INTRODUCTION:

Natural remedies are known, potent alternative strategies for treatment of diseases¹. Researches showed that medicinal plants with many beneficial constituents have potent biological activities, with low or negligible adverse side effects^2-7. In addition, there are reports that use of polyherbal formulations has better biological effects than individual herbs^8,9. Therefore, there is a great tendency to use herbal combinations for treatment of different diseases⁸, especially diabetes mellitus^8,10-14.

Afghanistan is famous as a mountainous country, with special dry climate and rich plant flora. This country has more than 5000 plant species that around 25-30% of which are endemic^15,16. Most of these plant species have valuable medicinal properties¹⁶. Since centuries, Afghan people are using traditional medicines for treatment and prevention of different diseases¹⁵.

Withania coagulans (W. coagulans) is an Afghan plant with nutritional and medicinal properties. It’s from Solanaceae family and found in Afghanistan, Iran, India and Pakistan^17,18. W. coagulans fruits are rich in alkaloids and steroidal lactones^17,19. The pharmacological properties of W. coagulans are anti-oxidative, anti-inflammatory²⁰, anti-hyperlipidemia¹⁷ and especially anti-diabetic^21-25. There are reports that W. coagulans fruit can alleviate fasting- and postprandial blood glucose, and also diabetes complications^26-28.

Ferula assa-foetida (F. assa-foetida) is one of Afghan plants, which is famous for its traditional uses. F. assa-foetida is belongs to Apiaceae family^9,29. Various parts of F. assa-foetida are used as Ayurvedic medicine. Its oleo gum resin is also an important part, which is rich in essential volatile oils, gum and especially resins^29,30. F. Assa-foetida possessed different properties, including anti-oxidant³¹, anti-inflammatory, anti-nociceptive^32,33, alleviating morphine withdrawal syndrome^9,29, and anti-diabetic^34,35. Studies report that extract of F. Assa-foetida oleo gum resin has antihyperglycemic and insulin-secreting effects on diabetic rats^34,36.

As W. coagulans and F. assa-foetida plants have significant anti-hyperglycemic effects^21,34, and polyherbal formulations showed much better activities than individual herbs⁸, this study determines the anti-hyperglycemic effect of each Afghan W. coagulans and F. assa-foetida extracts and their effective doses combinations on diabetic rats.

MATERIALS AND METHODS:

Materials:

The instruments and materials are including W. coagulans and F. assa-foetida extracts (Afghanistan), streptozotocin (Sigma-Aldrich, USA), and glucometer (On call plus, Acon, USA).

Plants extraction:

F. assa-foetida oleo gum resin from Daikundi province and W. coagulans fruits from Laghman province of Afghanistan, were collected. The fresh specimens were identified by Professor Nasim Sediqi, Department of Pharmacognosy, Kabul University in the herbarium of Faculty of science, Kabul University.

W. coagulans fruits were dried away from sunlight and then macerated in ethanol (70%) for 72 hours with occasionally shacking. The mixture was then filtered and it evaporated at the room temperature. As a result, W. coagulans ethanolic extract (WCEE) was obtained³⁷.

F. assa-foetida oleo gum resin that collected in mass form was dried away from sunlight and powdered. The powder was macerated in distilled water for 1 night and then filtered. As a result, F. assa-foetida aqueous extract (FAAE) was obtained^9,29.

Animals:

Male Sprague-Dawley rats (body weight: 200-225g) from Animal house of Khatam Al-Nabieen University Research and Technology Center were randomly selected for study. Animals were housed in Plexy-glass cages (15×40×30), with free access to food and water, in an air-conditioned room with temperature of 21-25°C and a periodical light/dark cycle. All experimental protocol was conducted according to the ethical guidelines of the National Institute of Health (NIH) guide for the care and use of laboratory animals and approved by ethic research board of Khatam Al-Nabieen University. Rats were handled to minimize unwanted stress.

