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Research J. Pharm. and Tech.

12(4): April 2019

ISSN 0974-3618 (Print) www.rjptonline.org


0974-360X (Online)

RESEARCH ARTICLE

Evaluation of In vitro Anti-Diabetic and Anti-Oxidant activities and


Preliminary Phytochemical screening of Gel, Epidermis and Flower extract
of Aloe vera
Spoorthy N. Babu, S. Govindarajan, M. A. Vijayalakshmi, Ayesha Noor*
Centre for Bio Separation Technology (CBST), Vellore Institute of Technology (VIT), Vellore, Tamil Nadu,
India-632 014
*Corresponding Author E-mail: spoorthy.n.babu@gmail.com, govind27382@yahoo.co.in,
ind_viji@yahoo.com, ayeshanoor17@yahoo.co.in

ABSTRACT:
Aloe vera (Aloe barbadensis Miller) is a perennial succulent plant belonging to the Aloeaceae family and has
been used for many centuries for its medicinal properties. Among the Aloe vera plant parts, gel is being widely
studied for their biological properties. However, epidermis and flowers are neglected and are not probed for their
importance. In this study, the methanolic extracts of Aloe vera gel, leaf and flower were evaluated for their anti-
oxidant and anti-diabetic potential in vitro and other phytochemical constituents. We observed that the Aloe vera
gel, leaf and flowers have shown significant anti-oxidant potential. When compared between leaf, gel and flower
of Aloe vera, the leaf showed better antioxidant activity in ABTS, DPPH, H2O2 radical scavenging activities,
Metal chelating activity and reducing power. However, the gel extract showed better anti-diabetic efficacy
compared to the flower extract with respect to α-amylase, α-glucosidase, DPP-IV inhibition and anti-glycation
activities. This study suggests that different parts of Aloe vera exhibit great potential for antioxidant and anti-
diabetic activity and may be useful in new health-care food supplements or nutraceutical.

KEYWORDS: Aloe vera, Gel, Epidermis, Flower, Phenolic compounds, antioxidant, antidiabetic.

