WORLD JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCES

Kaur et al. World Journal of Pharmacy and Pharmaceutical Sciences

SJIF Impact Factor 6.041

Volume 5, Issue 4, 1691-1706 Research Article ISSN 2278 – 4357

EVALUATION OF ANTI-HELICOBACTER PYLORI (DSMZ 10242)

ACTIVITY AND QUALITATIVE ANALYSIS OF QUERCETIN BY
HPLC IN PHYLLANTHUS NIRURI LINN.

Baljinder Kaur*1, Navneet Kaur2 and Vikas Gautam3

1,2
Department of Biotechnology, Punjabi University, Patiala, 147002, India.
3
Department of Clinical Microbiology, PGIMER (Post Graduate Institute of Medical
Education and Research), Chandigarh.

Article Received on
ABSTRACT
03 Feb 2016, Phyllanthus niruri Linn. is traditionally used in folk medicine for the
Revised on 24 Feb 2016,
Accepted on 16 March 2016 treatment of a variety of ailments such as asthma, arthritis, poor
DOI: 10.20959/wjpps20164-6476 appetite, constipation, cuts and bruises, corneal opacity, conjunctivitis,
flu and colds, blennorrhagia, colic, diabetes, dropsy, dysentery,

*Correspondence for dyspepsia, fever, flu, gout, gonorrhea, itch, jaundice, kidney aliments,
Author leucorrhea, malaria, menorrhagia, menstrual troubles/complaints,
Baljinder Kaur obesity, proctitis, stomachache, tenesmus, tumor, typhoid fever and
Department of
vaginitis. The present study was aimed to evaluate the anti-
Biotechnology, Punjabi
Helicobacter pylori and urease inhibition activities of hydroalcoholic
University, Patiala,
147002, India. extracts from different regions of Punjab using simple plate assays.
From the study it was reported that hydroalcoholic extract from Patiala
exhibited stronger anti-H. pylori activity (20 mm) than extracts from other regions of Punjab.
Moreover, quercetin was determined in all the extracts using HPLC and maximum
concentration of quercetin was also found in extract from Patiala region (574.8 mg/g).
Preliminary studies revealed that the inhibitory mechanism may involve quercetin based non-
competitive urease inhibition.

KEYWORDS: Anti-Helicobacter pylori, Phyllanthus niruri Linn., Urease inhibition,

Quercetin, HPLC, Anti-microbial activity.

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INTRODUCTION
Helicobacter pylori, is a Gram negative, spiral shaped bacterium recognized as class I
carcinogen by World Health Organization (WHO).[1] Infections due to H. pylori are more
prevalent in developing parts of the world due to the poor socioeconomic status and
overcrowded conditions.[2] However, this pathogen can persist in an acidic environment of
stomach (pH 2) of an individual for life long without causing any infection, only 10-20%
population are likely to get infected and 1-2% develop gastric carcinoma.[2,3] Various
pharmacological treatments are used for the eradication of H. pylori such as antibiotics
(amoxicillin, clarithromycin, metronidazole and tetracycline), bismuth salts, H2 blockers and
proton pump inhibitors in conjunction with antibiotics (in Dual, Triple or Quadruple
therapy).[4] The success of these treatment regimens is associated with various side effects
including treatment cost, effectiveness of antibiotics and resistance towards antibiotics.[4,5]
Considering these problems there is need to explore new drugs and to develop alternative
therapies with improved stability and low toxicity.[6,7]

The use of Traditional Asian Medicine (TAM) to combat H. pylori related disorders has been
widely studied. Phyllanthus niruri Linn. is a small annual herb widely used in traditional
Chinese and Ayurvedic medicine for the treatment of various disease such as asthma,
arthritis, poor appetite, constipation, cuts and bruises, corneal opacity, conjunctivitis, flu and
colds, blennorrhagia, colic, diabetes, dropsy, dysentery, dyspepsia, fever, flu, gout,
gonorrhea, itch, jaundice, kidney aliments, leucorrhea, malaria, menorrhagia, menstrual
troubles/complaints, obesity, proctitis, stomachache, tenesmus, tumor, typhoid fever and
vaginitis. This plant is rich source of bioactive constituents/phytochemicals such as alkaloids,
coumarins, flavonoids, lignans, saponins, tannins and terpenoids.[8]

