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:

Diabetes mellitus is a metabolic disorder that may lead to overproduction of reactive oxygen species (ROS) in endothelial cells which contributes the pathogenesis of various complications1. As the ROS level increases, there is disturbance in the anti-oxidant enzyme levels in the body, leading to oxidative stress. In diabetic condition, advanced glycation products are formed due to the reaction of glucose with plasma proteins, triggering further ROS production. Medicinal plants have wide range in phytochemicals such as flavonoids, polyphenols sterols, tannins and terpenoids etc. Provide protection against oxidative stress. These phytoconstituents scavenge the ROS by inhibiting the initiation and chain propagation2.

 

 

Medicinal plants have been reported to have anti-oxidant activities for overcoming the oxidative stress and cell damage caused due to diabetes mellitus3-4. The usage of herbal and natural drug products for the treatment of diabetes is growing worldwide, with the ancient Indian literature reports suggesting more than 800 plants having anti-diabetic activities5-6. One such medicinal plant is Aloe barbadensis Miller, commonly known as Aloe vera (Family: Aloeaceae). It has been used for centuries for its health, beauty, medicinal and skin care properties7. They are perennial succulents or xerophytes which are around 60 to 100 cm tall. Aloevera has green fleshy leaves covered by a thick cuticle or rind and an inner clear pulp (gel). Its flowers appear in the months of 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, anti-cancer, anti-inflammatory, anti-bacterial, anti-fungal properties8-9 and these have been attributed to synergistic effects of numerous bioactive compounds in Aloe vera. The phytochemical analysis of Aloe vera has shown the presence of tannin, saponins, flavonoids, steroids, terpenoids, and cardiac glycosides anthroquinones10-11. Many researchers have reported about different pharmacological properties either with entire plant or the gel of Aloe vera. There are few reports available with the epidermis and flower2. Little information is available about the comparative evaluation between the different constituents of gel, epidermis and flower of Aloe vera related to antidiabetic activity and antioxidant properties. So, in the present study comparative evaluation of anti-diabetic and anti-oxidant potential of gel, epidermis and flower of Aloe vera was assessed.

 

MATERIAL AND METHODS:

Chemicals:

Ascorbic acid, Gallic acid, quercetin, aluminium chloride, ferric chloride (FeCl3); Folin Ciocalteu reagent; bovine serum albumin (BSA); potassium persulphate; 2,2-diphenyl-1-picrylhy-drazyl (DPPH), trichloroacetic acid (TCA)

 

2,2′-Azino-bis (3-ethylbenzthiazoline-6-sulfonic acid (ABTS), Hydrogen peroxide (H2O2) were purchased from Sigma chemicals (St. Louis, MO, USA). All other chemicals were procured from Sisco Research Laboratory (Mumbai, India). Methanol (99.8%) used were of analytical grade and purchased from Merck Life Science Private Limited, Mumbai, India.

 

Plant collection, Identification, Extract preparation, Qualitative analysis of Aloe vera gel, flower and epidermis:

Aloe vera (Aloe barbadensis Miller) was collected from southern part of India and it was authenticated by the Botanical survey of India, Southern Regional Centre, Coimbatore, India (BSI/SRC/5/23/2018/Tech/728). The gel, epidermis and flower separately was refluxed with 95% ethanol and concentrated to powder in vacuum11. All the powder forms of gel, epidermis and flower were stored in 4°C. Further the extracts were dissolved in methanol, stirred for overnight, centrifuged at 5000rpm for 20 min. The supernatant was collected and used for further analysis. Various qualitative tests were also performed to know the presence of various phyto-chemical constituents.

 

Quantification of total phenolic and identification using RP-HPLC:

Total phenolic content in the methanolic extract of Aloe vera gel, epidermis and flower was determined with Folin-Ciocalteau colorimetric method12 using gallic acid as a standard reference  with absorbance measured at 725nm. The results were expressed at gallic acid equivalents (GAE) (mg GAE/g of dry extract). Aluminiumtrichloride colorimetric technique was used for flavonoid estimation of the extract with Quercetin as standard reference13 with absorbance measured at 415nm. The results were expressed as quercetin equivalents ((mg QE/g of dry extract). The identification of individual phenolic compounds was carried out by using Reverse Phase High-performance Liquid Chromatography (RP-HPLC)14.RP-HPLC C18 column (Waters, 150 X 3.9mm, i.d., 5µm) was used. Samples (20µl) were injected and analyzed at a flow rate of 1 ml/ min. The eluent was monitored at 255nm and 290nm. Identification of phenolic compounds was done by comparing retention time and area of peaks in the extracts against with that of the standard compounds from Sigma-Aldrich. Data acquisition and processing were performed using Breeze data (Waters) system. The quantity of each compound was calculated according to the formula15:

 

Quantity percent = (As * Wstd)/ (Ast *Ws) *purity of standard

 

Where As = sample peak area, Ast = Standard peak area, Ws = amount of sample Wstd = amount of standard

 

Total antioxidant capacity:

Total antioxidant capacity of the extracts was done using ascorbic acid as a standard reference compound16. An aliquot of 1 mg/ ml methanolic extract of gel, epidermis and flower were mixed with the reagent solution (0.6 M sulfuric acid, 28 mM sodium phosphate and 4 mM ammonium molybdate). The extract was incubated on boiling water bath at 95°C for 90 minutes then it cooled down and absorbance was measured at 695 nm against a blank contain 0.1 ml of methanol without plant extract. The results were expressed as ascorbic acid equivalents (AAE) (mg AAE/g of dry extract)

 

DPPH scavenging activity:

The free radical scavenging activity at various concentrations (0.1, 0.25, 0.5, 1, 2 mg/ml) of methanolic extract of gel, epidermis and flower were evaluated by DPPH assay17. The different concentrations of extract were vortexed with 1.0 ml of 0.1 mM of DPPH in methanol. The mixture was incubated in the dark for 30 min at room temperature. Degree of inhibition of DPPH was monitored by decrease in absorbance which was measured at 517 nm against methanol solvent blank. The DPPH solution without extract served as control. Scavenging activity was calculated as:

 

DPPH radical-scavenging activity (%) = [(Ac−As)/Ac] × 100,

 

where Ac denotes the absorbance of the control reaction (containing all reagents except sample), and As denotes the absorbance of the sample.

 

ABTS radical scavenging activity:

ABTS assay was performed by incubating ABTS radical in dark for 15 minutes at 37°C with different concentrations of gel, epidermis, flower extract as indicated above and the absorbance was noted at 734nm against methanol solvent blank. Ascorbic acid used as a standard reference compound. ABTS Scavenging activity was calculated as follows18:

 

ABTS radical-scavenging activity (%) = [(Ac− As)/Ac] × 100,

 

where Ac denotes the absorbance of the control reaction (containing all reagents except the sample), and As denotes the absorbance of the sample.

 

Hydrogen peroxide scavenging activity:

H2O2 assay was performed by adding The test solution contained ferrous ammonium sulphate, 1,10-phenanthroline, hydrogen peroxide along with different concentrations of gel, epidermis and flower extract tested for hydrogen peroxide scavenging activity and control solution contained only 1mM ferrous ammonium sulphate and 1mM 1,10-phenanthroline. Ascorbic acid used as a standard reference compound.H2O2 Scavenging activity was calculated as follows19:

 

H2O2 radical-scavenging activity (%) = [(Ac− As)/Ac] × 100,

 

where Ac denotes the absorbance of the control reaction (containing all reagents except the sample), and As denotes the absorbance of the sample.

 

Metal chelating assay:

Metal chelating activity was measured by adding 0.1 mMferrous sulphate (0.2 mL) and 0.25 mMferrozine (0.4 mL) subsequently into different concentration of extracts of gel, epidermis and flower. After incubating at room temperature for 10 min, absorbance was recorded at 562 nm with ascorbic acid as standard reference compound.The metal chelating activity was calculated as follows20:

 

Metal chelating activity = [(Ac− As)/Ac] × 100,

 

Where Ac denotes the absorbance of the control reaction (containing all reagents except the sample), and As denotes the absorbance of the sample.

 

DNA Protection Assay:

DNA Protection Assay was carried out with quercetin as a standard reference compound21. Plasmid DNA was isolated by Sigma GenELute Plasmid Miniprepkit Plasmid DNA was oxidized with H2O2and UV treatment in presence extracts and checked on 1% agarose. In brief, the reaction mixture consists of pRSETB plasmid DNA (100ng), H2O2was added at a concentration of 30mM with or without any sample. The reaction mixture was treated with UV irradiation for 2 minutes. 1% agarose gel was cast and the samples were loaded onto the wells and electrophoresis was carried out for 30 minutes at 70 Volts. Untreated pRSETB plasmid DNA was used as control, along with partial treatment (only UV and only H2O2) in each run. The gel was stained with ethidium bromide and was visualized under BIORAD Chemi Doc MP.

