Antihyperglycemic and antihyperlipidemic effects of Salvadora persica in streptozotocin-induced diabetic rats

Abstract

Context: Many synthetic antidiabetic components show toxic and/or mutagenic effects. Hence, attention has been given to naturally occurring antidiabetic components. Identification of effective antidiabetic components from plants origin is an ideal strategy for new drug development. The fresh root, bark, and leaves of Salvadora persica L. (Salvadoraceae) have been used in folk medicine for the treatment of a wide range of medical problems such as cough, asthma, scurvy, piles, rheumatism, leprosy, and gonorrhea disorders.

Objective: The S. persica root extract was investigated for the reduction of the risk of diabetes in diabetic rats.

Material and methods: The hydro-alcoholic root extract, 200 and 400 mg/kg, was fed to streptozotocin-induced diabetic rats for 21 d. Blood serum glucose, lipid profile, body weight, and food intake were monitored at 0, 7, 14, and 21 d after induction of diabetes.

Results: S. persica hydro-alcoholic root extract was not toxic at doses up to 1200 mg/kg. Significant reduction of blood glucose and lipid profile in diabetic rats treated with 400 mg/kg hydro-alcoholic root extract after 21 d versus diabetic control and glibenclamide-treated rats. The glibenclamide and root extract-treated group’s peak values of blood glucose significantly decreased from 281.50 to 106 mg/dL and 285.50 to 150.25 mg/dL, respectively. Hence, in this study, observations showed that root hydro-alcoholic reduced the blood glucose level in diabetic rats but values did not return to normal controls.

Conclusion: The research suggests that the root extract was significantly effective when compared with control and standard in the treatment of hyperlipidemia and hyperglycemia in diabetic rats. Therefore, it may be beneficial to diabetic patients.

• Diabetes mellitus
• lipid profile
• miswak
• natural health products
• OGTT
• toothbrush tree

Introduction

Diabetes mellitus is a multifaceted, dynamic expression of pathological disequilibria, resulting in various micro- and macro-vascular complications. Oxidation and production of free radicals are an integral part of normal cell metabolism. An imbalance between reactive oxygen species (ROS) and the antioxidant defense mechanisms (enzymatic and non-enzymatic) of a cell leads to excessive production of oxygen metabolites, creating a condition frequently termed as oxidative stress (Skaper et al., 1997). Excessive oxidative stress has been implicated in the pathology and complications of diabetes mellitus (Wolff, 1993). The increased blood glucose levels in diabetes produce superoxide anions, which generate hydroxyl radicals via Haber–Weiss reaction, resulting in per-oxidation of membrane lipids and protein glycation. This leads to oxidative damage to cell membranes. These radicals further damage other important biomolecules including carbohydrates, proteins, and DNA (Sato et al., 1979). Streptozotocin (STZ) selectively destroys β-cells of pancreas by generating excess ROS and carbonium ion (CH3+) leading to DNA breaks by alkylating DNA bases. The N-nitroso-N methyl urea portion of the molecule exhibits diabetogenic activity. Glucose may act as a carrier for this cytotoxic group (Verspohl, 2002). Low levels of plasma antioxidants implicated as a risk factor for the development of the disease hence antioxidants are important in the prevention of diseases (Facchini et al., 2000; Salonen et al., 1995).

Oxygen plays a vital role in diverse biological functions such as utilization of nutrition, electron transport to produce ATP, and the removal of xenobiotics (Hemnani & Parihar, 1998). While oxygen is essential for life, it also can provoke damaging oxidative events within cells. Oxygen, by its transformation to more reactive forms, i.e., superoxide radial (O₂), hydroxyl radical (OH), and hydrogen peroxide (H₂O₂), can nick DNA, can damage essential enzymes and structural proteins, and can also provoke uncontrolled chain reactions, such as lipid per-oxidation or auto-oxidation reactions (e.g., polymerization of catecholamines) (Bijlani, 1995; Halliwell & Cross, 1994). There is considerable interest in the role of ROS and reactive nitrogen species (RNS) as mediators of tissue injury in human diseases.