Flavonoids are the primary constituents of herbal medicines and possess anti microbial
activity. Flavonoids such as quercetin, rutin, quercitrin, astragalin and catechin have been
identified in extracts from P. niruri.[9] Out of these flavonoids, quercetin belonging to class
flavonols is widely studied bioflavonoid because of its health promoting advantages such as
anti-inflammatory, anti-nociceptive, anti-mutagenic, anti-leishmanial and anti-viral activity.
It acts as gastroprotective agent and also prevents osteoporosis.[10-16] Quercetin plays a very
important role in the prevention and treatment of peptic ulcer by promoting mucus secretion.
It has also been shown to inhibit the growth of H. pylori in in-vitro studies.[17]

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The study was undertaken, keeping in mind the need to identify the potential of this
medicinal plant against H. pylori. To the best of our knowledge hydroalcoholic extracts of P.
niruri haven't been screened yet for their anti-H. pylori activities. The extract exhibiting
promising anti-H. pylori activity was furthur tested for the presence of quercetin, as it seems
to play an important role in inhibition and prevention of colonization by H. pylori in stomach.

MATERIALS AND METHODS

Collection of Plant material
Different populations of Phyllanthus niruri were collected from various regions of Punjab,
India viz. Bathinda (Krishi Vigyan Kendra, Kheti Bhawan, Rose Garden and Chetak Park),
Amritsar (Guru Nanak Dev University, Khalsa College), Roopnagar (Chamkaur Sahib,
Bhakra Nangal dam and Gurudwara Sadabarat) and Patiala (Punjabi University, Baradari
Garden and Urban Estate). The plant samples were identified by Botanist Dr. Geetika
Sirhindi, Department of Botany, Punjabi University, Patiala. The collected populations were
washed thorougly under running water to remove soil and other extraneous matter. The
samples were then shade dried properly for atleast a month and crushed using electric grinder
to get fine powder.

Preparation of Hydroalcoholic Extracts

Air dried plant powder of P. niruri was minced and extracted with 50% ethanol and water in
1:3 ratio. The fraction was then macerated at room temperature (25±3°C) for 15 days. The
solvent was evaporated and the extract was concentrated to desired level using vaccum
evaporator and stored at -20°C until furthur analysis.[18]

Procurement and Maintenance of microbial culture:

H. pylori (DSMZ) 10242 was procured from DSMZ (Deutsche Sammlung von
Mikroorganismen und Zellkulturen), Germany. It was subcultured and maintained using
Brain Heart Infusion (BHI) media supplemented with 5-10% blood (HiMedia) at 37°C in 5%
CO2 atmosphere throughout the study as described by DSMZ. The culture used was
maintained as glycerol stock and stored at −20°C for further utilization. The work was carried
out using class II biosafety cabinet at Department of Clinical Microbiology, PGIMER (Post
Graduate Institute of Medical Education and Research), Chandigarh under Dr. Vikas Gautam.

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Antibacterial Assays of Plant extracts

The crude extracts were filtered through HEPA filters (0.47 micron membrane filter) and the
filterates were further used for in vitro testing of extracts using agar well diffusion assay and
disc diffusion assay. Each experiment was repeated thrice and the control consisted of 50%
ethanol and water in ratio 1:3 in each antimicrobial assay. The lowest concentration of each
of the extract which inhibited the bacterial growth was observed in triplicate analysis.

a) Well diffusion assay

For this assay, method of Divakar and co-workers[19] was followed along with some
modifications. Around 6 mm diameter wells were made using sterile cork borer on BHI agar
media having 5-10% v/v blood, overlaid with 5 ml soft BHI agar (0.8%) containing H. pylori
and each well was inoculated with 5-50 µl plant extracts. The plates were incubated for 48
hrs at 37°C in 5% v/v CO2 and inhibition zones around wells were measured. Inhibition was
scored positive if the zone was wider than 2 mm in diameter.

b) Disc diffusion assay

Antibacterial susceptibility using disc diffusion assay was assessed by the method of Yildrim
and Jhonson[20] with modifications. The filtered plant extracts (5-50 µl) were spotted on
sterile discs 8 mm in diameter on preseeded BHI agar plates containing 5-10% v/v blood.
Plates were preseeded with H. pylori using sterile swab, which was dipped into the respective
microbial suspensions and surplus removed by rotation of the swab against the sides of the
tube above the fluid level. Plates were incubated at 37°C for 48 hrs under microaerophilic
environment i.e. 5% v/v CO2. After incubation the plates were observed for inhibition zones
around the discs.

