Figure 1: Aerial parts of
Calico Plant, Joyweed Alternanthera dentata
Scheyground
Assamese name: Bifolyokoroni
Description: Alternanthera dentata Scheyground is a tropical perennial typically grown as an
annual, known for its colorful foliage that ranges from burgundy to purple.
Height & Width: 30-40cm high x 60-90cm wide.
Foliage: Stunning deep burgundy foliage.
Flowers: White flowers in spring.
Ethnomedicinal claims and uses: The leaves and young shoots are used in conditions like cuts
and wounds, inflammation, stomach pain and kidney stones.[14]
Plant 2: Calamus floribundus Griff.
Taxonomic classification of Calamus floribundus Griff.
Order: Arecales
Family: Arecaceae
Genus: Calamus
Species: Calamus floribundus Griff.
Common name: Lejaibet (Assamese)
Synonym: Palmijuncus leptospadix (Griff.) Kuntze.
Description: Calamus floribundus Griff. is a slender
cluster forming climber; stem thickened at joints, with
lead-sheaths 12-20 mm in diameter, naked stem smooth, 8
-10 mm in diameter at the internodes. Leaf sheaths are
green with grayish brown hairs, with scattered to densely Figure 1: Aerial parts of
arranged, brownish, flattened to 2.5 (to 5 at sheath apices) Calamus floribundus Griff.
cm long spines, those at sheath apices needlelike yellowish.
Flower: Inflorescence to 4m long, flagellate; bracts tubular, briefly open and spreading at the
apices.
Fruit: Globose to 1.5 cm diameter, white or yellowish.
The season for flowers and fruit is March-October.
Distribution: Found in North-eastern India, Bhutan, Nepal, Bangladesh and Myanmar.
Ethnomedicinal claims and uses: Calamus species are used in fever, piles, dyspepsia and
biliousness in the Ayurvedic system. Young shoots of the plant are used as vegetable. Flowers
are used as antiseptic, anti-bacterial, externally for cuts, burns, bruises, scalds. In Assam, folk
people believed that the tender shoots, leaves and seeds of this plant are used as vermicide.[15]
Objective and work plan

Aim of the study: To evaluate the Phytochemical constituents and Pharmacological activities
of Alternanthera dentata Scheygrond and Calamus floribundus Griff., with special reference
to Anti-inflammatory and Antiurolithiatic Activities.

Specific Objective:
To find out the antiurolithiatic and anti-inflammatory potential of the plants - Alternanthera
dentata Scheygrond and Calamus floribundus Griff., and to establish the ethnomedicinal
claims.

Plan of Work:
1. Literature survey on the selected plants.

2. Collection and authentication of selected plants material.

3. Study of the pharmacognostical parameters of the plants.

4. Extraction of the plant material with different solvents.

5. Phytochemical screening of the different solvent extracts of the plant material.

6. Thin layer chromatography of different solvent extracts.

7. In vitro anti-oxidant assay, in vitro antiurolithiatic assay, in vitro anti-inflammatory assay of

extracts using various models.

8. Acute toxicity studies of active extracts, which are to be tested on experimental animals.

9. Pharmacological evaluation of active extracts with special reference to anti-inflammatory

and antiurolithiatic activities.

10. In vitro cell line studies.

11. Isolation of phytoconstituents from active extract which shows highest and promising
pharmacological activity.

12. Spectroscopic and Chromatographic studies of isolated compounds.

13. Evaluation of isolated compound(s) for their relevant pharmacological activity.

Literature Review

1. Sarquis et al. 2019, reported that Decoction of leaves of Alternanthera

brasiliana, Alternanthera dentata and Alternanthera ficoidea, ingest 1 cup 4 x a day
for 20 days is taken for the Elimination of kidney stone, infections of the liver, stomach
pains by residents at the mouth of the Mazag˜ao River, State of Amap´a, Brazilian
Amazonia.[16]
2. Al-Alwani, et al. 2017, extracted natural dyes anthocyanin and chlorophyll from
Musa acuminata bracts and Alternanthera dentata leaves, respectively. The dyes were
then applied as sensitizers in TiO2-based dye-sensitized solar cells.[17]
3. Kumar et al. 2014, demonstrated a green rapid biogenic synthesis of silver
nanoparticles AgNPs using Alternanthera dentata (A. dentata) aqueous extract and
finally evaluated antibacterial effect of AgNPs against bacterial strains.[18]
4. Beatriz et al. 2006, reported the ethnomedicinal use of Aerial parts of
Alternanthera dentate as Anti-inflammatory and analgesic.[14]
5. Gogoi et al. 2021, reported that tribes in the Tinsukia district of Assam, India,
use a paste of the shoot and roots of Calamus floribundus to treat wounds.[19]
6. Myrchiang et. al. 2018, reported that the people of Nongtalang village, West
Jaintia Hills, Meghalaya, India, utilise root paste of Calamus floribundus to treat fever
and cough.[20]
7. P Jiji 2015, reported that the young shoot of Calamus floribundus is used as
medication for stomach problems by ethnic communities in the Sivasagar region of
Assam, India.[21]
8. T. N. Manohara's 2013, reported that the Calamus floribundus has a large
number of proteins, dietary fibres, and carbs. In dyspepsia, the ripe fruit pulp is
consumed with a little common salt and honey.[15]
Materials and Methods:
Collection, authentication and extraction of Plant material:
Plant parts of Alternanthera dentata Scheygrond and Calamus floribundus Griff. were
collected from Mangaldai, (Darrang district) in Assam state, INDIA. The plant specimen was
identified and authenticated by Dr. N. Odyuo, Scientist-D, Hoo, Botanical Survey of India,
Eastern Regional Centre, Shillong 793003. A voucher specimen of each plant was deposited in
the herbarium (Alternanthera dentata- DU/KD/2020/01) (Calamus floribundus -
DU/KD/2020/02).
The ariel part of Alternanthera dentata Scheygrond was thoroughly cleaned and dried under
shade and coarsely powdered. The dried powdered materials were then subjected to successive
extraction with solvents in increasing order of polarity- starting from Petroleum ether (PeAD),
chloroform (ChAD), ethyl acetate (EaAD) and finally methanol (MeAD).
The shoot part of Calamus floribundus Griff. was cut and the outer spiny leaf sheaths removed
followed by drying under shade and then the dried parts were coarsely powdered. The dried
powdered materials were then subjected to successive extraction with solvents in increasing
order of polarity- starting from Petroleum ether (PeCF), chloroform (ChCF), ethyl acetate
(EaCF) and finally methanol (MeCF).
Each extract was concentrated by distilling off the solvent and evaporated to dryness in Rotary
evaporator. The crude extracts were stored in air tight containers in 4°C until use.
Study of the pharmacognostical parameters of the plant material:
Transverse section and powder microscopy of both the plants were performed.
Phytochemical investigation of extracts:
The different extracts were separately tested for the presence of various plant constituents such
as alkaloids, flavonoids, glycosides, tannins, carbohydrates, saponins, steroids and
triterpenoids, fats and fixed oils.[22] The total phenolic and flavonoid content were
measured.[23]
Thin layer chromatography of the extracts:
The TLC of extracts were carried out in different solvent systems and separation of spots were
monitored. Spots were visualized under iodine vapour or UV light (254 nm and 366 nm).[24]
In vitro Antioxidant assay:
The plant extracts were screened for their antioxidant and free radical scavenging potential by
using various in vitro antioxidant assay methods like 1, 1-Diphenyl-2-picryl-hydrazyl
(DPPH)[23], nitric oxide radical scavenging activity[25].
In vitro evaluation:
a. Antiurolithiatic activity: in vitro antiurolithiatic activity of all the extracts of both the
plant was performed using (i) Nucleation assay, (ii) Aggregation assay and (iii) Single gel
diffusion assay.[26,27]
b. Anti-inflammatory activity: in vitro anti-inflammatory activity of all the extracts of both
the plant was performed using (i) Egg albumin denaturation assay and (ii) HRBC Membrane
stabilization assay.[28,29]
In vivo evaluation: The extracts which showed promising results in the in vitro studies were
further explored in the animal model.
The Institutional Animal Ethics Committee of Department of Pharmaceutical Sciences,
Dibrugarh University, Dibrugarh Assam authorized (IAEC/DU/199 Dated 19.05.2021) all of
the experimental protocols and methods utilized in this experiment. The animals were kept in
standardised conditions (temperature 27± 1°C, humidity 60±4%, natural lighting) and were
contained in polypropylene cages with regular supply of feed and water.
a. Acute toxicity study (oral): Acute oral toxicity studies according to the procedures
described in Organization for Economic Co-operation and Development (OECD) guidelines
425 on experimental animals was carried out.[30]
b. In vivo antiurolithiatic study: Two model namely Ethylene glycol and ammonium
chloride induced urolithiasis in rats (28 days model), Calculi-Producing diet induced (also
known as glycolic acid induced) urolithiasis model (42 days model) was employed to evaluate
the antiurolithiatic potential of the two plants.[26,31] Test extract 1 (high dose and low dose)
and test extract 2(high dose and low dose) were used. Cystone at a dose of 500 mg/kg b.w. was
used as standard in both the models. Body weight, water intake and the biochemical parameters
(Urine- phosphorus, calcium, and magnesium oxalate, uric acid, urea and citrate; serum-
phosphorus, calcium, urea, uric acid and creatinine; kidney homogenate- phosphorus, calcium
and oxalate) were recorded to examine the effect of the extracts in urolithiatic rats.
Histopathological examination of kidney of the rats was also performed.
c. In vivo anti-inflammatory study: For the evaluation of anti-inflammatory property was
evaluated using acute model namely Carrageenan induced paw edema model and chronic
model namely- Cotton pallet induced granuloma model was used.[32] Carrageenan induced
paw edema was monitored in the first model. Whereas in cotton pallet induced granuloma
model the activity was measured in terms of the inhibition of dry weight and transudative
weight of the cotton pallets. Diclofenac sodium at 10mg/kg bw was used as standard drug in
both the models.
d. Cell line studies: Normal rat epithelium‑derived renal tubular epithelial cells, i.e.,
NRK‑52E cells were procured from the National Centre of Cell Sciences, Pune (India). The
cells were cultured and maintained in Dulbecco's Modified Eagle Medium with High Glucose
(DMEM-HG) supplemented with 10% Foetal Bovine Serum (FBS). The cells were aspirated
into a 5ml centrifuge tube and cell pellet was obtained by centrifugation at 300 x g. The cell
count was adjusted, using RPMI medium containing 10% FBS, such that 50μl of suspension
contains approximately 10,000 cells.[33]
a. Cytotoxicity assay: MTT (3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium
bromide) method was employed to evaluate the toxicity of test extract towards NRK-52E cells.
Concentration of both the test extracts were 6.25, 12.5, 25, 50 & 100 µg/ml.[33]
b. Cytoprotective assay: MTT assay was employed to access the cytoprotective effect of
suitable test extract against sodium oxalate induced injury on NRK-52E cell lines. Sodium
oxalate at 1mM concentration was used to induce cell injury and the cytoprotective effect of
was monitored by test extract’s ability to inhibit the sodium oxalate induced toxicity and
improve cell viability.[33]
In silico antiurolithiatic study:
GC-MS analysis: The active extract was subjected to GC-MS analysis in order to access the
phytoconstituents present in it. GC-MS study was done using Shimadzu GCMS-TQ8030.
Compounds with the given codes are listed in table 21.
In silico study:
All the compounds identified from the GCMS data were loaded to the Discovery studio 2020
(DS 2020) molecular modelling software (Dassault Systèmes BIOVIA, San Diego, USA) and
three-dimensional structures of the compounds were generated using ‘Small Molecule’ tool of
the software. Then energy minimization of the compounds was carried out using ‘Full
Minimization’ protocol under ‘Small Molecule’ tool using CHARMM-based (Chemistry at
Harvard Macromolecular Mechanics) smart minimizer which performs 2000 steps of Steepest
Descent followed by Conjugate Gradient algorithm with an RMSD gradient of 0.01 kcal/mol.
Then compounds were saved in a single file for further studies.
X-ray crystal structure of the three target enzymes namely Xanthine dehydrogenase (Xdh)
(PDB ID: 1JRP), Tamm-Horesefall Protein (THP) (PDB ID: 4WRN) and Oxalate oxidase
(OxOx) (PDB ID: 2ETE) were obtained from the Protein Data Bank (PDB) websites
(www.rcsb.org). Native ligands to the selected proteins are Oxypurinol for Xdh and
Acetylglucosamine for OxOx and THP were chosen as control.[34]
The selected compounds were docked with the three targets using simulation-based docking
protocol CDocker of the DS 2020. The binding energies of the best poses were calculated using
‘Calculate Binding Energy’ protocol of the DS 2020. The compounds with greater docking
score to that of the control were then subjected to toxicity analysis using ‘Toxicity Prediction’
protocol under ‘Small molecules’ tool of the DS 2020.
Compounds filtered in the toxicity analysis were then finally analysed for ADMET
(Absorption, distribution, metabolism, elimination and toxicology) using ‘ADMET
Descriptors’.
Isolation of phytoconstituent from the active extract:
The active extract obtained from the in vitro and in vivo studies was subjected to isolation by
using column chromatography to isolate phytoconstituent(s). Initially the column was packed
with silica gel with mesh size 60-120. Fractionation was done by eluting the extract with
mixture of different solvents from lower to higher polarity in a gradient manner. The fractions
were collected and TLC was performed. The fractions with similar separation pattern and same
type of spots were combined and loaded in a small column packed with silica gel having mesh
size 100-200. Then the combined fractions were further eluted with solvent mixtures in gradient
manner. The compound(s) obtained from the fraction was further purified using different
techniques.[35]
Spectroscopic and Chromatographic studies of isolated compounds:
Physical properties of the isolated compound (colour, melting point, solubility) were analysed.
TLC of the isolated compound was performed. Spectroscopic methods particularly FTIR, 13C
and 1H NMR, HRMS were done to elucidate the isolated compound structure.
Evaluation of pharmacological activity of the isolated compound:
In vitro assay:
In vitro antiurolithiatic activity of isolated compound was evaluated by using nucleation,
aggregation and single gel diffusion assay. Further in vitro anti-inflammatory activity was also
done using egg albumin denaturation assay and HRBC membrane stabilization assay.
In silico assay:
In silico activity was performed using the same proteins (xanthine dehydrogenase (PDB:
1JRP), oxalate oxidase (PDB: 2ETE), and Tamm-Horsfall protein (PDB: 4WRN),) as
mentioned in the in silico evaluation of test extracts. The software used is Biovia Discovery
Studio (DS) 2022. After obtaining the structure of the isolated compound from spectroscopic
and spectrometric analysis, it was drawn using ChemDraw and saved in .sdf format. This
structure was later imported to the DS software to prepare for molecular docking study.
Results:
Pharmacognostical parameters:

Figure 1: TS and Powder microscopy of Alternanthera dentata and Calamus floribundus

Phytochemical investigation: The results of phytochemical investigation, yield of extracts are
listed below:
Phytochemical PeAD ChAD EaAD MeAD
Alkaloids - - - +
Glycosides - - + +
Carbohydrates - + + +
Flavonoids - - + +
Saponins - - - +
Tannins - - - -
Steroids and triterpenoids - - + +
Fats and oils + + - -
Table 1: Phytochemicals present in different extract of Alternanthera dentata (‘+’ indicates
present, ‘-’ indicates absent)
Extract Consistency Colour % Yield
PeAD Sticky solid Blackish Green 3.74 ± 1.22
ChAD Sticky solid Dark Green 15.46 ± 2.17
EaAD Sticky solid Brown 4.92 ± 0.97
MeAD Sticky solid Dark Brown 25.68 ± 1.54
Table 2: Physical characteristics and percentage yield of different solvent extracts of of
Alternanthera dentata.
Phytochemical PeCF ChCF EaCF MeCF
Alkaloids - - - +
Glycosides - - - +
Carbohydrates - - + +
Flavonoids - - + +
Saponins - - + +
Tannins - - + -
Steroids and triterpenoids - + + +
Fats and oils + + - -
Table 3: Phytochemicals present in different extract of Calamus floribundus (‘+’ indicates
present, ‘-’ indicates absent)
Extract Consistency Colour % Yield
PeCF Sticky solid Light brown 0.59 ± 0.21
ChCF Sticky solid Brown 0.31 ± 0.09
EaCF Sticky solid Brown 3.02 ± 0.22
MeCF Sticky solid Dark Brown 8.81 ± 0.78
Table 4: Physical characteristics and percentage yield of different solvent extracts of of
Calamus floribundus.
Thin layer chromatography of all the extracts: Figure 5 depicts TLC of all the extracts of
both the plants.
Figure 5: TLC of different extracts of Alternanthera dentata and Calamus floribundus. Hex-
Hexane, Tol- Toluene, Chl- Chloroform, EA- Ethyl acetate, GAA- Glacial Acetic acid, FA-
formic acid, Me- Methanol.
Total flavonoid and total phenolic content
The calibration curve of quercetin is shown in figure 6. The flavonoid content is listed in table
5.

Calibration curve of Quercetin

0.35
0.3 y = 0.002x + 0.1085
R² = 0.9933
Absorbance

0.25
0.2
0.15
0.1
0.05
0
0 20 40 60 80 100 120
Concentration (µg/ml)

Figure 6: Calibration curve for Quercetin for total flavonoid content

Total flavonoid (mg of Quercetin
equivalent/ g of plant extract)
EaAD 5.51± 2.39
MeAD 20.38± 4.33
EaCF 7.33± 1.82
MeCF 13.81± 1.22
Table 5: Total flavonoid content of extracts. (Values are represented as mean± SEM)
Gallic acid calibration curve (Figure 7) is used to measure the total phenolic content of EaAD,
MeAD, EaCF and MeCF.

Calibration curve of Gallic acid

1.2
y = 0.0102x - 0.0371
1 R² = 0.9952
Absorbance

0.8

0.6

0.4

0.2

0
0 20 40 60 80 100 120
Concentration (µg/ml)

Figure 7: Calibration curve of Gallic acid for total phenolic content

Total phenolic (mg of Gallic acid
equivalent/ g of plant extract)
EaAD 8.37± 1.21
 MeAD 28.62± 3.72
EaCF 9.11± 2.15
MeCF 17.70± 1.29
Table 6: Total phenolic content of extracts. (Values are represented as mean± SEM)
In vitro antioxidant studies: In the DPPH and NO scavenging assay it was seen that
methanolic extracts of Althernanthera dentata (MeAD) and the methanolic extract of Calamus
floribundus (MeCF) showed good antioxidant activity when compared with the standards. The
IC50 values depicts the same.
DPPH free radical scavenging activity:
Sample IC50 (μg/mL)
Ascorbic acid 39.48± 0.43
EaAD 89.46± 0.22
MeAD 55.42± 0.16
EaCF 65.48± 0.19
MeCF 57.35± 0.31
Table 7: IC50 values of extracts and standard in DPPH radical Scavenging activity. (Experiment
was done in triplicates and values are represented as mean± SEM)
Nitric oxide scavenging activity:
Sample IC50 (μg/mL)
Gallic acid 40.67± 0.28
EaAD 75.93± 0.23
MeAD 59.25± 0.17
EaCF 77.14± 0.38
MeCF 63.22± 0.32
Table 8: IC50 values of extracts and standard in NO radical Scavenging activity. (Experiment
was done in triplicates and values are represented as mean±SEM)
In vitro antiurolithiatic study:
a. Nucleation assay: The nucleation test used the solution's turbidity to measure the total
amount of crystals produced. The recorded absorbance of the control was deducted from the
absorbance obtained from the test. When incubated with sodium oxalate, there was a decline
in absorbance with a rise in the level of the test and standard. The percentage of nucleation
inhibition by standard and extracts increased in a dose-dependent manner (Figure 5 and Figure
6). The IC50 value is listed in table 9.
Figure 5: Percentage inhibition of nucleation by different extracts of Alternanthera dentata.
Values are expressed as mean± SEM, n=3. Data were tested with One way ANOVA followed
by Dunnet’s multiple comparison test. *p <0.0001 significant when compared with standard
group (cystone).

