Journal of Pharmacognosy and Phytochemistry 2022; 11(2): 222-228

E-ISSN: 2278-4136
P-ISSN: 2349-8234
[Link] Isolation, characterization and radical scavenging
JPP 2022; 11(2): 222-228
Received: 14-01-2022 activities of rutin isolated from leaves of Annona
Accepted: 18-02-2022
squamosa
Himesh Soni
DHS Bhopal, Madhya Pradesh,
India Himesh Soni and Jitender Malik
Jitender Malik
Abstract
Institute of Pharmacy, P.K.
Flavonoids are universally in photosynthesizing cells and are commonly found in various parts of nuts,
University, Shivpuri, Madhya
Pradesh, India fruit, vegetables, seeds, stems and flowers. Flavonoids are principal active constituents have been used to
treatment of various human diseases. Rutin (quercetin-3- rhamnosyl glucoside) as the main flavonoid
reported in leaves of Annona squamosa. It is a multipurpose tree with eatable fruits & is a source one of
the cosmetic products. Annona squamosa Linn is used in the treatment of diabetes, hepatotoxicity and in
cancer cell line, It has been recognized that flavonoids display anticancer, antiviral, anti-inflammatory,
and heart disease protective activities. The present study was carried out to isolation, characterization and
antioxidant activity of rutin from leaves of Annona Squamosa from 80% ethanolic extract. The chemical
compound isolated was analyzed by TLC, HPLC & IR. Several studies had been done for the isolation of
rutin by different chromatographic method. In this study rutin was isolated from leaves of Annona
squamosa by precipitation and fractional solubilization without the use of any chromatographic
technique. The radical scavenging activities were carried out by various In-vitro and In-vivo methods.
Rutin showed the most potent DPPH scavenging activity (IC50 = 3.17 μg/ml) slightly higher than
ascorbic acid (IC50=4.92 μg/ml). The IC50 value of lipid peroxidation assay for isolated rutin and BHT
were 18.27 μg/ml and 46.79 μg/ml respectively.

Keywords: [Link], rutin, HPLC, IR, Butylhydroxytoluene (BHT)