Many synthetic antidiabetic components have shown toxic and/or mutagenic effects. Hence attention has been given to naturally occurring antidiabetic components. Therefore, identification of effective antioxidants and antidiabetic components from plants origin is an ideal strategy for new drug development. Hence, the present study was designed to explore the antidiabetic activity of Salvadora persica L. (Salvadoraceae) in a prospective way. S. persica (SP) also known as miswak, toothbrush tree, and mustard tree, distributed mainly in tropical and sub-tropical Asia. The plant is a large, evergreen profusely branched shrub, or a small tree up to 4–6 m tall. SP has been used commonly as toothbrush to strengthen the gums (Sofrata et al., 2008). The fresh root bark and leaves have been used in folk medicine for the treatment of a wide range of medical problems such as cough, asthma, scurvy, piles, rheumatism, leprosy, gonorrhea, headache, and hepatic disorders (Kirtikar & Basu, 1975).

Various phytochemical studies on SP reported the presence of alkaloids salvadorine, trimethylaine, and salvadoricine (Malik et al., 1987), flavonoids (quercitin), triterpenes, phytosterols, and trace of vitamin C (Hamid et al., 1997). Essential oil from the roots of SP contains benzyl isothiocyanate (70%) with other components such as α-pinene, camphene, benzaldehyde, β-pinene, myrcene, δ-3-carene, limonene, terpinolene, benzyl nitrile, umbellulone, β-elemene, γ-muurolene, myristicin, β-caryophyllene, and longifolene (Bader & Flamini, 2002) Various pharmacological activities on SP including, in vivo antimicrobial activity especially on lactobacilli and streptococcus mutans (Almas & Al-Zeid, 2002), with moderate secretary activity significantly high acetyl cholinesterase inhibiting ability, antifertility activity in male rats (Darmani et al., 2003). The aqueous extract of SP leaves possesses analgesic activity and reduces carrageenan-induced inflammation in rat paw (Parihar et al., 2007). Aqueous and alcoholic extracts from leaves of SP reduce elevated urinary oxalate levels and deposition of stone-forming constituents in the kidneys of calculogenic rats (Geetha et al., 2010). In our previous study, we showed that root hydro-alcoholic extract of SP reduces carrageenan-induced inflammation in rats and scavenges 2,2-azino-bis-(3-ethyl benzo-thiazoline-6-sulphonic acid) (ABTS), superoxide radicals in a cell-free system (Hooda et al., 2006). The activities of hydro-alcoholic root extract of SP being a powerful antioxidant, free radical scavenger, and lipid per-oxidation inhibitor, and as an excellent total antioxidant capacity, on the bases of in vitro studies (Hooda & Singh, 2012). In the present study, we aimed to explore the antidiabetic activities of hydro-alcoholic root extract of SP in STZ-induced diabetic rats.

Materials and methods

Drugs and chemicals

Glucose oxidaseperoxidase strip (Accue-check* diagnostic kit, Roche Diagnostics India Pvt. Ltd, Mumbai, India), serum total cholesterol (TC), triglyceride (TG), and high-density lipoprotein (HDL)–cholesterol were estimated by using diagnostic kits (Erba Mannheim Cholesterol kit, Transasia Bio-Medicals Ltd., Daman, India), STZ HiMedia Laboratories Pvt. Ltd., were procured from Haryana Scientific & Engg. Corp. Rohtak, Haryana. All other drugs and chemicals used in these studies were of analytical grade.

Plant material

The root and stem of SP were collected in March–April 2011 from Kharainti, Meham Teh. Rohtak. District, Haryana (India), and authenticated by Dr. Ashok Sharma (M.D.) (Dravya Guna Vigyan), Prof. & Head of Department. Shri Baba Mastnath Ayurvedic Degree college, Asthal Bohar, Rohtak. Haryana. The root and stem of SP were collected; shade-dried at room temperature, and was crushed to a coarse powder and then subjected to continuous Soxhlet extraction.

Preparation of the extracts

The shade-dried plant material root was grinded and powdered material (100 g) was used for the extraction. The different solvent extracts were prepared by hot continuous percolation in a Soxhlet apparatus. The following extractions were collected separately and dried in vacuo: (1) root extracted with 99% ethanol (root-AA, yield: 09.15%); (2) root extracted with 70% ethanol (root-HA, yield: 11.65%); (3) root extracted water (root-W, yield: 12.25%); these extracts were condensed by re-distillation and dried in vacuum desiccators to obtain a final extract residue.

