In vivo Investigation of The Antidiabetic potential of Manilkara hexandra leaves and fruits extracts against streptozotocin-induced hyperglycaemia in rats

doi:10.21203/rs.3.rs-4824242/v1

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In vivo Investigation of The Antidiabetic potential of Manilkara hexandra leaves and fruits extracts against streptozotocin-induced hyperglycaemia in rats

https://doi.org/10.21203/rs.3.rs-4824242/v1

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Diabetes mellitus has been one of the most crippling diseases with huge social, health, and economic consequences. Manilkara hexandra (Roxb.) Dubard is a small to medium sized evergreen edible fruit-producing tree belonging to the family Sapotaceae and cultivated in Egypt. The main goal of the current study is to evaluate the effect of oral administration of M. hexandra different extracts including fruit methanolic soluble portion, leaf methanolic soluble portion, fruit aqueous methanolic extract, and leaf aqueous methanolic extract for 30 days at a dose of 1/10 of their LD₅₀ (2 g/kg b.w) on streptozotocin-induced diabetic rats. Glucose and insulin were assayed in blood while reduced glutathione (GSH), nitric oxide (NO), lipid peroxidation (TBARS), and thioredoxin reductase (TrxR) activity were measured in pancreatic tissues. Phytochemical investigation of M. hexandra leaf and fruit revealed the purification of five compounds among which three compounds were isolated from the leaf and identified as β-sitosterol, β-amyrin, and 3-O-β-D-glucopyranosy-protobassic acid, respectively. Moreover, two flavonoids were identified in M. hexandra fruit viz. quercetin 3-O-α-L-rhamnopyranoside and myricetin 3-O-α-L-rhamnopyranoside. The results obtained from this study revealed that streptozotocin administration resulted in significant elevation in the level of both lipid peroxidation and nitric oxide with depletion in the level of reduced glutathione as compared with control accompanied with hyperglycemia, hypo-insulinemia and low insulin sensitivity. Moreover, the activity of pancreatic TrxR was lower than the control group. Oral administration of the diabetic rats with the extracts normalizes the previous biochemical parameters reaching near the normal level. M. hexandra fruit and leaf aqueous methanolic extract and methanolic soluble portion exhibited significant antidiabetic activity and corrected the insulin level and sensitivity in experimentally induced diabetic rats.

Manilkara hexandra

Sapotaceae

hypoglycemic

nitric oxide

thioredoxin reductase

blood glucose

insulin

lipid peroxidation

Since ancient times, humans have received many benefits from natural plants and compounds. It has been generally recognized that traditional oriental medicine has a unique therapeutic role in the treatment of many human diseases. Oriental medicines containing a number of compounds which are known to demonstrate a variety of biological activities in both experimental and clinical settings [1]. Egypt is endowed with a rich floral biodiversity which is reflected in the existence of many thousand species of higher plants. Many of the plant species are used for ornamental, nutritional, symbolic, ritual, spiritual, and medicinal purposes.

Diabetes mellitus has been one of the most crippling diseases with huge social, health, and economic consequences, and its prevalence has risen dramatically over the past two decades [2]. Currently, 537 million adults (20–79 years) are living with diabetes. This number is predicted to increase to 643 million by 2030 and 783 million by 2045 [3]. Such rise in dia-betic patients is due to an increase in sedentary lifestyle, consumption of energy-rich diet, and obesity [4]. Type II diabetes mellitus (NIDDM) develops when the pancreas cannot produce enough insulin or when the body tissue becomes resistant to insulin and without insulin the body cannot process glucose resulting in too much sugar in the blood and not enough sugar in the body's cells [2]. This disturbance increases the risk of many disorders including obesity, hypertension, hyperuricemia, and dyslipidemia [5]. Synthetic hypoglycemic agents used in treatment of diabetes can produce serious side-effects, including hematological coma and disturbances of liver and kidney. In addition, they are not suitable for use during pregnancy [5]. Therefore, the search for more effective and safer hypoglycemic agents has continued to be an important area for research. Following the recommendations made by the WHO (1980) on the beneficial uses of medicinal plants for the treatment of diabetes mellitus, investigations of hypoglycemic agents from medicinal plants have also become more important [6].

Many plant extracts and plant products have been shown to have significant antioxidant activity [7] which may be an important property of plant medicines associated with the treatment of several ill-fated diseases including diabetes [8]. Thus, herbal plants are considered useful means to prevent and/or ameliorate certain disorders, such as diabetes, atherosclerosis and other complications [7]. Among these herbal resources, Manilkara hexandra is selected for the present study. M. hexandra (Roxb) Dubard (common name is Rayan/ Khirni) is a small to medium sized evergreen tree. It is native to South Asia and Tropical Countries and widely distributed in South, North and Central India mainly in Rajasthan, Gujrat, Madhya Pradesh and Maharashtra [7]. M. hexandra (Roxb) Dubard is cultivated in Egypt in the Zoo Garden, Giza, Egypt. Leaves, fruits, roots, and barks of M. hexandra are well known for their medicinal values. Its bark is used to treat a wide range of gastrointestinal symptoms like ulcers, dyspepsia, flatulence and colic also used for treatment of bronchitis, fever, helminthiasis and burning sensation [7] and it contains procyanidins, flavonoids, terpenoids and saponins [7, 9]. Fruits of M. hexandra are a good source of minerals, sugars, proteins, carbohydrates and Vitamin A and its consumption is also useful in hallucinations, loss of consciousness, anorexia, dipsia, bronchitis and antimicrobial property against different bacterial strains[7]. The leaf alcoholic and aqueous extracts were having antimicrobial property [7, 10], the stem bark methanolic extract has been shown to have antioxidant activity [11] and the seeds showed anti-inflammatory activity [9].

