Validation of a green extraction method based on Ultrasound Assisted Aqueous Extraction from Fresh Plants of Argemone mexicana

Abstract

Argemone mexicana is a well known medicinal plant abundantly grown in arable and non-arable land all over Bangladesh. The whole plant of A. mexicana was selected for the validation of a green extraction method conducted by ultrasound treatment. The proposed aqueous Ultrasound Assisted Extraction (UAE) from fresh plants of A. mexicana was compared with the conventional methanol and decoction extraction method. Higher extraction yield was observed in the UAE method with a maximum number of phytochemicals almost similar to the methanol extract. Both UAE and methanol extracts showed moderate antimicrobial sensitivity against Staphylococcus aureus and Salmonella typhi which was much higher than the extract obtained from the decoction method. Insignificant difference was observed in aqueous UAE crude extracts obtained both from fresh and dried plants give an option to avoid the time consuming drying stages of plant materials before extraction. Ultrasound mediated extraction successfully reduces the overall extraction time and cost as well as it allows aqueous solvent instead of hazardous organic solvent. Recent study indicates the suitability of the method both for laboratory and industrial setup.

Full text

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Validation of a green extraction method
based on Ultrasound Assisted Aqueous
Extraction from Fresh Plants of
Argemone mexicana
A F M Nazmus Sadat1,2*, Emad Hossain2, Gulam Kibria2, Shahidur Rahman2, Nazmus
Sakib2, Twyabatun Nahar Nila2, Roknuzzaman2 and Md. Rashedul Islam2
1 Institute of Environmental Science, Rajshahi University, Rajshahi 6205, Bangladesh.
2 Department of Pharmacy, University Of Development Alternative, Dhaka 1209, Bangladesh.
*Corresponding Email: afmnsadatbd@gmail.com
Abstract
Argemone mexicana is a well known medicinal plant abundantly grown in arable and non-
arable land all over Bangladesh. The whole plant of A. mexicana was selected for the validation
of a green extraction method conducted by ultrasound treatment. The proposed aqueous
Ultrasound Assisted Extraction (UAE) from fresh plants of A. mexicana was compared with
the conventional methanol and decoction extraction method. Higher extraction yield was
observed in the UAE method with a maximum number of phytochemicals almost similar to the
methanol extract. Both UAE and methanol extracts showed moderate antimicrobial sensitivity
against Staphylococcus aureus and Salmonella typhi which was much higher than the extract
obtained from the decoction method. Insignificant difference was observed in aqueous UAE
crude extracts obtained both from fresh and dried plants give an option to avoid the time
consuming drying stages of plant materials before extraction. Ultrasound mediated extraction
successfully reduces the overall extraction time and cost as well as it allows aqueous solvent
instead of hazardous organic solvent. Recent study indicates the suitability of the method both
for laboratory and industrial setup.
Key words: Argemone mexicana, Prickly poppy, Green extraction, Ultrasound, Staphylococcus
aureus, Salmonella typhi
.
Introduction:
Argemone mexicana L. is locally known as “Shiail Kata” and a very common annual herbaceous weed
extremely grow in the cultivated and abandoned land in all parts of Bangladesh[1]. The plant is belongs
to the family Papaveraceae[2] and commonly known as prickly poppy[3]. It contains 30-32 species, all
with prickly stems, leaves and capsules[4] and widely distributed in many tropical and subtropical
regions and has naturalized in the United States, Ethiopia, India and Bangladesh[3,5]. A. mexicana is a
prickly, glabrous, branching herb with eye-catching yellow flowers. It is a popular medicinal plant and
widely used in folk medicine to alleviate several ailments especially for its analgesic, antibacterial,
antimalarial, antispasmodic, sedative and narcotic effects[6]. Several studies specifically identified its
antioxidant [7], antihelmentic [8], anti-inflammatory[8], anti-bacterial[8], anti diabetic[9] activities.
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The plant is also useful for the treatment of warts, cold sores, cutaneous infections, skin diseases, itches,
dropsy, jaundice[10] and even sometime as an antidote to snake poisoning[11,12].