1. INTRODUCTION: Medicinal plants have been reported to have anti-oxidant


Diabetes mellitus is a metabolic disorder that may lead activities for overcoming the oxidative stress and cell
to overproduction of reactive oxygen species (ROS) in damage caused due to diabetes mellitus3-4. The usage of
endothelial cells which contributes the pathogenesis of herbal and natural drug products for the treatment of
various complications1. As the ROS level increases, diabetes is growing worldwide, with the ancient Indian
there is disturbance in the anti-oxidant enzyme levels in literature reports suggesting more than 800 plants having
the body, leading to oxidative stress. In diabetic anti-diabetic activities5-6. One such medicinal plant is
condition, advanced glycation products are formed due Aloe barbadensis Miller, commonly known as Aloe vera
to the reaction of glucose with plasma proteins, (Family: Aloeaceae). It has been used for centuries for
triggering further ROS production. Medicinal plants its health, beauty, medicinal and skin care properties7.
have wide range in phytochemicals such as flavonoids, They are perennial succulents or xerophytes which are
polyphenols sterols, tannins and terpenoids etc. Provide around 60 to 100 cm tall. Aloevera has green fleshy
protection against oxidative stress. These leaves covered by a thick cuticle or rind and an inner
phytoconstituents scavenge the ROS by inhibiting the clear pulp (gel). Its flowers appear in the months of
initiation and chain propagation2. October to December. Each flower looks pendulous,
having a 2–3 cm long yellow tubular corolla2.
Aloe vera has been reported to have significant
therapeutic effects such as anti-diabetic, anti-oxidant,
Received on 25.10.2018 Modified on 27.11.2018
Accepted on 31.12.2018 © RJPT All right reserved anti-cancer, anti-inflammatory, anti-bacterial, anti-fungal
Research J. Pharm. and Tech. 2019; 12(4):1761-1768. properties8-9 and these have been attributed to synergistic
DOI: 10.5958/0974-360X.2019.00295.6 effects of numerous bioactive compounds in Aloe vera.
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The phytochemical analysis of Aloe vera has shown the standard reference13 with absorbance measured at
presence of tannin, saponins, flavonoids, steroids, 415nm. The results were expressed as quercetin
terpenoids, and cardiac glycosides anthroquinones10-11. equivalents ((mg QE/g of dry extract). The identification
Many researchers have reported about different of individual phenolic compounds was carried out by
pharmacological properties either with entire plant or the using Reverse Phase High-performance Liquid
gel of Aloe vera. There are few reports available with the Chromatography (RP-HPLC)14.RP-HPLC C18 column
epidermis and flower2. Little information is available (Waters, 150 X 3.9mm, i.d., 5µm) was used. Samples
about the comparative evaluation between the different (20µl) were injected and analyzed at a flow rate of 1 ml/
constituents of gel, epidermis and flower of Aloe vera min. The eluent was monitored at 255nm and 290nm.
related to antidiabetic activity and antioxidant properties. Identification of phenolic compounds was done by
So, in the present study comparative evaluation of anti- comparing retention time and area of peaks in the
diabetic and anti-oxidant potential of gel, epidermis and extracts against with that of the standard compounds
flower of Aloe vera was assessed. from Sigma-Aldrich. Data acquisition and processing
were performed using Breeze data (Waters) system. The
MATERIAL AND METHODS: quantity of each compound was calculated according to
Chemicals: the formula15:
Ascorbic acid, Gallic acid, quercetin, aluminium
chloride, ferric chloride (FeCl3); Folin Ciocalteu reagent; Quantity percent = (As * Wstd)/ (Ast *Ws) *purity of standard
bovine serum albumin (BSA); potassium persulphate; Where As = sample peak area, Ast = Standard peak area,
2,2-diphenyl-1-picrylhy-drazyl (DPPH), trichloroacetic Ws = amount of sample Wstd = amount of standard
acid (TCA)
Total antioxidant capacity:
2,2′-Azino-bis (3-ethylbenzthiazoline-6-sulfonic acid Total antioxidant capacity of the extracts was done using
(ABTS), Hydrogen peroxide (H2O2) were purchased ascorbic acid as a standard reference compound16. An
from Sigma chemicals (St. Louis, MO, USA). All other aliquot of 1 mg/ ml methanolic extract of gel, epidermis
chemicals were procured from Sisco Research and flower were mixed with the reagent solution (0.6 M
Laboratory (Mumbai, India). Methanol (99.8%) used sulfuric acid, 28 mM sodium phosphate and 4 mM
were of analytical grade and purchased from Merck Life ammonium molybdate). The extract was incubated on
Science Private Limited, Mumbai, India. boiling water bath at 95°C for 90 minutes then it cooled
down and absorbance was measured at 695 nm against a
Plant collection, Identification, Extract preparation, blank contain 0.1 ml of methanol without plant extract.
Qualitative analysis of Aloe vera gel, flower and The results were expressed as ascorbic acid equivalents
epidermis: (AAE) (mg AAE/g of dry extract)
Aloe vera (Aloe barbadensis Miller) was collected from
southern part of India and it was authenticated by the DPPH scavenging activity:
Botanical survey of India, Southern Regional Centre, The free radical scavenging activity at various
Coimbatore, India (BSI/SRC/5/23/2018/Tech/728). The concentrations (0.1, 0.25, 0.5, 1, 2 mg/ml) of methanolic
gel, epidermis and flower separately was refluxed with extract of gel, epidermis and flower were evaluated by
95% ethanol and concentrated to powder in vacuum11. DPPH assay17. The different concentrations of extract
All the powder forms of gel, epidermis and flower were were vortexed with 1.0 ml of 0.1 mM of DPPH in
stored in 4°C. Further the extracts were dissolved in methanol. The mixture was incubated in the dark for 30
methanol, stirred for overnight, centrifuged at 5000rpm min at room temperature. Degree of inhibition of DPPH
for 20 min. The supernatant was collected and used for was monitored by decrease in absorbance which was
further analysis. Various qualitative tests were also measured at 517 nm against methanol solvent blank. The
performed to know the presence of various phyto- DPPH solution without extract served as control.
chemical constituents. Scavenging activity was calculated as:
Quantification of total phenolic and identification DPPH radical-scavenging activity (%) = [(Ac−As)/Ac] × 100,
using RP-HPLC: where Ac denotes the absorbance of the control reaction
Total phenolic content in the methanolic extract of Aloe (containing all reagents except sample), and As denotes
vera gel, epidermis and flower was determined with the absorbance of the sample.
Folin-Ciocalteau colorimetric method12 using gallic acid
as a standard reference with absorbance measured at ABTS radical scavenging activity:
725nm. The results were expressed at gallic acid ABTS assay was performed by incubating ABTS radical
equivalents (GAE) (mg GAE/g of dry extract). in dark for 15 minutes at 37°C with different
Aluminiumtrichloride colorimetric technique was used concentrations of gel, epidermis, flower extract as
for flavonoid estimation of the extract with Quercetin as indicated above and the absorbance was noted at 734nm