The present study was undertaken to investigate the effect of fruits methanolic soluble portion, leaves methanolic soluble portion, fruits aqueous methanolic extract, leaves aqueous methanolic extract of M. hexandra on streptozotocin (STZ)-induced hyperglycemic rats to evaluate the possible hypoglycemic activity of M. hexandra in diabetes mellitus.

Chemicals

All chemicals were purchased from sigma and Aldrich (Sigma Chemical Company, St. Louis, MO, USA). Facilities including animal housing and biochemical equipment were available by the National Research Centre (NRC), Cairo, Egypt.

Animals

The animal care and handling was done according to the guidelines set by the World Health Organization, Geneva, Switzerland, and according to approval from the committee for animals care at the National Research Centre, Egypt. Male Sprague-Dawley rats (body weight 150–200 g) were purchased from animal breeding center of National Research Centre, Egypt. The animals were kept under environmentally controlled conditions (constant temperature 25–27°C, with 12 h light/dark cycle) for one week prior to starting the experiments. All rats were kept in plastic cages, and they were provided with tap water and commercial diets.

Plant Materials

The aerial parts of Manilkara hexandra (leaves and fruits) were collected from the zoo garden, Giza, Egypt. The plant was botanically identified by Prof. Dr. Rim Hamdy, Prof. of Plant Taxonomy, Faculty of Science, Cairo University, Egypt. A voucher specimen (code: 20 Mhe/15). of both leaves and fruits were deposited at the Herbarium of Pharmacognosy Department, Faculty of Pharmacy, Helwan University, Helwan, Cairo, Egypt.

Plant Extracts

Leaves extracts

The air-dried powdered leaf (2500 g) of M. hexandra were macerated in 3 L 100% methanol with occasional stirring at room temperature for three days. The methanolic extract was concentrated and dried under vacuum (50 ^οC) to give dry aqueous methanolic extract (440 g). The dried residue (350 g) was re-dissolved in 500 mL methanol and poured onto 3 L acetone with continuous stirring to precipitate the crude saponins. The acetone precipitate was decanted then filtrated through centered glass filter. The acetone filtrate was filtrated to remove any traces of precipitated saponins, and then concentrated under vacuum (50 ^οC) to give dried methanolic soluble portion (170 g) which suggested containing polyphenolic content. Acetone filtrate then washed with chloroform 100 ml for three times to remove chlorophyll and other aglycones [7].

Fruits extracts

The air-dried powdered fruit (1100 g) of M. hexandra was subjected to extraction with 3 L of petroleum ether and the Marc was extracted by Ethyl acetate 3L to remove fats and then extracted with 100% methanol. The methanolic extract was dried under vacuum using rotary evaporator at 50 ^οC to give aqueous methanolic extract (230 g). The dried residue (190 g) was re-dissolved in 300ml methanol and poured onto 1.5 L acetone with continuous stirring in order to precipitate the crude saponins. The acetone precipitate was decanted then filtrated through centered glass filter. The acetone filtrate was filtrated to remove any traces of precipitated saponins, and then concentrated under vacuum (50 ^οC) to give methanolic soluble portion (80g) which suggested containing polyphenolic content [7].

Toxicity studies

The safe doses of the tested extracts were selected based on preliminary investigation according to Ghosh (2007) [12]. Each of the four extracts were administered orally to different groups of rats (n = 10) in doses ranging from 0 mg to 2 g/kg of body weight/day for one month. The rats were observed for any abnormal behaviors or mortality. No mortality was observed in any of the groups during the period of experiment, which revealed that the tested extracts are safe and non-toxic up to 2 g/kg.

Induction of diabetes

Diabetes was induced in rats as described by Ali and Abd El Kader (2004) [13]. Briefly, rats were intraperitoneally administrated with freshly prepared STZ in 0.1 M citrate buffer (pH 4.6) at a dose of 65 mg/kg body weight. After 72 h of STZ administration, blood glucose level was estimated in animals fasted overnight. Animals with a blood glucose level ranging between 200–300 mg/dl were considered diabetic and used for further experiment. The control rats received the same amount of 0.1 M citrate buffer (pH 4.6).

Extract dosing

Treatment with different tested extracts began on day 3, post-STZ treatment. Rats received oral doses of 1/10 0f the IC₅₀ once a day for thirty consecutive days by gavage. The diabetic control rats were given distilled water (orally) instead of the extracts.

Experimental design

Animals were divided into five groups of 10 animals each. Group (1) represents normal control rats were administrated 1ml of distilled water. Group (2, 3, 4, 5) serve as normal rats treated with tested extracts at (1/10 0f their IC₅₀ /day) orally by gavage for 30 days. Group (6) represent diabetic rats were administrated 1ml of distilled water. Group (7) serves as positive control was received tolbutamide, a standard hypoglycemic drug (100 mg/kg body weight /day) orally by gavage for 30 days. Group (8,9,10,11) diabetic rats were administrated with extracts at 1/10 0f the IC₅₀ /day orally by gavage for 30 days.

Analytical procedures

Overnight fasted animals were killed by cervical dislocation, 24 hours after the last injection. Blood was withdrawn from the retro orbital plexus into sterilized centrifuge tubes and allowed for clotting at room temperature, then centrifuged at 4000 rpm for 10 minutes for serum separation. Serum samples were stored in freezer at -20°C for analysis.

Portions of pancreas were washed in ice -cold saline, weighed and then homogenized in cold saline (0.90% NaCl). After centrifugation at 3,000 xg for 30 min at 4°C the supernatant was used for the biochemical investigations (all estimations were carried out on fresh 10% homogenate).