Introduction
In plants, Flavonoids are widely distributed as a naturally occurring polyphenols. Being both
dietary and biologically active compounds, flavonoids have attracted much attention of
investigators as potent species capable of affecting various biological processes in living
organisms. They are able to modulate various enzymes present in biological system.
(Alexander et al., 2007) [1]. The family (Annonaceae), is a large family which comprising
about 130 genera over 2000 species; the most important genera having a largest number of
species are Annona, with 120 species, from genera (Yoganarasiman et al., 2000) [19], Annona
squamosa also known as custard apple which is cultivated throughout India, mainly dietary
fruit. Annona squamosa syn. Arabic (gishta); Bengali (ata);German (Rahm Annone,
Rahmapfel, Zimtapfel, Süßsack); Hindi (sitaphal, ata, sharifa); Lao (Sino-Tibetan) (khièb);
Malay (nona sri kaya,sri kaya,buah nona); Mandarin (fan-li-chi); Portuguese (atta,fructa do
conde); Sanskrit (sitaphal); Spanish (candongo, chirimoya, fructo do conde, anón, anona
blanca, pinha, saramuya, anona).The plant is traditionally used for the treatment of epilepsy,
dysentery, cardiac problem, worm infection, constipation, hemorrhage, antibacterial infection,
dysuria, fever, and ulcer. It also has anti fertility, anti tumor and abortifacient properties (Soni
et al., 2012) [15]. Annona saponins, tannins, carbohydrates, flavonoids, proteins, phenolic
compounds, phytosterols, amino acids. The various chemical constituents isolated from
Annona squamosa including aporphine, coryeline, anonaine, isocorydine, glaucine. Leaves
contains Stigmasterol, 4-(2-nitro-ethyl 1)-1-6-((6-o-β- Dxylopyranosy1- β -D-
glucopyranosyl)- oxy)benzene, Borneol, Camphene, Camphor, car-3- ene, Anonaine, β-
Sitosterol, Benzyltetrahydroisoquinoline, Carvone, β- Caryphyllene, Farnesol, Geraniol, 16-
Hetriacontanone, Hexacontanol, Higemamine, Isocorydine, Limonine, Linalool acetate,
Menthone, Methyl anthranilate, Methylsalicylate, Methylheptenone, p-(hydroxybenzyl)-6,7-
(2- hydroxy,4-hydro) isoquinoline, n-Octacosanol, a- Pinene, b-Pinene, Eugenol Rutin,
Thymol and n- Triacontanol. Alkaloids, proteins & amino acids are vanished in the leaf extract
(Patel et al., 2008) [12]. Among these constituents, Rutin most widely used natural products
Corresponding Author:
Himesh Soni
present in leaves of [Link]. Rutin is the rhamnoglucoside of the flavonoid quercetin, and
DHS Bhopal Madhya Pradesh, found in many plants and used for treatment of various diseases related to the vascular
India
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(Toker et al., 1998). It is quercetin-3-rutinoside or 3,3',4', 5,7- and nucleic acids, and eventually cell death (Asres et al.,
pentahydroxy flavones-3-rutinoside, and has a chemical 2006) [3]. Antioxidant compounds in food play an important
formula [Link] by acting as antioxidants exhibited role as a health protecting factor. The main characteristic of
several beneficial effects, such as anti-inflammatory, anti- an antioxidant is its ability to trap free radicals. Highly
allergic, antiviral as well as an anticancer activity. They have reactive free radicals and oxygen species are present in
also been suggested to play a protective role in liver diseases, biological systems from a wide variety of sources. These free
cataracts, and cardiovascular diseases (Parabathina et al., radicals may oxidize nucleic acids, proteins, lipids or DNA
2010) [11]. and can initiate degenerative disease. Antioxidant compounds
like phenolic acids, polyphenols and flavonoids scavenge free
Pharmacological action of Rutin radicals such as peroxide, hydro- peroxide or lipid peroxyl
Rutin showed the most potent intrinsic activity, and produced and thus inhibit the oxidative mechanisms that lead to
the strongest inotropic responses among the different degenerative diseases (Jacobs & Joanne, 2000) [4]. Rutin act as
flavonoids. It exhibits antiulcer activity. Rutin and venorutin an inhibitor of iron-ion-dependent lipid peroxidation system
showed regenerative and hepato-protective effects in because it forms chelation of iron ions. The chelating
experimental cirrhosis. Quercetin and rutin have been used as mechanism of rutin blockage was more potent than quarcetin
effective constituents of several pharmaceuticals used for (Igor et al., 2002) [5].
treatment of capillary fragility and phlebosclerosis.
Flavonoids and esters of phenolic acids were investigated for Material and Method
their antibacterial, antifungal and antiviral activities. Rutin Plant material collection
active against Bacillus anthracis, Para influenza virus & The leaves were collected from botanical garden
Influenza virus (Narayana & Krishna, 2001) [6]. [Link] (M.P.) and authenticated (Voucher No.
004/bot/LNCP/10 & 004/bot/LNCP/11). They were dried in
shade for several days at room temperature and then grinded
as [Link] standard rutin was obtained as a gift sample
from Jamia hamdard, Delhi.

Extraction and isolation

Twenty grams of the powdered leaf was extracted by Soxhlet
apparatus with 250 ml of 80% ethanol till exhaustion. The
extract was filtered and concentrated by evaporation under
vacuum to about 10ml then mixed with 25ml distilled water,
and extracted with petroleum ether (50ml x3), then with
chloroform (50 ml x 3). After extraction, the aqueous layer
was collected and left to stand in a cold place for 72 hours; a
yellow precipitate separated out of the solution. The
precipitate was filtered and washed with a mixture of
chloroform: ethyl acetate: ethanol ([Link]). The un-
dissolved part of the precipitate was dissolved in hot methanol
and filtered; the filtrate was evaporated to dryness to give 110
mg yellow powder (rutin).
Fig 1: Annona squamosa

General and Physical Properties

Appearance, color, solubility and melting point of the isolated
constituents will be determined.

Chemical identification of Constituents

Little amount of the isolated constituent are dissolve in
methanol and perform the following test (Kokate, 2003) [7].

Shinoda Test (Magnesium Hydrochloride reduction test)

To the methanolic solution of rutin add few drops of Con.
HCl and 0.5 gm magnesium turning.