Experimental animals

Healthy adult Wister albino rats were selected for the study. Animals were housed in polypropylene cages and maintained under standard conditions (12 h light/dark cycle; 25 ± 30 °C; 45–55% humidity). They were fed with standard pellet diet (Hindustan Lever Ltd., Mumbai, India) and water ad libitum. The Institutional Animal Ethical Committee of Janta College of Pharmacy Butana, (Sonepat) Haryana, India (CPCSEA-667/02/c/CPCSEA), approved the studies.

Determination of acute toxicity of drug

Acute toxicity was determined in fasting mice. Animals were divided into groups of six each, and the extract was administrated orally with 1% CMC or 10, 30, 100, 300, 1000, and 1200 mg/kg body weight of SPHA. The mice were observed continuously for the 2nd, 4th, 6th, 12th, 24th, and up to 48 h (Ghosh, 1984).

Oral glucose tolerance test (OGTT) in normal rats

The OGTT was performed in overnight fasted normal rats. Rats divided into four groups (n = 6) were administered either drinking water or root-HA, 200 and 400 mg/kg. Glucose (2 g/kg) was fed 30 min after the administration of the extract. Glibenclamide (5 mg/kg) was used as the standard drug. Blood was withdrawn from tip of the tail, and blood glucose level was estimated on 30, 60, and 120 min of the glucose administration using glucose oxidase–peroxidase reactive strips and a glucometer (Accu-Chek, Roche Diagnostics, Indianapolis, IN) (Bonner-Weir, 1988).

STZ-induced diabetes mellitus in rats

The STZ solution fleshly prepared in 0.1 M ice cold sodium citrate buffer pH (4.5) was administered i.p. in overnight fasted (18 h) rats to induce diabetes, single dose STZ (50 mg/kg). The STZ was administered through i.p. route in dark room and day was considered as zero. Blood glucose level was determined on days 5 and 7 to assess incidence and extant of diabetes development. Animals showing average (on days 5 and 7) glucose levels of more than 200 mg/dL on day 7 were included in the study and those considered as diabetic on day 7 were divided in different groups. Drug pre-treatments were started 5 d prior to (STZ) and continued for further 15 d. The glucose, cholesterol, HDL, LDL, and TG were assessed before and after 15 d of drug treatments to assess the antidiabetic activity (Sarkhail et al., 2007).

Statistical analysis

The data were expressed as mean ± standard error mean (SEM). The significance of differences among the groups was assessed by using a one-way and multiple-way analysis of variance (ANOVA). The test was followed by Dunnet’s test and p values less than 0.05 were considered as significant.

Results

Acute toxicity study of drug

Acute toxicity studies revealed the non-toxic nature of the HA-root extract at doses up to 1200 mg/kg of SP. There were no lethality or toxic reactions found at any of the doses selected until the end of the study period. All the animals were alive, healthy, and active during the observation period.

Effect of root-HA of SP on OGTT in normal rats

OGTT in glucose-loaded normal rats showed that hypoglycemia was observed after 30 min with the maximum effect being seen at 120 min. OGTT indicates the efficacy of the extract in the maintenance of blood glucose levels in normal rats (Table 1).

Table 1. Effect of the root-HA of S. persica on oral glucose tolerance test in normal rats (OGTT).

		Plasma glucose concentration (mg/dL) in min
Group	Treatment	30	60	120
1	Control glucose (2 g/kg)	180.50 ± 0.64	150.00 ± 0.40	135.25 ± 0.47
2	Root-HA (200 mg/kg)	167.50 ± 0.64	136.50 ± 0.64*	120.25 ± 0.47*
3	Root-HA (400 mg/kg)	156.00 ± 0.40*	115.50 ± 0.64*	95.00 ± 0.40*
4	Glibenclamide (5 mg/kg)	150.00 ± 0.40*	101.50 ± 0.64*	72.50 ± 0.64*

Values are expressed in terms of mean ± SEM, n = 6 in each group. Root-HA, S. persica hydroalcoholic extract root; GT, glibenclamide treated. Results of test and standard were compared with the control group.

*p < 0.05.

