Download - Antidiabetic property of Aerva lanata (L.) Juss. ex Schult. is mediated by inhibition of alpha glucosidase, protein glycation and stimulation of adipogenesis 软毛白花苋 (L.)的降糖特性是通过对α葡萄糖甙酶与蛋白质糖基化的抑制以及脂肪生成的刺激达成的

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Antidiabetic property of Aerva lanata (L) Juss. ex Schult. is mediated by inhibition of alpha

glucosidase, protein glycation and stimulation of adipogenesis 1

Mariam Philip RIYAa, Kalathookunnel Antony ANTU

a, Savita PAL

b, Karuvakandy

Chandrasekharan CHANDRAKANTHa, Karunakaran Sasikala ANILKUMAR

c, Akhilesh Kumar

TAMRAKARd, Arvind Kumar SRIVASTAVA

b, Kozhiparambil Gopalan RAGHU

a*

aAgroprocessing and Natural Products Division, Council of Scientific and Industrial Research -

National Institute for Interdisciplinary Science and Technology (CSIR-NIIST),

Thiruvananthapuram, Kerala, India, Pin-695019.

bDivision of Biochemistry, CSIR - Central Drug Research Institute (CDRI ), Lucknow, Uttar

Pradesh, India, Pin-226001.

cMedicinal Chemistry division, CSIR-CDRI, Lucknow, Uttar Pradesh, India, Pin-226001.

dDivision of Pharmacology, CSIR-CDRI, Lucknow, Uttar Pradesh, India, Pin-226001.

*For

correspondence: Dr. K. G. Raghu, Agroprocessing and Natural Products Division,

Council

of Scientific and Industrial Research - National Institute for Interdisciplinary Science and

Technology (CSIR-NIIST), Thiruvananthapuram, Kerala, India, 695019. Tel: +919495902522,

Fax: +91-471-2491712/2491585.

Email: [email protected], [email protected]

Running title: Basis of antidiabetic property of Aerva lanata

This article has been accepted for publication and undergone full peer review but has not been through the

copyediting, typesetting, pagination and proofreading process, which may lead to differences between this

version and the Version of Record. Please cite this article as doi: 10.1111/1753-0407.12216

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Abstract

Background: Diabetes is the leading cause of morbidity and mortality, with a number currently

diagnosed as high as 371 million. Plant-based therapy could be an ideal choice because of fewer side

effects and wider acceptability. Hence, the antihyperglycemic potential of Aerva lanata, a herb

prescribed for diabetes in Ayurveda was evaluated to elucidate its possible mechanism of action.

Methods: HPLC analysis was employed for the characterization of 70% ethanolic (ALE) and ethyl

acetate (AEA) extracts. Further, they were evaluated for their antioxidant, alpha glucosidase

inhibition, protein glycation inhibition, dipeptidyl peptidase IV (DPP IV), protein tyrosine

phosphatase 1B (PTP1B), glucose uptake and glitazone like property (adipogenic potential) using in

vitro models. The promising alpha glucosidase inhibitory potential of ALE was further evaluated in

normal and streptozotocin (STZ) diabetic rats.

Results: ALE inhibited yeast (IC50- 81.76 μg/mL) and rat intestinal alpha glucosidase (IC50- 108.7

μg/mL), protein glycation, DPP IV enzyme (IC50- 118.62 μg/mL) and PTP1B (IC50- 94.66 μg/mL).

ALE stimulated maximal adipogenesis at 50 μg/mL and enhanced insulin mediated glucose uptake

(3 fold of basal) at 100 μg/mL in L6 myotubes. ALE (500mg/kg b.w) showed a significant

antihyperglycemic activity in sucrose loaded STZ normal (15.57%) and diabetic (18.44%) rats.

HPLC analysis of ALE revealed the presence of bioactives like alpha amyrin, betulin and beta

sitosterol.

Conclusions: Alpha glucosidase inhibition, antiglycation, and adipogenic potential significantly

contribute to the antidiabetic property of Aerva lanata. In addition, insulin sensitization and

antioxidant potential also enhance its therapeutic potential.

Significant findings of the study: Acc

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Antihyperglycemic potential of ALE in STZ diabetic rats via alpha glucosidase inhibition,

enhanced glitazone like potential in 3T3-L1 and in vitro antiglycation potential.

Promising glucose uptake in L6 myotubes, DPP IV and PTP1B inhibition.

What this study adds:

Alpha glucosidase inhibition, protein glycation and enhanced glitazone like potential are the key

mechanisms responsible for the antidiabetic potential of Aerva lanata.