Urease Inhibition Assay

Urease plate assay is based on the principle that either urease is located in the cytoplasmic
membrane or excreted by the bacteria under low-pH conditions, will convert urea to ammonia
and counter the low pH. Under these circumstances, due to rise in pH, bromocresol purple
will be converted to purple color. Thus, due to inhibition of urease activity, a yellow zone
would be observed. For this, urea (10mM) was added to the BHI medium containing agar
(3% w/v) supplemented with 5-10% v/v blood and bromocresol purple (0.01 g/liter). The
final pH of the medium was adjusted to 6.0. The BHI media was overlaid with H. pylori
using sterile swab as described earlier. Individual hydroalcoholic extracts from different
regions were added to sterile paper disks using a micropipette. Saturated disks (5-50 µl) were

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stored in dark at -20°C until use. BHI agar plates with H. pylori were spotted with saturated
discs and plates were incubated at 37°C for 48 h in CO2 incubator with 5% v/v CO2. The
plates were observed for the presence of yellow zones and diameter of each zone was
recorded.[21]

HPLC Analysis of Extracts for the Presence of Quercetin

The filtered hydroalcoholic extracts from different regions along with authentic quercetin
sample (1000 µg/ml) were injected in HPLC system equipped with PDA (Photo Diode array)
detector. Column was a Dynamax C18, with 250 x 4.6 mm dimensions and 5 μm particle
size. Qualitative analysis was made, in step gradient mode, with water, acetonitrile and acetic
acid as mobile phase in ratio 90:10: 0.2 at a flow-rate of 1 mL min-1. The injection volume
was 20 μL and the eluate was monitored at 339 nm.[22, 23] Percentage quercetin in each extract
was calculated using formula.[24]
% Quercetin= Area of test/Area of sample*100.
Conversion factor: 1 per = 10mg/g.

Statistical analysis
All experiments were performed in triplicates and the results are expressed as mean ±
standard deviation.

RESULTS
Antibacterial Assays of Plant extracts
The antibacterial activity of P. niruri, hydroalcoholic extracts from various regions of Punjab
was tested against H. pylori DSMZ 10242. Extracts were first diluted to different ratios
ranging from (1:99-1:999). However, none of the dilution inhibited growth of H. pylori. After
several attempts, the whole extracts were finally used for antibacterial assays as described
before without any dilution at different concentrations (5-50 µl).

a) Agar well diffusion assay

The hydroalcoholic extracts from different regions showed inhibitory activity against H.
pylori at concentration of 50 µl (Table 1 and Fig. 1). However, maximum inhibition was
shown by hydroalcoholic extract from Patiala region (13 mm), followed by Roopnagar (10
mm) and Amritsar (8 mm). Lowest inhibition was recorded in hydroalcoholic extract from
Bathinda (6mm).

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b) Disc diffusion assay

In this assay similar inhibition pattern was recorded as in agar well diffusion aasay (Table 1
and Fig. 1). Maximum inhibition was recorded in case of hydroalcoholic extract from Patiala
region (20 mm), followed by Roopnagar (15 mm) and Amritsar (10 mm). Again least
inhibition zone was shown by hydroalcoholic extract from Bathinda (5 mm).

Urease Inhibition Assay

Inhibitory effect of extracts over urease was tested and it was found that the hydroalcoholic
extracts from all the four regions inhibited urease. When the pH of the plates was adjusted to
6.0, the color of medium changed from purple to yellow. After inoculation as the bacteria
multiplied in number, the color of medium changed from yellow to purple by producing
ammonia through urease activity and thus countering the effect of pH in the medium.
However, the area around the paper disks didn't change to purple indicating that urease was
inhibited by the extracts. Larger the yellow zone, higher the inhibition by the extracts. The
hydroalcoholic extract from Patiala showed most potent inhibitory effect against urease by
giving maximum zone of inhibition i.e. 20 mm, followed by Roopnagar (14 mm), Amritsar (8
mm) and Bathinda (6 mm) (Table 1 and Fig. 1).

