PHYTOCHEMICAL AND PHARMACOLOGICAL EVALUATION OF

Alternanthera dentata SCHEYGROND AND Calamus floribundus GRIFF., TWO

ETHNOMEDICINAL PLANTS OF ASSAM WITH SPECIAL REFERENCE ANTI-
INFLAMMATORY AND ANTIUROLITHIATIC ACTIVITIES

PhD. Abstract Presentation

Presented By
Kangkan Deka
Under the Supervision of
Dr. Bibhuti Bhusan Kakoti
Department Of Pharmaceutical Sciences
Dibrugarh University
Dibrugarh, Assam 1
Aim: 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.

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.

2
Urolithiasis Percentage of Stone types
1%
The formation of stone in the urinary 7%
system, i.e. in the kidney, ureter, and in 12%
urinary bladder or in the urethra is called Calcium
Urolithiasis. Struvite

Stone formation occur in approximately Uric acid

Cystine
12% of global population
80%

3
Inflammation
• A protective action of body intended to eliminate the initial cause of
cell injury as well as necrotic cells and tissues resulting from that
injury.
• Infections, wounds, and any damage to tissue would not be able to heal
without an inflammatory response.

4
PLANT PROFILE 1

Scientific name: Alternanthera dentata Scheygrond

Order: Caryophyllales
Family: Amaranthaceae
Genus: Alternanthera
Species: Alternanthera dentata
Common/English names: Little Ruby, Joseph's coat,
Calico Plant, Joyweed
Assamese name: Bisholyokoroni
Fig 1. Aerial parts of
Alternanthera dentata
Distribution: Native to the West Indies and Brazil and Scheygrond
also found in some parts of Asia, Africa, and Australia.

Ethonomedicinal claims and uses: The leaves and young shoots are used in
conditions like cuts and wounds, inflammation, stomach pain and kidney stones.

5
PLANT PROFILE 2

Scientific name: Calamus floribundus Griff.

Order: Arecales
Family: Arecaceae
Genus: Calamus
Species: C. floribundus
Common name: Lejaibet (Assamese)
Synonym: Palmijuncus leptospadix (Griff.) Kuntze.

Distribution: Found in Northeastern India,

Fig 2. Arial parts of
Bhutan, Nepal, Bangladesh and Myanmar.
Calamus floribundus Griff.

Ethonomedicinal 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.

6
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.
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 suitable and selective 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.
7
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.
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.
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.
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] 8
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 Petri dish on water bath. The
crude extracts were stored in air tight containers in 4°C until use.

9
Phytochemical investigation
Phytochemical PeAD ChAD EaAD MeAD Phytochemical PeCF ChCF EaCF MeCF
Alkaloids - - - + Alkaloids - - - +
Glycosides - - + + Glycosides - - - +
Carbohydrates - + + + Carbohydrates - - + +
Flavonoids - - + + Flavonoids - - + +
Saponins - - - + Saponins - - + +
Tannins - - - - Tannins - - + -
Steroids and - - + + Steroids and - + + +
triterpenoids triterpenoids

Fats and oils + + - - Fats and oils + + - -

Extract Consistency Colour % Yield Extract Consistency Colour % Yield

PeAD Sticky solid Blackish Green 3.74 ± 1.22 PeCF Sticky solid Light brown 0.59 ± 0.21
ChAD Sticky solid Dark Green 15.46 ± 2.17 ChCF Sticky solid Brown 0.31 ± 0.09
EaAD Sticky solid Brown 4.92 ± 0.97 EaCF Sticky solid Brown 3.02 ± 0.22
MeAD Sticky solid Dark Brown 25.68 ± 1.54 MeCF Sticky solid Dark Brown 8.81 ± 0.78

10
TS and Powder microscopy of Alternanthera dentata

11
TS,LS and Powder microscopy of Calamus floribundus

12
13
14
In vitro antioxidant assay
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

