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Hypoglycemic effect of Egyptian Morus alba root bark extract: Effect on diabetes and lipid peroxidation of streptozotocin-induced diabetic rats
Journal of Ethnopharmacology 100 (2005) 333–338
Hypoglycemic effect of Egyptian Morus alba root bark extract: Effect on diabetes and lipid peroxidation of streptozotocin-induced diabetic rats Abdel Nasser B. Singab a,1 , Hesham A. El-Beshbishy b,2 , Makiko Yonekawa c , Taro Nomura c,3 , Toshio Fukai c,∗ a
c
Department of Pharmacognosy, Faculty of Pharmacy, Ain Shams University, Abbassia, Cairo, Egypt b Department of Biochemistry, Faculty of Pharmacy, Al-Azhar University, Nasr City, Cairo, Egypt Department of Biophysical Chemistry, School of Pharmaceutical Sciences, Toho University, 2-2-1 Miyama, Funabashi, Chiba 274-8510, Japan Received 30 September 2004; received in revised form 1 March 2005; accepted 24 March 2005 Available online 10 May 2005
Abstract The hypoglycemic activity of the flavonoids rich fraction of 70% alcohol extract of the Egyptian Morus alba root bark (MRBF-3) was evaluated after its oral administration to streptozotocin-induced diabetic rats. Diabetes was induced by injection of 60 mg kg−1 i.p. The administration of MRBF-3 to streptozotocin (STZ)-diabetic rats for 10 days in a dose of 200 and 400 mg kg−1 day−1 was not significant. However, administration of MRBF-3 for 10 days (600 mg kg−1 day−1 ) significantly reduced the amount of the glucose from control level (379 ± 9 mg/dl) to a lower level (155 ± 8 mg/dl) and significantly increased the insulin level from control (10.8 ± 0.3 U/ml) to a high level (15.6 ± 0.3 U/ml). The measurement of produced lipid peroxides (expressed as the amount of thiobarbituric acid (TBA) reactive substance, nmol TBARS/ml serum) indicated antiperoxidative activity of MRBF-3. The oral administration of MRBF-3 to STZ-diabetic rats significantly decreased the lipid peroxides from 6.3 ± 0.8 to 5.1 ± 0.7 nmol TBARS/ml serum. The phytochemical investigation of MRBF-3 resulted in the isolation of four hydrophobic flavonoids with one or two isoprenoid groups (log P = 5–9): morusin, cyclomorusin, neocyclomorusin, and kuwanon E, a 2-arylbenzofuran, moracin M, and two triterpenes, betulinic acid and methyl ursolate. The data obtained from this study revealed that MRBF-3 may protect pancreatic  cells from degeneration and diminish lipid peroxidation. However, this is the first biological screening of the Egyptian Morus alba root bark; further future merit studies including clinical study will be necessary in order to confirm the results obtained from this study. © 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Moraceae; Morus alba; Streptozotocin; Antidiabetic; Lipid peroxidation; Flavonoids; 2-Arylbenzofuran
1. Introduction The peroxidation of cellular membrane lipids can lead to cell necrosis and is considered to be implicated in a number ∗
Corresponding author. Tel.: +81 47 472 1792; fax: +81 47 476 6195. E-mail addresses: [email protected] (A.N.B. Singab), hesham [email protected] (H.A. El-Beshbishy), [email protected] (T. Fukai). 1 Present address: Department of Pharmacognosy, School of Pharmacy, Addis Ababa University, P.O. Box 1176, Addis Ababa, Ethiopia. 2 Present address: Biochemistry Department, Faculty of Science, Taibah University at Al-Madinah, P.O. Box 344, Saudi Arabia. 3 Present address: Nihon Pharmaceutical University, 10281 Komuro, Inamachi, Kita-Adachi-gun, Saitama 362-0806, Japan. 0378-8741/$ – see front matter © 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2005.03.013
of pathophysiological conditions (Kappus, 1987) as type 1 diabetes mellitus. This pathological condition is thought to occur as a result of the loss of insulin-producing pancreatic  cells by an environmentally triggered autoimmune reaction. It is likely that the free radicals and active oxygen species predominate in diabetes (Halliwell and Gutteridge, 1990). Substantial efforts have been made in recent years to identify both natural and synthetic antidiabetics. The screening of more effective and safe hypoglycemic agents has continued to be an important area. Furthermore, after the recommendations of WHO on diabetes mellitus (WHO Expert Committee on Diabetes Mellitus, 1980), investigation on hypoglycemic agents from medicinal plants has become more essential.
