Ruthisha et al., IJPSR, 2018; Vol. 9(12): 5177-5185.

E-ISSN: 0975-8232; P-ISSN: 2320-5148

IJPSR (2018), Volume 9, Issue 12 (Research Article)

Received on 04 April, 2018; received in revised form, 09 June, 2018; accepted, 18 June, 2018; published 01 December, 2018

PHYTOCHEMICAL, ANTIOXIDANT, ANTIBACTERIAL ACTIVITIES OF NERVILIA

CROCIFORMIS (ZOLL. & MORITZI) SEIDENF. AND NERVILIA INFUNDIBULIFOLIA
BLATT. & MC CANN (ORCHIDACEAE) COLLECTED FROM KASARGOD DISTRICT,
KERALA, INDIA
P. K. Ruthisha *, C. Swathi Krishna and K. M. Khaleel
Department of PG Studies and Research in Botany, Sir Syed College, Taliparamba, Kannur - 670142,
Kerala, India.
Keywords: ABSTRACT: The whole plant extracts of N. crociformis and N.
Phytochemicals, infundibulifolia was analyzed by priliminary phytochemical, total
Medicinal plants, Antioxidants, phenolics, flavonoid, carbohydrate and alkaloid content by standard
Antimicrobial, Nervilia methods. In-vitro antioxidant DPPH assay and Phosphomolybdenum
Correspondence to Author: assay developed by Braca et al., Prieto et al., respectively. In-vitro
P. K. Ruthisha antibacterial (agar well diffusion) activity by gram negative Pseudomonas
Department of PG Studies and aeroginosa (ATCC 27853), gram positive (Streptococcus mutans MTCC
Research in Botany, Sir Syed College, 890 & Staphylococcus aureus ATCC 25923, Esteritia coli ATCC 25922)
Taliparamba, Kannur - 670142, and antifungal activity by Aspergillus niger (ATCC 16404). The study
Kerala, India. shows that the petroleum ether, chloroform, ethanol and aqueous extract
E-mail: [email protected] of the plants contains a good number of phytochemicals such as
flavonoid, cardiac glycoside, saponin, alkaloid etc. as well as antioxidant
with respect to the scavenging activity against free radicals DPPH which
were roughly comparable to that of BHT. The plant extracts also
exhibited potent antimicrobial activity against S. aureus, P. aeruginosa,
S. mutans, E. coli and A. niger indicating its bactericidal and fungicidal
properties. It may be concluded that the plant Nervilia crociformis is
chemically more active than Nervilia infundibulifolia.
INTRODUCTION: India is a mega-biodiversity Among the 16 species Nervilia aragoana has
country, which is not only rich in medicinal plant subjected to various studies and the plant is
resource, but also rich in traditional knowledge commonly known as Orilathamara. But in many
about such medicinal plants. There are several part of the country traditional healers use other
studies to prove the presence of various species of Nervilia such as Nervilia crociformis,
bioactivities and bioactive compounds in Nervilia plicata and Habernaria diphylla were used
Orchidaeae plants. Nervilia is a genus of orchid instead of N. aragoana.
with about 65 species among them 16 occurring in
India. In Sanskrit the drug Padmacarini is reported hot,
QUICK RESPONSE CODE
bitter and astringent 1. It overcomes morbid kapha
DOI: and vatha and cures dysuria, urinary calculi, cough
10.13040/IJPSR.0975-8232.9(12).5177-85 and asthma 2. According to Rajanikhandu the drug
is aromatic and it cures vomiting, epilepsy,
Article can be accessed online on: diarrhoea, diabetis 3. The Padmacarini is a
www.ijpsr.com
constituent drug of Priyangvadi gana of Vagbhada.
DOI link: http://dx.doi.org/10.13040/IJPSR.0975-8232.9(12).5177-85 Vastyamayantaka ghrtam, Satavaryadi ghrtam and