In vitro studies have also shown the possible contribution of other associated mechanisms to its

therapeutic efficacy.

Key words: Aerva lanata, antioxidant, diabetes, dipeptidyl peptidase IV, protein tyrosine phosphatase 1B

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Introduction

Diabetes is a chronic metabolic disorder characterized by insulin resistance, impaired insulin

secretion and hyperglycemia1. Elevated postprandial glycaemia (PPG) is an important indicator of

pre-diabetes and hence is a valuable tool for its early diagnosis2. Persistent hyperglycemia is

associated with irreversible damage to eyes, kidneys, nerves and blood vessels. So targeting PPG

significantly aid in reducing the rapid onset of diabetic complications. Currently available drugs

include inhibitors of alpha glucosidase3, DPP IV

4, 5 and sensitizers like metformin

6 and

rosiglitazone7. PTP1B is an emerging drug target and its inhibition can improve insulin

sensitivity8. But, the cost of the prescribed drugs and their undesirable side effects (due to their

long-term use), emphasis the need for a traditional plant-based therapy.

Aerva lanata, commonly known as bhadrika in Sanskrit, is a prostrate decumbent, sometimes

erect herb, widely used in Ayurveda for the treatment of diabetes mellitus9, 10

. It is also consumed

as a vegetable in many of the Asian and African nations11, 12

. Pharmacological studies have shown

that Aerva lanata also possess diuretic, anti-lithiasis, anti-inflammatory, antimicrobial, antitumour

and antioxidant_ENREF_13 properties13,14

. The antidiabetic potential of aerial part and root have

been previously reported13, 15

. However, there has been no detailed study employing currently

explored drug targets for diabetes. In the present study, we have evaluated Aerva lanata (whole

plant) to unveil its possible mechanism of action using in vitro and in vivo models.

Methods

Plant material and preparation

Aerva lanata (L) Juss. ex. Schult. was collected from Thiruvananthapuram and identified by Dr

H. Biju, Taxonomist from the Jawaharlal Nehru Tropical Botanical Garden Research Institute

(JNTBGRI), Palode, Thiruvananthapuram, Kerala. After identification, voucher specimen (No. Acc

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66499) was deposited at JNTBGRI herbarium, for future reference. One kilogram of fresh whole

plants were air dried and sequentially extracted using different organic solvents (3.5-4 litres) in

their increasing order of polarity i.e. hexane (AHE), dichloromethane (ADE), ethyl acetate (AEA)

and 70% ethyl alcohol (ALE) until colourless. The resulting solvent fractions were concentrated

in a rotary evaporator (Laborota4010; Heidolph, Schwabach, Germany) at 40-45 °C. They were

lyophilized and stored at 4 °C until use.

Cell-culture and treatment

All cells were cultured at 37 °C in a humidified 5% CO2, 95% air atmosphere. HepG2 (human

hepatocellular carcinoma), L6 myotubes, 3T3-L1 murine preadipocytes were obtained from

ATCC (Manassas, VA, USA). HepG2 was maintained in low glucose (5.5mM) Dulbecco’s

Modified Essential Media (DMEM, Invitrogen, Carlsbad, CA, USA) supplemented with 10%

FBS and 1% antibiotic-antimycotic solution (10,000 U/mL). L6 myotubes were maintained in

alpha-MEM (Invitrogen, Carlsbad, CA, USA) supplemented with 10% FBS and 1% antibiotic-

antimycotic solution. Experiments were performed in differentiated myotubes (DMEM with 2%

FBS) 6-7 days after seeding. 3T3-L1 preadipocytes were cultured in DMEM (25 mM glucose)

supplemented with 10% FBS and antibiotics. Differentiation into adipocytes was induced by

switching to DMEM with 500 μM 3-isobutyl-1-methylxanthine, 10 μM dexamethasone and 500

nM insulin (Sigma, St Louis, MO, USA) for 48 h. Cells were then maintained in DMEM

containing 10% FBS and 500 nM insulin for 8 days.

Determination of cell viability

Cells were seeded at a density of 4 × 104 cells/well in 24 well plates and incubated for 24 or 48 h.

Cells were subsequently incubated with various concentrations (10, 50, 100 and 200 μg/mL) of Acc

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the extracts for 24 h or 48 h. Cell viability was evaluated using MTT assay kit (Cayman, USA).

Further cell line studies were based on cytotoxicity experiment.