Figure 5: Percentage inhibition of nucleation by different extracts of Calamus floribundus.

Values are expressed as mean± SEM, n=3. Data were tested with One way ANOVA followed
by Dunnet’s multiple comparison test. *p <0.0001 significant when compared with standard
group (cystone).
Nucleation inhibition IC50 (µg/ml)
Cystone 527.66± 2.12
PeAD 3937.38± 1.10
ChAD 3445.54± 2.49
EaAD 1087.60± 1.39
MeAD 780.81± 2.45
PeCF 2835.42± 1.23
ChCF 2580.24± 1.14
EaCF 1214.19± 2.19
MeCF 769.03± 1.22
Table 9: Nucleation assay- IC50 (Mean± SEM) value of cystone, and different extracts of
Alternanthera dentata and Calamus floribundus.
b. Aggregation assay: Similarly in the aggregation assay, concentration dependent
inhibition of the CaOx crystals aggregation was seen in the presence of extracts and standard
(cystone). The percentage inhibition is shown in Figure 6 and Figure 7. The IC50 is listed in
table 10.

Figure 6: Percentage inhibition of nucleation by different extracts of Alternanthera dentata.

Values are expressed as mean ± SEM, n=3. Data were tested with One way ANOVA followed
by Dunnet’s multiple comparison test. *p <0.0001, ● p <0.05 significant when compared with
standard group (cystone).
Figure 7: Percentage inhibition of nucleation by different extracts of Calamus floribundus.
Values are expressed as mean ± SEM, n=3. Data were tested with One way ANOVA followed
by Dunnet’s multiple comparison test. *p <0.0001, ● p <0.05 significant when compared with
standard group (cystone).
Aggregation inhibition IC50 (µg/ml)
Cystone 522.78± 1.32
PeAD 1986.28± 3.12
ChAD 1759.01± 1.31
EaAD 1005.14± 2.57
MeAD 762.37 ± 2.35
PeCF 2403.09± 1.41
ChCF 1616.58± 1.39
EaCF 932.98± 1.42
MeCF 784.89± 3.29
Table 10: Aggregation assay- IC50 (Mean± SEM) value of cystone, and different extracts of
Alternanthera dentata and Calamus floribundus.
c. Single gel diffusion assay:
Struvite crystals in the gel medium reduced in length when extracts and Cystone concentrations
rose. The perceived length of the struvite crystals, which were measured every 24 hours for
five days, is shown in Table 11 and 13. The percentage inhibition struvite crystals are
summarised in Table 12 and 14. X-shaped dendritic and needle-shaped struvite crystals
developed in the gel. Figure 8 shows gel medium-grown struvite crystals.
Figure 8: Shapes of different struvite crystals formed
 †
Cyst †Cyst †Cyst ▲PeA ▲ ▲
Ch †
†
MeA † ▲
Me
Time Contr PeA ●PeA ●ChA ChA †EaA †EaA †EaA MeA
one one one D AD D AD
(hr) ol D 1.0 D 2.0 D 0.5 D 2.0 D 0.5 D 1.0 D 2.0 D 1%
0.5% 1% 2% 0.5 1.0 0.5% 2%
1.192 0.952 0.972 0.938 0.985 1.044 1.159 1.132 1.091 0.821 0.775 0.887 0.943 1.021 0.973 1.291
24
±0.12 ±0.32 ±0.19 ±0.31 ±0.22 ±0.20 ±0.32 ±0.27 ±0.41 ±0.22 ±0.29 ±0.28 ±0.23 ±0.33 ±0.21 ±0.17
1.261 0.834 0.855 0.84 0.978 1.029 1.143 1.099 1.049 0.792 0.745 0.849 0.876 0.944 0.812 1.045
48
±0.29 ±0.22 ±0.43 ±0.12 ±0.19 ±0.33 ±0.27 ±0.34 ±0.27 ±0.17 ±0.41 ±0.33 ±0.17 ±0.43 ±0.18 ±0.49
1.315 0.713 0.749 0.662 0.961 1.004 1.131 1.023 0.991 0.74 0.715 0.782 0.763 0.816 0.734 0.932
72
±0.31 ±0.17 ±0.17 ±0.29 ±0.32 ±0.21 ±0.16 ±0.38 ±0.18 ±0.43 ±0.39 ±0.41 ±0.28 ±0.27 ±0.27 ±0.17
1.366 0.569 0.537 0.498 0.953 0.993 1.109 0.997 0.952 0.709 0.63 0.667 0.689 0.687 0.598 0.743
96
±0.26 ±0.28 ±0.18 ±0.31 ±0.27 ±0.32 ±0.36 ±0.42 ±0.36 ±0.42 ±0.17 ±0.27 ±0.37 ±0.32 ±0.15 ±0.44
1.406 0.428 0.419 0.387 0.945 0.985 1.091 0.953 0.891 0.645 0.521 0.579 0.576 0.541 0.477 0.619
120
±0.19 ±0.41 ±0.22 ±0.33 ±0.15 ±0.36 ±0.42 ±0.25 ±0.33 ±0.19 ±0.32 ±0.15 ±0.25 ±0.32 ±0.31 ±0.13
Table 11: Length of struvite crystals grown in single gel diffusion medium in different concentration of Cystone, PeAD, ChAD, EaAD, MeAD.
All the values have been tested using One-way ANOVA, Dunnett's multiple comparisons test, p<0.0001. Each value represents mean ± SD (n=3).
†
<0.0001 ▲<0.05 ●not significant.
Time Cysto Cysto Cysto PeAD PeAD PeAD ChAD ChAD ChAD EaAD EaAD EaAD MeAD MeAD MeAD
(hr) ne ne 1% ne 2% 0.5 1.0 2.0 0.5 1.0 2.0 0.5 1.0 2.0 0.5% 1% 2%
0.5%
48 12.39± 12.04 10.45 0.71 1.43 1.38 3.62 3.84 3.53 3.87 4.28 7.1 7.54 16.55 19.05
0.21 ±0.19 ±0.43 ±066 ±0.67 ±0.05 ±0.19 ±0.19 ±0.22 ±0.09 ±0.41 ±0.49 ±0.31 ±0.26 ±0.33
72 24.11 22.94 29.42 2.44 3.83 2.42 9.63 9.17 9.87 7.74 11.83 19.09 20.08 24.56 27.81
±0.32 ±0.17 ±0.32 ±0.65 ±0.14 ±0.21 ±0.75 ±0.23 ±0.17 ±0.19 ±0.46 ±0.22 ±0.15 ±0.41 ±0.19
96 40.23 44.75 46.80 3.25±0 4.88 4.31 11.92 12.74 13.64 18.71 24.8 26.93 32.71 38.54 42.45
±0.17 ±0.25 ±0.21 .31 ±0.50 ±0.40 ±0.58 ±0.16 ±0.19 ±0.38 ±0.27 ±0.31 ±0.21 ±0.27 ±0.22
120 55.04 56.89 58.74 4.06 5.65 5.87 15.81 18.33 21.43 32.77 34.72 38.91 47.01 50.98 52.05
±0.11 ±0.28 ±0.22 ±0.14 ±0.13 ±0.27 ±0.27 ±0.18 ±0.27 ±0.19 ±0.37 ±0.35 ±0.14 ±0.32 ±0.17
Table 12: Percentage inhibition of the struvite crystals in different concentration of Cystone, PeAD, ChAD, EaAD, MeAD.
 †
Cyst †Cyst †Cyst ▲PeC † ▲
Ch †
MeC †
Time Contr PeC †PeC †
ChC ●ChC †EaC †EaC ▲EaC MeC †MeC
one one one F CF F
(hr) ol F 1.0 F 2.0 F 1.0 F 2.0 F 0.5 F 1.0 F 2.0 F 1% F 2%
0.5% 1% 2% 0.5 0.5 0.5%
1.192 0.952 0.972 0.938 1.022 0.782 0.833 0.956 0.818 1.109 0.971 0.765 1.119 1.039 0.828 0.935
24
±0.12 ±0.32 ±0.19 ±0.31 ±0.34 ±0.31 ±0.16 ±0.18 ±0.17 ±0.22 ±0.19 ±0.31 ±0.29 ±0.33 ±0.21 ±0.17
1.261 0.834 0.855 0.84 0.993 0.769 0.812 0.932 0.799 1.079 0.921 0.701 0.989 0.983 0.719 0.823
48
±0.29 ±0.22 ±0.43 ±0.12 ±0.15 ±0.29 ±0.44 ±0.46 ±0.41 ±0.28 ±0.22 ±0.32 ±0.24 ±0.43 ±0.18 ±0.49
1.315 0.713 0.749 0.662 0.988 0.751 0.789 0.925 0.782 1.069 0.876 0.642 0.901 0.841 0.633 0.698
72
±0.31 ±0.17 ±0.17 ±0.29 ±0.24 ±0.34 ±0.43 ±0.45 ±0.26 ±0.33 ±0.32 ±0.18 ±0.34 ±0.27 ±0.27 ±0.17
1.366 0.569 0.537 0.498 0.979 0.746 0.772 0.919 0.775 1.048 0.822 0.578 0.845 0.722 0.529 0.518
96
±0.26 ±0.28 ±0.18 ±0.31 ±0.13 ±0.41 ±0.14 ±0.26 ±0.37 ±0.28 ±0.18 ±0.41 ±0.39 ±0.32 ±0.15 ±0.44
1.406 0.428 0.419 0.387 0.972 0.735 0.761 0.909 0.769 1.022 0.745 0.521 0.709 0.611 0.449 0.467
120
±0.19 ±0.41 ±0.22 ±0.33 ±0.19 ±0.18 ±0.12 ±0.34 ±0.43 ±0.14 ±0.19 ±0.21 ±0.27 ±0.32 ±0.31 ±0.13
Table 13: Length of struvite crystals grown in single gel diffusion medium in different concentration of Cystone, PeCF, ChCF, EaCF, MeCF. All
the values have been tested using One-way ANOVA, Dunnett's multiple comparisons test, p<0.0001. Each value represents mean ± SD (n=3).
†
<0.0001 ▲<0.05 ●not significant.
Cysto Cysto Cysto MeC
Time PeCF PeCF PeCF ChCF ChCF ChCF EaCF EaCF EaCF MeC MeC
ne ne ne F
(hr) 0.5 1.0 2.0 0.5 1.0 2.0 0.5 1.0 2.0 F 1% F 2%
0.5% 1% 2% 0.5%
12.39 12.04 10.45 2.84 1.66 2.52 2.51 2.32 2.71 5.15 8.37 11.62 5.39 13.16 11.98
48
±0.21 ±0.19 ±0.43 ±0.57 ±0.24 ±0.18 ±0.46 ±0.27 ±0.14 ±0.31 ±0.13 ±0.32 ±0.38 ±0.25 ±0.17
24.11 22.94 29.42 3.32 3.96 5.28 3.24 4.4 3.61 9.78 16.09 19.48 19.06 23.55 25.35
72
±0.32 ±0.17 ±0.32 ±0.68 ±0.41 ±0.21 ±0.26 ±0.18 ±0.53 ±0.28 ±0.43 ±0.16 ±0.39 ±0.42 ±0.39
40.23 44.75 46.80 4.21 4.60 7.32 3.87 5.26 5.5 15.34 24.44 24.49 30.51 36.11 44.59
96
±0.17 ±0.25 ±0.21 ±0.36 ±0.35 ±0.29 ±0.29 ±0.24 ±0.11 ±0.51 ±0.21 ±0.15 ±0.11 ±0.24 ±0.23
55.04 56.89 58.74 4.89 6.01 8.64 4.92 5.99 7.84 23.27 31.89 36.64 41.19 45.77 50.05
120
±0.11 ±0.28 ±0.22 ±0.23 ±0.23 ±0.34 ±0.31 ±0.22 ±0.25 ±0.49 ±0.42 ±0.86 ±0.46 ±0.29 ±0.48
Table 14: Percentage inhibition of the struvite crystals in different concentration of Cystone, PeCF, ChCF, EaCF, MeCF.
Characterisation of the Struvite crystals
Powder XRD pattern of struvite crystals with assigned planes is shown in Figure 9. The XRD
patterns were in good agreement with the struvite value reported by the Joint Committee on
Powder Diffraction Standards (96-900-7675). The study found that struvite crystallizes in the
orthorhombic system (Pmn21, space group) having lattice parameters a = 6.955 Å, b = 6.142
Å, c = 11.218 Å, and α = β = γ = 90°. The results obtained were consistent with previous
findings (Ferraris et al. 1986).[36] The struvite crystals' structural identity was validated by the
powder XRD investigations.