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Matsyakhyadi kashaya etc. are some of the Ltd., Thane, Maharashtra, India) for further
preparations using the drug 4, 16. Most of the analysis. Quantitative analysis and antioxidant
literature has illustrated the padmacarini as assays were done by dissolving 1 mg extract in 1
Hybanthus enneaspermus and Habernaria ml respective solvent. Antimicrobial study was
grandiflora. Traditional healers and ayurvedic done with well diffusion method by dissolving 1
physicians of Kerala have considered the single mg extract in 1 ml DMSO.
leafed plants orilathamara is same as padmacarini
and they used the plants Nervilia crociformis, Qualitative Phytochemical Screening: For
Nervilia plicata and Habernaria diphylla as preliminary phytochemical screening, plant extracts
padmacarini. The present study aims at qualitative of the whole plants of N. crociformis and N.
and quantitative phytochemical screening with infundibulifolia was subjected to various qualitative
special reference to the antioxidant and chemical tests to determine the presence of phyto-
antibacterial properties of the plant Nervilia constituents like glycosides, tannins, phytosterols,
crociformis which is known as padmacarini in proteins, amino acids, carbohydrates, flavonoids,
Sanskrit and a closely related species Nervilia phenolic compounds, oils and fats, and saponins 5.
infundibulifolia whole plant extracts. Total carbohydrate content of the selected plants
was done by anthrone method 6. The total phenolics
Indian species of Nervilia infundibulifolia have not of the different plant extracts were determined
yet been subjected to such phytochemical screening according to the method described by Makkar 7.
to understand the medicinal values. The selected The flavonoid contents of all the extracts were
plants were belonging to the family Orchidaceae. quantified according to the method described by
The plants have a single leaf and are ground Zhishen, et al. 8
hugging in case of N. crociformis and small and
with reddish patches in case of N. infundibulifolia. In-vitro Antioxidant Assays: Two different assays
These plants have a fleshy flower stem with only including free radical scavenging DPPH assay and
one flower and a leaf which arises later at the base Phosphomolybdenum assay were used to evaluate
of the dying flower stem. The flowers of the plants the antioxidant potential of the selected plants.
appear soon after the first heavy rain in monsoon Each experiment was done in triplicate and mean
season. values were taken. The antioxidant activity of the
extract was determined in terms of hydrogen
MATERIALS AND METHODS: donating or radical scavenging ability using the
Preparation of Extracts: The whole plants of N. stable radical DPPH, according to the method of
crociformis and N. infundibulifolia were collected Braca et al. 9, 10 The antioxidant activity of samples
from Kasargod District, Kerala, India. The plants was evaluated by the green phospho-molybdenum
were identified in department of PG studies and complex formation according to the method of
research in botany, Sir Syed College, Taliparamba. Prieto et al. 11
The collected plants were washed thoroughly with
tap water followed by distilled water for the Antibacterial Activity Test: The antibacterial
removal of dust and soil particles, cut into pieces activity was carried out on the extracts chloroform
and shade dried at room temperature for 15 days and ethanol against gram negative (Pseudomonas
then coarsely powdered using Panasonic Mixer and aeroginosa ATCC 27853) and gram positive
used for extraction. The powder (20 gm) was (Streptococcus mutans MTCC 890 &
extracted with petroleum ether, chloroform, ethanol Staphylococcus aureus ATCC 25923), Esteritia
in a Soxhlet apparatus (3840; Borosil Glass works coli (ATCC 25922) bacterial strains were used for
Ltd., Mumbai, India) and finally the dried powder the study. Antifungal activity by Aspergillus niger
was macerated using water with constant stirring (ATCC16404).
for 48 h using the orbital shaker (Rivotek; Riviera
The agar well diffusion method was employed for
Glass Pvt., Ltd., Mumbai, India) and the extract
the determination of antimicrobial activities of the
was filtered. The extracts were concentrated using a
selected plants. A suspension of the tested bacteria
rotary evaporator and stored at -20 ºC in the deep
(0.1 mL of 1 × 108 cells/mL) was spread on the
freezer (RQV- 300; plus, REMI electro technik