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against methanol solvent blank. Ascorbic acid used as a In-vitro anti-diabetic assays:
standard reference compound. ABTS Scavenging α-amylase inhibition assay:
activity was calculated as follows18: The α-amylase inhibition assay was performed using the
3,5-dinitrosalicylic acid (DNSA) method22. The extracts
ABTS radical-scavenging activity (%) = [(Ac− As)/Ac] × 100, were mixed with 0.25% starch azure and alpha
where Ac denotes the absorbance of the control reaction amylase(4U/mL) solution. After 3 minutes, DNS was
(containing all reagents except the sample), and As added and boiled for 15 minutes. The absorbance was
denotes the absorbance of the sample. noted at 540 nm. Acarbose was used as standard
reference compound. α-amylase inhibition was
Hydrogen peroxide scavenging activity: calculated as follows:
H2O2 assay was performed by adding The test solution
contained ferrous ammonium sulphate, 1,10- α-amylase inhibition activity= [(Ac− As)/Ac] × 100,
phenanthroline, hydrogen peroxide along with different where Ac denotes the absorbance of the control reaction
concentrations of gel, epidermis and flower extract tested (containing all reagents except the sample), and As
for hydrogen peroxide scavenging activity and control denotes the absorbance of the sample
solution contained only 1mM ferrous ammonium
α-glucosidase inhibition assay:
sulphate and 1mM 1,10-phenanthroline. Ascorbic acid
α-glucosidase inhibition of the extracts was measured
used as a standard reference compound.H2O2 Scavenging
with 1mg/mL 4-nitrophenyl-α-D-glucopyranoside and α-
activity was calculated as follows19:
glucosidase(0.3U)23. After incubating at 37 ºC for 30
H2O2 radical-scavenging activity (%) = [(Ac− As)/Ac] × 100, min, reaction was stopped by the addition of 50 mM
sodium hydroxide, and the absorbance was recorded at
where Ac denotes the absorbance of the control reaction 405 nm. Acarbose was used as standard reference
(containing all reagents except the sample), and As compound. α-glucosidase inhibition was calculated as
denotes the absorbance of the sample. follows:
Metal chelating assay: α-glucosidase inhibition activity= [(Ac− As)/Ac] × 100,
Metal chelating activity was measured by adding 0.1 where Ac denotes the absorbance of the control reaction
mMferrous sulphate (0.2 mL) and 0.25 mMferrozine (0.4 (containing all reagents except the sample), and As
mL) subsequently into different concentration of extracts denotes the absorbance of the sample.
of gel, epidermis and flower. After incubating at room
temperature for 10 min, absorbance was recorded at 562 Dipeptidyl peptidase IV (DPP-IV) inhibition assay:
nm with ascorbic acid as standard reference DPP-IV inhibition assay was done by adding different
compound.The metal chelating activity was calculated as concentrations of methanolic extracts of gel, epidermis
follows20: and flower which incubated with plasma (0.05Unit of
DPP-IV enzyme) for 15 minutes. 1mM Gly-Pro p
Metal chelating activity = [(Ac− As)/Ac] × 100, nitroanilidine was added and again incubated for 30
minutes. The absorbance was recorded at 410 nm with
Where Ac denotes the absorbance of the control reaction
sitagliptin as standard reference compound14. DPP-IV
(containing all reagents except the sample), and As
inhibition was calculated as follows:
denotes the absorbance of the sample.
DPP-IV inhibition activity= [(Ac− As)/Ac] × 100,
DNA Protection Assay:
where
DNA Protection Assay was carried out with quercetin as
21 Ac denotes the absorbance of the control reaction
a standard reference compound . Plasmid DNA was
(containing all reagents except the sample), and As
isolated by Sigma GenELute Plasmid Miniprepkit
denotes the absorbance of the sample.
Plasmid DNA was oxidized with H2O2and UV treatment
in presence extracts and checked on 1% agarose. In In vitro antiglycation activity:
brief, the reaction mixture consists of pRSETB plasmid In vitro antiglycation activity of the extracts was
DNA (100ng), H2O2was added at a concentration of examined by testing their ability to inhibit the
30mM with or without any sample. The reaction mixture fluorescence of BSA24. The reaction mixture of BSA and
was treated with UV irradiation for 2 minutes. 1% glucose in 0.1 M phosphate buffered-saline (PBS), pH
agarose gel was cast and the samples were loaded onto 7.4 containing 0.02% sodium azide was incubated at
the wells and electrophoresis was carried out for 30 37°C for 9 days. Then 10% TCA was added and
minutes at 70 Volts. Untreated pRSETB plasmid DNA centrifuged. The pellet was dissolved in PBS and the
was used as control, along with partial treatment (only fluorescence was measured at excitation (370nm) to
UV and only H2O2) in each run. The gel was stained emission(440nm) using Perkin ElmerEnSpire. Anti-