Induction of diabetes:

Streptozotocin (STZ) (60mg/kg) was prepared in cold citrate buffer (0.1 M) and administrated to fasted animals intraperitoneally (i.p.). 72 hours later, fasting blood glucose (FBG) was measured and rats with FBG of 250mg/dl or higher were considered as diabetic rats³⁸.

Experimental groups:

The rats were divided into 11 groups randomly (n = 6/ group): (1) Normal group (vehicle-treated); (2) Diabetic group (STZ-treated); (3-5) WCEE groups (treated with 100, 200 and 400mg/kg WCEE, i.p.); (6-8) FAAE groups (treated with 25, 50 and 100mg/kg FAAE, i.p.); (9) Combination 1 group (treated with 200mg/kg WCEE + 25mg/kg FAAE, i.p.); (10) Combination 2 group (treated with 200mg/kg WCEE + 12.5mg/kg FAAE, i.p.); and (11) Combination 3 group (treated with 100 mg/kg WCEE + 25mg/kg FAAE, i.p.). The treatment period of all groups was 21 days.

Procedures:

The FBG level of blood withdrawn from tail of rats was estimated using a glucometer on days 0 and 21 (of diabetes confirmation). The body weight was also measured on days 0 and 21.

Statistical analysis :

Graph Pad Prism (6.07) software was used to analyze the data. Two-way ANOVA was performed (P < 0.05). The results are showed as mean ± SEM.

RESULT:

Measurements of FBG on day 0 indicated that there is a notable difference between normal group and all diabetic groups (P < 0.0001) (see Table 1). On day 21, WCEE 200 (P < 0.0001) and 400mg/kg groups (P < 0.05) showed a significant reduction in FBG level comparing to diabetic group. However, there was not a considerable difference in FBG level between WCEE 100mg/kg and diabetic group (P > 0.05). Also, The FBG levels of FAAE 25 (P < 0.0001), 50 (P < 0.05) and 100mg/kg groups (P < 0.05) were significantly decreased than diabetic group. Therefore, the 200mg/kg dose of WCEE and 25mg/kg dose of FAAE were considered as their most-effective doses. The effective doses of WCEE and FAAE were combined in different proportions and their effect was evaluated on FBG level of diabetic rats. FBG levels in all combination groups were considerably decreased in comparison with diabetic group (P<0.0001). Figure 1 shows a comparison between WCEE and FAAE effective doses and their combination groups with diabetic group. The FBG levels was decreased significantly in each WCEE 200 mg/kg, FAAE 25mg/kg groups, and especially in combination 1, 2 and 3 groups, compared with diabetic group (P < 0.0001). Further, the comparison of body weight in different groups with diabetic group did not show a significant difference on day 0 (P > 0.05). A considerable body weight loss was seen in all diabetic groups, in comparison with normal group on day 21 (P < 0.0001). However, neither of extract-treated groups showed a considerable difference in body weight with diabetic group (P > 0.05) (see Table 1).

Figure 1. Effect of WCEE and FAAE effective doses and their combinations on FBG level of diabetic rats on day 0 and 21. FBG: Fasting blood glucose, WCEE: Withania coagulans ethanolic extract, FAAE: Ferula assa-foetida aqueous extract. Data are presented as mean±SEM.^**:P < 0.01, ^****: P < 0.0001 in comparison with diabetic group.

Table 1. Effect of WCEE and FAAE and their combinations on body weight and FBG of rats