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solid media plates. Wells of approximately 10 mm the zone of inhibition was measured after overnight
was bored using a well cutter and sample of 25, 50, incubation and compared with that of standard
and 100 µg concentrations were added. After antimycotic (Clotrimazole).
staying at 4 ºC for 2 h, the plates were incubated at
37 ºC for 24 h. The antibacterial activity was RESULTS:
assayed by measuring the diameter of the inhibition Phytochemical Screening: Screening of various
zone formed around the well (NCCLS, 1993) in phytochemicals of crude extracts reveals a good
centimetres. Streptomycin was used as a positive concentration of proteins, alkaloids, phenols,
control with the tested bacteria. In order to access tannins, amino acids, carbohydrates, saponins,
the biological significance and ability of the flavonoids, glycosides and fixed oils and fats in
sample, the antifungal activity was determined by petroleum ether, chloroform, ethanol and water
Agar well diffusion method. Potato Dextrose agar extracts of Nervilia crociformis Table 1 and
plates were prepared and overnight grown species Nervilia infundibulifolia Table 2. These chemical
of fungus, Aspergillus niger was swabbed. Wells of compounds are responsible for different medicinal
approximately 10 mm was bored using a well cutter properties of the plant extracts.
and samples of different concentration was added;
TABLE 1: RESULTS OF PRELIMINARY PHYTOCHEMICAL SCREENING N. CROCIFORMIS
S. no. Phytochemicals Pet. ether Chloroform Ethanol Water
1 Carbohydrate + ++ +++ +++
2 Protein + + + ++
3 Amino acid - - + +
4 Alkaloid - - + +
5 Flavonoid + + + +
6 Tannin - - + +
8 Saponin - - - +
9 Phenol + + + +
11 Cardiac Glycosides - - + -
12 Fixed oils and Fats - + + +

TABLE 2: RESULTS OF PRELIMINARY PHYTOCHEMICAL SCREENING N. INFUNDIBULIFOLIA

S. no. Phytochemicals Pet. ether Chloroform Ethanol Water
1 Carbohydrate + ++ +++ +++
2 Protein - - + +
3 Amino acid - - - +
4 Alkaloid - - + +
5 Flavonoid + + + +
6 Tannin - - + -
8 Saponin - - - +
9 Phenol + + + +
11 Cardiac Glycosides - - - +
12 Fixed oils and Fats - - + -
(+): Presence of chemical compound, (-): Absence of chemical compound; (+) < (++) < (+++): Based on the intensity of characteristic colour

Quantification of Total Carbohydrates: spectroscopic method. The phenolic content of the

TABLE 3: CARBOHYDRATE CONTENT IN THE whole plant extract of N. crociformis and N.
SELECTED PLANT SAMPLES infundibulifolia in different solvent extracts were
Plant sample Carbohydrate shown in Table 4.
Nervilia crociformis 152.93±.70
Nervilia infundibulifolia 311.53±5.29 TABLE 4: PHENOLIC CONTENT IN N. CROCIFORMIS
AND N. INFUNDIBULIFOLIA WHOLE PLANT
Total carbohydrate content was high in N. EXTRACTS
S. no. Extract Phenolics (GAE/1000mg)
infundibulifolia compared to N. crociformis and it
N. c N. i
was 311.53 ± 5.29 and in N. crociformis it was 1 Petroleum ether 24.28±.37 10.53±.44
152.93 ± .70. 2 Chloroform 17.23±.51 8.45±.27
3 Ethanol 60.49±2.03 27.43±.32
Quantification of Total Phenolics: The phenolic 4 Water 23.53±.82 21.03±19
content in the selected plants were studied by

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The chloroform extract shows lowest activity of the were investigated for antioxidant potential by using
two species studied and the ethanol extract shows three different methods. All extracts showed
highest activity. Of the two plants studied the antioxidant activity up to varying extent.
whole plant extracts of N. crociformis has highest
phenolic content than N. infundibulifolia. DPPH Radical Scavenging Activity:
TABLE 6: DPPH RADICAL SCAVENGING ACTIVITY
OF NERVILIA CROCIFORMIS AND NERVILIA
INFUNDIBULIFOLIA
S. no. Extract IC50 mg/ml
N. c N. i
1 Petroleum ether 48.16956 136.612
2 Chloroform 25.56237 88.65248
3 Ethanol 79.36508 111.1111
4 Water 24.11963 173.6111

FIG. 1: GRAPHICAL REPRESENTATION OF TOTAL

PHENOLICS IN N. CROCIFORMIS AND N. INFUNDI-
BULIFOLIA WHOLE PLANT EXTRACTS
Series 1; Pet. ether, Series 2; Chloroform, Series 3; Ethanol,
Series 4; Water