Blood glucose level

Blood glucose level was determined using a commercial kit obtained from Roche Diagnostics (Mannheim, Germany) based on the previously reported work [14].

Insulin level and Insulin resistance

Blood fasting insulin level was estimated using an ELIZA kit (Mercodia Ultrasensitive Insulin, Sweden). It is based on the direct sandwich technique in which two monoclonal antibodies are directed against separate antigenic determinants on the insulin. The color is read spectrophotometrically at wave length 450 nm. A homeostasis model assessment (HOMA-IR) was used for detecting insulin resistance. HOMA-IR was calculated from the fasting concentrations of insulin and glucose using the following formula: fasting plasma insulin / (22.5 e^{− ln fasting plasma glucose}) as described by Thirunavukkarasu et al. (2004) [15]. Low HOMA-IR values indicate high insulin sensitivity, whereas high HOMAIR values indicate low insulin sensitivity or insulin resistance [16].

Pancreatic lipid peroxidation (TBARS)

Pancreatic thiobarbituric acid reactive substances (TBARS) as a product of lipid peroxidation, was determined according to the method of Conrad et al. (2000). Concentration of TBARS was expressed in nmol of TBARS/mg tissue [17]. The level of lipid peroxidation is expressed as nmol TBARS/g tissue, using 1, 1, 3, 3-tetraethoxypropane as standard.

Pancreatic reduced glutathione (GSH)

Reduction of non-protein sulfhydryl levels was used as an indicator of total GSH content, which was determined using 5,5´ dithiobis-2-nitrobenzoic acid (DTNB). To precipitate the protein 0.50 ml of 50% TCA was added to 1 ml of homogenized tissue. The sample was then mixed and centrifuged at 9000×g for 10 min and the supernatant was used to determine GSH at 412 nm [6].

Pancreatic nitric oxide ((NO)

NO was determined in pancreatic tissues according to the methods of Granger et al. (1995) based on that NO production is detected in biological fluids via nitrite by using nitrite/nitrate assay using Roche Diagnostics kit (Germany). The nitrate present in the sample is reduced to nitrite by NADPH in the presence of the enzyme nitrate reductase. The nitrite formed reacts with sulfanilamide and N-(1-naphthyl)-ethylenediamine dihydrochloride to give a complex which measured at 550 nm [18].

Pancreatic thioredoxin reductase (TrxR) activity

This assay used the NADPH-dependent reduction of DTNB to 2-nitro-5-thiobenzoic acid by TrxR [19]. The assay mixture was prepared fresh in an ice bath and contained, 1 M of sodium phosphate buffer, pH 7.0, 0.2 mg/ml NADPH, 0.2 mg/ml bovine serum albumin, 5 mM DTNB, 0.01 M EDTA, 1% ethanol; 5µl of pancreatic homogenates were added to 200 µl of the assay mixture. The increase of absorbance was registered at 412 nm at 37°C.

Statistical Analysis

Data were expressed as mean ± standard error (S.E.) and analyzed statistically using a one-way analysis of variance (ANOVA) followed by the student t-test for comparison between groups. Differences were considered significant at P < 0.05.

Isolated compounds

Compound 1 was obtained as white amorphous powder (20 mg). ¹HNMR (400MHz, DMSO-d₆) of compound 1 showed signals at δ 3.4(1H, m, H-3), 5.27 (1H, m, H‐6), 0.96 (3H, d, J = 6.4 Hz, H-19), 0.92 (3H, t, H-24), 0.87 (3H, d, J = 6.5 Hz, H-26), 0.76 (3H, d, J = 6.5 Hz, H-27), 0.69(3H, s, H-28) and 1.08(3H, s, H-29) ppm. ¹³C NMR (125MHz, DMSO-d₆) of Compound 1 showed signal at 142.26 (C‐5), 46.51 (C‐22), 122.45 (C‐6), 72.85 (C‐3), 56.9 (C‐14), 56.30 (C‐17), 50.42 (C‐9), 34.34 (C‐20), 39.99 (C‐12), 42.40 (C‐13), 42.10 (C‐4), 37.42 (C‐1), 36.70 (C‐10), 32.52 (C‐8), 32.19 (C‐7), 29.15 (C‐25), 28.37 (C‐16), 30.26 (C‐2), 26.12 (C‐15), 19.47 (C‐28), 21.69 (C‐11), 20.26 (C-26), 19.63 (C‐27), 19.47 (C‐19), 26.90 (C‐21), 36.76 (C‐18), 12.16 (C-29), 12.50(C-24) and 23.81 (C-23). ¹H NMR spectra of compound 1 showed the presence of six methyl signals that appeared as two methyl sinlgets at δ 0.69, and 1.08; three methyl doublets that appeared at δ 0.87, 0.76, and 0.96; and a methyl triplet at δ 0.92. ¹H NMR data also revealed the presence of one olefinic proton at δ 5.27, a proton corresponding to the proton connected to the C-3 hydroxy group which appeared as multiplet at δ 3.53. ¹³C NMR has shown recognizable signals 142.26 and 122.745 ppm, which are assigned C-5 and C-6 double bonds, respectively, and the value at 19.47 ppm corresponds to angular carbon atom (C-19). ¹³C NMR Spectrum showed 29 carbon signals including six methyls, nine methylenes, eleven methane and three quaternary carbons. The structure of compound 1 was assigned as β-sitosterol (Fig. 1) by comparing the data to the reported literature values [20, 21].