Effect of the root-HA of SP on blood glucose level in diabetic rats

In STZ-induced diabetic rats, the blood glucose levels were in the range of 281–285 mg/dL, which was considered as severe diabetes. In the glibenclamide (5 mg/kg) and root-HA (400 mg/kg) treated groups, the peak values of blood sugar significantly decreased from 281.50 to 106 mg/dL and from 285.50 to 150.25 mg/dL on the 21st day, respectively. Hence, in this study, observations showed that root-HA reduced the blood glucose level in diabetic rats but values did not return to those of normal controls. Therefore, root-HA possesses significant (p < 0.05) antidiabetic activity, when compared with diabetic control. There was a marked reduction in blood glucose level (in 21 d) in STZ-induced diabetic animals. This effect of root-HA (400 mg/kg) is nearly equal to, if not better than, that of glibenclamide (5 mg/kg) (Table 2).

Table 2. Effect of the root-HA of S. persica on blood glucose level in STZ-induced in diabetic rats.

		Blood glucose (mg/dL) in day
Group	Treatment	0	7	14	21
1	Normal control (−)	93.25 ± 1.10	93.00 ± 0.91	93.00 ± 1.47	92.50 ± 1.19
2	Diabetic control (−)	281.25 ± 1.65	292.25 ± 2.05	305.00 ± 2.27	323.25 ± 1.88
3	Diabetic rats + root-HA (400 mg/kg)	285.50 ± 1.32	261.75 ± 1.25*	209.25 ± 1.54*	150.25 ± 2.28*
4	Diabetic rats + glibenclamide (5 mg/kg)	281.50 ± 2.90	246.50 ± 2.90*	178.00 ± 2.04**	106.00 ± 1.95**

Values are expressed in terms of mean ± SEM, n = 6 in each group. Root-HA, S. persica hydroalcoholic extract root; GT, glibenclamide treated. Results of test and standard were compared with the diabetic control group.

*p < 0.05, **p < 0.01.

Effect of the root-HA of SP on lipid profile in diabetic rats

Compared to the normal control group, the level of serum TC, TG, LDL, VLDL-cholesterol was significantly (p < 0.05) increased, whereas the level of HDL-cholesterol was significantly (p < 0.05) reduced in untreated diabetic rats (Table 3). After the treatment of root-HA, the level of serum TC, TG, LDL, and VLDL-cholesterol reduced significantly (p < 0.05), whereas, the level of serum HDL-cholesterol was significantly increased. Glibenclamide treatment reduced the level of TC, TG, LDL, and VLDL-cholesterol and enhanced the level of HDL-cholesterol.

Table 3. Effect of the root-HA of S. persica on lipid profile in STZ-induced diabetic rats.

		Lipid profile in STZ-induced diabetic rats (mg/dL)
Group	Treatment	TC	TG	HDL	LDL	VLDL
1	Normal control	83.25 ± 1.10	74.50 ± 0.64	28.50 ± 0.64	44.75 ± 1.10	17.50 ± 0.64
2	Diabetic control	173.25 ± 1.88	133.25 ± 1.49	18.50 ± 0.64	129.75 ± 1.93	34.75 ± 1.49
3	Root-HA (400 mg/kg)	102.75 ± 1.93*	91.50 ± 1.55*	26.50 ± 0.64*	53.00 ± 1.29*	19.00 ± 0.40*
4	Glibenclamide	75.25 ± 1.10**	61.50 ± 1.04*	29.50 ± 0.64*	46.50 ± 0.64*	16.50 ± 0.47**

Values are expressed in terms of mean ± SEM, n = 6 in each group. Root-HA, S. persica hydroalcoholic extract root; GT, glibenclamide treated. Results of test and standard were compared with the diabetic control group.

*p < 0.05, **p < 0.01.

Effect of the root-HA of SP on body weight in diabetic rats

Normal control group had an increase in their body weight, but diabetic rats showed significant reduction in body weight during the 14 d treatment. STZ caused body weight reduction, which was reversed significantly by root-HA after 14 d of treatment (Table 4).

Table 4. Effect of the root-HA of S. persica on body weight in diabetic rats.

		Changes in body weight (g) in day
Group	Treatment	0	7	14
1	Normal control	220.50 ± 0.64	234.00 ± 0.40	247.25 ± 0.47
2	Diabetic control	218.25 ± 0.47	207.25 ± 0.47	198.25 ± 0.85
3	Root-HA (400 mg/kg)	218.25 ± 0.85	209.25 ± 0.85	217.00 ± 0.81*
4	Glibenclamide	215.50 ± 0.64	208.50 ± 0.64	217.75 ± 0.47*

Values are expressed in terms of mean ± SEM, n = 6 in each group. Root-HA, Salvadora persica hydroalcoholic extract root; GT, glibenclamide treated. Results of test and standard were compared with the diabetic control group.