with ethidium bromide and was visualized under glycation activity was calculated as follows:
BIORAD Chemi Doc MP.
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Anti-glycation activity = [(Ac− As)/Ac] × 100, (25.5±1.4mgAAE/g) when compared to flower and gel
(22.1±0.7mgAAE/g and 12.8±1.89mg AAE/g)
where Ac denotes the absorbance of the control reaction
respectively (Fig.1). The methanolic extract of the
(containing all reagents except the sample), and As
epidermis showed higher content of polyphenols,
denotes the absorbance of the sample.
flavonoids and total anti-oxidant content when compared
Statistical Analysis: to the gel and flower extract. Our study corroborates
Statistical comparisons between groups were performed with previousreport10that the epidermis has higher
with Two-way ANOVA followed by Bonferroni method content of phenolics, anthrones, anthaquinones,
for independent observations using Graph Pad Prism 6. chromones compared to gel and flower and this maybe
Differences were considered significant at p≤ 0.05. Each one of the reason for the higher antioxidant potential of
experiment was repeated for three times. IC50 values, epidermis. The epidermis extract showed higher anti-
from the in vitro data, were calculated by non- linear oxidant potential in a concentration dependent manner
regression analysis. compared to flower and gel extracts with different anti-
oxidant assays.
RESULTS AND DISCUSSION:
Qualitative analysis, Total phenolic content,
flavonoid content and anti-oxidant content:
The phytochemical constituents of gel, epidermis and
flower were determined through both qualitative and
quantitative analysis. Through different qualitative
methods, it was observed that the epidermis and flower
extracts has shown more of terpenoids, phenols,
flavonoids compared to the gel extract. Sterols and
tannins were present more in the extract of epidermis
followed by flower and gel extracts respectively. The
Fig .1: Phytochemical analysis of Aloe veraGel, Epidermis and
presence of carbohydrates was shown more in the gel Flower extracts.
extracts compared to epidermis and flower. We have Values are expressed as mean±SD. All expermients performed in
also noticed the presence of proteins, alkaloids and triplicates
triterpenes in all the three extracts of Aloe vera (Table
1). Determination of constituents from RP-HPLC:
Different research groups have been reported for the
Table 1 Phytochemical Screening presence of various phenolic constituents of gel,
Constituents Gel Epidermis Flower
epidermis and flower. In our study the different phenolic
Terpenoids ++ ++++ ++++
Phenols ++ ++++ ++++ constituents present in the gel, epidermis and flower
Flavonoids ++ ++++ ++++ were analyzed using RP-HPLC. It was noticed that
Saponins + ++ + myrcetin, gallic acid, ascorbic acid and kaempferol were
Carbohydrates ++++ ++ ++ present in all the three extracts. Catechin was absent in
Sterols ++ ++++ ++++ gel where as it was present in both epidermis and flower.
Proteins + + +
Alkaloids + + +
Luteolin was present in gel and flower extracts, while
Tannins + ++ + naringenin and pelargonidin chloride were observed in
Triterpenes + + + gel and epidermis extracts (Table 2). In flower it was
Presence (+), Abundant (++), More abundant (++++); done in reported that26 there was absence of quercetin and gallic
triplicates acid in epidermis however it was present in our study.
The phenolic content in the epidermis, gel and flower The quantity percentage calculated through our HPLC
was 14.9±1.5 mg GAE/g, 7.21±2.5 mg GAE/g and analysis shown in (Table 2)15also indicated that the
5.1±2.1mg GAE/g respectively (Fig. 1). The phenolics epidermis extract has more quantity of different
are one of the important components of Aloe vera, which phenolics compared to gel and flower. This variation in
plays a major role in scavenging of free radicals. The phenolic compounds may be due to geographical origin.
total flavonoid content present in epidermis, gel and
ABTS and DPPH radical scavenging activity:
flower was 12.9±1.6mg QE/g, 3.71±1.9mg QE/g and
The ABTS scavenging activity of methanolic extracts of
1.31±1.3mgQE/g respectively (Fig .1). Flavonoids are
gel, epidermis and flower was evaluated at different
the most common polyphenolic groups found
concentrations (0.1, 0.25, 0.5, 1.0 and 2.0 mg/mL) as
ubiquitously in plants and in the human diet. Flavonoids
shown in (Fig. 2a). It was observed that the ABTS
are known to possess antioxidant, anti-inflammatory and
radical was scavenged in a dose dependent manner. The
antidiabetic properties25. The total antioxidant content in
epidermis extract showed higher scavenging activity as
epidermis was observed to be higher
compared to the flower and gel extracts. It was also
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observed that IC50valuesin epidermis was better (p ≤ Metal chelating activity:


0.001) when compared to flower and gel extract (Table In this study it was noticed that as the concentration of
3). The DPPH scavenging activity of the extracts in the the extracts increased the metal chelating activity was
varied concentrations (0.1, 0.25, 0.5, 1.0 and 2.0 mg/mL) also increased significantly in epidermis compared to gel
was determined. The DPPH radical was scavenged in and flower (Fig. 2d). The IC50 values in (Table 3)
concentration dependent manner as shown in (Fig. 2b). indicate that the epidermis has better metal chelating
The methanolic extract of epidermis has shown better activity (0.295±0.12mg/mL) compared to flower (p ≤
scavenging activity as the concentration increased 0.001) and gel extracts (p ≤ 0.001) respectively. From
compared to gel and flower extract. The IC50 values the results it was observed that the epidermis extract has
shown in (Table 3) indicate that the epidermis has better shown better anti-oxidant potential followed by gel and
scavenging activity of (0.578±0.05 mg/mL) compared to flower as indicated by the better IC50 values shown in
gel (p ≤ 0.01) and flower (p ≤ 0.001). It was also (Table 3). In this assay all the three extracts interfered
observed that the epidermis extract has better potential to with the formation of ferrous complex with ferrozine,
scavenge DPPH radical compared to gel and flower suggesting that they have chelating activity and capture
extracts. Our results corroborate with a previous the ferrous ion before ferrozine. Our study also
study26that the epidermis has better anti-oxidant potential corroborates with other study reporting that the presence
compared to flower. Studies related to the potential of of phenolic compounds in plant extracts has good
epidermis to scavenge the ABTS radical has not been capacity for iron chelating activity suggesting its action
reported so far. In this study it was observed that as an antioxidant28. In this study by different anti-oxidant
epidermis extract has shown scavenging ABTS activity assays, epidermis extract has shown better anti-oxidant
compared to gel and flower extracts. Our findings also potential compared to gel and flower extracts.
corroborates with previous study that flower extract can
scavenge ABTS and DPPH free radicals2. DNA Protection Assay:
Table 2: HPLC analaysis of Gel, Epidermis and flower compared In the DNA damage protection assay, it was noticed that
with standards the epidermis extract at a lower concentration of
Compounds Gel Epidermis Flowers 20μg/ml was able to protect the pRSETBplasmid DNA
Ascorbic Acid 0.46% 0.38% 0.20% damage against UV and 30mM H2O2 treatment
Gallic Acid 0.40% 0.23% 0.71% compared to the gel and flower extracts which required a
Catechin 0.12% 0.09% 0.18%
Naringin 0.03% 0.95% 0.05%
higher concentration of 80μg/ml to protect the plasmid
Taxifolin 0.064% 0.75% 0.05% DNA (Fig.3). The pRSETBplasmid DNA in the presence
Pelargonidin Chloride 0.02% 0.68% 0.04% of H2O2and UV converted to open circular and linear
Myrcetin 0.17% 2.90% 0.16% form (lane 4) compared to the control lane (lane 1).
Luteolin 1.50% 2.30% 0.07% However, in the presence of gel, epidermis and flower
Quercetin 0.08% 0.77% 0.002%
Naringenin 0.02% 0.10% -
extracts, plasmid DNA is protected from the damages
Kaempferol 0.03% 0.18% 0.002% taking place in the presence of UV and H2O2.The
All experiments are done in triplicates presence of different phenolic constituents present in all
the three extracts which was analyzed by RP-HPLC may
Hydrogen peroxide scavenging activity: suppress the formation of linear plasmid DNA and
Epidermis has shown better H2O2scavenging activity protected damage. The DNA protection assay also
compared to gel and flower extracts as shown in (Fig. demonstrated the presence of strong antioxidant
2c). The IC50 value for methanolic extract of epidermis properties of Aloe vera epidermis, gel and flower
for H2O2 scavenging activity was found to be 0.318±0.03 extracts. We observed that there is a correlation between
mg/mL, while in the gel extract it was 0.425±0.09 the antioxidant activity and phenolic constituents which
mg/mL and in flower extract with 0.804±0.15 mg/mL are mainly responsible for the antioxidant activity in all
respectively. Hydrogen peroxide is a weak initiator of the extracts of Aloe vera.
lipid peroxidation, however it produces active oxygen
species as a result of its ability to generate reactive Table 3: IC50 values of Aloe vera gel, epidermis and flower extracts
hydroxyl radicals. The ability to Aloe vera gel extract to by different antioxidant assays.
Extracts ABTS DPPH H2O2 Metal
scavenge has beenreported27but so far no reports are chelating
reported with flower and epidermis. We infer that the Gel 0.257±0.06 0.688±0.05 0.425±0.09 0.945±0.35
presence of phenolics in these two extracts confer them Epidermis 0.170±0.05 0.578±.05 0.318±0.03 0.295±0.12
the property to scavenge the formation of H2O2 radical. Flower 0.217±0.07 0.915±0.06 0.804±0.15 0.775±0.9
All experiments are done in triplicates. Values are expressed as
mean±SDof IC50 (mg/ml)