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

15
In vitro antiurolithiatic assay

Values are expressed as mean ± SEM, n=3. Data were tested with One way ANOVA followed by Dunnet’s multiple comparison test. *p
16<0.0001,
● p <0.05 significant when compared with standard group (cystone).
17
18
19
20
Nucleation inhibition IC50 (µg/ml) Aggregation inhibition IC50 (µg/ml)
Cystone 527.66± 2.12 Cystone 522.78± 1.32
PeAD 3937.38± 1.10 PeAD 1986.28± 3.12
ChAD 3445.54± 2.49 ChAD 1759.01± 1.31
EaAD 1087.60± 1.39 EaAD 1005.14± 2.57
MeAD 780.81± 2.45 MeAD 762.37 ± 2.35
PeCF 2835.42± 1.23 PeCF 2403.09± 1.41
ChCF 2580.24± 1.14 ChCF 1616.58± 1.39
EaCF 1214.19± 2.19 EaCF 932.98± 1.42
MeCF 769.03± 1.22 MeCF 784.89± 3.29

IC50 (Mean± SEM) value of cystone, and different extracts of Alternanthera dentata
and Calamus floribundus.

21
 Growth inhibition assay

Time Cyston Cysto Cysto PeAD PeAD PeAD ChAD ChAD ChAD EaAD EaAD EaAD MeAD MeAD MeAD
(hr) e 0.5% 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%
48 12.39±0 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
.21 ±0.19 ±0.43 ±066 ±0.05 ±0.19 ±0.19 ±0.22 ±0.09 ±0.41 ±0.49 ±0.31 ±0.26 ±0.33
±0.67
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 ±0.40 ±0.58 ±0.16 ±0.19 ±0.38 ±0.27 ±0.31 ±0.21 ±0.27 ±0.22
±0.50
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 ±0.19 ±0.37 ±0.35 ±0.14 ±0.32 ±0.17
±0.27
Time Cyston Cysto Cysto PeCF PeCF PeCF ChCF ChCF ChCF EaCF EaCF EaCF MeCF MeCF MeCF
(hr) e 0.5% 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%
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

Percentage inhibition (Mean±SD, n=3) of the struvite crystals in different concentration of standard and test extracts

22
In vitro anti-inflammatory assay

23
Percentage inhibition (Mean± SEM, n=3)
24
25
26
27
Egg albumin Denaturation assay HRBC Hypotonic
Sample IC50 Sample IC50
Diclofenac 223.03± 1.13 Diclofenac 275.16± 1.24
PeAD 1,307.89± 2.17 PeAD 1815.47± 2.11
ChAD 1,090.33± 1.92 ChAD 1140.86± 1.97
EaAD 431.35± 0.98 EaAD 606.48± 1.27
MeAD 331.05± 1.35 MeAD 437.96± 2.83
PeCF 1,683.83± 3.43 PeCF 903.59± 3.11
ChCF 1,234.55± 2.33 ChCF 735.78± 1.68
EaCF 559.66 ±1.78 EaCF 533.65± 1.25
MeCF 429.96 ± 2.12 MeCF 424.70± 2.18

IC50 value (Mean± SEM, n=3)

28
In vivo Antiurolithiatic study: Ethylene glycol induced model
Parameters Group 1 NC Group 2 DC Group 3 std Group 4 MAD D1 Group 5 MAD D2 Group 6 MCF D1 Group 7 MCF D2
12.15 ±1.03 19.72 ±0.92 14.86 ±0.83 15.68 ±1.13
Change in body weight 25.12 ± 0.91D▲ -5.91 ± 1.2 N▲ 21.99 ±0.82N▲D▲ N▲D▲ N▲D▲ N▲D▲ N▲D▲

12.84 ±0.41 11.92 ±0.33 11.23 ±0.28

Water intake (ml/24h) 8.9 ± 0.23D▲ 14.92 ± 0.29N▲ 10.22 ±0.17 N∎D▲ N▲D▲ N▲D▲ N▲D▲
10.3 ±0.19 N∎D▲
Urine (mg/dl)
11.28 ±0.32 12.12 ±0.43 11.24 ±0.32
Calcium U 7.23 ± 0.41D▲ 13.98 ± 0.49N▲ 9.11 ±0.27 N▲D▲ N▲D▲
10.1 ±0.47 N▲D▲ N▲D▲ N▲D▲

12.14 ±0.43 10.22 ±0.60 11.34 ±0.54

Phosphorus U 4.12 ± 0.53 D▲ 13.61 ± 0.38N▲ 7.24 ±0.67 N▲D▲ N▲D▲ N▲D▲ N▲D▲
9.32 ±0.03 N▲D▲