Figure 9: Powder XRD patter on the struvite crystals grown in the gel medium

The FTIR spectrum of the grown-up struvite crystals were recorded in the wave number range
4000-400 cm-1 and depicted in Figure 10. The absorption of all the four main constituents of
struvite viz. water of crystallization, PO4-3, NH4+, and metal-oxygen bonds were interpreted in
the recorded spectrum. A broad band observed between 2365 to 3200 cm-1 (approximately) can
be attributed to O-H stretching vibrations due to water of crystallization. The absorptions at
3493 and 3170 cm-1 are likely caused by H-O-H stretching vibrations, whereas the peak at 1581
cm-1 can be associated with H-O-H bending modes, indicating the presence of water of
crystallization. The peak at 2865 cm-1 represents the stretching vibration of N-H in NH4+.
Further, the sharp peak occurring at 1429 cm-1 corresponds to the bending vibrations of N-H in
NH4+. Additionally, at 752 cm-1, the symmetric stretching vibration of PO4-3 units was
identified, whereas at 985 cm-1, the asymmetric stretching vibrations of PO4-3 units was noted.
The absorption band at 681 cm-1 was attributed to bending vibration of PO4-3 units. Lastly, the
metal-oxygen bond due to the presence of magnesium is responsible for the absorption band
that appears at 565 cm-1.
Figure 10: Fourier Transform Infrared Spectrum of the Gel-Grown Struvite Crystals

In vitro anti-inflammatory study:

a. Egg albumin denaturation assay: Anti-inflammatory activity was evaluated against

heat-treated protein denaturation methods. A concentration dependent reduction in albumin
denaturation was observed (Figure 11 & Figure 12). IC50 listed in table 15.

Figure 11: Percentage inhibition (Mean± SEM, n=3) of egg albumin denaturation by different
extracts of Alternanthera dentata.
Figure 11: Percentage inhibition (Mean± SEM, n=3) of egg albumin denaturation by different
extracts of Calamus floribundus.

Egg albumin Denaturation assay

Sample IC50
Diclofenac 223.03± 1.13
PeAD 1,307.89± 2.17
ChAD 1,090.33± 1.92
EaAD 431.35± 0.98
MeAD 331.05± 1.35
PeCF 1,683.83± 3.43
ChCF 1,234.55± 2.33
EaCF 559.66 ±1.78
MeCF 429.96 ± 2.12

Table 15: Egg albumin denaturation assay- IC50 (Mean± SEM, n=3) value of diclofenac, and
different extracts of Alternanthera dentata and Calamus floribundus.

b. Human Red Blood Cell (HRBC) membrane stabilization assay- (Hypotonic Solution-
Induced Hemolysis) The extracts showed a concentration-dependent anti-inflammatory
activity and protected the human erythrocyte membrane exposed to the hypotonic solution
(Figure 12 and 13). IC50 is listed in table 16.
Figure 12: Percentage inhibition (Mean± SEM, n=3) of hypotonicity-Induced hemolysis by
different concentration of diclofenac sodium and extracts of Alternanthera dentata.

Figure 13: Percentage inhibition (Mean± SEM, n=3) of hypotonicity-Induced hemolysis by

different concentration of diclofenac sodium and extracts of Calamus floribundus.

HRBC Hypotonic
Sample IC50
Diclofenac 275.16± 1.24
PeAD 1815.47± 2.11
 ChAD 1140.86± 1.97
EaAD 606.48± 1.27
MeAD 437.96± 2.83
PeCF 903.59± 3.11
ChCF 735.78± 1.68
EaCF 533.65± 1.25
MeCF 424.70± 2.18

Table 16: Human Red Blood Cell (HRBC) membrane stabilization assay- IC50 value (Mean±
SEM, n=3) of diclofenac, and different extracts of Alternanthera dentata and Calamus
floribundus.

In vivo study:

Acute oral toxicity study:

A single dose of 2000 mg /Kg bw orally administered to mice and further observed for 14 days
for signs of toxicity and mortality. The extracts (MeAD and MeCF) are found to be safe upto
the dose of 2000 mg/kg bw and thus theLD50 is considered as > 2000 mg/kg. Hence 250 mg/kg
b.w. and 500 mg/kg b.w. are used for further studies.

Antiurolithiatic study:

Body weight, water intake, serum and urine parameters and results of the kidney homogenate
analysis are listed in Table 17 and Table 18 for Ethylene glycol induced and Glycolic acid
induced urolithiasis models respectively. The kidney histopathology images are given in figure
14 and 15 for ethylene glycol induced and glycolic acid induced urolithiasis models
respectively.
Ethylene glycol induced antiurolithiatic model:

Table 17: Changes in the body weight, water intake and the biochemical parameters in the
Ethylene Glycol induced urolithiasis model. Values are expressed as Mean ± SD (n=5), Two-
way ANOVA followed by Dunnett’s test, Statistical significance▲: P<0.0001, ∎: P<0.001,
●P<0.05, ♦: P>0.05 (not significant), N compared with normal control group, D compared with
disease control group.

Figure 14: Kidney histopathological study of Ethylene glycol induced urolithiasis model.
Yellow arrow shows crystal deposits.
Glycolic acid induced urolithiatic model:

Table 18: Changes in the body weight, water intake and the biochemical parameters in the
Ethylene Glycol induced urolithiasis model. Values are expressed as Mean ± SD (n=5), Two-
way ANOVA followed by Dunnett’s test, Statistical significance▲: P<0.0001, ∎: P<0.001,
●P<0.05, ♦: P>0.05 (not significant), N compared with normal control group, D compared with
disease control group.