Compound 2 was purified as white powder (15 mg); ¹H NMR (DMSO-d₆, 400 MHz) revealed signals at 3.17(1H, dd, J = 4.4, 10.8 Hz, H-3), 0.68 (1H, d, J = 11 Hz, H-5), 5.21(1H, t, J = 3.2, H-12), 0.72–1.24 (each 3H, s, H-23, 24, 25, 26, 27, 28, 29, 30). ¹³C NMR (DMSO, 125 MHz) showed carbon signals at 38.63(C-1), 121.77(C-12), 144.93 (C-13), 28.48(C-28), 79.10(C-3), 37.19(C-4), 28.18(C-2), 55.21(C-5), 18.43(C-6), 33.42(C-7), 39.75(C-8), 47.68(C-9), 36.73(C-10), 23.59(C-11), 41.90(C-14), 30.77 (C-20), 18.43 (C-26), 23.77 (C-30), 26.89(C-27), 16.86 (C25), 15.58 (C-24), 26.07 (C-15), 26.21(C16), 33.72 (C-21), 37.10 (C-22), 33.21 (C-29), 46.88(C-18), 28.16 (C23), 47.27 (C-19), and 34.79 (C-17). ¹H NMR showed the presence of eight methyl sinlgets at δ 0.72, 0.75, 0.87, 0.89, 0.96, 0.98, 1.08 and 1.24. The ¹H NMR spectra of compound 2 also showed a proton corresponding to the H-3 of a terpene moiety which appeared as a doublet of doublets at δ 3.17 and a proton at δ 5.24 as a triplet suggesting the presence of a trisubstituted olefinic bond. ¹³C NMR showed signals at 121.77 for C-12 and 144.93 for C-13 carbons which support the presence of double bond and C-3 at 79.10 suggest the presence of hydroxyl group. The ¹H and ¹³C NMR data supported the presence of oleanane triterpene skeleton having a hydroxyl group at C-3 position with a double bond at C-12/C-13 with eight methyl groups. A search in literature found that the spectral data of compound 2 was supportive to the structure of β-amyrin (Fig. 1). Thus, the structure of compound 2 was assigned as β-amyrin that was consistent to the reported literature values [21, 22].

Compound 3 was obtained as white powder (20 mg). ¹H NMR (DMSO-d₆, 400 MHz) revealed signals at 0.68, 0.75, 0.98, 1.17, 0.88, 0.87 (each 3H, s, H3 of C-29, C-30, C-27, C-25, C-24, C-26), 2.27 (lH, dd, J = 14.0, 4.0 Hz, H-18), 3.63 (lH, m, H-5 of glc.), 3.91 (lH, m, H-2 of glc.), 4.02 (lH, m, H-3 of glc.), 4.22 (lH, t, H-4 of glc.), 3.21 (lH, d, J = 3.7 Hz, H-3), 4.04 (lH, m, Hz, H-6 of glc.), 5.22 (lH, d, J = 8.0 Hz, H-l of glc.), and 5.29 (lH, t, H-12). ¹³C NMR data (DMSO-d₆, 125 MHz) showed signals at 15.65 (C-24), 17.14 (C-25), 73.84(C-16), 16.84 (C-26), 23.83 (C-30), 24.2 (C-11), 25.93 (C-27), 34.80 (C-15). 28.02 (C-20). 40.16 (C-7), 30.77 (C-22), 33.20 (C-29), 35.00 (C-21), 39.33 (C-8), 36.73 (s, C-10), 40.80 (C-18), 46.01 (C-l), 41.9 (C-14), 41.3 (C-4), 46.4 (C-19), 47.56 (C-9), 48.9 (C-17), 63.7 (C-23), 70.03 (C-2), 81.9 (d, C-3), 67.1 (C-6), 121.9 (C-12), 143.9 (C-13), 47.50 (C-5), and 175.71 (C-28). ¹H NMR data revealed the presence of six singlets between 0.93 and 1.64 ppm corresponding to six angular methyl and Ehylenic proton H-12(br.t) at δH 5.29 ppm. Four protons linked to oxygenated carbons: H-2, H-3 at δH 3.21 ppm, H-6, H-23, two germinal protons at 3.43 and 3.74 ppm. ¹³C NMR spectrum also showed characteristic signals for an ethylinic carbon C-12 at δC 121.93, quaternary carbon C-13 at δC 143.92 ppm and a carbonyl carbon C-28 at δ 177.1 ppm. The aglycone part of compound 3 was identified as protobassic acid, the structure was determined from the analysis of ¹H, ¹³C NMR spectra; the data were compared with that reported in literature [9, 22]. The identification evidence of sugar moiety as β-D-glucose was in turn reinforced from the presence of anomeric proton signal at 5.22 with large J value. The attachment of sugar unit at C-3 confirmed by spectral data of H-3at 3.21 ppm and C-3 carbon at 82 ppm which compared to literature review [9]. From the above data, by comparison with the reported data [9, 23] compound 3 was confirmed as 3-O-β-D-glucopyranosy-protobassic acid which was isolated for the first time from Manilkara hexandra.