Animals

Male albino Sprague Dawley (SD) rats (160±20 g, 7-8 week old) bred at animal facility of Central

Drug Research Institute of India, Lucknow were utilized for this study. All the procedures

complied with the guidelines of Committee for the Purpose of Control and Supervision of

Experiments on Animals (CPCSEA) formed by the Government of India in 1964 (Ethics

committee reference number IAEC/2008/63/Renewal 04 dated 16.05.2012). Rats were housed in

polypropylene cages under an ambient temperature of 23 ± 2 °C; 50-60% relative humidity; light

300 lux at floor level with regular 12 h light/dark cycle. Animals were maintained on a standard

pellet diet and water was provided ad libitum.

Chemical composition

Analysis of fibre and mineral content

Crude fibre was estimated by adopting the official method of analysis by Association of

Analytical Chemists (AOAC)16

. The mineral constituents were determined by digesting 1 g of the

sample in a mixture of nitric acid and hydrochloric acid (1:3)16

and quantified by atomic

absorption spectrophotometer (AAS; Perkin Elmer A Analyst 100; Perkin Elmer, MA, US).

Results are expressed as mg/g of sample.

Quantification of total phenolic, flavonoid and tannin content

Total phenolics17

, flavonoid18

and tannin content19

were determined spectrophotometrically.

Results were expressed as gallic acid equivalents/g extract (mg GAE/g) for total phenolics,

quercetin equivalents/g extract (mg QE/g) for total flavonoid and tannic acid equivalents/g extract

(TAE/g) for total tannin content.

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High-performance liquid chromatography (HPLC)

Betulin, beta sitosterol and alpha amyrin (Sigma, St Louis, MO, USA) were identified and

quantified using HPLC. Chromatographic separation was performed using Agilent HPLC 1100

series equipped with C-18 column (150 x 4.6, 5 µm). Acetonitrile: water in the ratio of (30:70)

v/v was used as the mobile phase at a constant flow rate of 1.0 mL/min. Peaks were detected at

220 nm with UV Detector.

Determination of antioxidant potential

The antioxidant activity was measured by DPPH20

and ABTS21

method. Percentage of radical

scavenging activity obtained was plotted against the corresponding concentration of the extract to

obtain IC50 value. Gallic acid for DPPH and Trolox for ABTS served as standards.

Measurement of hyperglycemia induced intracellular reactive oxygen species (ROS)

Cells were maintained in low glucose medium for 24 h, and then switched to high glucose (25

mM) medium with or without the extracts or quercetin for another 24 h. Treated cells were loaded

with H2DCFDA (10 μM)22

in serum free medium for 30 min at 37 °C. Subsequently the cells

were rinsed with prewarmed PBS and fluorescence intensity was measured (λex 490 nm; λem 525

nm). In addition, fluorescent images were also captured using BD 855 bio-imaging system (USA).

Alpha glucosidase inhibition

Alpha glucosidase (EC 3.2.1.20) inhibitory activity was assessed using yeast alpha glucosidase

and rat intestinal enzyme as reported elsewhere23

. Percentage inhibition was plotted against the

corresponding concentration to obtain the IC50 value. Acarbose served as the reference standard.

In vivo study

Induction of diabetes Acc

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Diabetes was induced by a single intraperitoneal injection of STZ at 60 mg/kg b.w (Sigma, St

Louis, MO, USA) dissolved in 0.1 M citrate buffer, pH 4.5. After 72 h, induction of diabetes was

confirmed and animals with blood glucose >270mg/dL were included in the study24,25

.

Oral sucrose tolerance test (OSTT) in normoglycemic and STZ diabetic rats

Both normal and diabetic rats fasted overnight were divided into 5 groups (n = 6 per group).

Animals of control and experimental groups were administered suspension of the vehicle or ALE

(100, 250 & 500 mg/kg b.w in 1.0% gum acacia)/acarbose (100 mg/kg b.w) orally by a gavage

needle (18-gauge, 38 mm long curved, with a 21/4 mm ball end). Sucrose load of 10 g/kg b.w for

normoglycemic and 2.5 g/kg b.w for diabetic rats was given 30 min after administration of the

vehicle/ALE/acarbose. Blood was collected from the tail vein and glucose levels were determined

using glucometer (Accu-check, Roche Diagnostics, USA) at 30 min interval for 2 h after the

sucrose load in normoglycemic24

and at 0, 30, 60, 90, 120, 180, 240 and 300 min respectively in

diabetic rats24, 26

. Percentage antihyperglycemic activity was calculated by comparing the area

under curve (AUC) of experimental and control groups.

Determination of antiglycation activity

BSA derived advanced glycation end products (AGEs) were measured as previously reported 27

with slight modifications. BSA (10 mg/mL) in the presence of ribose (500 mM) in phosphate

buffered saline (PBS) was used as control. Quercetin (100 μM) served as the reference standard.

