Hypoglycaemic activity of Mathurameha, a Thai traditional herbal formula aqueous extract, and its effect on biochemical profiles of streptozotocin-nicotinamide-induced diabetic rats

Diabetes mellitus (DM), a chronic metabolic disorder that is characterized by hyperglycaemia, continues to be a major health problem with that has seen a great increase in worldwide incidence due to behaviour and lifestyle changes [1]. In Thailand, there has been a rising trend in the hospitalization rate for patients with diabetes over the last decade, from 380.8 in 2003 to 1081.2 per 100,000 persons in 2013 [2]. Due to high cost of medical care and required treatments with long-term healing, diabetes is considered to be an economic burden and national public health problem that is of great concern [3]. According to the failure of conventional hypoglycaemic drugs to satisfactorily maintain normal glucose levels and some serious side effects [4‐6], significant interest in alternative and complementary medicine, especially herbal preparations, has been maintained [7, 8].

Mathurameha, a Thai traditional herbal formula established by Nirund Pongsoiphet and Foundation for the promotion of Thai Traditional Medicine Formula, has long been used by Ayuravedic (Chevagakomarapat) school to treat diabetes mellitus for more than 30 years. In recent years, it has been used as a complementary treatment for type 2 diabetes mellitus in Wangnamyen Community Hospital, Sa kaeo province [9]. The oral dosage form (350 mg capsule) of this herbal formula contains dried powder of 26 medicinal herb combinations (Table 1 ).

From previous studies, in vivo hypoglycaemic effects of 12 component herbs have been reported [10‐32], and 8 component herbs have been studied for their hypoglycaemic actions, including α-glucosidase inhibitory activity [30, 31, 33‐38], inhibition of glucose absorption [17], stimulation of insulin secretion from pancreatic β-cells [17, 24, 29, 39], stimulation of glucose uptake to fat and muscle cells [18, 40‐44], normalization of hepatic glucose output and glycogen synthesis [19, 43]. Twelve active hypoglycaemic compounds have been identified — these include andrographolide from Andrographis paniculata (Burm.f.) Nees [30, 45]; corosolic acid [36] and lagerstroemin [41, 42] from Lagerstroemia speciosa (L.) Pers; salacinol and kotalanol from Salacia chinensis (L.) [31]; gallic acid from Terminalia bellirica (Gaertn.) Roxb. [21]; chebulanin, chebulagic acid and chebulinic acid from Terminalia chebula Retz. [34]; and borapetoside C [35, 43] and borapetol B [39] from Tinospora crispa (L.) Hook.f. & Thomson. Therefore, the hypoglycaemic effects as well as the mechanism of actions of this anti-diabetic herbal formula are worth investigating.

Because of the high oral solid dosage form of this formula (5 capsules twice daily) as well as the unappreciable taste of the decoction, development of a preparation based on traditional knowledge might be helpful to reduce the administration dose [46]. Therefore, a comparison between the hypoglycaemic effects of the dried powder and extracts of the whole formula is needed to confirm the efficacy of the extract and to examine the most suitable extraction method. Although this herbal formula has already been used in diabetic patients without any sign or symptom of toxicity [9], the toxicity study of the whole formula extract remains necessary to ensure its safety for further usage.

The aims of this study are to observe the in vivo hypoglycaemic activity, effects on biochemical profiles as well as acute toxicity of the whole formula extracts to provide scientific evidence of its efficacy and safety, which might be useful for the further development of this herbal drug as well as for treating diabetes patients with fewer undesirable side effects.

In this study, diabetes in rats was induced by a streptozotocin-nicotinamide injection. Streptozotocin (STZ) has toxic effects on pancreatic β-cells based on its potent alkylating properties, which are related to cell death induction. STZ generates reactive oxygen species and acts as a nitric oxide donor, which contributes to DNA fragmentation and caused other changes that resulted in the destruction of pancreatic islet cells [52, 53]. The administration of suitable dosages of nicotinamide partially protects β-cells from the cytotoxic effect of STZ. Therefore, the diabetes syndrome induced in this experiment appears to be closer to human type 2 diabetes in that it demonstrated a significant response to glucose and sensitivity to sulfonylureas, a class of hypoglycaemic drug [49]. These drugs, including glibenclamide, stimulate insulin secretion from pancreatic β-cells by closing the ATP-dependent potassium channel (K_ATP) [54].