In the present study, the whole plant of A. mexicana was used to prepare crude extract by using
an optimized green extraction method known as “Aqueous Ultrasound Assisted Extraction
(UAE) from Fresh Plants” as per the process proposed by Sadat et al.[13]. The basic principle
of the UAE method is to utilize ultrasonic waves on the vegetal material that breaks the cells
and releases the cells’ contents into the extraction medium[14]. This method is capable of
reduceing the use of extraction solvents, processing time, and therefore, energy consumption
enhancing during the extraction of the desired biocomponents [15-18].
Botanical nomenclature [19]
Kingdom: Plantae
Division: Magnoliophyta
Class: Magnoliopsida Dicotyledons
Subclass: Magnoliidae
Order: Papaverales
Family: Papavaraceae
Genus: Argemone
Species: A. mexicana
Figure 1: Washing stage of freshly collected Argemone mexicana plants
Materials and Methods
Study protocol
Ultrasound was applied on the fresh plants of Argemone mexicana for facilatating the
extraction of phytochemicals in water and subsequently compared to the conventional
decoction and methanol cold extraction method (Table-1). Both fresh and shade dried plant
materials were used in the present study[13, 20-22]. Extraction yield (Eq. 1), presence of
common phytochemicals (Table-2) and antimicrobial sensitivity were the parameters set for
the comparison.
Collection of Plant Material
The whole plants of Argemone mexicana was collected from Botanical Pesticide Garden of the
Institute of Environmental Science (IES) of Rajshahi University (RU), Bangladesh and duly
identified by the professional taxonomist of the Department of Botany, RU and a voucher
specimen was deposited at the herbarium of the institute.
Extraction Procedure
Healthy plants of Argemone mexicana were collected from the field before sun rise and
immediately washed by the running tap water and distilled water. After shade drying of the
surface water, the plants were devided into five parts (Table 1). As per Toma et al.,[23] the
optimum material-solvent ratio is 1:5 for effective ultrasound extraction which is also
frequently followed in many conventional extraction methods[24-26]. Extraction from Part A
& B of the fresh plants were conducted immediately by using UAE and decoction method.
Whereas “Part-C, D and E” of fresh plants were first allowed for week-long drying and powder
form were used for UAE, decoction and methanol cold extraction. Dissolved phytochemicals
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were seperated from the debris by using five layers of polyester cloths and dried at 60oC in a
conventional water bath. The dried crude extracts were then stored in an air tight bottle with
identical labels and preserved in a cold chamber for further use. The efficiency of the extraction
process was mesured by comparing yield (Equation 1)[27] and the presence of common
phytochemicals (Table 2). The efficacy of the crude extracts were compaed by using
antimicrobial sensitivity study. All process were repeated three times for calculating
significance level. SPSS 16.0 was used for all types of statistical calculation.
Table 1: Extraction procedure of Argemone mexicana whole plants
Method
Plant parts
Treatment
Method
Ref.
A
Fresh Plants*
Ultrasound
Juice were placed in the ultrasonic bath (Power Sonic
405) for 30 minutes ultrasonic treatments at 40oC bath
temperature.
[13,
20-21]
B
Fresh Plants*
Decoction
Juice were allowed to boil for 5 minutes before extraction
[28-29]
C
Dried Plants**
Ultrasound
Fine powder was mixed with distiled water (1:5 ratio) and
treated in ultrasonic bath for 30 minutes at 40oC bath
temperature
[13,
20-21]
D
Dried Plants **
Decoction
Fine powder was mixed with distil water (1:5 ratio) and
allowed to boil for 5 minutes before extraction
[28-29]
E
Dried Plants **
Methanol
cold
extraction
Fine powder was mixed with methanol (1:5 ratio) and
allowed for cold extraction up to 72 hours with
intermittent shaking as per standard method
[24-26]
* Juice of fresh plants (100 gm) was prepared by conventional blender by adding distilled water q.s. to 500 ml.