Table 1: Anti-H. pylori activity of Hydroalcoholic extracts of Phyllanthus niruri from

different regions of Punjab.
Quercetin Zone of inhibition (mm)
Hydroalcoholic
S. No. content Well Diffusion Disc Diffusion Urease Inhibition
Extracts (Regions)
(mg/g) Assay Assay Assay
1. Amritsar 0 8.0±0.71 10.0±0.75 10.0±0.78
2. Bathinda 0 6.0±0.86 5.0±0.78 8.0±0.78
4. Patiala 574.8 13.0±0.74 20.0±0.62 20.0±0.83
3. Roopnagar 393 10.0±0.98 15.0±0.70 14.0±0.92
* Results are mean ± S.D.

Figure 1: Anti-H. pylori and urease inhibition aasay of Phyllanthus niruri Linn. from
Punjab where, A; Amritsar, B; Bathinda, P; Patiala, R; Roopnagar and C; Control.

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HPLC Analysis of Extract from Patiala for the Presence of Quercetin

The hydroalcoholic extracts from various regions were also tested for the presence of
flavonoid quercetin. The method provided a quick analysis of the hydroalcoholic extract. The
conditions used provided good separation of the peaks which were identified in the
chromatogram (Fig. 2) as quercetin (Rt= 5.32) in hydroalcoholic extract from Patiala and
(Rt= 5.23) in Roopnagar hydroalcoholic extract, by comparing them with the chromatogram
of the quercetin reference compound (Rt=5.3) (Fig. 2) obtained under same conditions on
line. However, in hydroalcoholics extracts of Amritsar and Bathinda region, quercetin was
not detected. Area percentage was calculated in all the four extracts and maximum area
percentage was found in extract from Patiala (574.8%) followed by Roopnagar (393%)
(Table 1). Thus, it was postulated that the inhibition activity of the extracts may be due to the
presence of quercetin.

Figure 2: HPLC Fingerprinting of Hydroalcoholic extracts of different P. niruri

populations from Punjab a) Authentic Quercetin standard b) Patiala c) Roopnagar d)
Amritsar e) Bathinda. Column: Dynamax C18 (250 x 4.6 mm, 5 μm); Eluent: Water:
Acetonitrile: Acetic acid, 90:10:1 (0-15 min); Flow-rate: 1 mL min-1; Detection
wavelength: 339 nm.

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Table 2: Plants tested for anti-H. pylori activity along with their mechanism of action.
S.No. Plant Plant Part Extract used Mechanism of Action References
Leaves and [6]
1. Acacia nilotica Aqueous Urease inhibition
flowers
Achillea Leaves/Root/ [26]
2. Ethanol/ Methanol Urease inhibition
millefolium Stem
[30]
3 Acorus calanus Root Methanol/water Urease inhibition
Acetone and [6]
4. Adhatoda vasica Whole plant Urease inhibition
Methanol
Adhatoda [26]
5. Root/ Stem Ethanol Urease inhibition
zeylanica
Reduced IL-8 expression in [31]
6. Allium sativum Dried fruits Water
infected AGS cells
Angelica [30]
7. Leaf Methanol/water Urease inhibition
archangelica
Aristolachia [26]
8. Leaves/Stem Ethanol/ Methanol Urease inhibition
bracteata
Artemisia [32]
9. Whole plant Methanol Urease inhibition
scoparia
Sulphoraphane increase
Brassica [3]
10. Sprouts - mammalian cytoprotective
oleracea
response
Urease inhibition and
Calophyllum Hydroethanol/ [33]
11. Stem bark modulation of endogenous
brasiliense dichloromethane
antioxidant system
Calotropis Leaves and Aqueous and [6]
12. Urease inhibition
procera flowers acetone
[34]
13. Camellia sinesis Leaf Ethanol Urease inhibition
Cassia [35]
14. Whole plant Ethanol Urease inhibition
obtusifolia
Casuarina [6]
15. Fruit Methanol Urease inhibition
equisetifolia
[36]
16. Cinnamom Powder Ethanol Urease inhibition
[34]
17. Citrus arantifolia Fruit Methanol/water Urease inhibition
Cuminum Reduced IL-8 expression in [31]
18. Dried fruits Water
cyminum infected AGS cells
[26]
19. Cuscuta reflexa Stem Ethanol Urease inhibition
Eucalyptus [26]
20. Leaves Ethanol Urease inhibition
globules
[6]
21. Fagonia arabica Whole plant Acetone Urease inhibition
[30]
22. Lauras nobilis Leaf Methanol/water Urease inhibition
Leaves/Root/ [26]
23. Malva parviflora Ethanol Urease inhibition
Stem
Mangolia [35]
24. whole plant Ethanol Urease inhibition
officinalis
Matricaria [34]
25. Flower Methanol/water Urease inhibition
recutita
[26]
26. Mentha Leaves/Root/ Ethanol/ Methanol Urease inhibition