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Fig. 2: Anti-oxidant acitivity of Aloe vera gel, epidermis and flower extracts by different assays. a)ABTS assay b) DPPH assay c) H 2O2
assay and d) metal chelating acitivity. Values expressed as mean±SD. All experiments are done in triplicates.

disaccharides to monoscahhrides5. The inhibitors of


these two enzymes can help in control of hyperglycemia.
These can delay the carbohydrate digestion thereby
reducing the glucose levels. We observed that the α-
amylase and α-glucosidase inhibited in a dose dependent
manner in gel and flower extracts. The IC50 values
indicated that the gel has higherα-amylase and α-
glucosidase inhibition activities (p≤ 0.001) compared to
Fig. 3: Effect of gel, epidermis and flower extracts on the flower extract (Table 4) (Fig. 4b). Phenolic compounds
protection of DNA damage induced by UV and H202 and flavonoids present in the plants have known to
possess anti-diabetic properties29. Our study has also
M: Marker- (DNA ladder lane 1), C: control
shown the presence of phenolics such as quercetin,
(pRSETBPlasmid DNA at concentration of 100ng per
luteolin, myrcetinetc in gel and flower extracts. These
lane) (lane 2), U: UV treated (lane 3), H: 30mM
phenolics may play a role in inhibiting the α-amylase
H2O2treated (lane 4), UH: UV+ 30mM H2O2 treated
and α-glucosidase activity thereby contributing to the
(lane 5), G: Gel extract (80μg/ml) treated with UV+
anti-diabetic property of Aloe vera gel and flower5,29.It
30mM H2O2(lane 6), E: Epidermis extract(20μg/ml)
was observed that though the epidermis is rich in
treated with UV+ 30mM H2O2 (lane 7), F: Flower
constituents such as polyphenols, flavonoids, chromones,
extract(80μg/ml) treated with UV+ 30mM H2O2 (lane
anthrones, anthraquinones, benzyl derivatives10 which
8), S: Standard Quercetin treated with UV+ 30mM H 2O2
are known to possess anti-oxidant potential did not show
(lane 9),
any significant anti-diabetic potential compared to gel
α-amylaseandα-glucosidase inhibition activities: and flower extracts. In addition to these constituents the
The α-amylase inhibition activity of methanolic extracts gel and flower extracts has also been reported for the
of gel, epidermis and flower were evaluated at different presence of phyto-constituents such as carbohydrates,
concentrations (0.1, 0.25, 0.5, 1.0 and 2.0 mg/mL) as proteins, phytosterols, mineral components 2,10. These
shown in (Fig. 4a). α-amylase and α-glucosidase are the phytoconstituents present in gel of Aloe vera may play a
main carbohydrate hydrolyzing enzymes responsible for role in antidiabetic activity30-31. It may be also due to the
postprandial hyperglycemia in diabetes. Amylase presence of sugars, proteins, phytosterols or the
hydrolyses the 1,4-glycosidic linkage of polysaccharides synergetic effects of the all these constituents. The gel
to disaccharides while α-glucosidase converts the and flower extracts has shown better anti-diabetic

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potential as well as anti-oxidant potential. However, in In vitro anti-glycation activity:


epidermis extract it may be due to the presence of more The glucose levels can also be controlled through
amounts of chromones, anthrones which masks the inhibiting the formation of glycated products seen in
phenolics and flavonoids. This may be one of the reason patients suffering from diabetes mellitus24.The formation
that the epidermis extract did not show any significant of advanced glycation end (AGE) products was observed
anti-diabetic property in spite of having better anti- by measuring the fluorescence intensity of BSA-Glucose
oxidant potential. solutions for a period of 9 days. There was a significant
increase in AGE’s formation when BSA was incubated
DPP-IV inhibition activity: with glucose. Both the gel and flower extracts were able
Glucose levels are regulated by one of the pathways is to inhibit the formation of glycated products
the incretin pathway. The GLP-1 (Glucagon like peptide- significantly (Fig. 4d). There was no significant activity
1) helps in release of insulin from pancreas. However, in the methanolic extract of epidermis. The IC50 values
the GLP-1 is inhibited by DPP-IV enzyme after a period in (Table 4) indicate the gel has better anti-glycation
of one and a half minutes. Inhibiting the DPP-IV enzyme activity when compared to flower (p ≤ 0.001). This may
can aid in prolonged action of GLP-1 leading to be due to the presence of phenolics and flavonoids in
secretion of insulin and lowering of glucose levels these extracts which may play a role in inhibiting the
during diabetic condition. In our study, the gel and AGEs formation thereby controlling the sugar levels.
flower extract has shown significant inhibition activity Studies have been reported with the plant phenolics and
(Fig. 4c) whereas the epidermis extract did not show any flavonoids having anti-glycation properties27. It has been
significant activity. It was observed that gel has better suggested that the anti-glycation property of plants
potential to inhibit the DPP-IV enzyme compared to the correlate with the scavenging of free radicals34. In this
flower extract (p ≤ 0.001) (Table 4)this was correlated study it indicates that the presence of phenolics in gel
with IC50 values. Our results correlates with previous and flower may contribute anti-glycation property
study14that the gel extract showed significant DPP-IV through decrease of free radicals formed through auto-
inhibition with better IC50.Plant phenolics and flavonoids oxidation of sugars.
has also been reported to possess DPP-IV inhibition
Table 4: IC values of Aloe vera gel, epidermis and flower extracts
property in alleviation of diabetes mellitus32-33. The by different50anti-diabetic assays.
presence of the various phenolics in gel and flower Extracts α-amylase α-glucosidase DPP-IV Anti-
extracts (Table 2) may contribute in inhibiting the DPP- inhibition inhibition inhibition glycation
IV enzyme. Gel 0.456±0.07 0.608 ±0.08 0.985±0.23 1.54±0.45
Flower 0.555±0.09 0.488±0.04 1.34±0.85 2±0.56
Values are expressed as mean±SDof IC50 (mg/ml). All experminets are
done in triplicates

Fig. 4:Anti-diabetic activity of Aloe veragel, epidermis and flower extracts by differernt assays.
a) α-amylase inhibition assay b) α-glucosidase inhibition assay c) DPP-IV inhibition assay and d) anti-glycation assay.
Values are expressed as mean±SD; All experments done in triplicates.