AGE fluorescence (λex370 nm; λem 440 nm) was measured (Biotek, USA) after 24 h. Samples were

analysed for changes in the complexity of glycated product on EVO 18 special edition model of

Carl Zeiss (Carl Zeiss, Munich, Germany) scanning electron microscope28, 29

. All images of

samples were captured at 16K x magnification for comparison.

DPP IV and PTP 1B inhibitory potential Acc

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DPP IV (EC 3.4.14.5) was measured using commercially available kit from Cayman (Ann Arbor,

Michigan, USA). PTP1B (EC 3.1.3.48) was measured using commercially available kit from

Calbiochem, Darmstadt, Germany). Diprotin A for DPP IV and suramin for PTP1B served as

standards. IC50 value was calculated as mentioned above.

2-deoxy glucose (2-DG) uptake

2-DG uptake in L6 myotubes was performed as reported elsewhere30, 31

with slight

modification_ENREF_25. Briefly, the myotubes were incubated with extracts or standards for 16 h.

Cells were serum deprived for 3 h before the experiment. For assessment of insulin stimulated

glucose uptake, cells were stimulated with 100 nM insulin for 20 min. Basal glucose uptake was

determined without insulin stimulation. Glucose uptake was assessed after incubation in HEPES-

buffered saline containing 10 µM 2-DG (0.5 µCimL-1 2-[3H] DG) at room temperature for 5 min.

Subsequently, cells were rinsed with ice-cold solution of 0.9% NaCl and 20 mM D-glucose and

lysed in 0.05 N NaOH. Radioactivity in the lysate was determined using scintillation fluid in a β

counter (Perkin Elmer, USA). Cytochalasin B (50 µM) was used to determine non-specific

glucose uptake. Results were expressed as fold induction with respect to unstimulated cells after

normalization to the total protein content. Metformin (10 mM) served as positive control.

Adipocyte differentiation assay

Oil Red O staining and quantitation

The adipogenic potential of the extracts was tested for triglyceride content in differentiating 3T3-

L1 preadipocytes over an 8 day period32

. Cells were stained with Oil Red O and triglyceride

content was measured as previously reported33

. Rosiglitazone (10 μM) was used as positive

control. Acc

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Determination of glycerol-3-phosphate dehydrogenase (GPDH) activity, diacylglycerol O-acyltransferase 1

(DGAT1) and adiponectin

On day 8, adiponectin level in the media of different treatment groups was determined using kit

from Cayman (Ann Arbor, Michigan, USA). Cells were washed twice with PBS and harvested

into 25 mM Tris buffer (pH 7.5) containing 1 mM EDTA and 1 mM DTT. Cells were disrupted

by sonication and then centrifuged at 12,000g for 20 min at 4 °C. The supernatants were assayed

for GPDH activity according to the method of Wise and Green 34

. DGAT1 level was determined

using kit from MyBiosource.com (San Diego, California, USA). Results were normalized to

protein content using BCA method.

Statistical analysis

Results are expressed as mean ± standard error of mean (SEM). IC50 value was interpolated from

three independent experiments with triplicates using non-linear regression. Quantitative glucose

tolerance was calculated by AUC using GNU PSPP (www.gnu.org/s/pspp/). AUC of the control

and the experimental groups were compared for percentage antihyperglycemic activity. Results

were analysed by one-way analysis of variance with Dunnett post hoc test. P<0.05 was considered

to be significant.

Results

Preliminary analysis

The yield for each of the extracts is as follows: AHE (4.34%), ADE (9.83 %), AEA (14.45%) and

ALE (39.51%) Total crude fibre in the dried plant material was found to be 55%. AAS analysis

revealed presence of various minerals like potassium (24.96 mg/g), calcium (20.00 mg/g), iron

(10.24mg/g), magnesium (3.56 mg/g), sodium (1.33 mg/g) and zinc (0.02mg/g).

Phytochemical screening

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ALE has highest amount of phenolics including flavonoids and tannins followed by AEA and

ADE (Table1). HPLC fingerprint of ALE clearly shows the presence of alpha amyrin (8.74 mg/g),

betulin (5.4 mg/g) and beta sitosterol (18.3 mg/g extract) (Supplementary Fig 1). ALE and AEA

were taken further in this study.