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longifolia Stem
Nasturtium [34]
27. Leaf Methanol/water Urease inhibition
officinale
[30]
28. Nigella sativa seed Methanol/water Urease inhibition
Inhibition of proline
Origanum [21]
29. Whole plant Water dehydrogenase and urease
vulgare
activity
Pelargonium
Inhibit adhesion on stomach [37]
30. sidedoides (EPs) Roots Ethanol
cells
7630
Reduced IL-8 expression in [31]
31. Piper nigrum Dried fruits Water
infected AGS cells
[1]
32. Pistacia lentiscus Bark Mastic gum Reduce colonization
Phyllanthus Inhibition of urease by Present
33. Whole plant Hydroalcoholic
niruri quercetin study
Inhibition of urease and
Fruit juice [38]
34. Prunus mume Fruit inhibition of motility by
concentrate
syringaresinol
[34]
35. Punica granatum Flower Methanol/water Urease inhibition
Inhibition of proline
Vaccinium [21]
36. whole plant Water dehydrogenase and urease
macrocarpon
activity
Zingiber [30]
37. Rhizome root Methanol/water Urease inhibition
officinale

Table 3: Characterized phytochemicals exhibiting urease inhibitory activity[4,37,40,41]

S. No. Phytochemical Plant IC50 value
1. (E)-2-haxenel Annacardium occidantale 50g/ml
2. 1,2,3,4,5,6-penta-O-galloyl-D-glucoside Paeonia lactiflora 72M
3. Allian diallyl thiosulphinate Garlic -
4. Anacardic acid Annacardium occidantale 125 /ml
Sterospermum
5. Atranorin 18.2µM
acuminatissimum
6. Baicalin Scutellariae baicalensis 2.7mM
7. Caseadine Corydalis govaniana 20.2µM
8. Caseamine Corydalis govaniana 66.7µM
9. Decarboxymethyl ligstroside aglycone Olive oil 1.3µg/ml
10. Genstein Pueraria thunbergiana 430/ml
11. Govaniadine Corydalis govaniana 38.9µM
12. Juglone Juglans nigra 4.8µM
13. Luteolin Lonicera japonica 35.5µM
14. Luteolin-7-O-glucoside Lonicera japonica 55.8µM
15. Methyl gallate Paeonia lactiflora 1.3mM
16. Myricetin Lonicera japonica 77.2µM
17. Myricitrin Lonicera japonica 98.7µM
Heliotropium
18. Ophiamide A 23.1µM
ophioglossum
19. Ophiamide B Heliotropium 12.6µM

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ophioglossum
20. Patchouli alcohol Pogostemon cablin 77g/ml
21. Protopine Corydalis govaniana 54.1µM
22. Quercetin Lonicera japonica 11.2µM
23. Quercetin-4'-O-D-glucoside Allium cepa 190
24. Rutin Lonicera japonica 67.6µM
25. Scutellarin Erigeron beriscapus 1.3mM
26. Shoreaphenol Hopea exalata 126.8µM
27. Sulphoraphane Brassica oleracea -
28. Tectorigenin Pueraria thunbergiana 0.43mg/ml
29. Vernonione Vernonia cinevascens 227.6µM
30. Zygophyloside A Zygophyllum fabago 500
31. Zygophyloside E Zygophyllum fabago 500
32. Zygophyloside G Zygophyllum fabago 500 µM

Figure 3: Non-competitive inhibition of enzyme urease by quercetin.