Zinc Hydrochloride Reduction Test

To the methanolic solution of rutin add a mixture of Zinc dust
and conc. Hydrochloric acid. Heat the solution and observe
Fig 2: Structure of Rutin the color.
Free radicals are atoms or groups of atoms that have at least Alkaline Reagent Test
one unpaired electron, which make them highly unstable and To the methanolic solution of rutin add few drops of sodium
reactive. Living organisms accumulate free radicals through hydroxide solution and observe the colour formation.
both normal metabolic processes and exogenous sources.
Although radicals have beneficial effect during energy TLC and paper chromatography
production and as antibacterial, excessively high levels of free Isolated rutin was compared with standard rutin using TLC
radicals cause damage to cellular proteins, membrane lipids method; a pre-coated aluminum sheet with silica gel GF254
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with the following mobile phases: ethyl acetate: butanone: with 2.5 ml water and is shaken with 0.5 ml of freshly
formic acid: water ([Link]), ethyl acetate: formic acid: prepared 0.1% ferric chloride. The absorbance was measured
acetic acid: water (100: 11: 11:27). In paper chromatography, at 700 nm. All tests were performed in triplicate and the graph
Whatmann No.1 filter paper was used as a stationary phase was plotted with the average of the three determinations
and mobile phases of: Acetic acid: water (15:85) and (Poonia et al., 2011) [13].
isopropyl alcohol: water (60:40) (Lederer, 1957) [8].
Thin Layer chromatography analysis of antioxidant
Spectrophotometric analysis constituents
The isolated rutin was dissolved in methanol and its UV TLC analysis of antioxidant constituents the antioxidant
absorption peaks were determined and compared with constituents were analyzed using thin layer chromatography
standard rutin. Spectrophotometric analysis was carried out in (TLC) followed by DPPH (2, 2- Diphenyl-1-picrylhydrazyl)
Shimadzu 1700 UV spectrophotometer. technique (Moore J., Yin J. and Yu L 2006). About 100 μg of
sample was applied on TLC plate (Merck, 10x 10 cm2). The
HPLC analysis plate was developed in 10% chloroform in methanol and
The HPLC analysis was carried out using a LC-100, Cyberlab Methanol: Chloroform: Hexane ([Link]) . Then developed
TM, Salo Torrace, Millburry, MAO 1527, USA with LC-UV- plate was air dried and observed under visible and UV
100 UV detector. A CAPCELL (C-18) HPLC-packed column light (240 and 300 nm). The Rf values was calculated. The
(4.6 mm I.D.X 250 mm), type MG 5 μm, number intensity of spot was observed after treatment with 0.05% of
AKAD/05245 was used for the chromatographic separations. DPPH solution in methanol. The active antioxidant
The mobile phase contain methanol: water (70:30). The flow constituents were detected as yellowish white spots produced
rate was 0 .5 mL/min, and a column temperature of 25°C. The by bleaching of DPPH by resolved bands on the TLC
injection volume was 25μl, and UV detection was achieved plates. Ascorbic acid was used as positive control.
at 280 nm.
In-Vitro Lipid Peroxidation Method
Antioxidant activity Lower concentrations (10, 20, 30, 40, 50 μg/ml) of isolated
DPPH Radical Scavenging Activity rutin sample were prepared by serially diluting stock
Ascorbic acid and rutin were weighed (25 mg each) and solutions. Similarly BHT stock solution (500 μg/ml) was
dissolved in 250 ml of methanol to get 500μg/ml stock prepared (50 mg BHT in 500 ml methanol). Lower
solutions separately. Lower concentrations of ascorbic acid concentrations (25, 50, 100, 200, 400 μg/ml) of BHT were
and rutin (2, 4, 6, 8, 10 μg/ml and 2, 4, 6, 8, 10 μg/ml prepared by serially diluting stock dilution. Normal male rats
respectively) were prepared by serially diluting stock (250 g) were used for the preparation of liver homogenate.
solutions. The stable DPPH radical was used for The perfused liver was isolated, and 10% (w/v) homogenate
determination of free radical-scavenging activity of the was prepared with homogenizer at 0-4°C with 0.15M KCl.
extracts. The 0.1 mM solution of DPPH in methanol (22.2 mg The homogenate was centrifuged at 8,000 rpm for 15 min,
in 1000ml) was freshly prepared. Different concentrations of and clear cell-free supernatant was used for the study with in
rutin were added at an equal volume (2ml) to methanolic vitro lipid peroxidation assay. Different concentrations
solution of DPPH. After 30 min at room temperature, the sample were added in test tubes and 1ml of 0.15M KCl and
absorbance was recorded at 517 nm. IC50 values denote the 0.5ml of rat liver homogenates were added. Peroxidation was
concentration of sample, which is required to scavenge 50% initiated by adding 100 μl of 0.2 mM ferric chloride. After
of DPPH free radicals. Radical scavenging activity was incubation at 37 °C for 30 min, the reaction was stopped by
calculated by the following formula. adding 2 ml of ice-cold HCl (0.25 N) containing 15% TCA,
0.38% TBA, and 0.5% BHT. The reaction mixtures were
% Radical Scavenging Activity = Acontrol- Asample /Acontrol * 100 heated at 80 °C for 60 min. The samples were cooled and
Where, Acontrol = Absorbance of control Asample = Absorbance centrifuged, and the absorbance of the supernatants was
of sample measured at 532 nm. The percentage inhibition of lipid
peroxidation is calculated by the formula:
The inhibition curve was plotted for duplicate experiments
and represented as % of mean Inhibition of lipid peroxidation (%) =1– (sample OD/blank
OD) × 100
Inhibition ± standard deviation.
IC50 value was determined from the plotted graph of
Ferric Reducing Antioxidant Power (FRAP) Assay scavenging activity against the different concentrations of
3mg of isolated rutin was dissolved in 30 ml of methanol to sample, which is defined as the total antioxidant necessary to