** Powder was prepared and mixed with respective solvents as per ratio 1:5.
Table 2: Phytochemical Screening Test of Argemone mexicana
Phytochemicals
Qualitative test
Pharmacological importance
Alkaloids
Mother solution + 2% of H2SO4 + Heat
+ few drops Dragendoff's reagent →
Orange red precipitate (This is known
as Dragendoff's test)[30-31]
Anaesthetics, CNS stimulants,
narcotics, poisons due to their potent
biological activities[4, 32]
Mother solution + 2% of HCl + Heat +
few drops Mayer’s reagent → turbidity
or yellow precipitation (This is known
as Mayer’s test)[30, 33]
Anthraquinones
Mother solution + benzene or
chloroform + 10% (v/v) ammonia
solution → pinkish or color change[31,
34]
Anthraquinones provide anticancer,
anti-inflammatory, diuretic,
antiarthritic, antifungal, antibacterial,
and antimalarial activities[35].
Flavonoids
Mother solution + dilute ammonia
Solution + Conc. H2SO4 → yellow
coloration that disappear on
standing[34]
Its antioxidant property provides
protection against diseases like
cancer, ageing, atherosclerosis,
inflammation[5, 32]
Mother solution + few drops of 1%
aluminium solution → yellow
coloration[34]
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Glycosides
Mother solution + 3 ml of glacial acetic
acid + 1 drop of 5% ferric chloride
Solution + 0.5 ml of Conc. H2SO4
→Brown or blue ring of the
interface[33-34]
Glycosides play numerous important
roles in living organisms, including
antioxidant, antiinflammatory,
antihypertensive, and antidiabetic
activities[36]
Saponins
Mother solution + equal volume water
+ vigorous shaken→ foam stable more
than 10 minutes[33]
Saponins provide hypolipidemic and
anticancer activity and are also used
in hypercholesterolemia,
hyperglycemia, antioxidant,
anticancer, anti-inflammatory and
weight loss etc.[5, 37-38]
Steroids /
Terpenoids
Mother solution + 2ml Chloroform +
Carefully added 3 ml Conc. H2SO4 to
form a layer → reddish brown colour of
the interface (This is known as
Salkowski test)[34]
steroids possess cardiotonic activity,
also insecticidal and antimicrobial
properties[5, 32]
Tannins
Mother solution + 1% FeCl3 solution →
dark green colour[39-40]
These are used for the treatment of
diseases like leucorrhoea,
rhinorrhoea and diarrhoea[5, 38]
Antimicrobial Study
Disc diffusion method[41-44] was used for antimicrobial sensitivity study on Staphylococcus
aureus (Gram +ve bacteria) and Salmonella typhi (Gram -ve bacteria). Microorganisms were
collected from the Microbiology Lab, Department of Biochemistry and Molecular Biology,
Rajshahi University, Bangladesh. The filter paper discs impregnated with the 400µg/disc of
extracts were placed on the surface of the inoculated nutrient agar media with the aid of
sterilized pair of forceps. After allowing 30 minutes of pre-diffusion, the petridish was then
placed in an incubator for 24 hours at 370C. The degree of sensitivity of the organisms to the
extracts was determined by measuring the diameter of visible zones of inhibition to the nearest
millimetre. The procedure was repeated thred times for each batch and the average result was
counted for statistical analysis.
Results and Discussion
High extraction yields were observed in both cases of fresh (27.83±1.09% ) and dried (24.54±
1.19%) plants of A. mexicana treated by ultrasounds. Insignificant difference (p>0.05)
observed in the above two methods giving space to avoid the time consuming drying stages
from the extraction method. Conventional extraction methods like decoction from fresh plants
(yield, 8.7± 0.47%), decoction from dried plants (yield, 9.81± 0.51%) and methanol cold
extraction from dried plants (yield, 15.41± 0.44%) were observed significantly (p<0.05) lower
than the ultrasound method of extraction. On the basis of above results the ultrasound assisted
extraction was proved better than the conventional decoction and cold extraction method.
It was observed that Argemone mexicana extract was rich in different phytochemicals (Table
4). Qualitative phytochemical analysis indicated the presence of alkaloid, anthraquinone,
flavonoids, glycoside, saponin, steroid/ terpenoid and tannin in the crude extracts obtained from
fresh and dried plants by ultrasound treatment and methanol extracts. Similarly extracts
obtained from decoction were rich of anthraquinones, flavonoid, saponin and steroid truly
matched with the work of Veni and Puspanathan [45].