The oral glucose tolerance test (OGTT), which examines changes in the plasma glucose levels in response to oral glucose administration, has long been used clinically for diabetes mellitus diagnosis and in research to evaluate the effectiveness of hypoglycaemic agents [17]. In the present study, the OGTT was employed in a single dose administration experiment to determine the plasma glucose lowering effects of the Mathurameha herbal formula and its extracts at the same equivalent dose of 350 mg/kg in both normal and diabetic rats.

In normal rats, the plasma glucose levels of all of the treated groups and the untreated control group at every time point after glucose administration were the same. This indicates that the herbal formula did not alter blood glucose regulation in normoglycaemic conditions. However, the hypoglycaemic effects of the herbal formula were observed in diabetic rats. A single dose of aqueous extract (50 mg/kg) exhibited outstanding plasma glucose lowering effects in diabetic rats (Fig. 1 ). The results suggested that the major components that possessed hypoglycaemic activity of this herbal formula could be polar compounds, which are soluble in water. Daily oral administration of various doses of aqueous extract to diabetic rats revealed that a dose of 50 mg/kg exhibited the most potent glucose lowering effect and lowered 2-hour postprandial plasma glucose (2h–PPG) level after a 1-week administration, and this effect remained after 2-week administration. While the powdered mixture suspension showed a non-dose dependent glucose lowering effect, aqueous extract exhibited this effect in a dose-dependent manner after 2 weeks (Fig. 2 ), possibly because of the difference in the intestinal absorption of actively hypoglycaemic components. In the powdered mixture suspension, the active components that are covered by plant cell wall lead to low consistency in intestinal absorption. By contrast, the active components in aqueous extract are more available for absorption, resulting in high consistency in intestinal absorption. Aqueous extract is preferred for developing an herbal drug formulation for diabetes treatment.

Furthermore, previous reports showed that aqueous extract of medicinal plants, including Abutilon indicum [17, 28], Derris reticulata [20], Lagerstroemia speciosa [16, 55], Orthosiphon aristatus [29], Solanum trilobatum [14], Terminalia chebula [23], and Tinospora crispa [24], which are the components of this herbal formula, have in vivo hypoglycaemic effects. It has been reported that Lagerstroemia speciosa contains polar active compounds, including ellagitannins, such as lagerstroemin and ellagic acid derivatives, which could stimulate glucose uptake into 3T3-L1 adipocytes [40‐42]. Ellagitanins from Terminalia chebula including chebulanin, chebulagic acid, and chebulinic acid, have been reported to have inhibitory effects against α-glucosidase [34]. Boropetoside C, which was isolated from Tinospora crispa, also showed α-glucosidase inhibitory effects [35], increased glucose utilization in peripheral tissues, and reduced hepatic gluconeogenesis [43]. Therefore, it is suggested that these compounds might contribute to the hypoglycaemic effects of this herbal formula aqueous extract.

In this study, an increase in the serum level of total cholesterol (TC) triglycerides (TG) was observed in diabetic rats. The elevation of the TC and TG levels in circulation could be due to the increase in cholesterogenesis, fatty acid uptake, and deposit as triglycerides in the liver [56, 57]. The altered TC levels were significantly restored to normal values by aqueous extract (25 mg/kg) treatment (Table 2 ). The extract might have hypocholesterolomic effects with clearance elevation and reduction of endogenous cholesterol production [57, 58]. However, an increase in the serum TG level was observed in diabetic rats that were treated with extract (Table 2 ). This indicates that the formula failed to reduce TG production and could not control mobilization of triglycerides from the liver to circulation [59].

Liver enzymes, including AST, ALT, and ALP, in circulation that were investigated in this study are indicators of hepatic damage as they are released into the bloodstream after cell membrane damage reflecting hepatocellular necrosis [57, 60, 61]. However, the serum AST and ALT levels of the diabetic rats used in this study were not significantly different from those of the normal control group (Table 2 ). This might be due to the protection of nicotinamide against the hepatotoxic effects of STZ [62, 63]. Moreover, the significant decrease in the ALT levels in the 25 mg/kg aqueous extract treated group might be from the gluconeogenesis inhibitory effects of the extract [64]. Nevertheless, the serum ALP levels of the diabetic control group were significantly increased, indicating leakage from liver damage. A decline in the serum ALP levels was observed in the diabetic groups that were treated with aqueous extract (12.5 and 25 mg/kg) (Table 2 ). This revealed that the extract could reduce the leakage of liver enzymes into the bloodstream.