Groups	FBG		BW
Groups	Day 0	Day 21	Day 0	Day 21	Day 0-21 (loss/ gain)
Normal	84.17±4.59^****	83.83±3.09^****	204.17±6.40	251.17±9.15^****	47.00± 4.15^****
Diabetic	349.83±13.20	509.67±29.34	192.67±2.06	144.50±0.85	-48.17± 2.04
WCEE 100 mg/kg	398.17±22.94	435.00±18.16	202.33±4.05	152.00±3.68	-50.33± 5.51
WCEE 200 mg/kg	400.50±17.61	354.00±27.57^****	195.33±3.35	160.17±8.44	-35.17±6.23
WCEE 400 mg/kg	391.33±22.91	406.83±44.15^*	199.33±2.89	155.17±4.25	-44.17± 2.50
FAAE 25 mg/kg	335.17±27.43	351.33±10.45^****	192.83±2.24	161.00±2.24	-31.83± 4.11
FAAE 50 mg/kg	331.17±18.68	436.83±19.71^*	205.17±3.24	165.50±7.25	-47.17± 10.43
FAAE 100 mg/kg	319.83±9.12	441.00±19.97^*	189.33±2.17	145.83±2.21	-43.50± 1.54
Combination 1	331.33±12.30	302.33±29.73^****	191.83±2.60	148.83±2.15	-43.00± 2.18
Combination 2	273.83±8.01	246.67±38.69^****	194.17±4.11	147.17±3.65	-47.00± 2.53
Combination 3	298.83±14.67	224.50±46.97^****	194.83±3.68	154.00±4.49	-40.83± 3.76

WCEE: Withania coagulans ethanolic extract, FAAE: Ferula assa-foetida aqueous extract, BW: Body weight. Data are showed as mean ± SEM. ^*:P<0.05, ^****:P<0.0001.

DISCUSSION:

Hyperglycemia is considered as the most important symptom of diabetes mellitus, which leads to multiple complications in diabetic persons^8,39-41. Control of hyperglycemia is an essential strategy in diabetes treatment⁴². Beneficial properties of Ayurvedic medicines combination caught attention of researchers to use herbal combinations for control of hyperglycemia^4,37. Therefore, the present study evaluated the anti-hyperglycemic effect of Afghan WCEE and FAAE either alone or in combinations in diabetic rats. As STZ administration selectively destroys pancreatic beta cells and produces hyperglycemia and body weight loss, STZ was used in this study as a diabetogenic agent^43,44. Administration of STZ in this study is also produced a significant body weight loss and hyperglycemia.

Moreover, for determination of the anti-hyperglycemic effect of Afghan WCEE, it administered for 21 days and FBG levels of diabetic rats were measured. Based on results, the high doses (200 and 400mg/kg) of WCEE could reduce the FBG levels after 21 days administration period. The anti-hyperglycemic effect of 200mg/kg dose of WCEE was specially considerable than its other doses. However, low dose (100mg/kg) of WCEE didn’t show any significant anti-hyperglycemic effect on diabetic rats. Also, neither of WCEE doses could compensate the body weight loss in diabetic rats. Therefore, 200mg/kg dose of Afghan WCEE was considered as its most-effective dose on diabetic rats.

There are also reports from previous studies on anti-diabetic property of W. coagulans. A study on the effect of ethanolic extract of W. coagulans on STZ-induced diabetic rats showed that its oral administration (500, 750 and 1000mg/kg) over 30 days showed significant antihyperglycemic effect. The 750mg/kg dose of W. coagulans considered as its effective dose on hyperglycemia²³. Other previous study was also evaluated the anti-diabetic effect of W. coagulans aqueous extract over a long-term administration period. They administered its aqueous extract (500, 750, 1000 and 1250mg/kg) by oral administration over 30 days. It also reported its significant effect on hyperglycemia and glucose tolerance and 1000mg/kg was considered as its effective-dose²². Also, some studies determined the anti-diabetic effect of W. coagulans aqueous extract on nicotineamide-STZ-induced diabetic rats. They administered 125, 250 and 500mg/kg doses of W. Coagulans over 30 days, and its 250mg/kg dose was considered as the most-effective dose of the W. Coagulans on biochemical parameters of diabetic rats^26,27. In addition, a study determined the anti-diabetic effect of W. coagulans hydro-alcoholic extract and glipizide combination on diabetic rats over 28 days. They reported a synergic effect of W. coagulans hydro-alcoholic extract and glipizide on diabetic rats²¹. Therefore, the previous studies confirm the results of this study, which showed anti-hyperglycemic activity of W. coagulans extract over long-term administration period, especially in high doses.