Magnesium U 3.88 ± 0.98 D▲ 0.74 ± 0.54N▲ 2.51 ±0.29 N∎D▲ 1.98 ±0.18 N▲D∎ 2.22 ±0.06 N▲D▲ 2.01 ±0.19 N▲D∎ 2.35 ±0.22 N▲D▲
Oxalate U 1.15 ± 0.78 D▲ 4.59 ± 0.29N▲ 2.52 ±0.61 N∎D▲ 3.06 ±0.75 N▲D▲ 2.83 ±0.72 N▲D▲ 2.99 ±0.23 N▲D▲ 2.78 ±0.43 N▲D▲
Uric acid U 3.37 ± 0.42 D▲ 7.01 ± 0.23N▲ 4.81 ±0.52 N▲D▲ 5.71 ±0.33 N▲D∎ 5.03 ±0.54 N▲D▲ 5.78 ±0.38 N▲D● 4.99 ±0.62 N▲D▲
17.51 ±0.16 15.13 ±0.18 16.78 ±0.11 14.92 ±0.09
Urea U 11.91 ± 0.03 D▲ 19.56 ± 0.19N▲ 13.32 ±0.11 N∎D▲ N▲D▲ N▲D▲ N▲D▲ N▲D▲

18.56 ±0.20 15.28 ±0.08 14.91 ±0.21 15.78 ±0.09 14.24 ±0.03
Citrate U 20.15 ± 0.09 D▲ 12.72 ± 0.12N▲ N▲D▲ N▲D▲ N▲D▲ N▲D▲ N▲D▲

Serum (mg/dl)
10.08 ±0.29 11.21 ±0.23
Calcium S 6.01 ± 0.45 D▲ 13.19 ± 0.23N▲ 7.43 ±0.21 N▲D▲ N▲D▲
9.31 ±0.38 N▲D▲ N▲D▲
9.9 ±0.19 N▲D▲

Phosphorus S 2.31 ± 0.21 D▲ 8.72 ± 0.29N▲ 5.41 ±0.26 N▲D▲ 7.11 ±0.21 N▲D▲ 6.52 ±0.18 N▲D▲ 7.36 ±0.31 N▲D∎ 6.91 ±0.25 N▲D▲
25.63 ±0.59 49.31 ±0.56 48.53 ±0.61 46.51 ±0.49 38.63 ±0.61
Urea S 22.62 ± 0.67 D▲ 57.9 ± 0.56N▲ N▲D▲ N▲D▲ N▲D▲ N▲D▲ N▲D▲

Creatinine S 0.32 ± 0.12 D▲ 2.73 ± 0.28N▲ 1.34 ±0.53 N●D∎ 1.62 ±0.10 N∎D● 1.42 ±0.21 N●D∎ 1.67 ±0.36 N∎D● 1.39 ±0.16 N●D∎
Uric acid S 1.26 ± 0.19 D▲ 5.38 ± 0.32N▲ 2.25 ±0.41 N●D▲ 4.09 ±0.24 N▲D∎ 3.13 ±0.28 N▲D▲ 4.16 ±0.42 N▲D● 3.42 ±0.19 N▲D▲
Kidney Homogenate (mg/dl)
Calcium KH 5.35 ± 0.64 D▲ 11.05 ± 0.31N▲ 6.54 ±0.73 N●D▲ 9.18 ±0.88 N▲D▲ 8.62 ±0.65 N▲D▲ 9.33 ±0.36 N▲D▲ 8.41 ±0.43 N▲D▲
Phosphorus KH 2.47 ± 0.21 D▲ 6.45 ± 0.54N▲ 3.65 ±0.12 N●D▲ 4.92 ±0.63 N▲D▲ 4.16 ±0.50 N▲D▲ 4.65 ±0.37 N▲D▲ 3.99 ±0.25 N▲D▲
Oxalate KH 2.09 ± 0.32 D▲ 6.73 ± 0.89N▲ 3.12 ±0.43 N●D▲ 4.32 ±0.40 N▲D▲ 3.68 ±0.32 N▲D▲ 4.16 ±0.19 N▲D▲ 3.57 ±0.82 N▲D▲

Values are expressed as Mean ± SD (n=5), Two way ANOVA followed by Dunnett’s test, Statistical significance▲: P<0.0001, ∎: 29P<0.001,
●P<0.05, ♦: P>0.05 (not significant), N compared with normal control group, D compared with disease control group.
Ethylene glycol
model-
Histopathological
images