Compound 4 was obtained as yellow amorphous powder (40 mg). Chromatographic properties: R_f values; 0.55(BAW), 0.64 (15%AW); dark purple spot under UV- light, turned to yellow fluorescence on exposure to ammonia vapors. It gave a deep green color with FeCl₃ spray reagent. Complete acid hydrolysis resulted in rhamnose in aqueous phase and quercetin in organic phase. ¹H-NMR (400 MHz, DMSO-d₆) δ ppm 7.3 (1H, d, J = 2.0 Hz, H-2`), 7.25(1H, dd, J = 8.4, 2.0 Hz, H-6`), 6.86 (1H, d, J = 8.0 Hz, H-5`), 6.40 (1H, d, J = 1.6 Hz, H-8), 6.21 (1H, d, J = 1.6 Hz, H-6), 5.21(1H, d, J = 1.6 Hz, H-1``), 3.98 (1H, br s, H-2``), 3.13–3.61 (remaining sugar protons), 0.81 (3H,d, J = 6 Hz, H-6``). ¹³C-NMR (125 MHz, DMSO-d₆) showed the presence of signals at δ ppm 178.19 (C-4), 164.80 (C-7), 161.75 (C-5), 156.92 (C-2), 157.75 (C-9), 148.93 (C-4`), 145.68 (C-3`), 134.67 (C-3), 121.56 (C-6`), 121.19 (C-1`), 116.12 (C-5`), 115.94 (C-2`), 104.50 (C-10), 102.29 (C-1``), 99.20 (C-6), 94.12 (C-8), 71.65 (C-4``), 71.05 (C-3``), 70.82(C-2``), 70.52 (C-5``), 17.96 (CH3-6``). Compound 4 was expected to be quercetin 3-O-glycoside based on its chromatographic properties. Detection of L-rhamnose and quercetin in their hydrolysis products was evidence for suggesting a quercetin 3-O-rhamnoside compound. ¹H-NMR data showed ABX spin coupling system at δ ppm 7.30, 7.25 and 6.86 for H-2`, H-6` and H-5` respectively of 3`, 4`, dihydroxy ring-B. Another AM coupling system of two meta coupled protons at δ ppm 6.40 and 6.21 for H-8 and H-6, respectively to indicate 5,7-dihydroxy ring-A. The sugar moiety was identified as α-L-rhamnose from its characteristic anomeric proton at δ ppm 5.21 together with doublet signal of CH3-6`` at δ ppm 0.81 (6 Hz). This evidence was confirmed from ¹³C-NMR (APT) spectrum of compound 4 that exhibited characteristic 15 ¹³C resonances for 3-O-substituted quercetin. Among the ¹³C-signals of quercetin the characteristic resonance of C-4`, C-3` and C-3 at δ ppm 148.93, 145.68 and 134.67, respectively. The sugar moiety was confirmed as rhamnose from the characteristic carbon resonance at δ ppm 102.29 and 17.96 for the anomeric carbon and CH₃-6`` respectively, together with the rest of carbon resonances for rhamnose carbons. According to the above discussed data as well as comparing with previous reported data [7, 24]. Compound 4 was confirmed as quercetin 3-O-α-L-rhamnopyranoside (quercetrin) (Fig. 1).

Compound 5 was purified as yellow amorphous powder (30 mg). Chromatographic properties: Rf values; 0.41(BAW), 0.52 (15%AW); dark purple spot under UV- light, turned to dark yellow fluorescence on exposure to ammonia vapours; It gave green colour with FeCl₃ spray reagents. Complete acid hydrolysis resulted in rhamnose in aqueous phase and myricetin in organic phase. ¹H-NMR (400 MHz, DMSO-d₆) showed signals at δ ppm 6.89 (2H, s, H-2`/6`), 6.37 (1H, d, J = 2.0 Hz, H-8), 6.20 (1H, d, J = 2.0 Hz, H-6), 5.20 (1H, d, J = 1.6 Hz, H-1``), 3.99 (1H,br s, H-2``), 3.33–3.59 (Hs- 3``, 4``, 5), 0.84 (3H, d, J = 6.12 Hz, H-6``). ¹³C-NMR (125 MHz, DMSO-d₆) revealed the presence of signals at δ ppm 178.25 (C-4), 164.67 (C-7), 161.78 (C-5), 156.88 (C-2/9), 146.24 (C-3`/5`), 136.93 (C-4`), 134.75 (C-3), 120.09 (C-1`), 108.38 (C-2`/6`), 104.50 (C-10), 102.40 (C-1``), 99.14 (C-6), 94.00 (C-8), 71.74 (C-4``), 71.02 (C-3``), 70.86 (C-2``), 70.48 (C-5``), and 17.99 (CH3-6``). Compound 10 was expected to be myricetin-3-O-glycoside according to the chromatographic properties. With complete acid hydrolysis, compound 10 gave L-rhamnose in aqueous phase and myricetin in organic phase (CoPC) suggesting myricetin 3-O-rhamnoside. ¹H-NMR spectra showed a myricetin aglycone from the characteristic singlet signal integrated to two protons at δ ppm 6.89 for H-2`/6` of 3`, 4`, 5`-trihydroxy ring-B and the two meta coupled doublets at δ ppm 6.38, 6.20 for H-8 and H-6, respectively to indicate a 5,7-dihydroxy ring-A. The identification evidence of sugar moiety as α –rhamnose was in turn reinforced from the presence of α-anomeric proton signal at 5.20 (1.6 Hz) together with doublet signal of CH3-6`` at 0.84 (6.12 Hz). ¹³C-NMR (APT) data revealed the characteristic signals at δ ppm 146.24, 108.38, 134.75 for C-3`/5`, C-2`/6`and C-3 respectively. In addition, typical 10 ¹³C- resonances for 3-O-substituted myricetin. The spectrum also showed typical six ¹³C-resonances for rhamnose moiety characterized by anomeric carbon at δ ppm 102.40 and CH3-6`` at 17.99. Configuration and confirmation of the rhamnose was proved to be α-₁C⁴ pyranose based on its δ and J-value in ¹H and ¹³C-NMR data. All other resonances were assigned based on their comparison with previous reported data [7, 25]. Accordingly, compound 4 was confirmed as myricetin 3-O-α-L-rhamnopyranoside (myricetrin) (Fig. 1).