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15. Sathyanarayanan S, Chandran R, Thankarajan S, Abrahamse H and


CONCLUSION: Thangaraj P. Phytochemical composition, antioxidant and anti-bacterial
In conclusion, the present comparative study of Aloe activity of Syzygium calophyllifolium Walp. fruit. Journal of Food Science
vera gel, epidermis and flower extracts showed and Technology. 2018;55(1):341-350.
16. Pavithra S, Banu N. Free Radical Scavenging Activity and Total
significant antioxidant and scavenging activities of Antioxidant Capacity of Tin Chlorophyllin from Morinda citrifolia L.
DPPH, ABTS, H2O2, metal chelating activity and Research Journal of Pharmacy and Technology. 2017;10(2):453-455.
17. Nikhat F, Satyanarayana D, Evs S. Isolation, Charectrisation and Screening
protected the DNA damage. In addition, the gel and of Antioxidant Activity of the Roots of Syzygiumcuminii (L) Skeel. Asian
flower showed antidiabetic properties by inhibiting alpha Journal of Research in Chemistry. 2009;2(2):218-221.
18. Menaka, Venkatasubramanian C. Free radical scavenging activity of
amylase, alpha- glucosidase, DPP-IV enzymes and Lyophilised Syzygium cumini (L) Skeels fruit pulp (Jamun). Research
formation of glycation products. The presence of these Journal of Pharmacy and Technology. 2017;10(4):986-990.
different polyphenols and flavonoids in these extracts 19. Mukhopadhyay D, Dasgupta P, Sinha Roy D, Palchoudhuri S, Chatterjee I,
Ali S and Dastidar SG. A Sensitive In vitro Spectrophotometric Hydrogen
contribute to their antioxidant and anti-diabetic Peroxide Scavenging Assay using 1,10-Phenanthroline. Free Radicals and
properties. These extracts might be more effective to Antioxidants. 2016;6(1):124-132.
20. Gülçin I, Elmastat M, Aboul-Enein HY. Determination of Antioxidant and
reduce or delay the diabetic complications subjected to Radical Scavenging Activity of Basil (Ocimum basilicum L. Family
further in vivo evaluation. Further studies are needed to Lamiaceae) Assayed by. Phytotherapy Research. 2007;21(4):354–361.
21. Kumar A, Chattopadhyay S. DNA damage protecting activity and
characterize the active components and their antioxidant potential of pudina extract. Food Chemistry. 2007;100(4):1377-
pharmacological properties so that they may be used as 1384.
nutraceuticals. 22. Pawar PS, Patil JT, Bendre RS. Synthesis, Characterization and In-Vitro
Antidiabetic Studies of Vanadium Complexes derived from N2O2 donor
Ligands. Asian Journalof Research in Chemistry. 2018;11(1):8-14.
ACKNOWLEDGEMENT: 23. Khamis M, Talib F, Rosli NS, Mohd KS, Dharmaraj S,Latif ZA,Utharkar
Authors thank Department of Science and Technology MRS andSrenivasan S.In vitro α-amylase and α-glucosidase inhibition and
increased glucose uptake of Morinda citrifolia fruit and scopoletin. Research
(IR/SO/LU-03/2004), New Delhi, India for the financial Journal of Pharmacy and Technology. 2015;8(2):189-193.
support. 24. Kazeem MI, Akanji MA, Hafizur RM, Choudhary MI. Antiglycation,
antioxidant and toxicological potential of polyphenol extracts of alligator
CONFLICT OF INTEREST: pepper, ginger and nutmeg from Nigeria. Asian Pacific Journal of Tropical
Biomedicine. 2012;2(9):727-732.
The authors declare no conflict of interest. 25. Tapas A, Sakarkar D, Kakde R. Flavonoids as Nutraceuticals: A Review.
Tropical Journal of Pharmaceutical Research. 2008;7(3):1089-1099.
REFERENCES: 26. López A, De Tangil MS, Vega-Orellana O, Ramírez AS and Rico M.
1. Gupta VK, Sharma SK. Plants as natural antioxidants. Indian Journal of Phenolic constituents, antioxidant and preliminary antimycoplasmic
Natural Products and Resources. 2014;5(4): 326-334. activities of leaf skin and flowers of Aloe vera (L.) Burm. f. (syn. A.
2. Debnath T, Ghosh M, Lee YM, Nath NCD, Lee KG and Lim BO. barbadensis Mill.) from the Canary Islands (Spain). Molecules.
Identification of phenolic constituents and antioxidant activity of Aloe 2013;18(5):4942-4954.
barbadensis flower extracts. Food and Agricultural Immunology. 27. Kaithwas G, Singh P, Bhatia D. Evaluation of in vitro and in vivo