In-vitro anti-diabetic assays:

α-amylase inhibition assay:

The α-amylase inhibition assay was performed using the 3,5-dinitrosalicylic acid (DNSA) method22. The extracts were mixed with 0.25% starch azure and alpha amylase(4U/mL) solution. After 3 minutes, DNS was added and boiled for 15 minutes. The absorbance was noted at 540 nm. Acarbose was used as standard reference compound. α-amylase inhibition was calculated as follows:

 

α-amylase inhibition activity= [(Ac− As)/Ac] × 100,

where Ac denotes the absorbance of the control reaction (containing all reagents except the sample), and As denotes the absorbance of the sample

 

α-glucosidase inhibition assay:

α-glucosidase inhibition of the extracts was measured with 1mg/mL 4-nitrophenyl-α-D-glucopyranoside and α-glucosidase(0.3U)23. After incubating at 37 ºC for 30 min, reaction was stopped by the addition of 50 mM sodium hydroxide, and the absorbance was recorded at 405 nm. Acarbose was used as standard reference compound. α-glucosidase inhibition was calculated as follows:

 

α-glucosidase inhibition activity= [(Ac− As)/Ac] × 100, where Ac denotes the absorbance of the control reaction (containing all reagents except the sample), and As denotes the absorbance of the sample.

 

Dipeptidyl peptidase IV (DPP-IV) inhibition assay:

DPP-IV inhibition assay was done by adding different concentrations of methanolic extracts of gel, epidermis and flower which incubated with plasma (0.05Unit of DPP-IV enzyme) for 15 minutes. 1mM Gly-Pro p nitroanilidine was added and again incubated for 30 minutes. The absorbance was recorded at 410 nm with sitagliptin as standard reference compound14. DPP-IV inhibition was calculated as follows:

 

DPP-IV inhibition activity= [(Ac− As)/Ac] × 100,

where

Ac denotes the absorbance of the control reaction (containing all reagents except the sample), and As denotes the absorbance of the sample.

 

In vitro antiglycation activity:

In vitro antiglycation activity of the extracts was examined by testing their ability to inhibit the fluorescence of BSA24. The reaction mixture of BSA and glucose in 0.1 M phosphate buffered-saline (PBS), pH 7.4 containing 0.02% sodium azide was incubated at 37°C for 9 days. Then 10% TCA was added and centrifuged. The pellet was dissolved in PBS and the fluorescence was measured at excitation (370nm) to emission(440nm) using Perkin ElmerEnSpire. Anti-glycation activity was calculated as follows:

 

Anti-glycation activity = [(Ac− As)/Ac] × 100,

 

where Ac denotes the absorbance of the control reaction (containing all reagents except the sample), and As denotes the absorbance of the sample.

 

Statistical Analysis:

Statistical comparisons between groups were performed with Two-way ANOVA followed by Bonferroni method for independent observations using Graph Pad Prism 6. Differences were considered significant at p≤ 0.05. Each experiment was repeated for three times. IC50 values, from the in vitro data, were calculated by non- linear regression analysis.

 

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 presence of carbohydrates was shown more in the gel extracts compared to epidermis and flower. We have also noticed the presence of proteins, alkaloids and triterpenes in all the three extracts of Aloe vera (Table 1).

 

Table 1 Phytochemical Screening

Constituents

Gel

Epidermis

Flower

Terpenoids

++ 

++++ 

++++ 

Phenols

++ 

++++ 

++++ 

Flavonoids

++

++++

++++ 

Saponins

 +

++ 

 +

Carbohydrates

++++

++ 

 ++

Sterols

++ 

++++ 

++++

Proteins

 +

 +

Alkaloids

Tannins

++ 

Triterpenes

+

Presence (+), Abundant (++), More abundant (++++); done in triplicates

 

The phenolic content in the epidermis, gel and flower was 14.9±1.5 mg GAE/g, 7.21±2.5 mg GAE/g and 5.1±2.1mg GAE/g respectively (Fig. 1). The phenolics are one of the important components of Aloe vera, which plays a major role in scavenging of free radicals. The total flavonoid content present in epidermis, gel and flower was 12.9±1.6mg QE/g, 3.71±1.9mg QE/g and 1.31±1.3mgQE/g respectively (Fig .1). Flavonoids are the most common polyphenolic groups found ubiquitously in plants and in the human diet. Flavonoids are known to possess antioxidant, anti-inflammatory and antidiabetic properties25. The total antioxidant content in epidermis was observed to be higher (25.5±1.4mgAAE/g) when compared to flower and gel (22.1±0.7mgAAE/g and 12.8±1.89mg AAE/g) respectively (Fig.1). The methanolic extract of the epidermis showed higher content of polyphenols, flavonoids and total anti-oxidant content when compared to the gel and flower extract. Our study corroborates with previousreport10that the epidermis has higher content of phenolics, anthrones, anthaquinones, chromones compared to gel and flower and this maybe one of the reason for the higher antioxidant potential of epidermis. The epidermis extract showed higher anti-oxidant potential in a concentration dependent manner compared to flower and gel extracts with different anti-oxidant assays.