Determination of in vitro antioxidant potential

Antioxidant potential was assessed using DPPH (IC50 value for ALE and AEA-124.24 μg/mL and

268.28 μg/mL respectively, IC50 for gallic acid - 6.5 µg/mL, Table 2) and ABTS (IC50 value for

ALE and AEA -167.91 μg/mL and 189.54 μg/mL respectively, IC50 for trolox-3.8 µg/mL, Table

2) assay. The extracts were found to be safe up to 200 μg/mL. ALE and AEA (10, 50 and 100

μg/mL) were further evaluated in cell based system (HepG2) to check their potential to prevent

hyperglycemia induced ROS generation. Hyperglycemia induced 155% increase in ROS

compared to vehicle control (Fig 1A & 1B a, b). ALE and AEA (100 μg/mL) demonstrated a

significant decrease in oxidative stress by 41% and 37% respectively compared to hyperglycemia

control. 52% decrease in ROS was observed after treatment with positive control (Fig 1A & 1B b,

c; P <0.05).

Alpha glucosidase inhibitory potential of ALE and AEA

ALE and AEA exhibited comparable dose dependent enzyme inhibition against yeast alpha

glucosidase with an IC50 value of 81.76 μg/ mL and 108.23 μg/ mL respectively (Fig 2A & B).

IC50 value of acarbose was 45 μg/mL. Both the extracts also dose-dependently inhibited rat

intestinal alpha glucosidase (IC50 value of ALE and AEA-108.7 and 208.04 μg/ mL respectively,

IC50 value of acarbose was 49.78 μg/ mL.

Antihyperglycemic potential of ALE in sucrose loaded normal and diabetic rats Acc

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Treatment with ALE at 100, 250 & 500 mg/kg b.w exhibited 10.09%, 13.57% and 15.57%

reduction in plasma glucose when compared to vehicle control (Fig 3A & B). Acarbose at 100

mg/kg b.w decreased blood glucose by 20.4% when equated to vehicle control. ALE treatment

(500 mg/kg b.w) prevented a sharp rise in postprandial blood glucose (18.44%) after 5 h of

treatment compared to STZ control (Fig 3C & 3D). Results were comparable with acarbose

treated group which showed 21.7% improvement in blood glucose profile compared to STZ

control.

Antiglycation property

Antiglycation potential was evaluated for their ability to inhibit the formation of AGEs using

fluorescence assay and scanning electron microscopy. ALE and AEA exhibited promising

antiglycation potential (IC50- 160.82 μg/mL and 268.99 μg/mL; Fig 4A). Control group showed

extremely granular agglomeration with uneven pores and complex microstructure (Fig 4B a).

ALE and AEA showed a noticeable decrease in complexity of the microstructure (Fig. 4B c-f).

Quercetin (IC50- 30.1 μg/mL) showed a significant decrease in fluorescence and complexity of

microstructure (Fig 4A & B).

DPP IV and PTP1B inhibitory potential

ALE showed promising inhibition against DPP IV enzyme in vitro. IC50 value of ALE and AEA

was 118.62 & 304.62 μg/mL respectively, whereas Diprotin A showed an IC50 value of 1.54

mg/mL (Fig. 5A). Extracts also showed promising PTP1B inhibition (IC50 value of ALE and AEA

was 94.66 & 150.73 μg/mL) respectively. IC50 value of Suramin was 14.01 µg/mL (Fig 5B).

Glucose uptake in L6 myocytes

ALE and AEA increased glucose uptake at 100 μg/mL (1.6 and 1.4 fold increase in basal glucose

uptake, Fig 6, P<0.05). 10 mM metformin which was used as standard showed 1.7 fold increase in Acc

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basal glucose uptake (Fig 6, P<0.05). The effect of ALE and AEA on insulin stimulated glucose

uptake was also investigated. Insulin alone showed a significant increase in glucose uptake (1.9

fold of basal control). Fold increase of 3 and 2.6 respectively was observed after pretreatment

with ALE and AEA (100 μg/mL) for 16 h prior to insulin stimulation (Fig. 6). 10 mM metformin

used as standard showed 3.8 fold increase in glucose uptake compared to insulin treated control

(Fig. 6, P<0.05).

Determination of glitazone like potential of ALE & AEA

Adipogenic potential of ALE and AEA was assessed using 3T3-L1 preadipocytes over an 8 day period.

ALE showed dose dependent (10, 25 and 50 μg/mL; Supplementary Fig 2) increase in adipogenesis

compared to MDI+ve (control) as evident from the phase contrast micrographs. Triglyceride content was

determined by Oil red O staining (Fig 7A & B). The GPDH activity (Fig 7C), DGAT1 (Fig 7D) and

adiponectin level (Fig 7E) also showed a significant increase after treatment with ALE. AEA did not

show any significant increase in adipogenesis for all the 3 doses (Fig 7B).