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The root bark of mulberry tree (Morus species, Moraceae) has been used by human beings for at least 4000 years. The earliest written reference to the use of mulberry tree is contained in the first Chinese dispensatory “Shen Nong Ben Cao Jing” whose original anonymous volume probably appeared by the end of the third century. In the book, the root bark of the plant is called as ‘Sang Bai Pi’ (Nomura, 1988; Nomura et al., 2003). The herb has been used as antiphlogistic, diuretic, expectorant and antidiabetic in traditional Chinese medicine (Nomura, 1988; Chen et al., 1995). A piperidine alkaloid (moranoline = 1-deoxynojirimycin) and glycoproteins (morans A and 20 K) were isolated as antidiabetic agents from Morus root bark and/or leaves (Yagi et al., 1976; Hikino et al., 1985; Kim et al., 1999). On the other hand, the antioxidant potency of some phenolic compounds (flavonoids, stilbenes and 2-arylbenzofurans) from Morus alba has been reported (Fukai et al., 2003; Fukai and Nomura, 1998; Jin et al., 2002; Nomura et al., 1977, 1980; Sharma et al., 2001). The present paper focused on the investigation of the hypoglycemic potential of the flavonoids rich fraction of 70% alcoholic extract of Egyptian Morus alba root bark (MRBF-3) as well as isolation of its main constituents. The hypoglycemic effect of MRBF-3 was investigated through the determination of serum glucose and insulin hormone levels in experimentally induced diabetic rats treated with streptozotocin that leads to complete destruction of pancreatic -cells (O’Brien et al., 1997). Also, the antiperoxidative effect of MRBF-3 (through the measurement of thiobarbituric acid reactive substance; TBARS, an indicative of lipid peroxidation) was carried out.
2. Materials and methods 2.1. Plant material The root bark of Morus alba L. was collected in delta region, Egypt, and was air-dried. The identification of the plant was verified by Dr. Abd El Salaam Mohamed Al-Nowiahi, Professor of Plant Taxonomy, Faculty of Science, Ain Shams University, Abbassia, Cairo, Egypt. A voucher specimen of the authenticated Morus alba L. root bark was deposited at the Department of Pharmacognosy, Faculty of Pharmacy, Ain Shams University, Abbassia, Cairo, Egypt. 2.2. Apparatus and chemicals used for phytochemical study Melting points were determined using a Yanaco MP-500V apparatus (GTR TEC Corp., Kyoto, Japan); they were uncorrected. The UV spectra were measured on Shimadzu UV-265 spectrophotometer (Shimadzu, Kyoto, Japan). The 1 H and 13 C NMR spectra were recorded on Jeol JNM EX-400 (Jeol Ltd., Akishima, Japan). Electron ionisation (EI) mass spectra (MS) were measured with a Joel JMS-AMM System II-50. The silica gel 60 (70–230 mesh) for column chromatography
(CC) was purchased from E-Merck (Darmstadt, Germany). Thin layer chromatography (TLC) was performed on precoated silica gel G 60 F254 sheets, 0.25 mm and pre-coated silica gel 60 G F254 , 2 mm thickness for preparative work (EMerck, Germany). The cellulose powder for CC was supplied from E-Merck, Germany. 2.3. Chemicals used for biological study Streptozotocin was purchased from Sigma (St. Louis, MO, USA), while thiobarbituric acid was purchased from Fluka (Buchs, Switzerland). 1-Deoxynojirimycin was obtained from Wako Pure Chem. Ind., Ltd. (Osaka, Japan). All remaining chemicals were of the highest grade commercially available. The rat insulin radioimmunoassay kit was purchased from Amersham Biosciences, (Piscataway, NJ, USA). Glucose oxidase kit was purchased from Diamond Diagnostics, Egypt. Glucotest (glucose urine strips) was purchased from Roche Diagnostics Germany (Mannheim, Germany). 2.4. Plant extraction and isolation The air-dried root bark of Morus alba (1 kg) was successively extracted with 70% ethanol. The extract was concentrated in vacuo to give a residue (220 g). Fifty grams of the extract was fractionated over cellulose CC (800 g), eluted with H2 O (100%), 50% MeOH and finally with 100% MeOH to give three fractions (F-1, F-2 and F-3). Each fraction was evaporated in vacuo to give 14.8 g (F-1), 10.9 g (F-2) and 23.5 g (F-3) of residue, respectively. TLC analysis of these fractions indicated that the third fraction (MRBF-3) was the richest one of flavonoids (FeCl3 detection) and did not contain 1-deoxynojirimycin. Ten grams of MRBF-3 was subjected to silica gel CC and eluted with a mixture of n-hexane and acetone, increasing the amount of acetone, to yield 40 fractions. Betulinic acid (22 mg) and methyl ursolate (20 mg) were obtained from fractions 12 and 14, respectively, by crystallization from the mixture of methanol and acetone. Their identification was carried out by co-TLC with authentic specimens and by the EI-MS data. Methyl ursolate was isolated for the first time from family Moraceae. Fraction 15 showed a yellow precipitate after addition of benzene. This fraction (150 mg) was purified over silica gel CC eluted with benzene saturated with H2 O to give five fractions. Fraction 4 was crystallized from mixture of n-hexane and diethyl ether to yield 50 mg of morusin (1), and then the mother liquor of fraction 5 was recrystallized from n-hexane:diethyl ether mixture to give 6 mg of cyclomorusin (2). Fraction 16 showed a major spot on TLC using n-hexane:acetone (98:2), it was subjected to preparative TLC using n-hexane:diethyl ether (9:1) to yield 20 mg of a known compound (3); the compound was called tentatively as compound A in the previous paper (Nomura, 1988), and thus we name here as neocyclomorusin for this compound. Fractions 18 and 19 were collected and evaporated to give 100 mg of residues. These materials were subjected to preparative TLC, development systems; n-hexane:diethyl
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Fig. 1. Structures of flavonoids (1–3 and 5) and a 2-arylbenzofuran (4) from root bark of the Egyptian Morus alba.