get 100 µg/ml stock solutions separately. Lower inhibit lipid peroxidation by 50 % (Arora & Singh, 2009) [2].
concentrations of rutin (20, 40, 60, 80, 100µg/ml) were
prepared by serially diluting stock solution. Ascorbic acid was Results and Discussion
weighed (5 mg) and dissolved in 50 ml of methanol to get 500 Rutin is a flavonol glycoside comprised of Quercetin and
µg/ml stock solutions Lower concentrations of ascorbic acid rutinose. Rutin was isolated from leaves of Annona squamosa
(20, 40, 60, 80, 100 µg/ml) were prepared by serially diluting by precipitation and fractional solubilization without the use
stock solution. Various concentrations of sample and standard of any chromatographic technique. Isolated rutin showed a
solutions (1ml each), 2.5 ml of phosphate buffer (pH 6.6) and melting point at 196°C which is identical with that reported
2.5 ml of 1% potassium ferricyanide were mixed separately for rutin (Merck index, 2006) [17].
and allowed to incubate at 50 º C for 30 min and 2.5 ml of The general physical properties observed in isolated rutin
10% TCA was added to the mixtures and centrifuged for 10 were tabulated in table [Link] identification of rutin
min at 3000 rpm. About 2.5 ml of the supernatant was diluted showed presence of flavonol moiety (table 2).Qualitative
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analysis of isolated rutin were carried out by TLC and paper free radical scavenging (Roshan et al., 2010) [14]. The result of
chromatography and the results revealed that Rf value were Ferric reducing power assay was showed in fig. 7 & 8. The
more or less similar to standard rutin (table 3). The UV absorbance of the sample and standard were tabulated in table
spectrum of rutin in methanolic solution shows three major 6. The TLC analysis of antioxidant constituent revealed the
absorption bands at 236, 257 & 358.5nm, which indicates the intense yellow spot after treated with DPPH (table 8). In-Vitro
presence of flavonol structure (fig. 3) IR analysis, were Lipid Peroxidation Method, the decrease in the MDA levels in
tabulated in table 5. IR Spectrum comparison of Rutin the presence of increased concentration of sample (rutin)
(standard) with sample showed in fig.4. The construction of indicates their role as antioxidants. TBARS assay was used to
chromatographic fingerprints plays an important role in the determine the anti lipid peroxidation properties of the rutin
quality control of complex herbal medicines. Chemical isolated from leaves of [Link]. Thus, rutin inhibit the
fingerprints obtained by chromatographic techniques are initiation of lipid peroxidation by scavenging the free radicals
strongly recommended for the purpose of quality control of that form alkyl peroxyl and alkoxyl radicals or can donate
herbal medicines. HPLC separations of isolated sample with hydrogen atom to alkyl peroxyl and alkoxyl radicals and thus
reference to standard were performed on a Cyber Lab C-18 stop chain propagation (Mruthunjaya & Hukkeri, 2010 [10].
column (250 x 4.0 mm, 5μ). A chromatographic fingerprint These observations also support our finding that rutin have
plays an important role in the quality control of herbal more antioxidant potential in comparison to with standard
medicines. HPLC separations of isolated sample with antioxidants. The result was tabulated in table 9.
reference to standard were performed on a Cyber Lab C-18
column (250 x 4.0 mm, 5μ). Thus chromatographic Conclusion
fingerprint should be considered as a tool to evaluate the The results of this study, it is clearly indicate that isolated
quality of herbal medicines. (Soni et al., 2011) [16]. The HPLC rutin from leaves of Annona squamosa have high radical
chromatogram of standard and isolated rutin showed 2.55 & scavenging activity against various antioxidant systems in
2.375 respectively (fig 5 & 6). The HPLC analysis was vitro & in vivo. These assays have important appositeness for
tabulated in table 4. DPPH is stable nitrogen centered free the food and pharmaceutical industry. Moreover rutin can be
radical which can be effectively scavenged by antioxidants used as an easily attainable source of natural antioxidants and
and shows strong absorbance at 517 nm. The change in as a possible food supplement.
absorbance of DPPH radical caused by the sample was due to
the reaction between the antioxidant molecules and the
sample, which resulted in the scavenging of the radical by
hydrogen donation. It was visually noticeable as a
discoloration from purple to yellow. Extent of DPPH radical
scavenged was determined by the decrease in intensity of
violet colour in the form of IC50 values. The antioxidant
potential rutin was tabulated in table 6 & fig. 9. Ferric
Reducing Antioxidant Power Assay The reductive capability
of the isolated rutin was measured with reference to ascorbic
acid. For the measurement of the reductive ability, we
investigated the ferric (Fe) - ferrous (Fe2+) transformation in
the presence of the rutin was investigated. In this method, the
rutin form a colored complex with potassium ferricyanide,
trichloroacetic acid and ferric chloride that was measured at
700 nm. The reducing capacity of the rutin may serve as the Fig 2: Isolated from leaves of [Link]
antioxidant activity, the reducing power of the rutin increased
with increasing the concentration. Increase in absorbance of Table 1: General Physical Properties of isolated Rutin
the reaction mixture indicates the increase in the reducing
power of the sample. The antioxidant activity can be S. No. Physical properties Inference
1. Appearance Powder
attributed to various mechanisms, among which are the
2. Color Pale Yellow
prevention of chain initiation, the binding of transition metal 3. Solubility Pyridine, methanol
ion catalysts, decomposition of peroxides, the prevention of 4. Melting point 1960C
continued hydrogen abstraction, the reductive capacity and