Figure 15: Kidney histopathological study of Glycolic acid induced urolithiasis model. Yellow
arrow shows crystal deposits.
In vivo anti-inflammatory study:
Carrageenan induced paw oedema: Figure 16 depicts the mean change in paw volume. The
data were tested with One-way ANOVA followed by Dunnet’s multiple comparison test. It was
found that the percentage inhibition for Test extract and standard was maximum 3rd Hour after
injecting the hind paw with carrageenan (Figure 17).

Figure 16: Effects of MeAD, MeCF and diclofenac on carrageenan‑induced paw oedema in
rats. Values are expressed as Mean± SEM, n=5. Data tested with One way ANOVA followed
by Dunnet’s test. ■- represents not significant, ●-represents p<0.0001, ▲-represents p<0.001,
⁕-represents p<0.05.
Figure 17: Percentage inhibition (Mean± SEM, n=5) of carrageenan induced paw oedema in
rats.

Cotton pellet induced granuloma: Chronic anti-inflammatory potential of MeAD and MeCF
was analysed by cotton pellet-induced granuloma in vivo model and their results are presented
in Table 19.

Wet weight of Dry weight of

Animal % Transudative %
cotton pellet cotton pellet
groups Inhibition weight (mg)* Inhibition
(mg)* (mg)*
Disease
223.32±2.02 68.96±1.92 - 154.36±1.89 -
Control
Diclofenac 98.84±1.38 31.42±1.23 54.44 67.42±1.03 56.32
MeAD D1 163.53±1.54 52.21±1.09 24.29 111.32±2.21 27.88
MeAD D2 149.32±1.04 44.13±1.32 36.01 105.19±1.39 31.85
MeCF D1 183.46±1.22 60.64±0.98 12.06 122.82±1.53 20.43
MeCF D2 168.92±1.98 54.15±1.21 21.48 114.77±1.42 25.65
Table 19: Effect of Diclofenac, MeAD and MeCF on the cotton pallet induced granuloma in
rats. Values are expressed as Mean± SEM, n=5 animals in each group. The results were
analysed using one way ANOVA. *p<0.05 was used to indicate statistical significance when
compared to control.
Cell line studies:

Cytotoxicity study:

The effects of various concentration of MeAD and MeCF on the viability of NRK-52E cells
are shown in Figure 17 and 19 respectively. MeAD showed mild cytotoxicity at the tested
concentration but MeCF showed severe cytotoxicity with a IC50 value of 11.1µg/ml. The
photomicrographic images of the cytotoxic effect of MeAD and MeCF on NRK-52E is shown
in Figure 20 and Figure 21 respectively.

Cytotoxicity study MeAD

100 96.82
100
84.81 84.73 85.30 82.40
80
% Viability

60

40

20

0
Untreated 6.25 12.5 25 50 100
Concentration (in µg/ml)

Figure 18: Percentage viability of NRK-52E cell line against MeAD

 Cytotoxicity study MeCF
100
100

80
69.99
% Viability

60
47.49

40

18.66 16.74 16.66

20

0
Untreated 6.25 12.5 25 50 100
Concentration (in µg/ml)

Figure 19: Percentage viability of NRK-52E cell line against MeCF

Sample name NRK-52E cell line IC5024h
MeAD NA (Sample showed mild toxicity at tested
concentrations)
MeCF 11.1µg/mL
Table 20: IC50 value of MeAD and MeCF in the cytotoxicity study.
Figure 20: Photomicrographic images of cytotoxicity of MeAD
Figure 21: Photomicrographic image of cytotoxicity of MeCF
Cytoprotective effect: The MTT test demonstrated a noteworthy preventive effect of MeAD
against oxalate-induced damage in NRK-52E cells. In the NRK-52E, sodium oxalate at a
concentration of 1 mM significantly harmed the cells and reduced their viability to 74.77%
(figure 22). In a concentration-dependent way, MeAD prevented the damage and markedly
raised the NRK-52E's viability. Figure 23 shows the photomicrographic pictures of the
cytoprotective effect.
 Cytoprotective activity
120
100
100
88.50
84.37
80 74.77
% Viability

60

40

20

0
Untreated Sodium Oxalate MeAD 6.25 µg/mlMeAD 12.5 µg/ml
1mM
Sodium Oxalate 1mM

Figure 22: Cytoprotective effect of MeAD on sodium oxalate induced toxicity

Figure 23: Photomicrographic images of cytoprotective effect of MeAD on sodium oxalate

induced injury.
In silico antiurolithiatic study:
GC MS Spectrum of MeAD: MeAD's GC–MS chromatogram (Figure 24) revealed 150 peaks
that corresponded to bioactive chemicals. These were identified by contrasting the mass spectra
of the compounds with their NIST library analogues. The compounds are listed in table 21.