*p < 0.05.

Discussion

Various plants have been traditionally used to treat diabetes, and some have been proven to have hypoglycemic effects. These studies have identified that compounds such as polysaccharides (Tomoda et al., 1985), flavonoids (Khanna & Jain, 1981), terpenoids, tannins (Reher et al., 1991), and steroids (Schimizu et al., 1984) are responsible for antidiabetic effect. SP root hydro-alcoholic extract also contains flavonoids, triterpenes, phytosterols, and trace of vitamin C, benzyl isothiocyanate, β-caryophyllene, and longifolene compounds. The observed hypoglycemic effects of this root could have resulted from the combined activity of these compounds present in the root extract.

In the present work, SP hydro-alcoholic root extract was administered to glucose-loaded normal rats and hypoglycemia was observed after 30 min with the maximum effect being seen at 120 min. OGTT indicates the efficacy of the extract in the maintenance of blood glucose levels in normal rats. STZ-induced hyperglycemia has been described as a useful experimental model to study the activity of hypoglycemic agents. STZ enhances free radical formation and/or produces defects in antioxidant defense system, resulting in the destruction of β-cells of the pancreas which may lead to hyperglycemia. In the present study, administration of hydro-alcoholic root extract of SP to diabetic rats (Group III) increased body weight significantly, which was comparable to the experiments carried out by Chakrobarty and Das (2010). Results of the present study are supported partially by the findings of Perez et al. (1998), who also observed a significant (p < 0.05) increase in the body weight after treatment with the extract of Ficus carica (fig tree) leaves in STZ-diabetic rats, which may be due to the lipid lowering activity of the extract or indirectly due to the influence on various lipid regulation systems.

Evaluation of the hydro-alcoholic root extract of SP in normoglycemic and STZ–hyperglycemic rats indicated that the extract possesses hypoglycemic and antihyperglycemic activities. Experiments by Chakrobarty and Das (2010) also support our finding that the administration of SP root extract in diabetic rats restored the level of blood glucose to near normal levels. Hyperlipidemia has been reported to accompany hyperglycemic states. A significant increase in the total cholesterol, TG, LDL, and VLDL levels were in accordance to the earlier studies (Bajaj & Srinivasan, 1998). Repeated administration of the hydro-alcoholic root extract of SP prevented the elevation of TC, TG, LDL, and VLDL-cholesterol level in diabetic rats, indicating the SP had a beneficial effect on the hyperlipidemia induced by STZ.

In order to protect the body from these ill effects of ROS, there exist antioxidant defensive mechanisms, which may be classified into primary defense against ROS and secondary defense against ROS.

The primary defense may be the group of antioxidant enzymes capable of catalytically removing the ROS (Fridovich, 1978); the secondary defense comprises the free radical scavengers. When these scavengers produce a lesser harmful radical, they are known as antioxidants. The activities of hydro-alcoholic SP root extract may be a powerful antioxidant, free radical scavenger, lipid per-oxidation inhibitor, and has excellent total antioxidant capacity, on the bases of in vitro studies (Hooda & Singh, 2012).

Our finding indicates that the SP commonly used in the management of different diseases in Unnani system of medicine may be useful for the treatment of diabetes associated with hyperlipidemia. The possible mechanism of action of the hydro-alcoholic root extract may be by promoting the insulin release from the undestroyed β-cells or its action may be like insulin, as reported by Chandola et al. (1980). Observations suggest that the extract could improve oral glucose tolerance by increasing the availability of insulin.

Conclusion

Hydro-alcoholic root extract of SP exhibits significant antihyperglycemic activities in STZ-induced rats. The extract also showed improvement in lipid profile, body weight, and OGTT results, hence might be valuable in diabetes, responsible for such effects powerful antioxidant, free radical scavenger, lipid per-oxidation inhibitor, and as an excellent total antioxidant capacity, on the bases of in vitro studies; in contrast, pharmacological activities were positive. Therefore, it appears useful for the treatment of diabetes and hyperlipidemia.

Declaration of interest

The authors declare no conflict of interest in this manuscript.