Model Aren Vinegar (Arenga pinnata Merr.) Phytochemical Analysis

and Hypoglycemic Effects in Streptozotocin-Niacinamide-Induced

Rats

Putu Indrayoni

, I Gusti Bagus Teguh Ananta

and Anak Agung Istri Mas Padmiswari

Bachelor of Clinical and Community Pharmacy, Faculty of Health, Institute of Technology and Health Bali, Indonesia

Applied Bachelor of Acupuncture and Herbal Medicine, Faculty of Health, Institute of Technology and Health Bali,

Indonesia

Keywords: Streptozotocin, Niacinamide, Hypoglycaemic Agent, Fermented Food.

Abstract: Fermented foods or drinks have potentials to prevent type 2 diabetes. Vinegar is a fermented product that has

been used empirically for treating various diseases, including DM. The use of aren vinegar is rarely

investigated. Meanwhile, aren vinegar as a hypoglycaemic agent has not been widely explored. This study

aimed to determine the phytochemical content and the effect of aren vinegar on hyperglycaemia conditions.

A total of 30 rats, divided into three treatment groups, were used in this study. The hyperglycaemia model

was obtained by the intraperitoneal induction of Streptozotocin-Niacinamide in male Wistar rats.

Phytochemical analysis shows that aren vinegar contains flavonoid compounds (1.03 mg/100 g QE) and

phenolic compounds (111.62 mg/100 g GAE). After receiving aren vinegar for four weeks, blood glucose

levels in hyperglycaemic mice dropped by 3.21%. One-way ANOVA and post hoc LSD tests used in the

statistical analysis of blood glucose levels show a significant difference between the aren-vinegar group and

the control group (p < 0.001). Aren vinegar has a hypoglycaemic effect as it produces flavonoid and phenol

compounds.

1 INTRODUCTION

Diabetes mellitus (DM) is a global health issue

causing a rapidly increasing prevalence. The

International Diabetes Federation (IDF) organization

estimated that at least 463 million people aged 20–79

years in the world suffered from diabetes in 2019,

equivalent to a prevalence rate of 9.3% of the total

population at the same age. Indonesia itself is ranked

7th among the ten countries with the highest number

of diabetes prevalence (Pangribowo, 2020). DM is a

metabolic disorder resulting from interrupted insulin

secretion, insulin action, or both. Diabetes is the state

of high plasma glucose levels (fasting plasma glucose

(FPG) of 126 mg/dL) (Fakhruddin et al., 2017) or

blood sugar levels two hours after eating (2-h PG) of

200 mg/dL during an OGTT (oral glucose tolerance

test) or an A1C of 6.5% (48 mmol/mol). Diabetes is a

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condition where a hyperglycemic crisis occurs with

randomized plasma glucose of 200 mg/dL (11.1

mmol/L) (American Diabetes Association, 2020).

Insulin deficiency triggers chronic hyperglycemia

with disturbances in carbohydrate, fat, and protein

metabolism (Pizzino et al., 2017). Factors that

contribute to hyperglycemia are decreased insulin

secretion, decreased glucose utilization, and

increased glucose production. Lifestyle interventions

for risk prevention of type 2 diabetes have been

explored in several studies. One of the lifestyle

interventions, such as the use of fermented foods, is

known to have a good effect on health.

Vinegar is a fermented food product that contains

sugar. One of the plants that produces vinegar is the

Aren plant (Arenga pinnata Merr.). Aren contains

various secondary metabolites and has an antioxidant

activity. Aren roots contain flavonoids, alkaloids,

Indrayoni, P., Ananta, I. and Padmiswari, A.

Model Aren Vinegar (Arenga pinnata Merr.) Phytochemical Analysis and Hypoglycemic Effects in Streptozotocin-Niacinamide-Induced Rats.

DOI: 10.5220/0011939200003576

In Proceedings of the 2nd Bali Biennial International Conference on Health Sciences (Bali BICHS 2022), pages 64-68

ISBN: 978-989-758-625-5

 2023 by SCITEPRESS – Science and Technology Publications, Lda. Under CC license (CC BY-NC-ND 4.0)

steroids, tannins, saponins, anthraquinones, and

terpenoids (Zainudin et al., 2015). The ethanol extract

of aren seeds is known to contain flavonoids,

triterpenoids, saponins, and tannins (Arief et al.,

2017).

Tapping male flower sap of aren plants can

produce sugar, which can turn into vinegar, drink and

alcohol (Lempang, 2012). The fermentation of sap

into aren wine generally lasts for a day with a mix of

yeast (Saccharomyces) and bacteria such as

Lactobacillus (Mussa, 2014). In aren wine, lactic

alcohol acetate fermentation occurs spontaneously by

involving lactic acid bacteria, yeast, and acetic acid

bacteria (Pradnyandari et al., 2017). Vinegar is

produced by the spontaneous fermentation of aren sap

over time.

Vinegar has been proven to lower blood glucose.

