INTRODUCTION:

One marker of oxidative stress is hyperglycemia⁽¹⁾. Oxidative stress is an imbalance between free radicals and antioxidants in the body. High levels of free radicals and decreasing amount of body oxidants trigger cellular damage and this may increase lipid peroxidase. In the long term, it can cause inflammation and insulin résistance. Another theory suggests that oxidative stress triggers ROS (reactive oxygen species) that underlie insulin resistance and beta langerhans cell dysfunction^(2,3). This mechanisms is one of the underlying factors for DM. The occurrence of dyslipidemia (increased TG & cholesterol) is mostly caused by chronic ongoing DM (Diabetic dyslipidemia)⁽⁴⁾.

Antioxidant compounds act as defense mechanisms against oxidative stress by various mechanisms. These mechanisms include: 1) Antioxidants act as preventive agents, 2) antioxidants can capture free radicals, 3) Antioxidants can repair cellular damage, 4) Antioxidants can increase cellular adaptation⁽⁵⁾.

Nigella sativa (N. sativa) is reported to have an antioxidant effect. The antioxidant effect is mainly facilitated by thymoquinone found in N. sativa. Thimoquionen (TQ) is reported to reduce nephrotoxic risk in gentamycin-induced mice⁽⁶⁾. TQ is also reported to reduce lipid peroxidation, improve antioxidant enzymes and cellular protein oxidation^(7,8). TQ is also reported to increase the activity of antioxidant enzymes and protect cells against various cancer cells⁽⁹⁾. Methanol N sativa extract has antioxidant activity by DPPH method at a dose of 0.5 mg /ml⁽¹⁰⁾.

MATERIALS AND METHODS:

Materials: Nigella sativa was found from Pasar Gede (market) in Surakarta, on Maret 2019. Wistar rats were taken from Pharmacology laboratory in Medical faculty of UMS, Indonesia. Alloxan (®sigma Aldirch).

Preparation of extract:

Nigella sativa seed was found in Pasar Gede Surakarta. The dried of sativa seed was macerated in 95% ethanol at ratio 1:10 for 4 days. Residue was re-macerated for 3 days. Crude extracts was evaporated by rotatory evaporator apparatus until semisolid extract. This semisolid extract was then suspended in water continued by extraction with ethyl acetate. The fractionation process is carried out using a separating funnel, by previously adding aqueous to the obtained thick extract and then adding ethyl acetate solvent to the mixture. Then the mixture was shaken and allowed to stand overnight (24 hours) in a separating funnel, and the separation process is carried out the next day. In the fractionation process using ethyl acetate solvent, there are two layers of solvent produced, where the bottom layer is aqueous and the top layer is the layer that contains ethyl acetate. All ethyl acetate layers obtained from the fractionation were collected and evaporated using an evaporator until a thick fraction was obtained, then evaporation was carried out using a water bath at 70°C until a dry fraction was obtained as a test sample.

Experiment Design:

A total of twenty five male rats (150–200 g) were obtained from pharmacology laboratory of Medical faculty universitas Muhammadiyah Surakarta. The animal experiments were carried out according to protocols approved by health research ethic committees Universitas Muhammadiyah Surakarta. Rats were kept for one week for acclimatization. The animals were then housed 5/cage and received normal basal diet and water ad libitum at a room temperature (28-29^oC). The rats were divided into five groups with 5 rats for each group. Group I was treated by glibenclamide 0.63 mg/kg bw (positive control); Group II was treated by aqueous 2,5 ml/200 g bw (negative control); and Groups III;IV and V were treated by ethyl acetate of ethanolic extract of N sativa seed at a dosage of 250 mg/kg bw;500 mg/kg bw and 1000mg/kg bw respectively. Before experimental, all rats were injected by alloxan at dose of 750 mg/kg bw ip. This method referred to Sutrisna’s research [11] on days 4, 11, and 15 all blood glucose levels of rats were measured. Data were analysis by one-way ANOVA followed by LSD test.

On days 15, after measuring of blood glucose, all rats was killed by decapitation. The pancreas were dissected out for hystopathological examination.

This research was approved by Health Research ethics committee of Medical Faculty of UMS with number 2064/A.2/ KEPK-FKUMS/III/2019.