Effect of extracts on plasma glucose, insulin levels and insulin resistance in diabetic rats

As shown in Table 1 and Fig. 2, the plasma glucose level was significantly increased by 254.36% (from 80.47 ± 9.11 to 285.16 ± 29.75a mg/dl, P < 0.05) in the diabetic group in comparison with the control group. However, when diabetic rats orally administrated with the extracts (fruits methanolic soluble portion, leaves methanolic soluble portion, fruits aqueous methanolic extract and leaves aqueous methanolic extract), the levels of blood glucose significantly decreased (P < 0.05) by 56.20, 61.40, 53.30 and 60.41% respectively as compared with diabetic rats reaching near the level of glucose in tolbutamide treated group. Concerning, plasma insulin level was significantly decrease (P < 0.05) in diabetic rats by 35.91% (from 11.20 to 7.18 ng/dl) in comparison with the control group. While the administration of the extracts to diabetic rats resulted in significantly increase (P < 0.05) in the insulin level by 25.41, 35.80, 26.73 and 29.91% respectively as compared to diabetic group (Table 1, Fig. 3). HOMA-IR value was significantly higher (P < 0.05) in diabetic rats than control group by 178.92% (from 4.6 to 12.83), indicating low sensitivity or insulin resistance. Meanwhile, the resistance value was reduced (P < 0.05) in diabetic rats treated with the extracts (fruits methanolic soluble portion, leaves methanolic soluble portion, fruits aqueous methanolic extract and leaves aqueous methanolic extract) by 53.21, 59.51, 53.20, and 54.11%, respectively (Table 1, Fig. 4). It is clear from the results that the Leaves methanolic soluble portion exhibited a potent significant amelioration on the glucose, insulin levels and insulin resistance.

Table 1

Effect of fruits methanolic soluble portion, leaves methanolic soluble portion, fruits aqueous methanolic extract and leaves aqueous methanolic extract of M. hexandra on plasma glucose, insulin levels and HOMA-IR value in normal and STZ- induced diabetic rats.
Groups	Blood Glucose mg/dl		Plasma Insulin ng/ml			^*HOMA-IR
1. Control	80.47 ± 9.11		11.20 ± 1.33			4.60 ± 0.48
2. fruit methanolic soluble portion	80.50 ± 9.32		11.28 ± 1.18			4.60 ± 0.46
3. Leaves methanolic soluble portion	76.34 ± 7.85		11.50 ± 1.21			4.32 ± 0.49
4. fruits aqueous methanolic extract	81.00 ± 8.66		11.24 ± 1.32			4.75 ± 0.63
5. Leaves aqueous methanolic extract	78.90 ± 8.13 ^b	11.20 ± 1.15 ^b					4.50 ± 0.49 ^b
6. Diabetic rats	285.16 ± 29.75^a			7.18 ± 9.00^a	12.83 ± 1.37 ^a
7. Diabetic + Tolbutamide	115.87 ± 12.77^{a, b}			10.16 ± 1.12^a,b	5.86 ± 0.65 ^a,b
8. Diabetic + fruit methanolic soluble portion	125.00 ± 13.76^{a, b}			9.00 ± 0.96 ^{a, b}	6.00 ± 0.70 ^{a, b}
9. Diabetic + Leaves methanolic soluble portion	110.23 ± 12.10^a,b			9.75 ± 1.14 ^{a, b}	5.20 ± 0.61 ^a,b
10. Diabetic + fruits aqueous methanolic extract	133.20 ± 15.10^{a, b}			9.10 ± 1.40 ^{a, b}	6.00 ± 0.75 ^{a, b}
11. Diabetic + Leaves aqueous methanolic extract	113.00 ± 11.60^{a, b}			9.33 ± 0.87 ^{a, b}	5.90 ± 0.66 ^{a, b}
Values are given as mean ± S.E. for groups of ten animals each.
*HOMA-IR (Homeostasis model assessment of insulin resistance).
^{a, b} Values are statistically significant at p < 0.05.
^a Significantly different from control. ^b Significantly different from STZ diabetic group.

Effect of extracts on oxidative stress in diabetic rats

Table 2 shows biochemical parameters indicative of pancreatic oxidative stress. The results revealed that diabetic rats produced significant increase (P < 0.05) in the levels of pancreatic TBARS by 146.51% (from 85.30 ± 9.12 to 210.27 ± 22.75 nmol/g tissue) and NO by 96.91% (from 1.60 to 3.15 µg /g tissue) as compared with control group. While, the activity of TrxR and level of GSH revealed were significantly decrease (P < 0.05) as compared with control group by 41.41% (from 2.91 to 1.70 U/g tissue) for TrxR and by 40.60% (from 3.20 to 1.90 nmol/g tissue) for GSH Figs. 5 and 6. On the other hand, treatment of diabetic rats with the extracts showed significantly decrease (P < 0.05) in the levels of TBARS (by 45.20, 49.80, 44.91 and 46.6%) and NO (by 47.60, 50.81, 45.71 and 49.21% ) as compared with diabetic group. While the activity of TrxR and level of GSH showed significantly increase (P < 0.05) by (47.11, 55.90, 52.91 and 55.90%) for TrxR activity and by (78.90, 92.63,71.12 and 89.50%) for GSH level as compared with diabetic group.