DISCUSSION
Due to therapeutic failure of some antibiotic resistant H. pylori strains, research has been
shifted to potential use of plant secondary metabolites/ phytochemicals as new anti-ulcer
drugs. Phytotherapy has recently gained significant attention due to less side effects and
effective disease control. Plants exhibiting anti-H. pylori activity along with their mechanism
of action is given in table 2.

In the present study the data revealed that hydroalcoholic extracts from all the four regions of
Punjab has significant inhibitory effect against H. pylori. In a previous study, aqueous extract
of P. niruri from Ecuador and Peru has been tested by Ranilla and co-workers [9] for anti-H.

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pylori activity and it was found that both the extracts inhibited H. pylori ATCC 43579 in dose
dependent manner. The inhibitory effect was accredited to the presence of ellagitannins,
geraniin or corilagin, previously identified in the leaves of P. niruri. The study postulated that
these ellagitannins were partially hydrolysed after the hot water extraction following the
formation of ellagic acid, gallic acid and other ellagitannin derivatives. Similarly, Phyllanthus
urinaria was studied earlier for anti-H. pylori activity in AGS cells (Human gastric epethelial
cells) and found to inhibit invasion and adhesion by H. pylori. The extract also inhibited H.
pylori-induced nuclear factor (NF)-kappa B activation, and the subsequent release of
interleukin (IL)-8 in AGS cells. The chemopreventive activity was accredited to group of
flavanoids namely β-sitosterol-3-O-β-d-glucopyranoside, methyl gallate, methyl
brevifolincarboxylate, rhamnocitrin, rutin, trimethyl-3,4-dehydrochebulate, phyllanthin,
phyltetralin and quercitrin.[25]

Urease enzyme is considered as the main virulence factor of H. pylori. It hydrolyzes urea into
ammonia, thereby creating a friendly environment for the survival of this gut pathogen.[26]
Many urease inhibitors have been described earlier such as fluorofamide, hydroxyurease and
hydroxamic acids etc. but their use has been prohibited due to toxicity and instability. Active
phytochemicals from medicinal plants with urease inhibitory activity are less toxic potential
therapeutics with higher stability (Table 3). In the present study inhibition of H. pylori urease
by extracts of P. niruri from various regions of Punjab were tested using simple plate assay.
The hydroalcoholic extracts of P. niruri from different regions of Punjab were found to
exhibit urease inhibition activity and maximum zone of inhibition i.e. 20 mm was shown by
extract from Patiala region. HPLC of extracts was further performed to find active principle
behind inhibition and quercetin was regarded as the reason behind it.

The results are supported by the study of Xiao et al.[27], they tested 20 flavonoids, out of
which quercetin showed maximum potency with IC50 of 11.2+/- 0.9 mu M against urease
from H. pylori ATCC 43504. The research group suggested quercetin as non-competitive
urease inhibitor. Moreover, 3-, 5-OH as well as 3', 4'-dihydroxyl groups were regarded as key
structural components for active compounds and removal of these groups led to a sharp
decrease in inhibition activity of quercetin. The diagramatic representation of inhibition of
urease activity by quercetin is given in Fig 3.

In another study Shin et al.[28] studied the effect of quercetin on the growth and vacuolation of
H. pylori. The results suggested that quercetin inhibited H. pylori Vac A vacuolation in HeLa

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cells with IC50 value of 0.046 mM. vac A gene encodes Vac A protein which is strongly
associated with damage of epethelial lining of gastric tissue and gastric ulcers.[29] It also
inhibited procaspase-3 activation to caspase-3 induced by H. pylori Vac A toxin and resulted
in induction of cell death by activating a cascade of proteolytic caspases.

Quercetin is a dietary flavonoid possessing strong antioxidant properties. However, in vivo

studies on the safety and efficacy of P. niruri extracts are extremely important in our ongoing
research work to formulate new herbal drug and make it available for further clinical use.
Currently the intake of plants containing quercetin is recommended to prevent incidents of
gastric ulcers caused due to H. pylori.

ACKNOWLEDGEMENTS
The authors thank Dr. Ashok Kumar Malik and Ms. Heena Rekhi, Research Scholar
(Department of Chemistry, Punjabi University, Patiala) for providing excellent technical
assistance while performing HPLC.

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