Types of vinegar used for diabetes treatment are

produced by salak, apple, grape, pineapple skin, sugar

cane, coconut, and palm (Soltan & Shehata, 2012;

Gheflati et al., 2019; Hermawati, Sitasiwi, Jannah,

2020). Vinegar has a glucose-lowering effect on

patients with glucose abnormalities, but the

mechanism of this effect is unclear. Although some

studies confirm that aren vinegar is practically useful,

further investigation on whether aren vinegar is

potential as a hypoglycemic agent has not received

much attention. Therefore, this study aimed to

ascertain the phytochemical composition, antioxidant

capacity, and hyperglycemic effects of aren vinegar.

The chemical content of aren vinegar was determined

through phytochemical screening. The DPPH method

was used to identify antioxidant activity in aren

vinegar, while the effect of hypoglycemia was

determined by measuring the blood glucose of

experimental animals. This study used hyperglycemic

albino Wistar rats that were given aren vinegar for 28

days. The last day of the experiment was the period

of measuring the rats’ blood glucose levels.

2 MATERIALS AND METHODS

2.1 Materials and Chemicals

Aren sap is obtained from Buleleng district, Bali. The

fermentation of sap into vinegar was carried out

spontaneously at a room temperature. The reagents

used included Folin-Ciocalteau's, methanol (E.

Merck), CaCO3, AlCl3, acetate buffer, gallic acid (E.

Merck), quercetin, Streptozotocin (Sigma-Aldrich),

niacinamide (Sigma-Aldrich), citrate buffer, and

aqueous solution NaCl 0.9%.

2.2 Phytochemical Screening

Alkaloids, saponins, flavonoids, phenols, and amino

acids are the phytochemicals in aren vinegar under

investigation. Using Mayer's reagent, the alkaloid

content was determined by looking at the appearance

of a white precipitate which indicated the presence of

alkaloids. When the sample was shaken for 15

minutes, the procedure produces 2-cm foam that

contained saponins. The presence of flavonoid

content was determined using the alkaline reagent

assay. While the presence of protein and amino acid

content was assessed using the ninhydrin test, the

phenol content test was conducted by administering

FeCl3 solution (De Silva et al., 2017).

The overall phenol and flavonoid in the vinegar

were measured using Widodo's method with slight

modifications (Widodo, Sismindari, Asmara, &

Rohman, 2019). A 40 μl of vinegar (1 mg/ml; 1 mg

dissolved in 1 ml methanol) was mixed with 360 μl of

distilled water; 100 μl of Folin-Ciocalteau and the

solution were shaken and left for two minutes. The

reaction was neutralized using 500 μl of 10% CaCO3

and mixed until the solution was homogeneous. The

mixture was incubated for 20 minutes at 40°C. A 150-

μl test solution was included in the microplate, and

the absorbance was measured at a wavelength of 732

nm. The total phenol content was expressed as mg of

gallic acid equivalent to g of the extract through linear

regression prepared from gallic acid at various

concentrations (0, 5, 10, 15, 20, and 25 μg/ml).

The total flavonoid content was measured by a

mixture of 100 μl vinegar, 150 μl solution of 0.1 M

AlCl3 (blank without AlCl3 and replaced with

methanol 150 μl), 350 μl of aquadest, 250 μl acetate

buffer (pH 3.8), and added with methanol up to a total

volume of 1,250 μl. The test solution was incubated

at 35°C for 30 minutes, and the absorbance was

measured with a UV-Vis spectrophotometer at 435

nm. Total flavonoid content was expressed as

quercetin equivalents per g extract by generating a

standard curve with a series of concentrations from 0

to 100 μg/ml of quercetin.

2.3 Antioxidant Activity

The 0.4 mM DPPH solution was obtained by

dissolving 15.8 mg of DPPH in 100 mL of methanol,

and 1 mL of the solution was taken. It was mixed with

4 mL of the extract. The standard blank solution used

methanol and quercetin. The test solution was shaken

and remained to stand at a room temperature for 30

minutes (Permatasari et al., 2019). The absorbance

was observed at a wavelength of 515.5 nm using a

Model Aren Vinegar (Arenga pinnata Merr.) Phytochemical Analysis and Hypoglycemic Effects in Streptozotocin-Niacinamide-Induced

Rats

UV-visible spectrophotometer (Biochrome SN

133467).

2.4 Hyperglycemia Induction

The hyperglycaemia model was obtained through the

induction of streptozotocin-niacinamide according to

the Furman method (2015). Niacinamide was

dissolved in a 0.9% NaCl solution to a concentration

of 230 mg/ml. As much as 32.5 mg streptozotocin

(STZ) was put in a microcentrifuge tube and covered

with aluminum foil. Next, inject niacinamide i.p at a

dose of 230 mg/kg (1 ml/kg). Niacinamide injection

was performed for 15 minutes before STZ

administration. After niacinamide injection, STZ

solution was immediately dissolved in 50 mM sodium

citrate buffer (pH 4.5) at a concentration of 32.5

mg/ml. Then the STZ solution was injected i.v. at a

dose of 65 mg/kg (2 ml/kg).