Discussion

Hyperglycemia induced oxidative stress is responsible for the early onset of secondary

complications in diabetic patients35

. Increased ROS accelerate formation of AGEs leading to

neuropathy, nephropathy and retinopathy27, 36,

36

. Therefore, therapeutic measures to reduce

hyperglycemia induced oxidative stress could be beneficial for diabetic patients. Exposure of

HepG2 cells to high glucose stress (25 mM glucose) caused a significant increase in ROS. Both

ALE and AEA reduced ROS generation compared to hyperglycemia control (Fig. 1A & B). In

addition, ALE and AEA exhibited promising scavenging potential against DPPH and ABTS

radicals (Table 2). Phenolic and flavonoid content of the extracts (Table 1) could be partly

responsible for the antioxidant potential. Acc

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Alpha glucosidase inhibitors are regarded as the first line of treatment for preventing the early-

onset of diabetes complications by suppressing postprandial hyperglycemia37, 38

. They inhibit the

action of carbohydrate digestive enzymes in the small intestine and thereby slow down the

liberation of glucose from oligosaccharides and disaccharides3, 39

. Cinnamon40

, kotalanol41

from

Salacia reticulata, triterpenes42

from Syzygium aromaticum are some of the promising alpha

glucosidase inhibitors from plants. ALE showed significant reduction in enzyme activity both in

vitro (Fig 2A & B) and in vivo (Fig 3A & B). ALE (500 mg/kg b.w) suppressed the postprandial

blood glucose AUC0-300 min in STZ induced diabetic rats, whereas control animals showed elevated

blood glucose even after 5 h of sucrose load (Fig 3A & B) which was comparable with acarbose.

Thus, ALE could be beneficial in reducing postprandial hyperglycemia via digestive enzyme

inhibition.

AGEs represent a heterogeneous group of entities resulting from non-enzymatic reaction between

reducing sugars and proteins, lipids and nucleic acids. Numerous studies have shown that build-

up of cross linked AGEs on long lived proteins accelerate secondary complications in diabetic

patients 36,43

. Aminoguanidine44

has demonstrated powerful ability to inhibit or break AGEs, but

has not been successful due to concerns regarding their toxicity in patients45

. Numerous studies

suggest that compounds with combined antiglycation and antioxidant properties could be more

beneficial. Carnosine46

, tannins in green tea47

, garcinol from Garcinia indica fruit rind48

and S-

allyl cysteine from aged garlic extract49

have been found to possess promising antiglycation and

antioxidant activity47

. Likewise, the present study shows that ALE possesses both the

antiglycation (Fig 4A & B) and antioxidant activity (Table 1, Fig 1A & B).

DPP IV inhibitors decrease hyperglycemia and improve glucose metabolism by glucose

dependent insulin secretion from pancreas50

. They prolong the action of endogenous incretin and Acc

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hence beneficial for diabetic patients51

. Results from similar studies 52

show that DPP IV

inhibition is an important target for diabetes. ALE inhibited the DPP IV enzyme significantly in a

dose dependent manner compared to AEA (Fig 5A). PTP1B inhibitors increase insulin sensitivity

by blocking the PTP1B mediated negative insulin signalling pathway53

. In the present study, ALE

exhibited significant inhibition of the enzyme over AEA (Fig 5B).

Reduced uptake of glucose in insulin sensitive tissues like skeletal muscle54

also contributes to

postprandial hyperglycemia. We have screened ALE and AEA for their ability to stimulate

glucose uptake in L6 under both basal and insulin stimulated conditions. ALE and AEA enhanced

basal glucose uptake (1.6 fold and 1.4 fold respectively at 100 μg/mL; Fig 6) compared to 10 mM

metformin (1.7 fold increase; Fig 6). Insulin stimulated control exhibited 1.9 fold increase of

glucose uptake. Pretreatment with ALE and AEA (100 μg/mL) for 16 h prior to insulin

stimulation, enhanced glucose uptake (3 fold of basal and 2.6 fold of basal; Fig 6). Results were

compared to metformin (3.8 fold of basal; Fig 6).

Lipid storage in adipocytes improve glucose homeostasis and helps prevent peripheral

lipotoxicity55

. Activators of PPAR γ like thiazolidinediones (TZDs) commonly referred as

glitazones improve insulin sensitivity by stimulating lipid accumulation in adipose leading to a

decrease in lipid content in liver and muscle56

. TZDs promote maturation and redistribution of

adipose and improve adiponectin profile. They also negates the action of TNF alpha and IL-6.