Quantification of Total Flavonoids:

TABLE 5: TOTAL FLAVONOID CONTENT IN N.
CROCIFORMIS AND N. INFUNDIBULIFOLIA WHOLE
PLANT EXTRACTS FIG. 3: GRAPHICAL REPRESENTATION OF DPPH FREE
S. no. Extract Flavonoids (RE/1000mg) RADICAL SCAVENGING ACTIVITY IN N. CROCIFORMIS
AND N. INFUNDIBULIFOLIA WHOLE PLANT EXTRACTS.
N. c N. i
N. i: Nervilia infundibulifolia; N. c: Nervilia crociformis
1 Petroleum ether 115.49±.96 178.06±2.55
2 Chloroform 128.65±.56 147.26±1.92 The petroleum ether extract of Nervilia crociformis
3 Ethanol 124.79±1.47 128.01±4.41
4 Water 114.85±3.09 142.77±2.94 whole plant sample exhibit a significant dose
N. i: Nervilia infundibulifolia; N. c: Nervilia crociformis dependent inhibition of DPPH activity with 50% of
inhibition (IC50) at concentration of 48.16956
mg/ml and IC50 value of BHT was 36.79176
mg/ml. The chloroform extract shows 50%
inhibition at the concentration 25.56237 mg/ml,
ethanol extract inhibit the free radical scavenger at
a concentration of 79.36508 mg/ml and water
extract shows an IC50 value of 24.11963 mg/ml.
In the case of Nervilia infundibulifolia the
chloroform extract shows the lowest value and the
FIG. 2: GRAPHICAL REPRESENTATION OF TOTAL chloroform extract of the selected plant was a good
FLAVONOID IN N. CROCIFORMIS AND N. radical scavenger than all other extract and the
INFUNDIBULIFOLIA WHOLE PLANT EXTRACTS concentration was 88.65248 mg/ml. The petroleum
Series 1; Pet. ether, Series 2; Chloroform, Series 3; Ethanol,
ether extract of Nervilia infundibulifolia whole
Series 4; Water
plant sample exhibit 50% inhibition at the
Comparatively the plant N. infundibulifolia extracts concentration 136.612, the ethanol extract shows an
may contain more flavonoid content than N. IC50 value of 111.1111 and water extract shows
crociformis extracts. 50% of inhibition (IC50) at concentration 173.6111.

Antioxidant Assays: In present study, the 4 Based on the demonstrated results the crude extract
extracts of N. crociformis and N. infundibulifolia of Nervilia crociformis can be categories as more

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free radical scavenging activity than Nervilia infundibulifolia shows an average of 183.7 ± 6.12,
infundibulifolia. Basically, a higher DPPH radical- ethanol extract shows a value of 264.26 ± 1.7 and
scavenging activity is associated with a lower IC50 water extract shows a result of 325.93 ± 1.4.
value. Nervilia crociformis shows an average of 191.67 ±
1.5 in petroleum ether extract, 348.52 ± 1.2 in
Phosphomolybdenum Assay: The antioxidant chloroform extract, 154.63 ± 3.9 in ethanol extract
activity of samples was evaluated by the green and 293.89 ± 6.3 in water extract.
phosphomolybdenum complex formation according
to the method of Prieto (1999). This method is
based on the reduction of phosphomolybdic acid to
phosphomolybdenum blue complex by sodium
sulfide. The obtained phosphomolybdenum blue
complex is oxidized by the addition of nitrite and
this causes a reduction in intensity of the blue
colour.
TABLE 7: RESULT OF PHOSPHOMOLYBDENUM ASSAY
S. no. Extract N.i N.c
1 Petroleum ether 209.44±2.55 191.67±1.5 FIG. 4: GRAPHICAL REPRESENTATION OF PHOSPHO-
2 Chloroform 183.7±6.12 348.52±1.2 MOLYBDENUM ASSAY IN N. CROCIFORMIS AND N.
3 Ethanol 264.26±1.7 154.63±3.9 INFUNDIBULIFOLIA WHOLE PLANT EXTRACTS. N. i:
4 Water 325.93±1.4 293.89±6.3 Nervilia infundibulifolia; N. c: Nervilia crociformis
N. i: Nervilia infundibulifolia; N. c: Nervilia crociformis
Antimicrobial Activity: Antimicrobial activity is
The petroleum ether extract shows an average of observed high in chloroform and ethanol extracts
209.44 ± 2.55, chloroform extract of Nervilia compared to petroleum ether and water.