ether (4:1), to yield 5 mg of moracin M (4). Fractions 20 and 21 were combined and purified with preparative TLC, using CHCl3 :ethyl acetate (5:1) to give 6 mg of (±)-kuwanon E (5). These known phenolic compounds (1–5, Fig. 1) (Nomura, 1988) were identified by the comparison with the authentic specimens.
blood was collected into heparinised chilled tubes containing sodium fluoride (to inhibit glycolysis). Serum was separated by centrifugation at 4 ◦ C and stored at −20 ◦ C until determination of serum glucose, lipid peroxides and serum insulin concentrations. 2.7. Measurements of the biochemical parameters
2.5. Animals and induction of diabetes Male Wister rats weighing 170–260 g were obtained from the experimental animal care centre of Faculty of Pharmacy, Al-Azhar University. The STZ was dissolved in 0.1 ml of citrate buffer (pH 4.5). Animals were made diabetic by injection of a single dose of STZ (60 mg/kg) intraperitoneally. STZ-treated rats were given 5% glucose in their drinking water for the first 24 h to counter any initial hypoglycemia. Control animals were similarly injected with vehicle only. On the third day, the animals were checked for the presence of glucose in the urine using enzymatic test strips. The animals were maintained under standard conditions of temperature 24 ± 5 ◦ C and 55 ± 5% relative humidity with a regular 12 h light:12 h dark cycle and allowed free access to standard laboratory food (Purina Chow) and water. All animals were treated humanely in accordance with the guideline for care of animals as set by WHO.
Blood samples were centrifuged at 4 ◦ C for 15 min at 3000 rpm and the sera obtained were used for biochemical analyses. Serum glucose was estimated by the glucose oxidase method (Trinder, 1969). Serum immunoreactive insulin was determined by the radioimmunoassay method using Amarsham insulin RIA kit using rat insulin as the standard (Gordon et al., 1985). Lipid peroxides (thiobarbituric acid reactive substance, TBARS production) were determined according to the previous method (Ohkawa et al., 1979). 2.8. Statistical analysis Data are expressed as means ± S.E.M. Statistical comparison between different groups were done using one-way analysis of variance (ANOVA) followed by the Tukey–Kramer multiple comparison test, to judge the difference between various groups. Significance was accepted at P < 0.05.
2.6. Experimental protocol for the biological study 3. Results The rats were divided at random into three groups of 10 animals each. The first group was the control (received citrate buffer alone), the second was STZ-diabetic control, while the third was the STZ-diabetic rats fed orally with MRBF-3 with a daily dose (600 mg/kg b.wt.) for 10 successive days (the administration of MRBF-3 to STZ-diabetic rats for 10 days in a dose of 200 and 400 mg/kg b.wt. showed no significant results). On the eleventh day, the rats were subjected to light ether anaesthesia and killed by cervical dislocation. Trunk
The i.p. injection of streptozotocin in a dose of 60 mg/kg b.wt. into rats resulted in loss of body weight from 230 to 207 g, P < 0.05 as compared with the normal control rats. The administration of MRBF-3 (600 mg kg−1 day−1 ) for 10 days to STZ-diabetic rats significantly restored the loss in body weight to reach a value of 226 g. Regarding the serum glucose level, the STZ-diabetic rat elicited a significant rise in serum glucose from 144 to 379 mg/dl, P < 0.05, as compared
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Table 1 Effect of Morus alba (MRBF-3) oral administration on body weight (g), serum glucose (mg/dl), serum insulin hormone (U/ml) and lipid peroxides production, described as nmol TBARS/ml serum in STZ-diabetic treated rats Control (untreated) Body weight (g) Serum glucose (mg/dl) Serum insulin (U/ml) Lipid peroxides
230 143.45 17.18 5.29
± ± ± ±
3.77 7.84 1.49 0.72
STZ-diabetic rats 207 378.77 10.75 6.26
± ± ± ±
2.49* 8.72* 0.28* 0.81*
STZ-diabetic rats fed on MRBF-3 225.98 155.07 15.58 5.13
± ± ± ±
2.06† 8.35† 0.25† 0.67†
Results are expressed as means ± S.E.M. (n = 10). * Significantly different from controls (P < 0.05). † Significantly different from diabetic control (P < 0.05).