Ultrasound treated extracts from fresh and dried plants and methanol extracts of Argemone
mexicana were found promising antimicrobial activities on Staphylococcus aureus (Gram +ve)
and Salmonella typhi (Gram –ve) presented in the Chart-1. Statistically insignificant
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antimicrobial differences were observed on the microorganisms indicating the same efficacy
of the applied extracts obtained both from the ultrasound treated and methanol extracts (Table-
5). Extracts obtained by decoction method from fresh and dried plants showed comparatively
poor sensitivity.
Chart 1: Antimicrobial sensitivity study (Mean±S.D.) of the crude extracts of A.mexicana plants.
(Here, * method of extraction as per Table-1)
Table 3: Yield variation of different extaction methods
Method of
Extraction
Starting
materials
(gm)
Weight (gm)
after drying
(Mean±SEM)
p
(Weight
variation)
Solvent used
(ml)
% Yield
p
(Yield
variation)
A*
100
-
q.s. to 500
27.83±1.09
-
B*
100
-
q.s. to 500
8.7±0.47
0.001x
C*
100
41.90±1.36
5 times to the
dry wt
24.54±1.19
0.266x
D*
100
40.57±1.52
0.211a
5 times to the
dry wt
9.81±0.51
0.008x
0.005y
E*
100
40.60±1.10
0.615a
5 times to the
dry wt
15.41±0.44
0.005x
0.030y
* Method of extraction as per Table-1. Mean calculated by considering successive 3 studyis.
a Significance level comparison with “C”, where, p ≥ 0.05, is statistically insignificant.
x Significance level comparison with “A”, where, p ≥ 0.05, is statistically insignificant.
y Significance level comparison with “C”, where, p ≥ 0.05, is statistically insignificant.
Table 4: Phytochemical screening study of crude extracts of A. mexicana
Phytochemical Tests
Crude extract
A*
B*
C*
D*
E*
1.Alkaloid,(i) Dragendroffs’ test
+
+
+
+
+
(ii) Mayer’s test
+
+
+
+
+
2. Anthraquinones
+
-
+
-
+
3. Flavonoid (i): by H2SO4
+
-
+
-
+
(ii): by aluminum
+
-
+
-
+
4. Glycoside
+
+
+
+
+
5. Saponin
+
-
+
-
+
6. Steroid/ Terpenoid
+
-
+
-
+
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* Method of extraction as per Table-1.
Here, (+) indicated presence of compound, and (–) indicated absence of compound
Table 5: Antimicrobial sensitvity study of crude extracts of A. mexicana
Method*
of
extraction
Zone of inhibition (mm)
Staphylococcus aureus
Salmonella typhi
Mean ±SEM
p
Mean ±SEM
p
A
13.78±0.46
-
13.89±0.66
-
B
8.22±0.28
0.000a
8.56±0.24
0.000a
C
13.56±0.69
0.782a
14.11±0.66
0.801a
D
8.11±0.26
0.000a; 0.000b
8.33±0.50
0.000a; 0.000b
E
14.22±0.77
0.609a; 0.299b
14.78±2.49
0.442a; 0.563b
* Method of extraction as per Table-1. Mean calculated by considering successive 3 studyis.
a Variation compared to method “A”, p≤0.05indicate significant variation
b Variation compared to method “C”, p≤0.05indicate significant variation
Conclusion:
Aqueous ultrasound assisted extraction from fresh plants of Argemone mexicana have many
points of green extraction without compromising the efficiency and efficacy obtained from
conventional extraction methods. The proposed method was proved cost effective and
environment friendly which successfully reduced the overall processing time and hazardous
organic solvents for extraction procedure. Results obtained from phytochemical and
antimicrobial studies indicate the potential medicinal values of the studied plants. Statistical
similarity with conventional methanol extraction proved the justification of using the
ultrasound on fresh plant’s material.