1 2 3

Group 1 NC Group 2 DC Group 3 std

4 5 6 7

Group 4 MAD D1 Group 5 MAD D2 Group 6 MCF D1 Group 7 MCF D2

30
In vivo Antiurolithiatic study: Glycolic acid induced model
Parameters Group 1 NC Group 2 DC Group 3 std Group 4 MAD D1 Group 5 MAD D2 Group 6 MCF D1 Group 7 MCF D2

Change in body weight 24.73 ±1.02 D▲ -4.68 ±0.81 N▲ 18.78 ±0.78 N▲D▲ 14.19 ±1.32 N▲D▲ 20.18 ±0.88 N▲D▲ 15.99 ±0.92 N▲D▲ 17.26 ±1.32 N▲D▲

Water intake (ml/24h) 9.3 ±0.19 D▲ 13.67 ±0.21 N▲ 10.03 ±0.23 N♦D▲ 12.17 ±0.51 N▲D∎ 11.67 ±0.26 N▲D▲ 12.29 ±0.19 N▲D● 11.54 ±0.43 N▲D▲
Urine (mg/dl)
Calcium U 6.92 ±0.55 D▲ 14.12 ±0.39 N▲ 9.34 ±0.25 N▲D▲ 11.85 ±0.33 N▲D▲ 10.45 ±0.51 N▲D▲ 12.56 ±0.37 N▲D∎ 11.1 ±0.44 N▲D▲

Phosphorus U 4.63 ±0.43 D▲ 13.92 ±0.48 N▲ 7.85 ±0.77 N▲D▲ 12.75 ±0.36 N▲D● 10.16 ±0.72 N▲D▲ 11.49 ±0.66 N▲D▲ 10.36 ±0.91 N▲D▲

Magnesium U 3.88 ±1.01 D▲ 0.86 ±0.31 N▲ 2.53 ±0.20 N●D▲ 1.89 ±0.21 N▲D● 2.62 ±0.19 N●D▲ 1.92 ±0.24 N▲D● 2.27 ±0.54 N▲D∎

Oxalate U 1.27 ±0.76 D▲ 4.26 ±0.37 N▲ 2.29 ±0.54 N●D▲ 3.13 ±0.67 N▲D● 2.71 ±0.77 N∎D∎ 2.62 ±0.40 N●D▲ 2.34 ±0.77 N●D▲

Uric acid U 3.21 ±0.43 D▲ 6.91 ±0.21 N▲ 4.78 ±0.48 N∎D▲ 5.32 ±0.39 N▲D∎ 5.15 ±0.65 N▲D▲ 5.56 ±0.43 N▲D● 5.03 ±0.64 N▲D▲

Urea U 12.03 ±0.08 D▲ 18.78 ±0.15 N▲ 14.25 ±0.19 N▲D▲ 17.15 ±0.20 N▲D▲ 16.53 ±0.15 N▲D▲ 17.43 ±0.29 N▲D● 15.19 ±0.75 N▲D▲

Citrate U 19.97 ±0.21 D▲ 13.54 ±0.29 N▲ 17.16 ±0.31 N▲D▲ 15.35 ±0.12 N▲D▲ 14.53 ±0.29 N▲D● 16.82 ±0.98 N▲D▲ 14.71 ±0.29 N▲D●
Serum (mg/dl)
Calcium S 6.1 ±0.39 D▲ 12.45 ±0.25 N▲ 7.32 ±0.49 N●D▲ 9.97 ±0.22 N▲D▲ 9.11 ±0.47 N▲D▲ 10.62 ±0.64 N▲D▲ 9.13 ±0.11 N▲D▲

Phosphorus S 2.22 ±0.31 D▲ 9.15 ±0.44 N▲ 5.62 ±0.12 N▲D▲ 7.31 ±0.34 N▲D▲ 6.35 ±0.28 N▲D▲ 8.13 ±0.23 N▲D● 6.78 ±0.37 N▲D▲

Urea S 21.59 ±0.76 D▲ 55.09 ±1.01 N▲ 24.92 ±0.98 N▲D▲ 41.6 ±1.20 N▲D▲ 40.91 ±0.64 N▲D▲ 46.22 ±0.76 N▲D▲ 37.32 ±0.98 N▲D▲

Creatinine S 0.49 ±0.09 D▲ 2.93 ±0.21 N▲ 1.4 ±0.45 N♦D∎ 1.89 ±0.19 N∎D● 1.46 ±0.29 N●D∎ 1.78 ±0.29 N●D● 1.49 ±0.28 N●D∎