 

 

Fig .1: Phytochemical analysis of Aloe veraGel, Epidermis and Flower extracts.

Values are expressed as mean±SD. All expermients performed in triplicates

 

Determination of constituents from RP-HPLC:

Different research groups have been reported for the presence of various phenolic constituents of gel, epidermis and flower. In our study the different phenolic constituents present in the gel, epidermis and flower were analyzed using RP-HPLC. It was noticed that myrcetin, gallic acid, ascorbic acid and kaempferol were present in all the three extracts. Catechin was absent in gel where as it was present in both epidermis and flower. Luteolin was present in gel and flower extracts, while naringenin and pelargonidin chloride were observed in gel and epidermis extracts (Table 2). In flower it was reported that26 there was absence of quercetin and gallic acid in epidermis however it was  present in our study. The quantity percentage calculated through our HPLC analysis shown in (Table 2)15also indicated that the epidermis extract has more quantity of different phenolics compared to gel and flower. This variation in phenolic compounds may be due to geographical origin.

 

ABTS and DPPH radical scavenging activity:

The ABTS scavenging activity of methanolic extracts of gel, epidermis and flower was evaluated at different concentrations (0.1, 0.25, 0.5, 1.0 and 2.0 mg/mL) as shown in (Fig. 2a). It was observed that the ABTS radical was scavenged in a dose dependent manner. The epidermis extract showed higher scavenging activity as compared to the flower and gel extracts. It was also observed that IC50valuesin epidermis was better (p ≤ 0.001) when compared to flower and gel extract (Table 3). The DPPH scavenging activity of the extracts in the varied concentrations (0.1, 0.25, 0.5, 1.0 and 2.0 mg/mL) was determined. The DPPH radical was scavenged in concentration dependent manner as shown in (Fig. 2b). The methanolic extract of epidermis has shown better scavenging activity as the concentration increased compared to gel and flower extract. The IC50 values shown in (Table 3) indicate that the epidermis has better scavenging activity of (0.578±0.05 mg/mL) compared to gel (p ≤ 0.01) and flower (p ≤ 0.001). It was also observed that the epidermis extract has better potential to scavenge DPPH radical compared to gel and flower extracts. Our results corroborate with a previous study26that the epidermis has better anti-oxidant potential compared to flower. Studies related to the potential of epidermis to scavenge the ABTS radical has not been reported so far. In this study it was observed that epidermis extract has shown scavenging ABTS activity compared to gel and flower extracts. Our findings also corroborates with previous study that flower extract can scavenge ABTS and DPPH free radicals2.

 

Table 2: HPLC analaysis of Gel, Epidermis and flower compared with standards

Compounds

Gel

Epidermis

Flowers

Ascorbic Acid

0.46%

0.38%

0.20%

Gallic Acid

0.40%

0.23%

0.71%

Catechin

0.12%

0.09%

0.18%

Naringin

0.03%

0.95%

0.05%

Taxifolin

0.064%

0.75%

0.05%

Pelargonidin Chloride

0.02%

0.68%

0.04%

Myrcetin

0.17%

2.90%

0.16%

Luteolin

1.50%

2.30%

0.07%

Quercetin

0.08%

0.77%

0.002%

Naringenin

0.02%

0.10%

-

Kaempferol

0.03%

0.18%

0.002%

All experiments are done in triplicates

 

Hydrogen peroxide scavenging activity:

Epidermis has shown better H2O2scavenging activity compared to gel and flower extracts as shown in (Fig. 2c). The IC50 value for methanolic extract of epidermis for H2O2 scavenging activity was found to be 0.318±0.03 mg/mL, while in the gel extract it was 0.425±0.09 mg/mL and in flower extract with 0.804±0.15 mg/mL respectively. Hydrogen peroxide is a weak initiator of lipid peroxidation, however it produces active oxygen species as a result of its ability to generate reactive hydroxyl radicals. The ability to Aloe vera gel extract to scavenge has beenreported27but so far no reports are reported with flower and epidermis. We infer that the presence of phenolics in these two extracts confer them the property to scavenge the formation of H2O2 radical.