References:
1
Apu AS, Al-Baizyd AH, Ara F, Bhuyan SH, Matin M and Hossain MF. (2012). Phytochemical
analysis and bioactivities of Argemone mexicana Linn. Leaves. Pharmacologyonline. 3:16 - 23
2
Bais Y, Chaudhari SB, Belani S and Umarkar AR. (2013). Valuation of Antimicrobial Activity of
Plant Leaf Argemone Mexicana. International Journal of Pharmacy and Biological Sciences, 3(1):
41-45
3
Nayak P, Kar DM and Maharana L. (2011). Antidiabetic Activity of Aerial Parts of Argemone
mexicana Linn. in Alloxan Induced Hyperglycaemic Rats. Pharmacologyonline 1: 889-903 (2011)
4
Desai SS, Kolachal SS, Badiger PM, Toragall VB, Magdum BK and Hungund BS. Phytochemical
analysis and antimicrobial activity of extracts from medicinal plant Argemone mexicana Linn.
Conference: Conference on Exploring Basic & Applied Sciences for Next Generation Frontiers
(EBAS-2014) organized by Lovely Professional University (ISBN: 978-93-5107-313-0, Elsevier)
At: Lovely Professiona University
5
Rahman MM, Alam MJ, Sharmin MA, Rahman MM, Rahman A and Alam MF. [2009]. In Vitro
Antibacterial Activity of Argemone mexicana L. (Papaveraceae). CMU. J. Nat. Sci. 8(1): 77-84.
6
Singh SK, Pandey VD, Singh A, Singh C. Antibacterial activity of seed extracts of Argemone
mexicana L. on some pathogenic bacterial stra
7
S. K. Wong, Y.Y. Lim, E.W. Chan, Antioxidant properties of Hibiscus species variation, altitudinal
change costal influence and floral colour change. J. Tro. For. Sci. (2009) 307-315
8
R.Nair, T. Kalariya, S. Chanda. Antibacterial activity of some selected Indian medicinal flora.
Turk. J. Biol, 29 (2005) 41- 47.
7. Tannin
+
+
+
+
-
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ISSN 2229-5518
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9
N. Singh, M. Gupta, Effect of ethanolic extract of Syzygium cumuni seed powder on pancreatic
islets of alloxen diebetic rats. Indian J. Exp. Biol. (2007) 861-867.
10
Singh SK, Pandey VD, Singh A and Singh C. [2009]. Antibacterial activity of seed extracts of
Argemone mexicana L. on some pathogenic bacterial strains. African Journal of Biotechnology.
8(24): 7077-7081
11
Chopra RN, Nayar SL and Chopra IC (1986). Glossary of Indian Medicinal plants (Including the
Supplement), Council of Scientific and Industrial Research, New Delhi.
12
Bhattacharjee I, Chatterjee SK, Chatterjee S, Chandra G (2006). Antibacterial potential of
Argemone mexicana solvent extracts against some pathogenic bacteria. Mem Inst Oswaldo Cruze
Rio de Janeiro 6: 645- 648.
13
Sadat AFMN, Mizan BB, Sutana A, Rahman MM and Azad MAK (2018). Comparative study of
the Antimicrobial Activity of Methanol Extract and Ultrasound Assisted Water Extract of the
Leaves of Azadirachta indica. Rajshahi University Journal of Environmental Science, 7: 40-47
14
Toma M, Vinatoru M and Mason TJ. 2001. Investigation of the effects of ultrasound on vegetal
tissues during sovent extraction. Ultrasonics Sonochemistry, 8: 137-142.
15
Chemat, F., Huma, Z., & Khan, M. (2011). Application of ultrasound in food technology:
processing, preservation and extraction. Ultrasonics Sonochemistry, 18(4), 813-835.
http://dx.doi.org/10.1016/j. ultsonch.2010.11.023.
16
Li, Y., Fabiano-Tixier, A. N., Tomao, V., Cravotto, G., & Chemat, F. (2013). Green ultrasound-
assisted extraction of carotenoids based on the bio-refinery concept using sunflower oil as an
alternative solvent. Ultrasonics Sonochemistry, 20(1), 12-18. http://dx.doi.
org/10.1016/j.ultsonch.2012.07.005.