Streptococcus mutans

Staphylococcus aureus

Pseudomonas aeruginosa

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Escheritia coli

Aspergillus niger
FIG. 5: ANTIMICROBIAL ACTIVITY OF N. CROCIFORMIS ETHANOL AND CHLOROFORM EXTRACTS
AGAINST DIFFERENT STRAINS

TABLE 8: ZONE OF INHIBITION OF N. CROCIFORMIS CHLOROFORM AND ETHANOL EXTRACT AGAINST

DIFFERENT BACTERIAL STRAINS
Sample Concentration (μg) Zone of inhibition (cm)
E. coli P. aeroginosa S. aureus S. mutans
chloroform 25 1.0 nil nil 1.0
50 1.1 1.4 1.0 1.0
100 1.6 2.3 1.5 1.3
ethanol 25 Nil Nil Nil 1.0
50 1.0 1.2 nil 1.4
100 1.4 1.7 1.3 2.0
Streptomycin 50 3.0 3.7 3.0 2.7

TABLE 9: ZONE OF INHIBITION OF N. CROCIFORMIS CHLOROFORM AND ETHANOL EXTRACT AGAINST A. NIGER
Sample Concentration (μg) Zone of inhibition (cm)
chloroform 25 nil
50 1.0
100 1.3
ethanol 25 Nil
50 nil
100 1.4
Clotrimazole 50 2.0

FIG. 6: GRAPHICAL REPRESENTATION OF FIG. 7: GRAPHICAL REPRESENTATION OF

ANTIMICROBIAL ACTIVITY AGAINST DIFFERENT ANTIMICROBIAL ACTIVITY AGAINST DIFFERENT
BACTERIAL STRAINS IN CHLOROFORM EXTRACT BACTERIAL STRAINS IN ETHANOL EXTRACT
Series 1 concentration 25 μg, Series 2 50 μg, Series 3 100 μg, Series 1 concentration 25 μg, Series 2 50 μg, Series 3 100 μg,
Series 4 antibiotics Series 4 antibiotics

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The present study investigated the in-vitro In the case of antibacterial activity, the average IZ
antibacterial and antifungal activity of two different of the chloroform extract were highest against the
solvent extracts in Nervilia crociformis whole plant growth of Pseudomonas aeroginosa and least in
sample against four different strains of bacteria like staphylococcus aureus. The average IZ of the
E. coli, Pseudomonas aeroginosa, Staphylococcus ethanol extract were highest against the growth of
aureus, Streptococcus mutans and a fungal strain streptococcus mutans and least in Staphylococcus
Aspergillus niger. The potency of the extracts and aureus. The inhibition zone against different
their antibacterial sensitivity were assessed bacterial strains in ethanol and chloroform extracts
quantitatively by determining the IZs given in shows in the Table 8 Fig. 7 and 8. Antifungal
Table 8 and the antifungal activity were assessed activity of the two extracts was shown in Table 9.
and IZs given in Table 9.

S. mutans P. aeruginosa

E. coli A. niger S. aureus

FIG. 8: ANTIMICROBIAL ACTIVITY OF N. INFUNDIBULIFOLIA ETHANOL EXTRACT AGAINST DIFFERENT STRAINS

TABLE 10: ZONE OF INHIBITION OF N. INFUNDIBULIFOLIA ETHANOL EXTRACT AGAINST DIFFERENT

BACTERIAL STRAINS
Sample Concentration (μg) Zone of inhibition (cm)
E. coli P. aeroginosa S. aureus S. mutans A. niger
Ethanol 25 1.0 Nil Nil Nil Nil
50 1.2 Nil Nil 1.2 Nil
100 1.7 1.6 1.1 1.6 1.1
Antibiotics 50 3.0 3.7 3.0 2.7 2