2017;29(1):27-38. antioxidant potential of polysaccharides from Aloe vera (Aloe barbadensis
3. Karpagam GN, Gayathri. R, Vishnupriya. V. Bioactivity analysis of Miller) gel. Drug and chemical toxicology. 2014;37(2): 135–143.
lemongrass oil. Research Journal of Pharmacy and 28. Shukla S, Mehta A, Bajpai VK, Shukla S. In vitro antioxidant activity and
Technology.2016;9(7):903-906. total phenolic content of ethanolic leaf extract of Stevia rebaudiana Bert.
4. Rajendran R, Saleem Basha N, Ruby S. Evaluation of in vitro Antioxidant Food and Chemical Toxicology. 2009;47(9):2338-43.
Activity of Stem barks and Stem wood of Premna serratifolia Linn., 29. Kim J-S, Kwon C-S, Son KH. Inhibition of Alpha-glucosidase and Amylase
(Verbenaceae). Research Journal of Pharmacognosy and Phytochemistry. by Luteolin, a Flavonoid. Bioscience, Biotechnology and Biochemistry.
2009;1(1):11-14. 2000;64(11):2458-61.
5. Hullatti K, Telagari M. In-vitro α-amylase and α-glucosidase inhibitory 30. Yagi A, Hegazy S, Kabbash A, Wahab EA-E. Possible hypoglycemic effect
activity of Adiantum caudatum Linn. and Celosia argentea Linn. extracts of Aloe vera L. high molecular weight fractions on type 2 diabetic patients.
and fractions. Indian Journal of Pharmacology. 2015;47(4):425-429. Saudi Pharmaceutical Journal. 2009;17(3):209-215.
6. Pattabiraman K, Muthukumaran P. Antidiabetic and Antioxidant Activity of 31. Misawa E, Tanaka M, Nomaguchi K, Yamada M, Toida T, Takase M,
Morinda tinctoria roxb Fruits Extract in Streptozotocin-Induced Diabetic Iwatsuki K and Kawada T. Administration of phytosterols isolated from
Rats. Asian Journal of Pharmacy and Technology. 2011;1(2):34-39. Aloe vera gel reduce visceral fat mass and improve hyperglycemia in
7. Hamman JH. Composition and applications of Aloe vera leaf gel. Zucker diabetic fatty (ZDF) rats. Obesity Research and Clinical Practice.
Molecules. 2008;13(8):1599-616. 2008;2(4):239-245.
8. Boudreau MD, Beland FA. An evaluation of the biological and toxicological 32. Gupta A, Jacobson GA, Burgess JR, Jelinek HF, Nichols DS, Narkowicz
properties of Aloe barbadensis (Miller), Aloe vera. Journal of environmental CK and Al-Aubaidy HA. Citrus bioflavonoids dipeptidyl peptidase-4
science and health. Part C, Environmental carcinogenesis and ecotoxicology inhibition compared with gliptin antidiabetic medications. Biochemical and
reviews. 2006;24(1):103-154. Biophysical Research Communications. 2018;503(1):21-25.
9. Patel DS, Shah PB, Managoli NB. Evaluation of In-vitro Anti-oxidant and 33. Nicolle E, Souard F, Faure P, Boumendjel A. Flavonoids as Promising Lead
Free Radical Scavenging activities of Withania somnifera and Aloe vera. Compounds in Type 2 Diabetes Mellitus: Molecules of Interest and
Asian Journal of Pharmacy and Technology. 2012;2(4):143-147. Structure-Activity Relationship. Current Medicinal Chemistry.
10. Cock IE. The genus aloe: Phytochemistry and therapeutic uses including 2011;18(17):2661-72.
treatments for gastrointestinal conditions and chronic inflammation. 34. Kaewnarin K, Niamsup H, Shank L, Rakariyatham N. Antioxidant and
Progress in drug research. 2015; 70:179-235. antiglycation activities of some edible and medicinal plants. Chiang Mai
11. Noor A, Gunasekaran S, Soosai Manickam A, Vijayalakshmi MA. Journal of Science. 2014;41(1):105-116.
Antidiabetic activity of Aloe vera and histology of organs in streptozotocin-
induced diabetic rats. Current Science. 2008;94(8):1070-1076.
12. Elias J, Rajesh M, Anish N, Jayan N. In Vitro Antioxidant Activity of the
Methanolic Extract of Simaruba glauca DC. Asian Journal of Research in
Chemistry. 2010;3(2):312-315.
13. Soares L, Silva L, Pezzini B. Spectrophotometric determination of the total
flavonoid content in Ocimum basilicum L. (Lamiaceae) leaves.
Pharmacognosy Magazine. 2015;11(41):96-101.
14. Raja C, Venkataraman K. Aloe vera phytochemicals inhibits dipeptidyl
peptdiase IV (DPP-IV), an anti-diabetic target. International Journal of
Pharma and Bio sciences. 2016;7(3):120-128.

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