17
Chemat, F., Rombaut, N., Sicaire, A. G., Meullemiestre, A., Fabiano-Tixier, A. N., & Abert-Vian,
M. (2017). Ultrasound assisted extraction of food and natural products. Mechanisms, techniques,
combinations, protocols and applications: a review. Ultrasonics Sonochemistry, 34, 540-560.
http://dx.doi.org/10.1016/j.ultsonch.2016.06.035.
18
Jacotet-Navarro, M., Rombaut, N., Deslis, S., Fabiano-Tixier, A. N., Pierre, F. X., Bily, A., &
Chemat, F. (2016). Towards a “dry” biorefinery without solvents or added water using microwaves
and ultrasound for total valorization of fruit and vegetable by-products. Green Chemistry, 18(10),
3106-3115. http://dx.doi.org/10.1039/ C5GC02542G.
19
Sharanappa R and Vidyasagar GM. 2014. Plant Profile, Phytochemistry And Pharmacology of
Argemone Mexicana Linn. A Review. International Journal of Pharmacy and Pharmaceutical
Sciences, 6 (7): 45-53.
20
Sadat AFMN, Hasan MM, Islam MS, Sharma D, Islam MR, Sultana A and Azad MAK (2021).
Validation of Aqueous Ultrasound Assisted Extraction Method by Using Fresh Leaves of Moringa
oleifera with Conventional Extraction Method. IJRDO - Journal of Applied Science. 7 (10): 27-
33.
21
Sadat AFMN, Ali M, Sultana A, Hasan MM, Sharma D, Rahman MA and Azad MAK (2021).
Comparative Study of a Proposed Green Extraction Method Named Aqueous Ultrasound Assisted
Extraction from Fresh Leaves of Acacia nilotica with Conventional Extraction Method.
International Journal of Innovative Science & Research Technology, 6(10): 946-951. ISSN-2456-
2165
22
Cheenickal M and Mendez RM. (2017). Phytochemical Screening and the Antimicrobial Activity
of the Leaves of Azadirachta Indica, A. Juss. International Journal of Scientific & Engineering
Research, 8(5): 221-224
23
Toma M, Vinatoru M, Paniwnyk L and Mason TJ (2001). Investigation of the effects of ultrasound
on vegetal tissues during solvent extraction. Ultrason Sonochem, 8: 137-142.
24
Hussain F and Hussain MM (2012). Cytotoxic Effect of Crude Extracts of Acacia Nilotica.
International Journal of Pharmaceutical Sciences and Research, 3(6): 1652-1655.
25
Bashir H S, Mohammed AM, Magsoud AS, Shaoub AM (2014). Isolation and Identification of
Two Flavonoids from Moringa oleifera (Leguminosae) Leaves. Journal of Forest Products &
Industries, 3(5), 211-215.
26
Latha CR, Kavitha S and Sathya S (2015). Antimicrobial Efficacy of Azadirachta indica Leaf
Extracts. International Journal of Science and Nature, 6(3): 432-440
IJSER

International Journal of Scientific & Engineering Research Volume 13, Issue 3, March-2022 858
ISSN 2229-5518
IJSER © 2022
http://www.ijser.org
27
Terblanche U, Semakalu CC, Mtunzi F and Pillay M. (2017). Screening of Variables Influencing
Extraction Yield of Cotyledon orbiculata: 23 Full Factorial Design. International Journal of
Pharmacognosy and Phytochemical Research, 9(3): 303-312
28
Madjos GG and Luceño AM (2019). Comparative Cytotoxic Properties of Two Varieties of Carica
papaya leaf extracts from Mindanao, Philippines using Brine Shrimp Lethality Assay. Bull. Env.
Pharmacol. Life Sci., 8 (2): 113-118
29
A F M Nazmus Sadat, Shamima Ahsan, Md. Sohrab Hosen, Nishat Rayhana, Debobrata Sharma,
Alamgir Sahriar, Md. Rashedul Islam, and Afroza Sultana. (2021). Validation of an Optimized
Ultrasound Assisted Green Extraction Method by using Fresh Leaves of Carica papaya.