Table 2

Fruits methanolic soluble portion, leaves methanolic soluble portion, fruits aqueous methanolic extract and Leaves aqueous methanolic extract on indicators of oxidative stress in pancreatic tissue of normal and STZ- induced diabetic rats.
Groups	TBARS (nmol/g tissue)	GSH level (nmol/g tissue)	NO (µg /g tissue)	TrxR activity (U/g tissue)
1. Control	85.30 ± 9.12	3.20 ± 0.37	1.60 ± 0.19	2.90 ± 0.34
2. fruit methanolic soluble portion	86.22 ± 9.34	3.37 ± 0.42 ^a	1.70 ± 0.18	2.92 ± 0.34
3. Leaves methanolic soluble portion	81.50 ± 9.11	3.60 ± 0.40 ^a	1.50 ± 0.19	3.06 ± 0.35
4. fruits aqueous methanolic extract	87.00 ± 8.45	3.40 ± 0.38 ^a	1.70 ± 0.17	2.90 ± 0.27
5. Leaves aqueous methanolic extract	84.44 ± 8.80	3.52 ± 0.40 ^a	1.61 ± 0.16	3.00 ± 0.28
6. Diabetic rats	210.27 ± 22.75 ^a	1.90 ± 0.27 ^a	3.15 ± 0.42 ^a	1.70 ± 0.19 ^a
7. Diabetic + Tolbutamide	94.85 ± 10.70 ^a,b	3.25 ± 0.33 ^b	1.70 ± 0.2 ^b	2.60 ± 0.30 ^b
8. Diabetic + fruit methanolic soluble portion	115.00 ± 12.75^a,b	3.40 ± 0.38 ^b	1.65 ± 0.21 ^b	2.50 ± 0.26 ^b
9. Diabetic + Leaves methanolic soluble portion	105.65 ± 11.87 ^a,b	3.66 ± 0.40 ^a,b	1.55 ± 0.11 ^b	2.65 ± 0.29 ^b
10. Diabetic + fruits aqueous methanolic extract	115.93 ± 12.44^a,b	3.25 ± 0.44 ^b	1.71 ± 0.20 ^b	2.60 ± 0.27 ^a,b
11. Diabetic + Leaves aqueous methanolic extract	112.20 ± 13.07^a,b	3.60 ± 0.39 ^a,b	1.60 ± 0.19 ^b	2.65 ± 0.34 ^b
Values are given as mean ± S.E. for groups of ten animals each.
^{a, b} Values are statistically significant at p < 0.05.
^a Significantly different from control. ^b Significantly different from STZ diabetic group.

Phytochemical investigation of Manilkara hexandra leaf and fruit revealed the purification of five compounds among which three compounds were isolated from the leaf and identified as β-sitosterol, β-amyrin, and 3-O-β-D-glucopyranosy-protobassic acid. Moreover, two flavonoids were identified in M. hexandra fruit viz. quercetin 3-O-α-L-rhamnopyranoside and myricetin 3-O-α-L-rhamnopyranoside. Previous studies on M. hexandra revealed its richness in flavonoids, phenolic compounds, and saponins [7, 9]. Dietary flavonoids are considered biologically essential molecules and play a crucial role in treatment besides management of chronic diseases like diabetes mellitus, liver diseases owing to their antioxidant capacity which enhance their anti-diabetic activities [26]. Also, increasing scientific suggestion recommends their anti-inflammatory and antioxidant properties [27]. Previous antidiabetic studies on apigenin and luteolin derivatives showed several mechanisms of action of flavonoids including decrease in gluconeogenic and lipogenic capacity[28, 29]. Quercetin has demonstrated in vitro and in vivo antidiabetic properties by several mechanisms including improving of oral glucose tolerance and sensitize pancreatic β-cell to secrete insulin, inhibits α-glucosidase and suppresses the release of pro-inflammatory markers [30]. β-sitosterol, a phytosterol found plentiful in plants and possesses several biological actions such as immunomodulatory, antimicrobial, anticancer, anti – inflammatory, lipid lowering effect, hepatoprotective, antioxidant and anti-diabetic activities [31]. Moreover, M. hexandra is a rich source of saponins with potential antidiabetic activity [32]. Several previous studies revealed that M. hexandra bark and leaves extracts showed potential antidiabetic effects[33, 34].

Type 2 diabetes is currently one of the main threats of human health worldwide, and its incidence is increasing even in children and adolescents[5]. In the present study, we aimed to evaluate the hypoglycemic effect of M. hexandra extracts in STZ –induced diabetic rats. The data obtained revealed that STZ treatment causes an increase in the blood glucose level and insulin concentration with increase in the insulin resistance (Table 1). These results can be explained by the data reported by Aybar et al. (2002) who proved that STZ at the doses (75 mg/ kg) produces destruction of pancreatic β-cells with permanent diabetes condition[35]. The results of the present work indicate an increase in the production of TBARS and NO an indicator of lipid peroxidation in STZ treated group as compared with control group, proving that STZ administration possibly develops an oxidation stress to cause deleterious effects on the pancreas. These effects could be one of the mechanisms by which STZ causes diabetes mellitus. These results were in agreement with Ali et al. (2009) who proved in their study excessive production of NO and TBARS in pancreatic tissue of the STZ treated rats [6]. STZ-induced oxidative stress [36]has been suggested to be due to the generation of NO, which can cause DNA damage and activate poly (ADP-ribose) polymerase[36], a major cause of islet cell damage in diabetes. In addition, nitric oxide reacts with superoxide radicals to form the noxious peroxynitrite that contribute in the pathogenesis of diabetic complications. Recently, Stadler et al. (2003) reported an increased level of NO in kidneys of streptozotocin-induced diabetic rats, thus supporting the role of NO, reactive oxygen species (ROS), and peroxynitrite-derived species in the development of early diabetic tissue injury [37].