Figure 24: GCMS chromatogram of MeAD

Sl Ret Area Area% Name Compound
no time code
1 5.895 10515 0.06 5-Ethyl-2-tridecen-4-one MeAD 52
2 7.097 137389 0.84 Isovaleric acid, undecyl ester MeAD 102
3 8.48 15338 0.09 2H-Pyran, 2-(2-heptadecynyloxy)tetrahydro- MeAD 28
4 8.53 34502 0.21 3.alpha.,7.beta.-Dihydroxy-5.beta.,6.beta.-epoxycholestane MeAD 38
5 10.315 11955 0.07 5-Methylene-1,3a,4,5,6,6a-hexahydropentalen-1-ol MeAD 54
6 10.985 22259 0.14 Testolactone MeAD 132
7 12.213 46176 0.28 Cyclopropane,1,1-dichloro-2,2-dimethyl-3-(2- MeAD 81
methylpropyl)-
8 13.15 72163 0.44 6-Epishyobunone MeAD 56
9 13.3 22989 0.14 (7R,8S)-Ethyl 8-hydroxy-trans-bicyclo[4.3.0]-3-nonene-7- MeAD 4
carboxylate
10 13.57 62534 0.38 Decaborane(14) MeAD 83
11 13.675 20522 0.13 Cyclodeca[b]furan-2(3H)-one,9-(acetyloxy)-3a,4,5,8,9,11a- MeAD 78
hexahydro-4-hydroxy-6,10-dimethyl-3-methylene-
12 14.275 31257 0.19 Alloaromadendrene oxide-(1) MeAD 63
13 14.798 54689 0.34 3-(methylselanyl)propanal MeAD 36
14 15.021 44315 0.27 4-hydroxytestosterone MeAD 44
15 15.56 53957 0.33 1H-Indene-1,2-diol, 2,3-dihydro-1-methyl-, cis- MeAD 14
16 15.651 27854 0.17 4,4-Difluororetinol (all-trans) MeAD 42
17 16.04 40236 0.25 Silane, dichloro-bis(methoxy)- MeAD 125
18 16.869 125795 0.77 Phenol, 2,4-bis(1,1-dimethylethyl)- MeAD 114
19 17.3 108196 0.66 Acetic acid, 7-hydroxy-1,3,4,5,6,7-hexahydro-2H- MeAD 62
naphthalen-4a-ylmethyl ester
20 17.442 53840 0.33 Tricyclo[7.2.0.0(2,6)]undecan-5-ol,2,6,10,10-tetramethyl- MeAD 141
(isomer 3)
21 17.525 76782 0.47 Silane, dichloro(2-methoxyethyl)methyl- MeAD 124
22 17.637 125399 0.77 1-Methyl-8-propyl-3,6-diazahomoadamantan-9-ol MeAD 15
23 18.125 39431 0.24 Indol-2(3H)-one, 3-acetylamino-1-(2-phenylthioethyl)- MeAD 99
24 18.247 153467 0.94 10-Methyltricyclo[4.3.1.1(2,5)]undecan-10-ol MeAD 10
25 18.399 203490 1.25 Silane, dimethyl(3-methylbut-3-enyloxy)hexyloxy- MeAD 128
26 18.731 194544 1.19 2-Oxabicyclo[3.3.0]oct-7-en-3-one, 7-(1-hydroxypentyl)- MeAD 32
27 18.995 40070 0.25 L-Serinamide, 1-methyl-5-oxo-L-prolyl-N,1-dimethyl-L- MeAD 103
histidyl-N,1-L-tryptophyl-N,N,N2,O-tetramethyl-
28 19.052 136645 0.84 Retinal MeAD 121
29 19.179 172566 1.06 1,2(equat)-dimethyl-trans-decahydroquinol-4-one MeAD 6
30 19.205 83671 0.51 Pyrrolizidine-3-one, 5-acetylmethyl- MeAD 120
31 19.712 45255 0.28 2,6-Octadienoic acid, 4-isopropylidene-6,7-dimethyl-, MeAD 24
methyl ester
32 19.905 11705 0.07 Silane, triethoxy- MeAD 129
33 19.97 179699 1.1 trans-Acrylic acid, 3[(3-(2,2-dimethylcyclopropyl)-2,2- MeAD 136
dimethylcyclopropyl)]-, methyl ester
34 20.016 62454 0.38 Acetamide, N-(thiazol-2-yl)-2-p-tolyloxy- MeAD 60
35 20.04 79484 0.49 1,2-Naphthalenediol, 1-ethyl-1,2,3,4-tetrahydro-, cis- MeAD 8
36 20.11 276446 1.69 2-(5-Isopropyl-2-methylphenyl)ethanol MeAD 20
37 20.14 81862 0.5 Propionic acid, 2-[(2-methyl-4,5,6,7-tetrahydrobenzofuran- MeAD 119
3-carbonyl)amino]-3-phenyl-
38 20.17 34799 0.21 Thiazole, 4-(4-iodophenyl)-2-(3-tolylamino)- MeAD 133
39 20.2 15749 0.1 Nitrone, N-(p-chlorophenyl)-.alpha.-(o-methoxyphenyl)- MeAD 111
40 20.434 87173 0.53 3-Isopropyltricyclo[4.3.1.1(2,5)]undec-3-en-10-ol MeAD 39
41 21.12 35574 0.22 Tricyclo[5.3.1.1(2,6)]dodecan-11-ol,11-methyl-12- MeAD 140
methylene-
42 21.48 45011 0.28 Azuleno[4,5-b]furan-2(3H)-one, decahydro-7,9-dihydroxy- MeAD 68
6,9a-dimethyl-3-methylene-, [3aS-
(3a.alpha.,6.beta.,6a.alpha.,7.alpha.,9
43 21.538 47925 0.29 4'-Hydroxybutyrophenone oxime MeAD 43
44 21.641 64494 0.4 Patchouli alcohol MeAD 113
45 21.864 77441 0.47 Thiazolidine-4-carboxylic acid, 2-(2-isopropoxyphenyl)- MeAD 134
46 22.235 13153 0.08 Silane, dimethyl(2-methoxyethoxy)pentyloxy- MeAD 127
47 22.275 69267 0.42 5,6-Dicarbadecaborane(12), 5,6-dimethyl- MeAD 48
48 22.41 68915 0.42 Dasycarpidan-1-methanol, acetate (ester) MeAD 82
49 22.645 37775 0.23 Hexasiloxane, 1,1,3,3,5,5,7,7,9,9,11,11-dodecamethyl- MeAD 98
50 22.69 29387 0.18 1-n-Butoxy-1-chloro-1-silacyclopentane MeAD 16
51 22.729 24452 0.15 Heptasiloxane, hexadecamethyl- MeAD 94
52 23.03 10114 0.06 Acetic acid, 2,2-di(2-buten-1-yl)-2-phenylsulfonyl-, methyl MeAD 61
ester (E,E-, E,Z-, Z,Z-)
53 23.053 11735 0.07 Ergostane-5,25-diol, 3,6,12-tris[(trimethylsilyl)oxy]-, 25- MeAD 89
acetate, (3.beta.,5.alpha.,6.beta.,12.beta.)-
54 23.125 21634 0.13 Silane, (2-chloroethyl)triethoxy- MeAD 122
55 23.526 39853 0.24 2-(2-Nitrophenoxy)benzaldehyde 4-isopropyl-3- MeAD 18
methylphenoxyacetylhydrazone
56 24.235 25987 0.16 Butanoic acid, 4-(2-hydroxycyclohexyl)-, methyl ester MeAD 72
57 24.285 112932 0.69 Phytol MeAD 115
58 24.394 225881 1.38 2-Pentadecanone, 6,10,14-trimethyl- MeAD 33
59 24.465 8859 0.05 8.beta.-Podocarp-12-ene-14-carboxylic acid, 8,13-dimethyl-, MeAD 57
methyl ester
60 24.49 35467 0.22 Decaborane, ethyl- MeAD 84
61 24.67 292859 1.79 2-Propenoic acid, 3-(4-hydroxy-3-methoxyphenyl)-, methyl MeAD 34
ester
62 24.799 96678 0.59 Urea, N-(3-chlorophenyl)-N'-[2-(1,3-dihydro-1-oxo-2H- MeAD 143
isoindol-2-yl)ethyl]-
63 24.96 23544 0.14 Tricyclo[5.2.2.0(1,6)]undecan-3-ol, 2-methylene-6,8,8- MeAD 139
trimethyl-
64 25.061 55112 0.34 Silane, (silylmethyl)[(trimethylsilyl)methyl]- MeAD 123
65 25.17 24938 0.15 Cyclodeca[b]furan-2(3H)-one, 9-(acetyloxy)-3a,4,5,8,9,11a- MeAD 78
hexahydro-4-hydroxy-6,10-dimethyl-3-methylene-
66 25.395 28764 0.18 2,2-Dimethyl-6-methylene-1-[3,5-dihydroxy-1- MeAD 21
pentenyl]cyclohexan-1-perhydrol
67 25.51 29849 0.18 8.beta.-Podocarp-12-ene-14-carboxylic acid, 8,13-dimethyl-, MeAD 57
methyl ester
68 25.645 55966 0.34 .alpha.-D-Galactopyranose, 1,2,3-tris-O-(trimethylsilyl)-, MeAD 5
cyclic methylboronate
69 25.806 14069 0.09 cis-7,10,13,16-Docosatetraenoic acid, trimethylsilyl ester MeAD 75
70 25.854 11609 0.07 Tricyclo[5.2.2.0(1,6)]undecan-3-ol, 2-methylene-6,8,8- MeAD 139
trimethyl-
71 25.875 37325 0.23 Anthracene, 9,10-diethyl-9,10-dihydro- MeAD 66
72 26.042 99193 0.61 Ethyl 9-hexadecenoate MeAD 90
73 26.183 1498014 9.18 Hexadecanoic acid, methyl ester MeAD 97
74 26.29 7190 0.04 4-hydroxytestosterone MeAD 44
75 26.315 8457 0.05 1-Phenanthrenecarboxylic acid, 1,2,3,4,4a,5,8,9,10,10a- MeAD 17
decahydro-1,4a-dimethyl-7-(1-methylethyl)-, methyl ester,
[1R-(1.alpha.,
76 26.395 98828 0.61 Diphosphoric(III,V) acid, tetraethyl ester MeAD 86
77 26.468 37676 0.23 3,7-Dimethyl-8-(phenylthio)octa-2,6-dien-1-ol MeAD 37
78 26.525 36189 0.22 Cyclopropane, 1-(2,2-dichloroethenyl)-2,2-dimethyl-3-(3,5- MeAD 80
dimethylpyrazol-1-yl)carbonyl-
79 26.745 51191 0.31 Diphosphoric(III,V) acid, tetraethyl ester MeAD 86
80 26.865 20886 0.13 Ingol 3,8,12-triacetate MeAD 100
81 26.986 177642 1.09 1,2-Benzenedicarboxylic acid, bis(2-methylpropyl) ester MeAD 7
82 27.099 96289 0.59 2-Trichloromethyl-5-[.alpha.,.alpha.,.alpha.-trichloro-p- MeAD 35
tolyl]-1,3,4-oxadiazole
83 27.195 49492 0.3 Fumaric acid, decyl propargyl ester MeAD 91
84 27.309 442518 2.71 n-Hexadecanoic acid MeAD 110
85 27.41 20620 0.13 Propanoic acid, 2-[5-(2-hydroxypropyl)tetrahydrofuran-2- MeAD 118
yl]-, 1-[5-(1-methoxy-1-oxopropan-2-yl)tetrahydrofuran-2-
yl]propan-2-yl
86 27.83 21011 0.13 Morpholine-4-methanamine, N-cyclohexyl-.alpha.- MeAD 109
(cyclohexylimino)-
87 27.91 895137 5.49 Hexadecanoic acid, ethyl ester MeAD 96
88 28.059 49102 0.3 2-[5-(2-Methoxy-ethyl)-7-(6-methoxymethyl-2,2-dimethyl- MeAD 27
7-oxa-bicyclo[4.1.0]hept-1-yl)-3,5-dimethyl-hepta-2,6-
dienylidene]-3,3-d
89 28.099 31384 0.19 Hexacyclo[7.2.2.2(4,7).0(3,8).0(12,14).0(13,15)]pentadecan- MeAD 95
1-ol-2-one
90 28.158 30754 0.19 Androstan-3-one, 17-hydroxy-1,17-dimethyl-, MeAD 65
(1.alpha.,5.alpha.,17.beta.)-
91 28.185 13720 0.08 Strophanthidin MeAD 130
92 28.27 36909 0.23 2-(4-Chlorophenylthio)-N'-(2,3- MeAD 19
dimethoxybenzylidene)acethydrazide
93 28.339 97717 0.6 4-Isopropenyl-1-methylbicyclo[4.1.0]heptan-2-one oxime MeAD 45
94 28.759 1141063 6.99 5,9-Propano-7H-benzocycloheptene-7,11-dione, 5,6,8,9- MeAD 49
tetrahydro-
95 28.89 129800 0.8 N-Methyl-1,2,3,4-tetra-O-acetyl-beta-d- MeAD 112
glucopyranuronamide
96 28.97 18167 0.11 5,6,7,8,9,10-hexahydro-9-phenyl-spiro[2H-1,3-benzoxazine- MeAD 47
4,1'-cyclohexane]-2-thione
97 29.135 66198 0.41 5-Ethyl-1,3-dioxane-5-methanol, tert-butyldimethylsilyl MeAD 51
ether
98 29.877 27521 0.17 2,6-Octadien-4-ol, 8-(4''-carboxy-6''-t-butyldimethysilyloxy- MeAD 23
2'',3'',3a'',4'',7'',7a''-hexahydro-3a''-hydroxy-5''-
methylbenzofu
99 30.079 26546 0.16 9,12,15-Octadecatrienoic acid, 2,3- MeAD 58
bis[(trimethylsilyl)oxy]propyl ester, (Z,Z,Z)-
100 30.785 15045 0.09 9-Acetoxy-1-propyl-3,6-diazahomoadamantane MeAD 59
101 30.94 76610 0.47 Thionazin MeAD 135
102 31.111 777894 4.77 Methyl 10-trans,12-cis-octadecadienoate MeAD 105
103 31.355 1040433 6.38 E,E,Z-1,3,12-Nonadecatriene-5,14-diol MeAD 88
104 31.902 2140701 13.14 Phytol MeAD 115
105 32.425 165660 1.02 Methyl 14-methyl-eicosanoate MeAD 106
106 32.515 24418 0.15 Benzo-1,4-dioxane, 2-phosphinomethyl- MeAD 70
107 32.61 33251 0.2 4-(7-Allyl-7-hydroxy-2,2 dimethyltetrahydro[1,3]dioxolo MeAD 40
[4,5-c]pyran-4-yl)-3-methylbut-2-enoic acid, methyl ester
108 33.75 22720 0.14 Verrucarol MeAD 144
109 33.824 40966 0.25 Tricyclo[20.8.0.0(7,16)]triacontane, 1(22),7(16)-diepoxy- MeAD 138
110 33.94 12425 0.08 1H-Cyclopropa[3,4]benz[1,2-e]azulene-5,7b,9,9a-tetrol, MeAD 13
1a,1b,4,4a,5,7a,8,9-octahydro-3-(hydroxymethyl)-1,1,6,8-
tetramethyl-, 5,
111 33.995 27533 0.17 Benz[a]anthracene, 1,2,3,4,7,7a,8,9,10,11,11a,12- MeAD 69
dodecahydro-
112 34.085 83272 0.51 Picras-2-en-1-one, 11,13,16-trihydroxy-2,12-dimethoxy-, MeAD 116
(11.alpha.,12.beta.)-
113 34.118 175686 1.08 Methyl trans-2-octadecenoate MeAD 108
114 34.322 12569 0.08 cis-7-Ethoxycarbonyl-bicyclo(4,3,0)non-3,7-diene MeAD 76
115 34.422 53716 0.33 6.beta.,6.beta.-Dibromo-6,7-methylenetestosterone MeAD 55
116 34.69 38958 0.24 Methyl 7,10,13,16-docosatetraenoate MeAD 107
117 34.76 10581 0.06 2H-Quinolizin-2-ol, octahydro-, trans- MeAD 29
118 35 44392 0.27 2-Methyl-E,E-3,13-octadecadien-1-ol MeAD 31
119 35.065 110614 0.68 Trispiro[4.2.4.2.4.2.]heneicosane MeAD 142
120 35.173 162124 0.99 5-(7a-Isopropenyl-4,5-dimethyl-octahydroinden-4-yl)-3- MeAD 46
methyl-pent-2-enal
121 35.36 40515 0.25 5-Cholesten-3,26-diol di[3,5-dinitrobenzoate]- MeAD 50
122 35.399 35354 0.22 Astaxanthin MeAD 67
123 35.445 52262 0.32 10,12-Tricosadiynoic acid, trimethylsilyl ester MeAD 9
124 35.746 8674 0.05 2-Isopropylphenyl-thiourea MeAD 30
125 35.795 16694 0.1 2-[2-[2-(Naphthalen-2-yloxy)-ethylsulfanyl]-benzoimidazol- MeAD 25
1-yl]-ethanol
126 36.074 9695 0.06 (3-Methoxycarbonylmethyl-5-oxocyclohexyl)acetic acid, MeAD 3
methyl ester
127 36.284 Table
5753221: GCMS
0.35 peakTricyclo[10.2.2.2(5,8)]octadeca-5,7,12,14,15,17-hexaene,
data for MeAD (compound code assigned for the In silico6-study)
MeAD 137
nitro-
128 36.385 21205 0.13 Card-20(22)-enolide, 3,5,14,19-tetrahydroxy-, MeAD 73
(3.beta.,5.beta.)-
129 36.46 17291 0.11 4,4,6a,6b,8a,11,12,14b-Octamethyl-docosahydropicene- MeAD 41
3,13-diol
130 36.475 68856 0.42 5-Fluoro-2-trifluoromethylbenzoic acid, nonadecyl ester MeAD 53
131 36.566 78759 0.48 1-Cyclohexene-1-butanal, .alpha.,2,6,6-tetramethyl- MeAD 12
132 37.215 48296 0.3 16-Nitrobicyclo[10.4.0]hexadecan-1-ol-13-one MeAD 11
133 37.34 46037 0.28 Cholest-5-en-3-ol (3.beta.)-, 3-phenyl-2-propenoate MeAD 74
134 37.44 137809 0.84 Corynan-17-ol, 18,19-didehydro-10-methoxy-, acetate (ester) MeAD 77
135 37.6 18830 0.12 2-[4-methyl-6-(2,6,6-trimethylcyclohex-1-enyl)hexa-1,3,5- MeAD 26
trienyl]cyclohex-1-en-1-carboxaldehyde
136 37.654 26917 0.16 T-2 Tetraol MeAD 131
137 38.025 37219 0.23 Ambrosin MeAD 64
138 38.106 76452 0.47 (3-Methoxycarbonylmethyl-2-pent-2- MeAD 2
enylcyclopentylideneaminooxy)acetic acid, methyl ester
139 40.269 17302 0.11 Germacrene A, 9-(methylthio)- MeAD 93
140 41.089 64536 0.4 i-Propyl 9-tetradecenoate MeAD 101
141 41.34 18126 0.11 2,6,10,15,19,23-Hexamethyl-tetracosa-2,10,14,18,22- MeAD 22
pentaene-6,7-diol
142 41.42 34407 0.21 Astaxanthin MeAD 67
143 41.53 7860 0.05 Bufa-20,22-dienolide, 3,14-dihydroxy-, (3.beta.,5.beta.)- MeAD 71
144 41.865 54223 0.33 Pregn-4-en-17,21-diol-3,20-dione, 9,11-epoxy- MeAD 117
145 42.012 9834 0.06 Diazoprogesterone MeAD 85
146 42.585 23726 0.15 D-Mannitol, 2,4:3,5-bis-O-(phenylmethylene)- MeAD 87
147 43.791 95494 0.59 (3-Bromo-phenoxy)-acetic acid (2-methoxy-benzylidene)- MeAD 1
hydrazide
148 43.88 31215 0.19 Menthol, 1'-(butyn-3-one-1-yl)-, (1R,2S,5R)- MeAD 104
149 44.575 29267 0.18 Silane, dimethyl(2,2,2-trichloroethoxy)propoxy- MeAD 126
150 44.959 61252 0.38 Furane-2-carboxaldehyde,5-nitro-,1- MeAD 92
phenylthiosemicarbazone
In silico antiurolithiatic study