to the normal control rats (Table 1). On the contrary, the STZdiabetic rats fed on MRBF-3 showed a significant decline in serum glucose level to a value of 155 mg/dl, P < 0.05 (which was nearly similar to those of normal control group) as compared to STZ-diabetic rats (Table 1). Consequently, serum insulin level had been significantly improved after administration of MRBF-3 to STZ-diabetic rats (15.6 U/ml; P < 0.05), as compared with STZ-induced diabetic rats (10.8 U/ml; P < 0.05) (Table 1). The production of lipid peroxides was significantly decreased in MRBF-3 treated STZ-diabetic rats from 6.3 to 5.1 nmol TBARS/ml serum; P < 0.05. The lipid peroxides was 5.3 nmol TBARS/ml serum for normal control rats, P < 0.05 (Table 1).
4. Discussion Asian and Egyptian mulberry tree (Morus alba, Morus australis, Morus bombycis, Morus mongolica and Morus lhou) is a candidate of promising drugs, because the plant has been widely cultivated in many countries and has been traditionally used in treatment of several diseases (Nomura, 1988; Chen et al., 1995). Fractionation of 70% alcohol extract of Morus alba root bark over cellulose column chromatography using H2 O, 50% MeOH and MeOH yielded three fractions, F-1, F-2 and F-3, respectively. TLC investigations of the three fractions indicated that F-3 was the richest one of flavonoids and was moranoline free. Therefore, this fraction (MRBF-3) was subjected to in vivo screening. It showed a promising hypoglycemic and inhibition of lipid peroxidation activities. So, it was subjected to chromatographic purification using different techniques in order to isolate its main constituents. Four main flavonoids with one or two isoprenoid groups (log P = 5–9)4 as well as a 2-arylbenzofuran (log P = 2.4) were isolated and identified. These compounds may be attributed to the fact that the phenolic contents of Morus alba root bark extract act as free radical scavengers (Fukai et al.,
4 1-Octanol-water partition coefficient (log P): data from database of Chemical Abstract Service (SciFinder Scholar). These log P were calculated using Advanced Chemistry Development (ACD) Software Solaris v.4.67.
2003; Nomura et al., 1977, 1980; Sharma et al., 2001)5 that arise as a result of STZ-intoxication, resulting in alleviating the state of diabetes mellitus found in the STZ-induced diabetic rats (Furusho et al., 2002). Similar antihyperglycemic fraction containing 2-arylbenzofurans (moracin M and glycosides of 2-arylbenzofuran with an isoprenoid group) was prepared from the leaves of Argentine mulberry tree, Morus insignis (Basnet et al., 1993). Furthermore, some Morus flavones with a prenyl group at C-3 position have inhibitory potency against aldose reductase (Yamaguchi et al., 1991). It could be considered that hypoglycemic action of Morus root bark (Chen et al., 1989, 1995) is due to synergistic or additive action of moranoline (1-deoxynojirimycin), morans (glycopeptides), hydrophobic flavonoids (flavones and flavanones) and 2-arylbenzofurans. The i.p. injection of streptozotocin in a dose of 60 mg/kg b.wt. into rats resulted in a state of type 1 diabetes mellitus (Sharifi et al., 2004). Ten days after STZ treatment, the diabetic rats exhibited a state of severe hyperglycemia as compared with normal control group as shown in Table 1. Poor general conditions of the STZ-diabetic treated rats were observed. A significant decrease in body weight (↓−11%) was observed 10 days after STZ treatment when compared with intact normal control rats. It was observed that oral administration of MRBF-3 for 10 days normalized the body weight loss (↑+9%) elicited by STZ. The MRBF-3 treated STZ-diabetic rats showed significant rise in body weight to a value that was nearly similar to the intact control rats. This finding was in close agreement with that of other investigators (Junod et al., 1969; Craft and Failla, 1983; Failla and Kiser, 1981; Schwechter et al., 2003), who noticed a body weight gain upon improvement of the diabetes status. The metabolism of glucose, proteins and lipids is abnormal in diabetes due to insulin secretion defect, leading to various metabolic disorders (Genuth, 1973; Goldstein et al., 2004) and complications (Saudek and Eder, 1979; Kannel and McGee, 1979). The STZ-diabetic rats elicited a signifi5 The main flavonoid of MRBF-3, morusin (1), gives a minor pyranoflavone (±)-neocyclomorusin (3) by a radical reaction via a hydroperoxide with same skeletal structure of 3. On the other hand, (±)-cyclomorusin (2) is derived from 1 by the radical reaction under oxygen-free conditions. One of the roles of morusin (1) in the plants is presumably an antioxidant, a quencher of radical chain reactions.