Studies reported that the main mechanism underlying the anti-hyperglycemic effect of W. coagulans is increased glucose utilization in peripheral tissues. Also, it can activate the insulin gene expression and increase the insulin levels in diabetic rats^21,45. The beneficial effect of W. coagulans may resulted from its antioxidant constituents including alkaloids, flavonoids, and steroidal compounds and also its mineral contents such as Calcium and Magnesium^22,23,26,27.

Further, the present study also determined the anti-hyperglycemic activity of Afghan FAAE over 21 days administration period. According to the results, all doses of FAAE showed anti-hyperglycemic effect, but not weight-gaining effect on diabetic rats. However, its low dose (25mg/kg) was especially effective on FBG level of diabetic rats. Therefore, the 25mg/kg dose was considered as FAAE most-effective dose on FBG level of diabetic rats.

Most of past studies also reported the anti-hyperglycemic effect of F. assa-foetida in diabetic rats. A study determined the anti-diabetic effect of F. assa-foetida ethyl acetate extract (25, 50 and 100mg/kg) over 28 days, which given by i.p. administration. They reported that all doses of F. assa-foetida have significant anti-diabetic, especially anti-hyperglycemic effect on diabetic rats. However, its low dose effect was much greater than other doses³⁶. In addition, another study showed anti-hyperglycemic effect of F. assa-foetida after administration of its aqueous extract for 30 days⁴⁶. Also, administration of FAAE (50, 100 and 300 mg/kg) combined with drinking water for 28 days showed marked anti-hyperglycemic effect in diabetic rats, especially in its low dose³⁵. Hence, previous studies are also confirming our results that F. assa-foetida has a marked antihyperglycemic effect, especially in low doses.

Studies on the mechanism of action of F. assa-foetida on diabetic rats revealed that it has a significant insulin-enhancing effect on diabetic rats. It is proposed that the insulin-enhancing activity of F. assa-foetida may greatly depend to its anti-oxidant compounds, such as ferulic acid^34-36.

Furthermore, after determination of WCEE and FAAE effective doses on hyperglycemia, the effect of three different combinations formula of WCEE and FAAE effective doses was evaluated on FBG level of diabetic rats over 21 days. Based on results, all three proportions of WCEE and FAAE combinations have a significant anti-hyperglycemic effect. Their effect was much greater in combination 2(200mg/kg WCEE + 12.5mg/kg FAAE) and especially in combination 3 (100mg/kg WCEE + 25 mg/kg FAAE) groups, which are received the lower proportions of each WCEE and FAAE effective doses. If compare the anti-hyperglycemic effect of WCEE and FAAE combinations with each individual herbs, we that these two herbs combinations effect on FBG level of diabetic rats is more potent than that of individual ones. Therefore, one can conclude that WCEE and FAAE combinations have a potent synergic anti-hyperglycemic activity. Studies reported that polyherbal formulations from herbs that have different mechanisms of action might produce synergic effects^4,8. Hence, WCEE and FAAE combinations synergic effect may be due to their different underlying mechanism for reducing hyperglycemia, since W. coagulans increases the peripheral glucose utilization and F. assa-foetida has insulin-enhancing property.

CONCLUSION:

Long-term administration of WCEE and FAAE either alone or in combination in this study showed a significant effect on hyperglycemia of diabetic rats. WCEE and FAAE Combinations had a synergic effect on FBG level of diabetic rats.

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

ACKNOWLEDGMENTS:

The Kavosh nonprofit Educational-Research Institute, Kabul, Afghanistan, supports this work. We thank all officials for their valuable support to provide the equipment and facilities for this study. We also thank Mr. Mustafa Ansari for his collaboration in data acquisition.

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Received on 18.10.2021 Modified on 25.11.2021

Research J. Pharm. and Tech 2022; 15(10):4715-4719.

DOI: 10.52711/0974-360X.2022.00792