Compounds identified in GCMS (Total 150) were given codes from MeAD 1 to MeAD 144
(similar compounds appearing twice were given the same code) and all the compounds were
subjected to in silico analysis to find out their possible role in antiurolithiatic activity. Xanthine
dehydrogenase (Xdh) (PDB ID: 1JRP)[34], Tamm-Horesefall Protein (THP) (PDB ID: 4WRN)[37],
and Oxalate oxidase (OxOx) (PDB ID: 2ETE)[38] are the three target enzymes. Native ligands to the
selected proteins are Oxypurinol for Xdh and Acetylglucosamine for OxOx and THP were chosen as
control.[39] From the docking study, 67 compounds against 1JRP, 49 compounds against 2ETE
and 41 compounds against 4WRN showed better docking score in comparison to their
respective co-crystal ligands. Nine of these chemicals were confirmed to be non-toxic in terms
of their carcinogenic and mutagenic properties after they underwent further toxicity
investigation. These compounds were further evaluated for different ADMET parameters
(Figure 26) where, 5 compounds showed acceptable results. Out of these 5 compounds 2
compounds named MeAD1 and MeAD123 showed interactions with all the three target
proteins. The docking pose of these 2 compounds with 1JRP, 2ETE and 4WRN are depicted in
figure 25. Hence there is a possibility that these compounds may show desired therapeutic
activity via multi-target mechanism approach.
Figure 25: Docking poses of MeAD 1 and MeAD 123 with 1JRP, 2ETE and 4WRN.
Figure 26: ADME parameters of MeAD

Isolation of phytoconstituents from the active extract:

The Column chromatography fractionations of the active extract and
purification yields a compound, Compound 1-KD_AD1
Compound 1: KD_AD1
Colour: Yellow
Physical nature: Solid
Melting point: 277.6°C
Solubility: Ethanol, DMSO
TLC of Compound 1: TLC of the compound was done using the solvent Figure 27: TLC of
KD_AD1
system- Toluene: Ethyl Acetate: Glacial Acetic Acid: 5.5:4:0.5. Single
spot was observed at Rf value 0.8.
Spectral Analysis of KD_AD1:

Figure 28: 1H NMR Spectra of KD_AD1 (DMSO-d6, 500 MHz)

1
H NMR (500 MHz, DMSO-d6) δ 12.48 (s, 1H), 10.20 (s, 2H), 9.46 (s, 1H), 8.05 (d, J = 8.6
Hz, 2H), 6.93 (d, J = 8.7 Hz, 2H), 6.44 (s, 1H), 6.20 (s, 1H).
Figure 29: 13C NMR Spectra of KD_AD1 (DMSO-d6, 125 MHz)
13
C NMR (125 MHz, DMSO) δ 175.83 (s), 163.84 (s), 160.64 (s), 159.12 (s), 156.11 (s), 146.73
(s), 135.58 (s), 129.43 (s), 121.61 (s), 115.36 (s), 102.97 (s), 98.13 (s), 93.40 (s).
SAIF,PANJAB UNIVERSITY,CHANDIGARH SYNAPT-XS#DBA064 09-Aug-2023
16:25:45
KANGKAN_KD_AD1 10 (0.205) 1: TOF MS ES+
287.0585 1.01e6
100
%

288.0622

338.3447

169.0161
409.0568
437.1958
0 m/z
60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 620 640 660 680 700 720 740 760 780 800 820 840 860 880 900 920 940 960 980

Figure 30: HRMS of KD_AD1

HRMS: Mass Spectroscopy of the KD_AD1 is done in methanol solvent with MicroMass Q-
TOF (Figure 30). HRMS (ESI-Positive) m/z: [M+H]+ calculated for C15H10O6 287.0477;
Found 287.0585.
FTIR Data: The characteristic band at 3306.33 cm-1 is attributed for stretching vibration of
phenolic O-H groups. Absorption band can be observed within the region 2950 cm-1— 2850
cm-1 due to the C-H stretching vibrations. Another characteristic band for C=O stretching
vibrations can be seen at 1657.27 cm-1.