ALE showed a positive adipogenic potential (Fig 7A-E) with an increase of TG57

, GPDH34, 57

,

DGAT157

and adiponectin58

, the hall marks of adipogenesis. Siveen and Kuttan 201159

have

shown that ethanolic extract of Aerva lanata stimulate bone marrow cells, which in turn can

reduce the incidence of bone fracture observed with TZDs therapy60, 61

. Previous studies have

shown that Aerva lanata contains a number of pharmacologically active compounds such as alpha Acc

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amyrin24, 62-64

, betulin64, 65_ENREF_43, beta sitosterol

13, 63, 64, 66-69 and alkaloids like canthine-6-one

and β carboline15, 59, 70

. We have identified and quantified alpha amyrin, betulin and beta sitosterol

in ALE by HPLC (Supplementary Fig 1). We have also shown the presence of significant amount

of fibre besides minerals like potassium, zinc_ENREF_5071 and magnesium

72, 73 which also possibly

contribute to its antidiabetic potential.

Therefore, we believe that significant alpha glucosidase inhibition, antiglycation and adipogenic

potential play an important role in the antidiabetic property of Aerva lanata. In addition, moderate

antioxidant, DPP IV and PTP1B inhibition along with glucose uptake in skeletal myotubes also

possibly contribute to its efficacy. Presence of bioactives like alpha amyrin, betulin and beta

sitosterol adds to its therapeutic advantage. So, Aerva lanata deserves special attention as a

potential therapeutic/adjunct for diabetes. A detailed investigation is in process to understand its

efficacy in ameliorating diabetes induced secondary complications.

Acknowledgement

Riya MP and Antu KA acknowledge CSIR and Indian Council of Medical Research (ICMR) and

UNDO CSIR (BSC103) 12th

Five Year Plan Project for financial assistance. We thank Director,

CSIR-NIIST and CSIR-CDRI for providing necessary laboratory facilities. We also thank

Dr.H.Biju, JNTBGRI for identification of the plant material.

Disclosure

Authors declare that there is no conflict of interest.

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Figure legend

Figure 1 A & B ALE and AEA attenuated high glucose-induced intracellular ROS levels in HepG2 cells.

(A) The quantitative analysis of fluorescence from 3 doses of ALE and AEA (10, 50 & 100 µg/mL) (B)

representative microscopic scans a-e (VC - Vehicle control, HG - High glucose, Q – quercetin, HG + Q

(10µg/mL), HG + ALE (10µg/mL & 100µg/mL), HG + AEA (10µg & 100µg/mL). Values are means

with SEM of 3 independent experiments, each performed in triplicate. * denote P<0•05 vs. HG group.

Figure 2A & B Inhibition of alpha glucosidase by ALE and AEA. (A) The dose-response curve of ALE

and AEA against yeast alpha glucosidase. IC50 value of ALE (81.76 µg/mL) & AEA (108.23 µg/mL) for

yeast alpha glucosidase enzyme inhibition (B) The dose-response curve of ALE & AEA against rat

intestinal alpha glucosidase. IC50 value of ALE (108.7 µg/mL) & AEA (208.04 µg/mL) for rat intestinal

alpha glucosidase enzyme inhibition. Values are mean ± SEM of 3 independent experiments, each

performed in triplicate.

Fig 3A & B Inhibitory effects of ALE on blood glucose after sucrose loading in normal rats. (A) The

glycaemic response curve in normal rats after sucrose challenge. (B) AUC0-120 min in normal rats after

sucrose administration. Data are expressed as mean ± SEM with n=6 in each group. * denote P<0.05 vs.

control (A) The glycaemic response curve in diabetic rats after sucrose challenge (B) AUC0-300min and

AUC0-300 min in diabetic rats after sucrose administration. Data are expressed as mean ± SEM, with n=6 in

each group. *denote P<0.05 vs. control.

Figure 4 A & B AGE inhibitory potential of ALE and AEA; Ex λ370 nm, Em 440 nm, IC50 - ALE-

160.82 µg/mL, AEA- 268.99 µg/mL, quercetin 30.1 μg/mL) was used as reference (IC50- 30.11. µg/mL)

(B) Scanning electron micrographs: a-d (Control, standard compound quercetin, ALE (500 mg and 1 mg),

AEA (500 mg and 1 mg). Values are mean ± SEM of 3 independent experiments, each performed in

triplicate.

Figure 5A & B DPP IV and PTPIB inhibitory potential of ALE and AEA. (A) The dose-response curve

of ALE & AEA for DPP IV inhibition. IC50 value of ALE (118.62 µg/mL) and AEA (304.62 µg/mL). (B)

The dose-response curve of ALE and AEA against PTP IB. IC50 value of ALE (94.66 µg/mL) and AEA

(150.73 µg/mL). Values are mean ± SEM of 3 independent experiments, each performed in triplicate.