Nitric oxide has also been proposed to play a significant role in diabetes mellitus. Mechanistic studies showed that the NO attack results in mitochondrial dysfunction and chromosomal DNA damage in pancreatic islet cells eventually damaging the cells and hence leading to failure in insulin secretion [38]. The increased oxidative stress experienced by tissues in diabetic patients as a result of hyperglycemia leads to loss of control over the NO production pathway. The overproduction of NO in such patients induces various complications and damage in many organs including the eye, kidney, and cardiovascular systems in both type I and type II diabetes [39].

Our results suggesting that STZ possibly develops an oxidative stress to cause deleterious effects on the endocrine pancreas by increasing the expression of protein kinase, a key component of the insulin activated signal transduction cascade [40]. The suppression of protein kinase expression/phosphorylation by STZ associated with significant inhibition of insulin-dependent glucose uptake by adipocytes supports our evidence of hyperinsulinemia and hyperglycemia. Also generation of ROS and reactive nitrogen species (RNS) ultimately activate stress-induced pathways (nuclear factor (NF-_KB), stress kinases and hexosamines) which will burn out the β-cells and induce apoptosis and/or necrosis [41]. In this study, STZ treatment led to depletion of pancreatic GSH content which is an endogenous antioxidant that acts as a first line defense system against prooxidant status [40]. Depletion of GSH could significantly affect the overall redox potential of the cell. Previous studies have documented that GSH level was reduced in diabetic patients and in experimental models [36]. Anathan et al. (2003) showed a significant reduction in plasma GSH levels in experimental diabetic rats [42]. Similarly, depleted GSH levels have been repeatedly reported in several tissues of experimental diabetic animals, including eye, aorta, kidney as well as small intestine. Furthermore, significant decreases in plasma as well as erythrocyte GSH levels have been documented in diabetic patients [43]. The depletion in the pancreatic GSH content was in consistent with El-Alfy et al. (2005) and can be explained by the fact that in the diabetes there is overproduction of ROS and GSH consume by acting as a substrate of the ROS defense enzyme GSH peroxidase and the GSH transferase family of detoxification enzymes to get rid of the excess ROS [44].

Thioredoxin reductase and thioredoxin (Trx) system represents a molecular target candidate in response to agents that induce oxidative stress. Trx plays an essential role in cell function by limiting oxidative stress directly via antioxidant effects and indirectly by protein–protein interactions with key signaling molecules such as Trx-interacting protein (TrxIP). Examples include the findings that hyperglycemia and diabetes induce TrxIP and decrease Trx activity [45]. ROS-scavenging function through p38 MAPK-mediated induction of function by TrxIP. In this study, a pancreatic decrease in TrxR activity in diabetic rats is observed (Table 2). This decrease of TrxR concentration is being interpreted as a marker of oxidative stress and/or modification of signaling pathways that could affect insulin secretion. In addition a reduction in plasma glucose concentration with stimulation of insulin secretion that is inconsistent with the result mentioned by El-Missiry and El-Gindy (2000) who reported that the active principles from plant sources might act by several mechanisms such as stimulation of insulin secretion, an increasing the antioxidative capability, enhancing the effect of insulin and adrenaline as well as increasing repair/proliferation of pancreatic β-cells which confirmed in our study by gradual increasing the level of insulin content in pancreatic tissues with increasing the extract doses [46].

The protective effect of M. hexandra leaf and fruit extracts on STZ-induced diabetes in rats was introduced herein. Phytochemical investigation of M. hexandra leaf revealed the identification of β-sitosterol, β-amyrin, and 3-O-β-D-glucopyranosy-protobassic acid, respectively. Moreover, two flavonoids were identified in M. hexandra fruit viz. quercetin 3-O-α-L-rhamnopyranoside and myricetin 3-O-α-L-rhamnopyranoside. M. hexandra extracts revealed protective effect on STZ-induced diabetic rats with significant reduction in blood glucose level, elevation in insulin level and reduction in insulin sensitivity. Morover, M. hexandra extracts caused significant reduction in oxidative stress markers indicating their protective effects. Further biological investigation of M. hexandra organs is recommended in future studies.

Data Availability Statement

All data generated or analyzed during this study are included in this published article [and its supplementary information files].

Author contributions

Samyah Alanazi: Methodology, Writing-original draft. Mostafa H. Baky Data curation, Methodology, Writing-original draft, Writing-review and editing. Amel M. Kamal, Methodology, Writing-review and editing. Mohamed R. Elgindi, Methodology, Writing-review and editing. Eman G. Haggag, Methodology, Writing-review and editing Mamdouh M. Ali Conceptualization; Supervision; Writing-review and editing. Mohamed F. Barghash: Data curation, Methodology, Writing-original draft.

Acknowledgments

None

Ethics approval and consent to participate

The experimental protocol was authorized by the Ethical Committee of approval from the committee for animal’s care at the National Research Centre, Egypt (ethic No. 11062).

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.

Funding

None

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In vivo Investigation of The Antidiabetic potential of Manilkara hexandra leaves and fruits extracts against streptozotocin-induced hyperglycaemia in rats

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Abstract

Figures

Introduction

Materials And Methods

Chemicals

Animals

Plant Materials

Plant Extracts

Leaves extracts

Fruits extracts

Induction of diabetes

Extract dosing

Experimental design

Analytical procedures

Blood glucose level

Insulin level and Insulin resistance

Pancreatic lipid peroxidation (TBARS)

Pancreatic reduced glutathione (GSH)

Pancreatic nitric oxide ((NO)

Pancreatic thioredoxin reductase (TrxR) activity

Statistical Analysis

Results

Isolated compounds

Effect of extracts on plasma glucose, insulin levels and insulin resistance in diabetic rats

Effect of extracts on oxidative stress in diabetic rats

Discussion

Conclusion

Declarations

References

Additional Declarations

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