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cant elevation of serum glucose by ↑+265% associated with a decrease in serum insulin by ↓−58%, indicating that their pancreatic  cells were irreversibly damaged. Our data indicated that the oral administration of MRBF3 for 10 days (600 mg kg−1 day−1 ) to the STZ-diabetic rats showed a significant reduction in serum glucose by ↓−41% and an increase in serum insulin level by +44% as compared to STZ-diabetic rats. Lipid peroxidation is a marker of cellular oxidative damage initiated by reactive oxygen species (Farber et al., 1990). It was reported that diabetics are highly sensitive to oxidative stress (Pritchard et al., 1986; Baynes, 1991; Urano et al., 1991). In STZ-diabetic animals, the STZ generates nitric oxide, which is a powerful free radical oxidant (Kwon et al., 1994) results in an increase in serum level of lipid peroxides due to oxidation of cells (Wakame, 1999). The STZ-diabetic rats exerted a significant elevation of lipid peroxides (expressed as nmol TBARS/ml serum) by ↑+15% as compared to the normal control. The production of lipid peroxides was significantly decreased in MRBF-3 treated STZ-diabetic rats by ↓−22% as compared to STZ-diabetic rats (Table 1). In this experiment, the oral administration of the extract of Morus alba root bark (MRBF-3) suppresses the elevation of lipid peroxides in STZ-diabetic rats compared with control. The data included in this work suggested that MRBF-3 prevents cellular damage induced by STZ via inhibition of lipid peroxidation possibly through antioxidant mechanisms due to its high flavonoids content, which is in accordance with the reported data on this kind of compounds (Oh et al., 2002), as well as it preserves the capability of insulin secretion. Also, this finding was in accordance with the work done by Coskun et al. (2005) who reported that a natural antioxidant flavonol (quercetin) has protective effect in diabetes by decreasing oxidative stress and preservation of pancreatic  cell integrity. In our study however, we demonstrated the presence of five phenolic compounds in the active extract; the data of these compounds as hypoglycemic agents are not reported here. So, further detail experiments are required to clarify the role of these flavonoids and 2-arylbenzofuran as hypoglycemic agents.
5. Conclusions The present study indicated that the flavonoids-rich fraction of 70% alcohol extract of the Morus alba root bark can recover from STZ-induced diabetes in rats. The fraction may protect pancreatic  cells from degeneration and diminish lipid peroxidation of cells. However, further merit investigations including clinical study are necessary in the future to confirm this hypothesis.
References Basnet, P., Kadota, S., Terashima, S., Shimizu, M., Namba, T., 1993. Two new 2-arylbenzofuran derivatives from hypoglycemic activity-bearing
337
fractions of Morus insignis. Chemical & Pharmaceutical Bulletin 41, 1238–1243. Baynes, J.W., 1991. Role of oxidative stress in development of complications in diabetes. Diabetes 40, 405–412. Chen, F.-J., Nakashima, N., Kimura, I., Kimura, M., 1989. Hypoglycemic activity of mulberry leaves (Folium Mori) and Cortex Mori radicis in streptozotocin-induced diabetic mice fasted and none fasted. Wakan Iyakugaku Kaisshi 6, 374–375. Chen, F.-J., Nakashima, N., Kimura, I., Kimura, M., 1995. Hypoglycemic activity and mechanisms of extracts from mulberry leaves (Folium Mori) and Cortex Mori radicis in streptozotocin-induced diabetic mice. Yakugaku Zasshi 115, 476–482. Coskun, O., Kanter, M., Korkmaz, A., Oter, S., 2005. Quercetin, a flavonoid antioxidant, prevents and protects streptozotocin-induced oxidative stress and -cell damage in rat pancreas. Pharmacological Research 51, 117–123. Craft, N.E., Failla, M.L., 1983. Zinc, iron, and copper absorption in the streptozotocin-diabetic rat. American Journal of Physiology—Endocrinology and Metabolism 244, E122–E128. Failla, M.L., Kiser, R.A., 1981. Altered tissue content and cytosol distribution of trace metals in experimental diabetes. The Journal of Nutrition 111, 1900–1909. Farber, J.L., Kyle, M.E., Coleman, J.B., 1990. Biology of disease: mechanisms of cell injury by activated oxygen species. Laboratory Investigation 62, 670–679. Fukai, T., Nomura, T., 1998. Proof against 2 -hydroxy-3-prenylflavoneoxygen complex by laser desorption/ionization time-of-flight mass spectrometry. Rapid Communications in Mass Spectrometry 12, 1945–1951, and references cited therein. Fukai, T., Satoh, K., Nomura, T., Sakagami, H., 2003. Antinephritis and radical scavenging activities of prenylflavonoids. Fitoterapia 74, 720–724. Furusho, T., Kataoka, E., Yasuhara, T., Wada, M., Innami, S., 2002. Administration of -carotene suppresses lipid peroxidation in tissues and improves the glucose tolerance ability of streptozotocin-induced diabetic rats. International Journal for Vitamin and Nutrition Research 72, 71–76. Genuth, S.M., 1973. Plasma insulin and glucose profiles in normal, obese, and diabetic persons. Annals of Internal Medicine 79, 812– 822. Goldstein, D.E., Little, R.R., Lorenz, R.A., Malone, J.I., Nathan, D., Peterson, C.M., Sacks, D.B., 2004. Test of glycemia in diabetes. Diabetes Care 27, 1761–1773. Gordon, C., Yates, A.P., Davies, D., 1985. Evidence for a direct action of exogenous insulin on the pancreatic islets of diabetic mice: islet response to insulin pre-incubation. Diabetologia 28, 291–294. Halliwell, B., Gutteridge, J.M., 1990. Role of free radicals and catalytic metal ions in human disease: an overview. Methods in Enzymology 186, 1–85. Hikino, H., Mizuno, T., Oshima, Y., Konno, C., 1985. Validity of the oriental medicines. 80. Antidiabetes drugs. 4. Isolation and hypoglycemic activity of moran A, a glycoprotein of Morus alba root bark. Planta Medica, 159–160. Jin, W.Y., Na, M.K., An, R.B., Lee, H.Y., Bae, K.H., Kang, S.S., 2002. Antioxidant compounds from twig of Morus alba. Natural Product Sciences 8, 129–132 (Chemical abstracts 139, 106237.). Junod, A., Lambert, A.E., Stauffacher, W., Renold, A.E., 1969. Diabetogenic action of streptozotocin: relationship of dose to metabolic response. The Journal of Clinical Investigation 48, 2129–2139. Kannel, W.B., McGee, D.L., 1979. Diabetes and cardiovascular disease. The Framingham study. The Journal of the American Medical Association 241, 2035–2038. Kappus, H., 1987. Oxidative stress in chemical toxicity. Archives of Toxicology 60, 144–149. Kim, E.S., Park, S.J., Lee, E.J., Kim, B.K., Huh, H., Lee, B.J., 1999. Purification and characterization of Moran 20 K from Morus alba. Archives of Pharmacal Research 22, 9–12.
338
A.N.B. Singab et al. / Journal of Ethnopharmacology 100 (2005) 333–338
Kwon, N.S., Lee, S.H., Choi, C.S., Kho, T., Lee, H.S., 1994. Nitric oxide generation from streptozotocin. The FASEB Journal 8, 529–533. Nomura, T., 1988. Phenolic compounds of the mulberry tree and related plants. In: Herz, W., Grisebach, H., Kirby, G.W., Tamm, Ch. (Eds.), Progress in the Chemistry of Organic Natural Products, vol. 53. Springer, Vienna, pp. 87–201, and references cited therein. Nomura, T., Fukai, T., Hano, Y., 2003. Chemistry and biological activities of isoprenylated flavonoids from medicinal plants (moraceous plants and Glycyrrhiza species). In: Atta-ur-Rahman (Ed.), Studies in Natural Product Chemistry, vol. 28: Bioactive Natural Products (Part I). Elsevier, Amsterdam, pp. 199–256, and references cited therein. Nomura, T., Fukai, T., Katayanagi, M., 1977. Oxidative cyclization of morusin with manganese dioxide. Heterocycles 6, 1847–1854. Nomura, T., Fukai, T., Matsumoto, J., 1980. Studies on the constituents of the cultivated mulberry tree. 6. Oxidative cyclization of morusin. Journal of Heterocyclic Chemistry 17, 641–646. O’Brien, B.A., Harmon, B.V., Cameron, D.P., Allan, D.J., 1997. Apoptosis is the mode of beta-cell death responsible for the development of IDDM in the nonobese diabetic (NOD) mouse. Diabetes 46, 750– 757. Oh, H., Ko, E.-K., Jun, J.-Y., Oh, M.-H., Park, S.-U., Kang, K.-H., Lee, H.-S., Kim, Y.-C., 2002. Hepatoprotective and free radical scavenging activities of prenylflavonoids, coumarin, and stilbene from Morus alba. Planta Medica 68, 932–934. Ohkawa, H., Ohishi, N., Yagi, K., 1979. Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Analytical Biochemistry 95, 351–358. Pritchard Jr., K.A., Patel, S.T., Karpen, C.W., Newman, H.A., Panganamala, R.V., 1986. Triglyceride-lowering effect of dietary vitamin E in streptozotocin-induced diabetic rats. Increased lipoprotein lipase activity in livers of diabetic rats fed high dietary vitamin E. Diabetes 35, 278–281.