Table 2: Chemical identification of Constituents

S. No. Test Observation Inference
1. Shinoda Test Green to blue color appears after few minutes Flavonol are present
2. Zinc Hydrochloride reduction test It gives red color after few minutes Flavonol are present
Formation of an intense yellow color, which turns to Colorless on addition
3. Alkaline reagent test Flavonol are present
of few drops of dil. Acid.

Table 3: TLC and Paper chromatography Analysis

TLC Paper Chromatography
Solvent System Rf(standard) Rf(isolated) Solvent System Rf(standard) Rf(isolated)
Ethyl acetate: butanone: formic acid: water
0.29 0.30 Acetic acid: water (15:85 0.57 0.61
(([Link])
Ethyl acetate: formic acid: acetic acid:water(100:
0.38 0.34 Isopropyl alcohol: water(60:40) 0.79 0.81
11: 11:27)
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Table 4: HPLC Analysis of Rutin

S. No Sample Height Area Conc. RT Inference
1. Standard Rutin 59566 1014993 97.4524 2.55 Rutin
2. Isolated Rutin 14631 152866 94.4032 2.375 Rutin

Table 5: IR Analysis of rutin

Table 6: 50% inhibition (IC50) for Rutin by DPPH method
IR Values Inference
3330 OH (bonded) S. No Sample IC50 (μg/ml)
2920 CH stretch 1. Ascorbic acid 3.17
1660 C=O 2. Rutin 4.92
1620 C=C
1600 Aromatic structure Table 7: Total reducing power of isolated rutin
1510 C=C aromatic
1360 C-O-C Concentration(µg/ml) Absorbance
1295 C-O-C Ascorbic acid(standard) Isolated rutin
1200 C-O-C 20 0.130 0.245
1060 C-O-C 40 0.221 0.268
810 Substituted aromatics 50 0.302 0.614
Other fingerprint bands characteristic to rutin are seen following 970, 80 0.745 0.702
880, 730 and 700 100 0.850 0.908

Table 8: Thin Layer chromatography analysis of antioxidant constituents

Rf value Colour intensity of spot
Solvent system
Isolated rutin Ascorbic acid Isolated rutin Ascorbic acid
Chloroform : methanol(10:90) 0.62 0.60 yellow pale yellow
Methanol: Chloroform: Hexane([Link]) 0.63 0.57 yellow yellowish white

Table 9: 50% inhibition (IC50) for Rutin by TBARS method

S. No Sample IC50 (μg/ml)
1. BHT 46.79
2. Rutin 18.27

Fig 3: UV Spectrum

.
Fig 4: IR Spectrum comparsion of Rutin (standard) with sample.

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Fig 5: HPLC Chromatogram of isolated rutin Fig 6: HPLC Chromatogram of standard rutin

Fig 7: Reducing Power of Ascorbic acid (standard) Fig 8: Reducing Power of isolated Rutin

Fig 9: Antioxidant activity of Isolated Rutin & Ascorbic acid by DPPH Method

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