Figure 31: FTIR Spectrum of KD_AD1

Molecular formula: C15H10O6
After spectral analysis the isolated compound KD_AD1 is identified as Kaempferol
Molecular weight: 286.23 (Figure 31)

Figure 31: KD_AD1- Kaempferol.

Evaluation pharmacological activity of KD_AD1: Pharmacological activity of the isolated
compound was evaluated by performing in vitro antiurolithiatic and in vitro anti-inflammatory
study followed by in silico evaluation of antiurolithiatic potential.
In vitro Antiurolithiatic activity
1. Nucleation assay:
Figure 32: Percentage inhibition (Mean ± SEM, n=3) of nucleation by different concentration
of KD_AD1.
Nucleation inhibition IC50
Cystone 576.26± 1.82
KD_AD1 924.32± 2.47
Table 22: IC50 value (Mean ± SEM, n=3) of nucleation inhibition by standard(cystone) and
KD_AD1.
2. Aggregation assay:

Figure 33: Percentage inhibition (Mean ± SEM, n=3) of aggregation by different concentration
of KD_AD1.
Aggregation inhibition IC50
Cystone 573.69± 1.54
KD_AD1 976.95± 2.17
Table 23: IC50 value (Mean ± SEM, n=3) of aggregation inhibition by standard(cystone) and
KD_AD1.
In vitro anti-inflammatory activity:
1. Egg albumin denaturation assay:

Figure 34: Percentage inhibition of egg albumin denaturation by different concentration of

KD_AD1.
Egg albumin Denaturation assay
Sample IC50
Diclofenac 218.01± 0.96
KD_AD1 372.99± 1.16

Table 24: Egg albumin denaturation assay- IC50 value (Mean ± SEM, n=3) of diclofenac and
KD_AD1.

2. Human Red Blood Cell (HRBC) membrane stabilization assay- (Hypotonic Solution-
Induced Hemolysis)

Figure 35: Percentage inhibition (Mean ± SEM, n=3) of hypotonicity-Induced haemolysis by

different concentration of diclofenac and KD_AD1.
 HRBC Hypotonic
Sample IC50
Diclofenac 267.26± 1.44
KD_AD1 376.79± 1.09

Table 25: Human Red Blood Cell (HRBC) membrane stabilization assay- IC50 value (Mean ±
SEM, n=3) of diclofenac, and KD_AD1.

In silico antiurolithiatic study of KD_AD1:

The predicted binding energies of KD_AD1 and the standard drugs (Oxypurinol and Acetyglucosamine)
with their respective proteins viz. 1JRP, 2ETE and 4WRN are shown in Table 26. The table demonstrates
that KD_AD1 has shown a higher binding affinity towards all the three receptors in comparison to their
respective co-crystallized ligands (Oxypurinol for 1JRP and Acetylglucosamine for 2ETE and 4WRN).
This indicates that KD_AD1 may bind to xanthine dehydrogenase, oxalate oxidase, and uromodulin
receptors more strongly and potentially exhibit therapeutic implications via multi-target mechanism
approach. Figure 36 shows different docking poses of KD_AD1 at the active sites of the corresponding
proteins. The figure highlights conformational changes and possible interactions that may occur when
KD_AD1 interacts with the binding domain of each protein. Further, on evaluation of in silico toxicity
prediction, KD_AD1 was found to be non-carcinogenic and non-mutagenic against all the toxicity
predictors. In addition, analysis of ADMET (Figure 37) properties revealed that KD_AD1 exhibits
acceptable results.

Ligand Name Binding Energy (kcal/mol)

1JRP 2ETE 4WRN
KD_AD1 -170.5052 -55.4969 -178.6746
Oxypurinol -86.5658 - -
NAG - -19.3488 -47.3272

Table 26: Binding energies of KD_AD1 with 1JRP, 2ETE and 4WRN and comparison with
their standards.
 2D pose Protein

1JRP

2ETE

4WRN

Figure 36: 2D Docking poses of KD_AD1 with 1JRP, 2ETE and 4WRN.
Figure 37: ADME prediction of KD_AD1.

Discussion and Conclusion:

This research validates ethnomedicinal claims about Alternanthera dentata and Calamus
floribundus' antiurolithiatic and anti-inflammatory properties. The plant samples were
extracted, and phytochemical investigations revealed the presence of secondary metabolites
such as flavonoid, alkaloids, phenols, and glycosides. The extracts were evaluated for in vitro
antiurolithiatic activity, which was found to inhibit the formation of calcium oxalate crystals.

The study also investigated protein denaturation and stabilization of human red blood cell
membranes to determine the mechanism by which the extracts exert their anti-inflammatory
effects. The extracts effectively prevented the denaturation of protein/albumin and
demonstrated efficacy in stabilizing red blood cell membranes or suppressing hypotonicity-
induced hemolysis at varying doses.
The methanolic extracts of both plants (MeAD and MeCF) were observed to possess better in
vitro antiurolithiatic and anti-inflammatory activity than all other tested extracts. These extracts
were further used in in vivo tests.

Two models of urolithiasis were used: one induced by glycolic acid and the other by a
combination of ethylene glycol and ammonium chloride. The experimental rats exhibited a
decrease in body weight and water consumption due to the development of urolithiasis, but
these values returned to their typical levels after administration of both standard and test
medicine.

The composition of urine plays a crucial role in identifying the specific kind of crystals and the
presence of large molecules on their surfaces. Hypercalciuria, a condition characterized by the
formation of calcium oxalate crystals in urine, is a significant factor in the development of
urolithiasis. The excretion of phosphate, calcium, and oxalate in urine is affected by the
excretion of inorganic phosphate, calcium, and oxalate. In this research, MeCF and MeAD
were found to decrease the excretion of these elements, inhibiting the formation of calculi. The
study also found that rats with urolithiasis had increased uric acid excretion, which reduces the
inhibitory effect of glycosaminoglycans and disrupts the solubility of calcium oxalate. The
administration of both standard and test treatments effectively preserved levels of uric acid in
the disease control group. Urolithiasis, a medical condition characterized by the presence of
calculi that obstruct the urinary system, was reduced in the disease control group. Co-
administration of test and standard treatment reduced calcium oxalate deposits, dilatation of
renal tubules, and damage to renal tubules in a dose-dependent manner.

The most commonly used approach for evaluating the anti-inflammatory effect of plant extracts
is paw oedema generated by carrageenan. The results demonstrated that carrageenan-induced
inflammation was considerably reduced by MeAD and MeCF during the third hour of the
experiment. A common technique to evaluate chronic anti-inflammatory effects of natural
substances is the cotton pellet-induced granuloma model. The administration of MeAD and
MeCF inhibited the production of granulomatous tissue, which in turn decreased the weight of
wet cotton pellets in a dose-dependent manner.

Methodological tests on the cytotoxic effect of MeAD and MeCF on the NRK-52E cell line
revealed that MeAD showed mild cytotoxicity at the tested concentration. However, after
MeAD treatment, the vitality increased, and it was shown that MeAD at the studied dose
exhibited cytoprotective capability against oxalate-induced cell injury.

After successful isolation of KD_AD1 via column chromatography, the FTIR, 13C and 1H
NMR and HRMS it was confirmed to be Kaempferol. It was further subjected to in vitro anti-
inflammatory, in vitro antiurolithiatic study followed by in-silico studies. The potential was
found to be comparable to that of the standards used.

From the research it can be drawn that both the plant possesses anti-inflammatory and
antiurolithiatic potential and thus confirming the ethnomedicinal claims.
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Seminar & Conference Presentations from Ph.D research work :

1. Presented poster entitled- “Antiurolithiatic potential of Alternanthera dentata

Scheygrond against ethylene glycol induced kidney stone, and NRK 52E cell protection
from oxalate injury” at 2nd National Conference on Novel Innovations in Biomedical
Sciences (NIBS)-2023, organized by NEMCARE Group of Institutions in collaboration
with The Association of Pharmaceutical Teachers of India, Rajiv Gandhi University,
Arunachal Pradesh, The Assam Nurses’ Midwives’ & Health Visitors’ Council,
Guwahati Biotech Park and NEMCARE Hospitals on 14th and 15th March 2023.
2. Presented poster entitled- “Antiurolithiatic potential of leaf extracts of Alternanthera
dentata Scheygrond” at National Symposium on Crosstalk Between Animal Research
and Alternatives, organized by CSIR-NIEST, Jorhat, Assam on 07th-9th September
2023.

Publication Details:

1. Kangkan Deka, Bibhuti Bhusan Kakoti, Ngurzampuii Sailo, Zonunmawii, Dev Jyoti
Kalita, Rajashri Bezbaruah; “Assessing The Antiurolithiatic Potentials of Calamus
floribundus Griff. An in vitro and in vivo Approaches” accepted at Pharmacognosy
Research.