Uric acid S 1.19 ±0.22 D▲ 5.3 ±0.42 N▲ 2.69 ±0.43 N∎D▲ 3.92 ±0.23 N▲D● 3.24 ±0.21 N▲D▲ 3.78 ±0.30 N▲D∎ 3.19 ±0.15 N▲D▲
Kidney Homogenate (mg/dl)
Calcium KH 5.49 ±0.69 D▲ 11.82 ±0.45 N▲ 6.35 ±0.76 N♦D▲ 9.21 ±0.76 N▲D▲ 8.46 ±0.49 N▲D▲ 8.95 ±0.63 N▲D▲ 7.22 ±0.54 N▲D▲

Phosphorus KH 2.28 ±0.30 D▲ 6.68 ±0.83 N▲ 3.62 ±0.09 N●D▲ 5.35 ±0.56 N▲D● 4.63 ±0.60 N▲D▲ 4.78 ±0.87 N▲D▲ 3.87 ±0.21 N∎D▲

Oxalate KH 2 ±0.27 D▲ 6.59 ±0.54 N▲ 3.28 ±0.65 N●D▲ 5.01 ±0.59 N▲D∎ 3.57 ±0.21 N∎D▲ 4.92 ±0.32 N▲D▲ 3.41 ±0.97 N∎D▲

Values are expressed as Mean ± SD (n=5), Two way ANOVA followed by Dunnett’s test, Statistical significance▲: P<0.0001, ∎: P<0.001,
31
●P<0.05, ♦: P>0.05 (not significant), N compared with normal control group, D compared with disease control group.
Glycolic acid
model-
Histopathological
images

1 2 3

Group 1 NC Group 2 DC Group 3 std

4 5 6 7
Group 4 MAD D1 Group 5 MAD D2 Group 6 MCF D1 Group 7 MCF D2

32
In vivo Anti-inflammatory study

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

33
34
35
In vivo Anti-inflammatory study

Wet weight of
Dry weight of cotton Transudative
Animal groups cotton pellet % Inhibition % Inhibition
pellet (mg)* weight (mg)*
(mg)*

Disease
223.32±2.02 68.96±1.92 - 154.36±1.89 -
Control

56.32
Diclofenac 98.84±1.38 31.42±1.23 54.44 67.42±1.03
163.53±1.54 24.29 111.32±2.21 27.88
MeAD D1 52.21±1.09
149.32±1.04 36.01 105.19±1.39 31.85
MeAD D2 44.13±1.32
183.46±1.22 12.06 122.82±1.53 20.43
MeCF D1 60.64±0.98
168.92±1.98 21.48 114.77±1.42 25.65
MeCF D2 54.15±1.21

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.

36
In vitro cell line studies: Cytotoxicity study

Cytotoxicity study MeAD Cytotoxicity study MeCF

100 96.82 100
100 84.81 84.73 85.30 82.40 100 Sample NRK-52E cell line IC5024h
80 80 69.99 name

% Viability
% Viability

60 60 47.49 MeAD NA (Sample showed mild

40 40 toxicity at tested
18.66 16.74 16.66 concentrations)
20 20
MeCF 11.1µg/mL
0 0

Concentration (in µg/ml) Concentration (in µg/ml)

Percentage viability of NRK-52E cell line against MeAD and MeCF

In vitro cell line studies: Cytoprotective study

Cytoprotective activity
120
100
100 84.37 88.50
74.77
80
% Viability

60
40
20
0
Untreated Sodium Oxalate MeAD 6.25 MeAD 12.5
1mM µg/ml µg/ml
Sodium Oxalate 1mM
37
In silico antiurolithiatic study of active extract:

GCMS chromatogram of MeAD

• Compounds identified in GCMS (Total 150) were given codes from MeAD 1 to MeAD 144
• Xanthine dehydrogenase (Xdh) (PDB ID: 1JRP), Tamm-Horesefall Protein (THP) (PDB ID: 4WRN), and Oxalate oxidase
(OxOx) (PDB ID: 2ETE) 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.
• 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 where, 5 compounds showed acceptable results.
38
• Out of these 5 compounds 2 compounds named MeAD1 and MeAD123 showed interactions with all the three target proteins.
 CDOCKER ENERGY (-kcal/mol) towards
Ligand Name
1JRP 2ETE 4WRN
Oxypurinol 13.9096 - -
Acetylglucosamine - 1.70537 25.2219
MAD_1 35.6504 5.46349 30.73
MAD_123 41.7636 18.6301 38.5753

39
Docking poses of MeAD 1 and MeAD 123 with 1JRP, 2ETE and 4WRN.
ADMET parameters of MeAD
40
Isolation of phytoconstituents
The Column chromatography fractionations of the active extract and purification yields a compound, Compound 1-KD_AD1

Colour: Yellow
Physical nature: Solid
Melting point: 277.6°C
Solubility: Ethanol, DMSO

TLC of KD_AD1
Solvent System-Toluene:
Ethyl Acetate: Glacial
Acetic Acid: 5.5:4:0.5.
Single spot was observed
at Rf value 0.8.

1H 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).
41
 13C 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
%

HRMS of the KD_AD1 is done in methanol

solvent with MicroMass Q-TOF. HRMS (ESI-
288.0622
Positive) m/z: [M+H]+ calculated for C15H10O6
287.0477; Found 287.0585.
338.3447

42
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
 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.

Molecular formula: C15H10O6

After spectral analysis the isolated compound KD_AD1 is identified as Kaempferol

Molecular weight: 286.23

Structure of isolated compound KD_AD1- Kaempferol 43

Evaluation of Pharmacological activity of KD_AD1
In vitro antiurolithiatic activity of KD_AD1

Percentage inhibition (Mean ± SEM, n=3) of Percentage inhibition (Mean ± SEM, n=3) of
nucleation by different concentration of aggregation by different concentration of
KD_AD1. KD_AD1.

Nucleation inhibition IC50 Aggregation inhibition IC50

Cystone 576.26± 1.82 Cystone 573.69± 1.54
KD_AD1 924.32± 2.47 KD_AD1 976.95± 2.17

IC50 value (Mean ± SEM, n=3) of nucleation IC50 value (Mean ± SEM, n=3) of aggregation
inhibition by standard(cystone) and KD_AD1. inhibition by standard(cystone) and KD_AD1.

44
In vitro anti-inflammatory activity of KD_AD1

Percentage inhibition of egg albumin Percentage inhibition (Mean ± SEM, n=3) of

denaturation by different concentration of hypotonicity-Induced haemolysis by different
KD_AD1. concentration of diclofenac and KD_AD1.

Egg albumin Denaturation assay HRBC Hypotonic

Sample IC50 Sample IC50
Diclofenac 218.01± 0.96 Diclofenac 267.26± 1.44
KD_AD1 372.99± 1.16 KD_AD1 376.79± 1.09
Egg albumin denaturation assay- IC50 value Human Red Blood Cell (HRBC) membrane
(Mean ± SEM, n=3) of diclofenac and stabilization assay- IC50 value (Mean ± SEM,
KD_AD1. n=3) of diclofenac, and KD_AD1

45
In silico antiurolithiatic activity of KD_AD1

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

2D Docking poses of KD_AD1 with 1JRP, 2ETE and 4WRN.

1JRP 2ETE 4WRN

46
ADME prediction of KD_AD1

47
Discussion and Conclusion
• 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 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
• In vitro results were supported by the in vivo studies pertaining to antiurolithiatic and anti-
inflammatory studies.
• The in vivo studies were followed by evaluating the cytotoxicity and cytoprotective effect of
the extract on NRK-52E cell line using MTT assay.
• The active extract was then subjected to in silico studies for evaluating its potential to bind
and inhibit the protein targets responsible for causing urolithiasis.
• Compound KD_AD1 was isolated from the active extract using column chromatography. The
spectroscopic analysis confirmed that isolated compound KD_AD1 is Kaempferol.
• KD_AD was further studied for in vitro antiurolithiatic and in vitro anti-inflammatory
studies, followed by the in silico studies targeting the proteins responsible for causing kidney
stone.
• It can be concluded that this research validates ethnomedicinal claims about Alternanthera
dentata and Calamus floribundus' antiurolithiatic and anti-inflammatory properties.
48
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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.

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