Phytochemical Analysis:

Identification of flavonoids⁽¹²⁾:

500 mg of extract was put into a test tube and 70% 2 mL of ethanol were added. The mixture is then stirred repeatedly. After that, 500 mg of magnesium and 3 drops of concentrated Hcl was added in this solution.

Identification of saponin⁽¹²⁾:

500 mg of extract was put into a test tube. A 70% ethanol solution of 2 mL is then added and stirred, after that, 20 mL aquadest was added. The solution was shaked repeatedly. The solution was left for 15-20 minutes. Then were add 500 mg Magnesium Powder and 3 drops of concentrated Hcl. The foam formation indicated the presence of saponins.

Identification of alkaloids⁽¹²⁾:

A total of 500 mg of extract was put into a test tube and 70% 2mL of ethanol was added and then stirred. A total of 5 mL of Hcl 2N was added and heated in a water bath. After it cools, the mixture was filtered and some filtrate was added with Meyer reagent drops. The presence of alkaloids, characterized by the presence of orange red precipitate

Identification of triterpenoids⁽¹²⁾:

500 mg of extract was put into a test tube. As much as 2 mL of 70% ethanol was added to the test tube then was stirred. The solution was added with chloroform 1 mL and anhydrous acetate 1 mL. This mixture was cooled. After it getting cools, a few drops of H₂SO₄ are added. The red ring indicated triterpenoids.

Tanin identification⁽¹²⁾:

A total 500 mg of extract was put in a cup. After that was added ethanol 70% 2 mL then was stirred. A total 3 drops of FeCl3 was added. The blue, black blue, green or blue green sediment indicated the presence of tannins

RESULTS:

The effect of ethyl acetate fraction of ethanol extract of Nigela sativa (EANS) on fasting blood glucose is presented in table 1.

From this table, we can see that the EANS at dose of 500mg/kg bw can reduce blood glucose level. On day 15, rats was killed by cervical decapitation. Pancreas was staining for hystopathological examination (Figure 1).

Figure 1. Pancreas Hystopathological examination on day 15 treatment group (A) and control group (B).

From the hystopathological in figure 1, can be seen that there is no necrosis of pancreas treatment group, but on control group was found necrosis. The ethyl acetate of ethanol extract of Nigella Sativa seed maybe inhibit pancreas damage. We do not know exactly the active compound of NS seed that decrease fasting blood glucose level. From the phytochemical analysis, ethyl acetate fraction of ethanol extract of Nigella sativa (EANS) contains alkaloid, flavonoid, and saponin (table 2).

Table 2. Active compounds of Etyl Asetat fraction of Ethanolic extract of Nigela Sativa

Active compounds	Hasil
Alkaloid	Positive
Flavonoid	Positive
Tannin	Negative
Saponin	Positive
Triterpene	Negative
Steroid	Negative

From table 2, we can see that the EANS contains alkaloid, flavonoid and saponin

DISCUSSION:

In this study, ethyl acetate ethanol extract Nigella sativa dose of 500 and 1000 mg / kg for 5 days was able to reduce blood sugar levels in alloxan-induced mice. This study in line with several previous research. Zaoui et al found that N. sativa 1 mL / kg body weight for 12 weeks can reduce blood glucose⁽¹³⁾. N sativa dose 100-300 mg/day. N. sativa for 4 days can reduce blood glucose significantly⁽¹⁴⁾. Nigella sativa seeds methanol extract can reduced fasting blood glucose n streptozotocin-induced diabetic male rats⁽¹⁵⁾. The hydro alcoholic extract of N sativa (NS) (5 mg/kg B.W, IP) reduced fasting blood glucose level on rats induced by streptozotocin⁽¹⁶⁾. Research by Karima et al, found that Aqueous extract of NS significantly decreased blood glucose level, TG, T-cholesterol, LDL-c, and TBARs and increase in HDL-c in female wistar rats Diabetes was induced by alloxan at dose of 200 mg/kg of bw⁽¹⁷⁾. Research by Khalid et al, 2019 found that Nigella sativa seeds 4g 2x/day for 2days, decrease fasting blood glucose level among 10 healthy non diabetic adults from 99.4±3.1 mg/dl to 93.1±4.6 mg/dL⁽¹⁸⁾. This effect of N Sativa seed was agedly related which the active comppund was isolated among others: thymoquinone, thymol, thymohydeoquinone nigellidinen and nigellicine^(19-22⁾.