Saudek, C.D., Eder, H.A., 1979. Lipid metabolism in diabetes mellitus. The American Journal of Medicine 66, 843–852. Schwechter, E.M., Vel´ısˇkov´a, J., Vel´ısˇek, L., 2003. Correlation between extracellular glucose and seizure susceptibility in adult rats. Annals of Neurology 53, 91–101. Sharifi, A.M., Mousavi, S.H., Larijani, B., 2004. Study of interaction between nitric oxide and ACE activity in STZ-induced diabetic rats: role of insulin. Pharmacological Research 50, 261–266. Sharma, R., Sharma, A., Shono, T., Takasugi, M., Shirata, A., Fujimura, T., Machii, H., 2001. Mulberry moracins: scavengers of UV stressgenerated free radicals. Bioscience, Biotechnology, and Biochemistry 65, 1402–1405. Trinder, P., 1969. Determination of blood glucose using an oxidaseperoxidase system with a non-carcinogenic chromogen. Journal of Clinical Pathology 22, 158–161. Urano, S., Hoshi-Hashizume, M., Tochigi, N., Matsuo, M., Shiraki, M., Ito, H., 1991. Vitamin E and the susceptibility of erythrocytes and reconstituted liposomes to oxidative stress in aged diabetics. Lipids 26, 58–61. Wakame, K., 1999. Protective effects of active hexose correlated compound (AHCC) on the onset of diabetes induced by streptozotocin in the rat. Biomedical Research 20, 145–152. WHO Expert Committee on Diabetes Mellitus, Diabetes Mellitus Second Report. Technical Report Series 646, World Health Organization, Geneva, 1980. Yagi, M., Kouno, T., Aoyagi, Y., Murai, H., 1976. The structure of moranoline, a piperidine alkaloid from Morus species. Nippon Nogei Kagaku Kaishi 50, 571–572. Yamaguchi, T., Sato, S., Mitsuhashi, H., Nomura, T., 1991. Aldose reductase inhibitors containing benzopyrans for treatment of diseases associated with diabetes. Japan Kokai Tokkyo Koho 1991. 19910325 (Chemical abstracts 116, 46283).
Hypoglycemic effect of Egyptian Morus alba root bark extract: Effect on diabetes and lipid peroxidation of streptozotocin-induced diabetic rats Abdel Nasser B. Singab a,1 , Hesham A. El-Beshbishy b,2 , Makiko Yonekawa c , Taro Nomura c,3 , Toshio Fukai c,∗ a
c
Department of Pharmacognosy, Faculty of Pharmacy, Ain Shams University, Abbassia, Cairo, Egypt b Department of Biochemistry, Faculty of Pharmacy, Al-Azhar University, Nasr City, Cairo, Egypt Department of Biophysical Chemistry, School of Pharmaceutical Sciences, Toho University, 2-2-1 Miyama, Funabashi, Chiba 274-8510, Japan Received 30 September 2004; received in revised form 1 March 2005; accepted 24 March 2005 Available online 10 May 2005
Abstract The hypoglycemic activity of the flavonoids rich fraction of 70% alcohol extract of the Egyptian Morus alba root bark (MRBF-3) was evaluated after its oral administration to streptozotocin-induced diabetic rats. Diabetes was induced by injection of 60 mg kg−1 i.p. The administration of MRBF-3 to streptozotocin (STZ)-diabetic rats for 10 days in a dose of 200 and 400 mg kg−1 day−1 was not significant. However, administration of MRBF-3 for 10 days (600 mg kg−1 day−1 ) significantly reduced the amount of the glucose from control level (379 ± 9 mg/dl) to a lower level (155 ± 8 mg/dl) and significantly increased the insulin level from control (10.8 ± 0.3 U/ml) to a high level (15.6 ± 0.3 U/ml). The measurement of produced lipid peroxides (expressed as the amount of thiobarbituric acid (TBA) reactive substance, nmol TBARS/ml serum) indicated antiperoxidative activity of MRBF-3. The oral administration of MRBF-3 to STZ-diabetic rats significantly decreased the lipid peroxides from 6.3 ± 0.8 to 5.1 ± 0.7 nmol TBARS/ml serum. The phytochemical investigation of MRBF-3 resulted in the isolation of four hydrophobic flavonoids with one or two isoprenoid groups (log P = 5–9): morusin, cyclomorusin, neocyclomorusin, and kuwanon E, a 2-arylbenzofuran, moracin M, and two triterpenes, betulinic acid and methyl ursolate. The data obtained from this study revealed that MRBF-3 may protect pancreatic  cells from degeneration and diminish lipid peroxidation. However, this is the first biological screening of the Egyptian Morus alba root bark; further future merit studies including clinical study will be necessary in order to confirm the results obtained from this study. © 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Moraceae; Morus alba; Streptozotocin; Antidiabetic; Lipid peroxidation; Flavonoids; 2-Arylbenzofuran
1. Introduction The peroxidation of cellular membrane lipids can lead to cell necrosis and is considered to be implicated in a number ∗
Corresponding author. Tel.: +81 47 472 1792; fax: +81 47 476 6195. E-mail addresses: [email protected] (A.N.B. Singab), hesham [email protected] (H.A. El-Beshbishy), [email protected] (T. Fukai). 1 Present address: Department of Pharmacognosy, School of Pharmacy, Addis Ababa University, P.O. Box 1176, Addis Ababa, Ethiopia. 2 Present address: Biochemistry Department, Faculty of Science, Taibah University at Al-Madinah, P.O. Box 344, Saudi Arabia. 3 Present address: Nihon Pharmaceutical University, 10281 Komuro, Inamachi, Kita-Adachi-gun, Saitama 362-0806, Japan. 0378-8741/$ – see front matter © 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2005.03.013
of pathophysiological conditions (Kappus, 1987) as type 1 diabetes mellitus. This pathological condition is thought to occur as a result of the loss of insulin-producing pancreatic  cells by an environmentally triggered autoimmune reaction. It is likely that the free radicals and active oxygen species predominate in diabetes (Halliwell and Gutteridge, 1990). Substantial efforts have been made in recent years to identify both natural and synthetic antidiabetics. The screening of more effective and safe hypoglycemic agents has continued to be an important area. Furthermore, after the recommendations of WHO on diabetes mellitus (WHO Expert Committee on Diabetes Mellitus, 1980), investigation on hypoglycemic agents from medicinal plants has become more essential.