The decline of plasma proteins in diabetes, albumin and globulin, which are mainly synthesized by the liver, might be from the augmentation of protein catabolism or microproteinuria, indicators of diabetic nephropathy [64]. This study demonstrated that the serum total protein, albumin, and globulin levels of diabetic rats were significantly lower than those of the normal control group. The significant increase in these serum proteins was observed in diabetic groups that were treated with the 12.5 mg/kg aqueous extract (Table 2 ). These results are in agreement with decreasing the serum ALP levels by aqueous extract, indicating the protective effects of herbal formulas against liver damage that are associated with hyperglycaemia. Considering the evidence implicating diabetes mellitus in hepatic dysfunction, this hepatoprotective effect may be due to the hypoglycaemic effects of the whole formula aqueous extract investigated in this study [65].

Diabetic hyperglycaemia induces elevation of serum blood urea nitrogen (BUN), creatinine, and uric acid, which are considered to be significant markers of renal dysfunction [66, 67]. A significant increase in the serum BUN, creatinine and uric acid levels was observed in diabetic rats compared to the normal control group. Aqueous extract could not restore the altered BUN levels in diabetic rats (Table 2 ). This might indicate that the extract could not reduce the accumulation of urea nitrogen that occurred due to the enhanced breakdown of both liver and plasma proteins and that it did not play a role in protein metabolism [68]. Additionally, the treatment with all doses of aqueous extract significantly reduced the serum creatinine and uric acid levels of diabetic rats to normal levels (Table 2 ). This indicates improvement of the kidney excretion ability of creatinine, a byproduct of the breakdown of creatine and phosphocreatine, which are energy storage compounds in muscle, as well as uric acid, an end product of purine metabolism [68]. The normalization of the serum creatinine and uric acid levels indicates the role of this herbal formula in renal function improvement [67].

Overall, the results suggested that the aqueous extract is a good candidate for diabetes treatment because no undesirable effects on liver and kidney functions were observed, as it could be generated by long-term treatment with anti-diabetic agents. Although, the most potent dose of aqueous extract in regard to hypoglycaemic activity was 50 mg/kg, lower doses (12.5 and 25 mg/kg) exhibited more beneficial effects on the lipid profiles and liver and kidney functions. Therefore, 25 mg/kg was considered to be the most suitable dose because it has both potent hypoglycaemic activity and biochemical profile improvement effects in diabetic rats. Therefore, this dose should be used in the experimental design of further pharmaceutical, pharmacological and clinical studies on this herbal formula.

Aqueous extract did not exhibit acute toxicity to either sex of normal rats. Its LD₅₀ was estimated to be higher than 5 g/kg. This result agreed with those of previous studies that reported the aqueous extracts of Abutilon indicum [17, 69], Acanthus ebracteatus [70], Harrisonia perforata [71], Imperata cylindrica [13], and Terminalia chebula [72] showed no acute toxicity in animals.

The plasma glucose lowering effect in both single and daily repeated oral administration of whole formula aqueous extract observed in STZ-nicotinamide-induced diabetic rats revealed its potent hypoglycaemic activity. Overall, the results suggested that aqueous extract is a good candidate for diabetes treatment due to the lack of observed undesirable effects on liver and kidney functions. The extract was not only unaltered, it improved most biochemical profiles, including total cholesterol, alkaline phosphatase, total proteins, albumin, globulin, creatinine, and uric acid. It was considered to have hypocholesterolomic, hepatoprotective, and renoprotective effects during diabetic hyperglycaemia. However, the elevation of serum triglycerides levels of diabetic rats treated with aqueous extract should be considered. Aqueous extract has no acute toxicity on male and female rats at a dose of 5 g/kg.

This study confirms the efficacy and safety of the Mathurameha herbal formula treating type 2 diabetes mellitus. The revealed hypoglycaemic activity, beneficial effects to biochemical profiles and non-acute toxicity of the entire formula aqueous extract provided valuable insight for next-steps research of this herbal formula. Further investigations on the in vitro hypoglycaemic actions of the whole formula aqueous extract and chemical compounds responsible for its effect should be performed to clarify the mechanisms and active compounds.