 

 

 

Metal chelating activity:

In this study it was noticed that as the concentration of the extracts increased the metal chelating activity was also increased significantly in epidermis compared to gel and flower (Fig. 2d). The IC50 values in (Table 3) indicate that the epidermis has better metal chelating activity (0.295±0.12mg/mL) compared to flower (p ≤ 0.001) and gel extracts (p ≤ 0.001) respectively. From the results it was observed that the epidermis extract has shown better anti-oxidant potential followed by gel and flower as indicated by the better IC50 values shown in (Table 3). In this assay all the three extracts interfered with the formation of ferrous complex with ferrozine, suggesting that they have chelating activity and capture the ferrous ion before ferrozine. Our study also corroborates with other study reporting that the presence of phenolic compounds in plant extracts has good capacity for iron chelating activity suggesting its action as an antioxidant28. In this study by different anti-oxidant assays, epidermis extract has shown better anti-oxidant potential compared to gel and flower extracts.

 

DNA Protection Assay:

In the DNA damage protection assay, it was noticed that the epidermis extract at a lower concentration of 20μg/ml was able to protect the pRSETBplasmid DNA damage against UV and 30mM H2O2 treatment compared to the gel and flower extracts which required a higher concentration of 80μg/ml to protect the plasmid DNA (Fig.3). The pRSETBplasmid DNA in the presence of H2O2and UV converted to open circular and linear form (lane 4) compared to the control lane (lane 1). However, in the presence of gel, epidermis and flower extracts, plasmid DNA is protected from the damages taking place in the presence of UV and H2O2.The presence of different phenolic constituents present in all the three extracts which was analyzed by RP-HPLC may suppress the formation of linear plasmid DNA and protected damage. The DNA protection assay also demonstrated the presence of strong antioxidant properties of Aloe vera epidermis, gel and flower extracts. We observed that there is a correlation between the antioxidant activity and phenolic constituents which are mainly responsible for the antioxidant activity in all the extracts of Aloe vera.

 

Table 3: IC50 values of Aloe vera gel, epidermis and flower extracts by different antioxidant assays.

Extracts

ABTS

DPPH

H2O2

Metal chelating

Gel

0.257±0.06

0.688±0.05

0.425±0.09

0.945±0.35

Epidermis

0.170±0.05

0.578±.05

0.318±0.03

0.295±0.12

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)


 

 

 

Fig. 2: Anti-oxidant acitivity of Aloe vera gel, epidermis and flower extracts by different assays. a)ABTS assay b) DPPH assay c) H2O2 assay and d) metal chelating acitivity. Values expressed as mean±SD. All experiments are done in triplicates.

 


 

Fig. 3: Effect of gel, epidermis and flower extracts on the protection of DNA damage induced by UV and H202

 

M: Marker- (DNA ladder lane 1), C: control (pRSETBPlasmid DNA at concentration of 100ng per lane) (lane 2), U: UV treated (lane 3), H: 30mM H2O2treated (lane 4), UH: UV+ 30mM H2O2 treated (lane 5), G: Gel extract (80μg/ml)  treated with UV+ 30mM H2O2(lane 6), E: Epidermis extract(20μg/ml) treated with UV+ 30mM H2O2 (lane 7), F: Flower extract(80μg/ml)  treated with UV+ 30mM H2O2 (lane 8), S: Standard Quercetin treated with UV+ 30mM H2O2 (lane 9),

 

α-amylaseandα-glucosidase inhibition activities:

The α-amylase inhibition activity of methanolic extracts of gel, epidermis and flower were evaluated at different concentrations (0.1, 0.25, 0.5, 1.0 and 2.0 mg/mL) as shown in (Fig. 4a). α-amylase and α-glucosidase are the main carbohydrate hydrolyzing enzymes responsible for postprandial hyperglycemia in diabetes. Amylase hydrolyses the 1,4-glycosidic linkage of polysaccharides to disaccharides while α-glucosidase converts the 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 flower extract (Table 4) (Fig. 4b). Phenolic compounds and flavonoids present in the plants have known to possess anti-diabetic properties29. Our study has also shown the presence of phenolics such as quercetin, luteolin, myrcetinetc in gel and flower extracts. These phenolics may play a role in inhibiting the α-amylase and α-glucosidase activity thereby contributing to the anti-diabetic property of Aloe vera gel and flower5,29.It was observed that though the epidermis is rich in constituents such as polyphenols, flavonoids, chromones, anthrones, anthraquinones, benzyl derivatives10 which are known to possess anti-oxidant potential did not show any significant anti-diabetic potential compared to gel and flower extracts. In addition to these constituents the gel and flower extracts has also been reported for the presence of phyto-constituents such as carbohydrates, proteins, phytosterols, mineral components2,10. These phytoconstituents present in gel of Aloe vera may play a role in antidiabetic activity30-31. It may be also due to the presence of sugars, proteins, phytosterols or the synergetic effects of the all these constituents. The gel and flower extracts has shown better anti-diabetic potential as well as anti-oxidant potential. However, in epidermis extract it may be due to the presence of more amounts of chromones, anthrones which masks the phenolics and flavonoids. This may be one of the reason that the epidermis extract did not show any significant anti-diabetic property in spite of having better anti-oxidant potential.

 

DPP-IV inhibition activity:

Glucose levels are regulated by one of the pathways is the incretin pathway. The GLP-1 (Glucagon like peptide-1) helps in release of insulin from pancreas. However, the GLP-1 is inhibited by DPP-IV enzyme after a period of one and a half minutes. Inhibiting the DPP-IV enzyme can aid in prolonged action of GLP-1 leading to secretion of insulin and lowering of glucose levels during diabetic condition. In our study, the gel and flower extract has shown significant inhibition activity (Fig. 4c) whereas the epidermis extract did not show any significant activity. It was observed that gel has better potential to inhibit the DPP-IV enzyme compared to the flower extract (p ≤ 0.001) (Table 4)this was correlated with IC50 values. Our results correlates with previous study14that the gel extract showed significant DPP-IV inhibition with better IC50.Plant phenolics and flavonoids has also been reported to possess DPP-IV inhibition property in alleviation of diabetes mellitus32-33. The presence of the various phenolics in gel and flower extracts (Table 2) may contribute in inhibiting the DPP-IV enzyme.

 

 

 

In vitro anti-glycation activity:

The glucose levels can also be controlled through inhibiting the formation of glycated products seen in patients suffering from diabetes mellitus24.The formation of advanced glycation end (AGE) products was observed by measuring the fluorescence intensity of BSA-Glucose solutions for a period of 9 days. There was a significant increase in AGE’s formation when BSA was incubated with glucose. Both the gel and flower extracts were able to inhibit the formation of glycated products significantly (Fig. 4d). There was no significant activity in the methanolic extract of epidermis. The IC50 values in (Table 4) indicate the gel has better anti-glycation activity when compared to flower (p ≤ 0.001). This may be due to the presence of phenolics and flavonoids in these extracts which may play a role in inhibiting the AGEs formation thereby controlling the sugar levels. Studies have been reported with the plant phenolics and flavonoids having anti-glycation properties27. It has been suggested that the anti-glycation property of plants correlate with the scavenging of free radicals34. In this study it indicates that the presence of phenolics in gel and flower may contribute anti-glycation property through decrease of free radicals formed through auto-oxidation of sugars.

 

Table 4: IC50 values of Aloe vera gel, epidermis and flower extracts by different anti-diabetic assays.

Extracts

α-amylase inhibition

α-glucosidase inhibition

DPP-IV inhibition

Anti-glycation

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.


CONCLUSION:

In conclusion, the present comparative study of Aloe vera gel, epidermis and flower extracts showed significant antioxidant and scavenging activities of DPPH, ABTS, H2O2, metal chelating activity and protected the DNA damage. In addition, the gel and flower showed antidiabetic properties by inhibiting alpha amylase, alpha- glucosidase, DPP-IV enzymes and formation of glycation products. The presence of these different polyphenols and flavonoids in these extracts contribute to their antioxidant and anti-diabetic properties. These extracts might be more effective to reduce or delay the diabetic complications subjected to further in vivo evaluation. Further studies are needed to characterize the active components and their pharmacological properties so that they may be used as nutraceuticals.

 

ACKNOWLEDGEMENT:

Authors thank Department of Science and Technology (IR/SO/LU-03/2004), New Delhi, India for the financial support.

 

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

 

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Received on 25.10.2018         Modified on 27.11.2018

Accepted on 31.12.2018         © RJPT All right reserved

Research J. Pharm. and Tech. 2019; 12(4):1761-1768.

DOI: 10.5958/0974-360X.2019.00295.6