In the clinical study, several research stated that N sativa can decreased blood glucose level. N Sativa oil at dose of 0.7g/day for 40 day reduced fasting blood glucose level and increased insulin level on Type 2 diabetic mellitus (DM)⁽²³⁾. Research by Bamosa et al found that N sativa seed at dose 2 g/day for 12 weeks improve diabetic control when combined oral anti-diabetic on 94 Type 2 DM patients⁽²⁴⁾. Research by El-Shamy et al. with subjects Type 2 DM patients showed consumption of N sativa tea extract at dose 5g/day for 6 months decrease blood glucose level on 66 T2DM (25). N sativa oil 5ml/day (combined with oral antidiabetic drug) for 3 months decreased fasting blood glucose levels, and HbA1c on 72 patients with type 2 DM⁽²⁶⁾. Kaatabi et al, found that N sativa capsul 2g/day for 3 months decreased FBG, HbA1c, insulin resistance (IR) when combined with oral antidiabetic drug on 114 patients with type 2 DM⁽²⁷⁾, meanwhile research by Bamosa et al., found that N. sativa capsul 2g/day for 1 years decreased HbA1c, when combined with oral antidiabetic drug⁽²⁸⁾. Nigella sativa oil in soft gel capsule 3 g/day for 12 weeks significantly decreased FBG and HbA1c when combined with oral ant diabetic drug on 72 Type 2 DM⁽²⁹⁾.

From hystopatological examination, this extract was allegedly inhibit pancreas damage. This is in line with research by previous research by Samad et al. Samad et al, stated that this extract may be has protective pancreatic islets damage (Samad Alimohammadi).

The effect hypoglycemic of NS maybe contributed by antioxidant activity of this extract⁽³⁰⁾. From this research, we found that the ethyl acetate of NS contain alkaloid, flavonoid and saponin. The ethanol extract of NS seed have antioxidant with IC₅₀ values 548.5 ± 9.4, with of DPPH scavenging activity⁽³¹⁾. Research by Jerrine et al, found that methanol extracts and ethyl acetate extracts of NS seed have antioxidant activity with of DPPH radical scavenging 63.32 µg/ml and 37.41 µg/ml. The chemical compound of this extract are total tannin, flavonoid and alkaloid⁽³²⁾.

thymoquinone, thymohydroquinone, dithymoquinone, thymol,

carvacrol, nigellimine-N-oxide, nigellicine, nigellidine

thymoquinone, thymohydroquinone, dithymoquinone, thymol,

carvacrol, nigellimine-N-oxide, nigellicine, nigellidine

thymoquinone, thymohydroquinone, dithymoquinone, thymol,

carvacrol, nigellimine-N-oxide, nigellicine, nigellidine

CONCLUSION:

Etyl Asetat fraction of ethanol extract of Nigela Sativa Linn. (EANS) at dose of 500mg/kgbw can reduce fasting blood glucose level. This fraction has potencies anti-diabetic.

ACKNOWLEDGEMENT:

The authors thank to Ministry of Research and Technology of the Republic of Indonesia for funding our research

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Received on 22.10.2020 Modified on 28.11.2020

Research J. Pharm. and Tech 2022; 15(1):381-384.

DOI: 10.52711/0974-360X.2022.00062

Groups	Blood glucose level (Mean±SE)
	Day 0	Day 5	Day 10	Day 15
Positive control (glibenclamide 0.63 mg/kg bw)	104.60±4.16	108±12.28*	87.75±11.56*	75.66±5.20*
Negative control (aqueous)	120.00±6.68	158.25±7.19	135.00±11.78	126.00±1.52
EANS at dose of 250mg/kgbb	128.80±17.79	137.60±24.12	141.75±9.19	116.75±8.06
EANS at dose of 500mg/kgbb	98.60±8.28	111.00±6.36*	131.40±10.53	108.50±0.28*
EANS at dose of 1000mg/kgbb	115.20±7.61	112.80±3.73*	138.80±8.78	127.20±5.48