Fig 6 Effect of AEA and ALE on 2-DG uptake in L6 myotubes. Concentration dependent effect of AEA

and ALE on 2-DG uptake in L6 myotubes. Results are mean ± SEM of three independent experiments

performed in triplicate. *p < 0.05 relative to control.

Fig 7A-E Lipid accumulation in differentiating 3T3-L1 adipocytes treated for 8 days with 10, 25 and 50

µg/mL ALE or AEA or rosiglitazone (10µM) or with vehicle only (0.1% DMSO in differentiation

medium). (A) Photomicrographs and (B) Relative lipid content with Oil red O staining at 492nm. (C)

GPDH activity, (D) DGAT1 level and (E) Adiponectin was determined for ALE. Results were compared

with control (MDI+ve). Rosiglitazone (10 μM) served as positive control. Values are mean ± SEM of 3 Acc

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independent experiment, each performed in triplicate. *p < 0.05 relative to MDI-ve, #P<0.05 relative to

MDI+ve.

Table

Table 1 Total phenolics, flavonoid and tannin content in different extracts of Aerva lanata

(†ALE - ethanolic extract, ‡AHE - hexane extract, ¶ADE - dichloromethane extract, §AEA - ethyl acetate

extract. Results for total phenolics were expressed as gallic acid equivalents/g extract (mg GAE/g) or

quercetin equivalents/g extract (mg QE/g) or tannic acid equivalents/g extract (TAE/g).

Table 2 DPPH & ABTS radical scavenging activity of various extracts obtained by sequential extraction

from Aerva lanata

(†ALE-ethanolic fraction, ‡AHE-hexane fraction, ¶ADE- dichloromethane fraction, §AEA- ethyl acetate

fraction. IC50 value for DPPH and ABTS radical scavenging assay is expressed as μg/mL which indicates

its radical scavenging potential).

Supplementary Figures

Supplementary Figure 1 HPLC chromatogram (A) Betulin (r.t. -16.72 min), (B) beta-sitosterol (r.t. –

11.57 min), (C) alpha amyrin (r.t. – 4.07 min) & (D) 70 % Ethanolic extract (ALE) detected at 220 nm.

Supplementary Figure 2 Lipid accumulation in 3T3-L1 adipocytes in the presence of MDI and 10, 25

and 50 µg/mL of ALE or AEA or with vehicle only (0.1% DMSO in differentiation medium) for 8 days.

Rosiglitazone (10µM) was used as a positive control. Phase contrast micrographs of (a) MDI-ve, (b)

control (MDI+ve), (c) rosiglitazone (10 µM) and (d-i) ALE and AEA (10, 25 and 50 µg/mL) treated cells

showing lipid droplets at 10X.

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Table 1 Total phenolics, flavonoid and tannin content in different extracts of Aerva lanata

(†ALE - ethanolic extract, ‡AHE - hexane extract, ¶ADE - dichloromethane extract, §AEA - ethyl acetate

extract. Results for total phenolics were expressed as gallic acid equivalents/g extract (mg GAE/g) or

quercetin equivalents/g extract (mg QE/g) or tannic acid equivalents/g extract (TAE/g).

Table 2 DPPH & ABTS radical scavenging activity of various extracts obtained by sequential extraction

from Aerva lanata

Extracts DPPH radical scavenging

activity IC50 (μg/mL)

ABTS radical scavenging

activity IC50 (μg/mL)

ALE† 124.24 167.91

AHE‡ 150.81 178.8

ADE¶ 224.56 237.6

AEA§ 268.28 189.54

Gallic

acid

6.5 --

Trolox -- 3.8

(†ALE-ethanolic fraction, ‡AHE-hexane fraction, ¶ADE- dichloromethane fraction, §AEA- ethyl acetate

fraction. IC50 value for DPPH and ABTS radical scavenging assay is expressed as μg/mL which indicates

its radical scavenging potential).

Fractions Total phenolics (mg

GAE/g)

Total flavonoid (mg

QE/g extract)

Total tannin (mg

TAE/g extract)

ALE† 55.46 44.6 19.91

AHE‡ 21.55 15.8 --

ADE¶ 34.82 26.0 --

AEA§ 51.54 39.9 12.85

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Download - Antidiabetic property of Aerva lanata (L.) Juss. ex Schult. is mediated by inhibition of alpha glucosidase, protein glycation and stimulation of adipogenesis 软毛白花苋 (L.)的降糖特性是通过对α葡萄糖甙酶与蛋白质糖基化的抑制以及脂肪生成的刺激达成的

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