The results as diameter of the inhibition zone in DISCUSSION: Many species of orchids having
ethanol extract have been given in the Table 10 helpful phytoconstituents are currently being used
and Fig. 9. The chloroform extract of the plant as drugs in the Indian system of medicine. Being
shows little activity against selected microbial members of a highly advanced family, orchids have
strains so the results would be discarded. IZ of N. a major role to play in the genetic engineering of
infundibulifolia whole plant ethanol extract is new forms that may be useful in floriculture,
maximum in the case of E. coli and minimum in S. pharmacology and other, as yet unexplored fields
aureus. of science. Classical texts highlighted that orchid
drugs like Jeevaka, Rishabhaka, Riddhi as
Streptomycin was used as control in the case of Rasayana 12 and Vajeekarana agents 13 and used
antibacterial activity and Clotrimazole in the case mainly in the treatment of Vata dominant diseases
of antifungal activity. (which involves nervous system and musculo-

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skeletal disorders mainly) 13. Certain conditions Out of five test strains, all the strains namely S.
like rheumatism, fractures, ear ache, nervous aureus (gram +ve, inhabit the respiratory tract is a
disorders, wounds, sexual problems, swellings, facultative pathogen) and P. aeruginosa, (gram -ve,
worm infestation, headache are found to be cause infection in blood and lungs), E. coli (gram -
managed by the traditional practitioners with ve, intestinal infection and fever), S. mutans (gram
simple formulations consisting of orchids. +ve, inhabiting in the mouth of humans) were
found to be inhibited by the 100 µg ethanol and
The results obtained from the present study provide chloroform extract of Nervilia crociformis and
certain informations with respect to the phyto- ethanol extract of Nervilia infundibulifolia. The
chemical constituents, antioxidant property, results obtained were found to be encouraging as
antibacterial activity of the two species of compared to that of Streptomycin. A. niger was the
medicinal orchid Nervilia namely Nervilia fungal strain studied and the results were
crociformis and Nervilia infundibulifolia. These satisfactory in the case of the extracts of the two
two species are closely related in their morphology studied plants.
and characteristics. In this study the authors try to
identify the differences in chemical properties of CONCLUSION: Hence, orchids were commonly
these two species because these two species were used as traditional medicine many of the medicinal
used interchangeably for a certain disease kidney orchids were yet to be explored for their medicinal
stone. properties. In the present study indicated that
Nervilia crociformis and Nervilia infundibulifolia
As evident from the phytochemical analysis Table two medicinal orchids possess a good phyto-
1 and 2, Out of the 12 phytochemicals studied chemical, antibacterial and antioxidant activities.
ethanol and water extracts of Nervilia crociformis Further, work is required to find out the active
contains a good number of phytochemicals principle from the plant extracts and to carry out
compared to Nervilia infundibulifolia. But pharmaceutical studies.
quantitatively carbohydrate and flavonoid content
was high in Nervilia infundibulifolia. Surprisingly ACKNOWLEDGEMENT: We are thankful to the
all the extracts of Nervilia infundibulifolia studied faculties and Head of the department of PG studies
show good result than that of Nervilia crociformis. and research centre in botany Sir Syed College,
In the case of total phenolic content the case was Taliparamba, Kannur, Kerala for providing
just reversed, Nervilia crociformis shows good necessary laboratory facilities, and also thankful to
results than Nervilia infundibulifolia in all extracts. the Kannur University for the financial assistance
to carry out this research.
The results of free radical scavenging activity of
the plants shows that Nervilia crociformis extracts We acknowledge the research scholars of the Sir
were good free radical scavenger compared to Syed College, Taliparamba for giving us support.
Nervilia infundibulifolia. The presence of free
radical scavenging property in high amount in the CONFLICT OF INTEREST: Nil
plant extract supported by its rich phytochemicals
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How to cite this article:

Ruthisha PK, Krishna CS and Khaleel KM: Phytochemical, antioxidant, antibacterial activities of Nervilia crociformis (zoll. & moritzi)
seidenf. and Nervilia infundibulifolia blatt. & mc cann (orchidaceae) collected from Kasargod district, Kerala, India. Int J Pharm Sci &
Res 2018; 9(12): 5177-85. doi: 10.13040/IJPSR.0975-8232.9(12).5177-85.
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MURALI Date: 2024.03.14

18:58:55 +05'30'

International Journal of Pharmaceutical Sciences and Research 5185