International Journal of Innovative Science and Research Technology. 6(10): 1022-1029
30
Trease GE and Evans WC (1989). Pharmacology. 11th Edition. BailliereTindall Ltd., London,
pp:60 – 75. 16. Maxson ED and Rooney LW (1972).Evaluation of methods for Tannin analysis in
Sorghum Grain.American association of cereal Cherrism, inc., Minnesota 56121. 11pages.
31
Ajayi AO and Fadeyi TE (2015). Antimicrobial Activities and Phytochemical Analysis of Moringa
oleifera Leaves on Staphylococus aureus and Streptococcus species. American Journal of
Phytomedicine and Clinical Therapeutics, 3(10): 643-653.
32
[19] A. Kar, Pharmaocgnosy and Pharmacobiotechnology (Revised-Expanded Second Edition).
New Age International Publishres, New Delhi, 2007, pp 332-600.
33
Dash SP, Dixit S and Sahoo S (2017). Phytochemical and Biochemical Characterizations from
Leaf Extracts from Azadirachta Indica: An Important Medicinal Plant. Biochem Anal Biochem, 6:
323.
34
Ayoola GA, Coker HAB, Adesegun SA, Adepoju-Bello AA, Obaweya K, Ezennia EC and
Atangbayila TO (2008). Phytochemical Screening and Antioxidant Activities of Some Selected
Medicinal Plants Used for Malaria Therapy in Southwestern Nigeria. Tropical Journal of
Pharmaceutical Research, September,; 7 (3): 1019-1024.
35
Diaz-Munoz G, Miranda IL, Sartori SK, Rezende DC and Diaz MAN. 2018. Anthraquinones: An
Overview, Chapter in Studies in Natural Products Chemistry. DOI: 10.1016/B978-0-444-64056-
7.00011-8
36
Kren V and Martinkova L. 2001. Glycosides in Medicine: “The Role of Glycosidic Residue in
Biological Activity”. Current Medicinal Chemistry 8(11):1303-28.
DOI: 10.2174/0929867013372193
37
[16] P.D. McDonald , M. Antolovich and K. Robards,. Phenolic content and antioxidant activity
of olive extracts. Food Chem. (2001) 73-84.
38
[18] Y. Huang, J. Wang, G. Li, Z. Zheng and W. Su, Anti-tumor and Anti fungal activities in
endophytic fungi isolated from pharmaceutical plants, FEMS Immunology Med. Microbio. 31
(2001) 163-167.
39
Maxson ED and Rooney LW (1972). Evaluation of methods for Tannin analysis in Sorghum
Grain.American association of cereal Cherrism, inc., Minnesota 56121. 11pages.
40
Hazali NB, Ali MABM, Ibrahim MB and Masri M (2015). Determination of phytochemicals and
vitamin content of underutilized Baccaurea angulata fruit. Journal of Pharmacognosy and
Phytochemistry, 4(4): 192-196
41
Baker J, Liu JP, Robertson EJ and Efstratiadis A (1993). Role of Insulin-like Growth Factors in
Embryonic and Postnatal Growth. Cell, 75: 73-82.
42
Mukhtar MD and Tukur A. (2000). Antibacterial Activity of Aqueous and Ethanolic Extracts of P.
stratiotes. Journal of the Nigerian Society for Experimental Biology, 1: 51-59
43
Servan ES, Ionescu M, Matinca D, Maier CS and Bojita MT (2011). Screening of the Antibacterial
and Antifungal Activity of Eight Volatile Essential oils. Farmacia, 59(3): 440-446.
44
Hossain MA, Hitam S and Ahmed SHI (2019). Pharmacological and toxicological activities of the
extracts of papaya leaves used traditionally for the treatment of diarrhea. Journal of King Saud
University-Science, 32 (2020): 962-969.
45
Veni T and Pushpanathan T. 2014. Investigations of Antimicrobial And Phytochemical Analysis
of Argemone Mexicana Medicinal Plant Extracts Against Bacteria With Gastrointestinal
Relevance. Asian Journal of Pharmaceutical and Clinical Research, 7(2): 93-97
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