2.5 Determination of Rat Blood

Glucose Levels

This study used 24 male albino Wistar rats weighing

150–200 grams. The inclusion criteria of the

experimental animals were rats with blood glucose

levels of > 150 mg/dl and rats without any anatomical

abnormalities. Sick mice, those with passive

movement, and those died during treatment were

excluded from the samples. The blood glucose levels

of experimental animals were determined by the

glucose oxidase biosensor method using a

commercial glucometer kit. Measurements were

made after the rats had fasted for 12 hours. The rats’

blood samples were taken from the lateral tail vein

and dripped into a glucometer strip.

3 RESULTS

The results of the phytochemical screening of aren

vinegar are shown in Table 1 and 2.

Table 1: Phytochemical screening of aren vinegar.

Content Test Reagent Results

Fenol FeCl

Amino acid/protein Ninhydrin -

Flavonoid Alkaline reagent +

Alkaloid Mayer -

Saponin -

Table 2: Total flavonoid, total phenol and DPPH test of aren

vinegar.

Average

Flavonoid

Content

(mg/100g QE)

Average

Phenol

Content

(mg/100 g

GAE)

IC 50 Antioxidant

Activity

1,03 111,62 7.879,43 Wea

The hypoglicemic activity of aren vinegar is shown in

table 3.

Table 3: Blood glucose levels.

Grou

I II III

Average

Blood

Glucose

Levels

(mg/dL)

Day 0 267.5 ±

26.30

233.75 ± 34 99 ±

16.59

Day 7 255.75 ±

38.46

230.75 ±

29.81

101.75

± 15.88

Day 14 250.75 ±

36.18

230.25 ±

30.58

103.25

± 16.5

Day 21 245.75 ±

35.76

229 ± 28.90 105.5 ±

16.30

Day 28 235.75 ±

30.48

226.25 ±

30.99

105.75

± 15.84

Differences

in Blood

Glucose

Levels

(

)

11.87 3.21 6.82

Group I: group of hyperglycemic rats given glibenclamide

Group II: group of hyperglycemic rats given 1 mL of aren

vinegar

Group III : a group of normal rats given aquadest

4 DISCUSSION

Phytochemical screening shows the presence of

phenolic and flavonoid compounds in aren vinegar

(Table 1). An alkaline reagent test was used to

determine the presence or absence of flavonoids in

aren vinegar. Aren vinegar dripped with NaOH

became a solution with an intense yellow color. Then,

it became colorless when added with dilute acid,

indicating the presence of flavonoids. The phenol

content test was carried out by dripping FeCl3

solution, and the appearance of a bluish-black color

indicated the presence of phenol. The continuation of

the phytochemical screening was aimed to determine

the levels of total flavonoids and phenols in aren

vinegar. In addition to determining the total levels of

Bali BICHS 2022 - The Bali Biennial International Conference on Health Sciences

flavonoids and phenols, the antioxidant activity in

aren vinegar was weak according to the IC50 test

results.

Aren contains various secondary metabolites and

have antioxidant activity. Aren sap contains water,

carbohydrates, ash, protein, fat, and organic acids

(citric, tartaric, malic, succinic, lactic, fumaric, and

pyroglutamic acids) (Karouw and Lay, 2006).

Spontaneous fermentation of aren sap from time to

time will produce vinegar, with acetic acid as the

main component.

The aren-vinegar group experienced a decrease in

blood glucose levels. Vinegar might serve as a

protective measure to avoid excessive body weight

gains and high plasma concentrations of glucose,

triglycerides and cholesterol (Dios Lozano et al.,

2012). Vinegar ingestion may enhance satiety (Darzi

et al., 2013).

Glucose metabolism was likely affected by the

consumption of vinegar. Many studies on vinegar

consumption support this finding. For instance, the

study of Hu et al. (2020) found that butyric acid and

acetic acid can increase islet and beta cell viability.

At a concentration of 1 mM, these two short-chain

fatty acids can stop apoptosis, decreased viability,

mitochondrial dysfunction, and the overproduction of

ROS and NO caused by streptozotocin. According to

Gheflati et al. (2019), individuals with diabetes and

dyslipidaemia who took apple cider vinegar had a

lower glycaemic index and less oxidative stress.

According to a study by Soltan and Shehata (2012),

diabetic rats were benefited from receiving several

vinegars for six weeks, including apple cider vinegar,

wine vinegar, cane vinegar, coconut vinegar, palm

vinegar, and artificial vinegar. These types of vinegar

also have a hhypocholesterolaemia effect.

5 CONCLUSIONS

In conclusion, aren vinegar contains flavonoid and

phenolic compounds, while its antioxidant activity is

classified as weak. Aren vinegar also has a

hypoglycaemic activity. Further testing is needed to

determine the chemical content of aren vinegar and

its mechanism in lowering blood glucose levels.

ACKNOWLEDGEMENTS

The authors would like to thank the Institute

Technology and Health Science Bali (ITEKES Bali)

for giving financial support.

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