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The root bark of mulberry tree (Morus species, Moraceae) has been used by human beings for at least 4000 years. The earliest written reference to the use of mulberry tree is contained in the first Chinese dispensatory “Shen Nong Ben Cao Jing” whose original anonymous volume probably appeared by the end of the third century. In the book, the root bark of the plant is called as ‘Sang Bai Pi’ (Nomura, 1988; Nomura et al., 2003). The herb has been used as antiphlogistic, diuretic, expectorant and antidiabetic in traditional Chinese medicine (Nomura, 1988; Chen et al., 1995). A piperidine alkaloid (moranoline = 1-deoxynojirimycin) and glycoproteins (morans A and 20 K) were isolated as antidiabetic agents from Morus root bark and/or leaves (Yagi et al., 1976; Hikino et al., 1985; Kim et al., 1999). On the other hand, the antioxidant potency of some phenolic compounds (flavonoids, stilbenes and 2-arylbenzofurans) from Morus alba has been reported (Fukai et al., 2003; Fukai and Nomura, 1998; Jin et al., 2002; Nomura et al., 1977, 1980; Sharma et al., 2001). The present paper focused on the investigation of the hypoglycemic potential of the flavonoids rich fraction of 70% alcoholic extract of Egyptian Morus alba root bark (MRBF-3) as well as isolation of its main constituents. The hypoglycemic effect of MRBF-3 was investigated through the determination of serum glucose and insulin hormone levels in experimentally induced diabetic rats treated with streptozotocin that leads to complete destruction of pancreatic -cells (O’Brien et al., 1997). Also, the antiperoxidative effect of MRBF-3 (through the measurement of thiobarbituric acid reactive substance; TBARS, an indicative of lipid peroxidation) was carried out.
2. Materials and methods 2.1. Plant material The root bark of Morus alba L. was collected in delta region, Egypt, and was air-dried. The identification of the plant was verified by Dr. Abd El Salaam Mohamed Al-Nowiahi, Professor of Plant Taxonomy, Faculty of Science, Ain Shams University, Abbassia, Cairo, Egypt. A voucher specimen of the authenticated Morus alba L. root bark was deposited at the Department of Pharmacognosy, Faculty of Pharmacy, Ain Shams University, Abbassia, Cairo, Egypt. 2.2. Apparatus and chemicals used for phytochemical study Melting points were determined using a Yanaco MP-500V apparatus (GTR TEC Corp., Kyoto, Japan); they were uncorrected. The UV spectra were measured on Shimadzu UV-265 spectrophotometer (Shimadzu, Kyoto, Japan). The 1 H and 13 C NMR spectra were recorded on Jeol JNM EX-400 (Jeol Ltd., Akishima, Japan). Electron ionisation (EI) mass spectra (MS) were measured with a Joel JMS-AMM System II-50. The silica gel 60 (70–230 mesh) for column chromatography
(CC) was purchased from E-Merck (Darmstadt, Germany). Thin layer chromatography (TLC) was performed on precoated silica gel G 60 F254 sheets, 0.25 mm and pre-coated silica gel 60 G F254 , 2 mm thickness for preparative work (EMerck, Germany). The cellulose powder for CC was supplied from E-Merck, Germany. 2.3. Chemicals used for biological study Streptozotocin was purchased from Sigma (St. Louis, MO, USA), while thiobarbituric acid was purchased from Fluka (Buchs, Switzerland). 1-Deoxynojirimycin was obtained from Wako Pure Chem. Ind., Ltd. (Osaka, Japan). All remaining chemicals were of the highest grade commercially available. The rat insulin radioimmunoassay kit was purchased from Amersham Biosciences, (Piscataway, NJ, USA). Glucose oxidase kit was purchased from Diamond Diagnostics, Egypt. Glucotest (glucose urine strips) was purchased from Roche Diagnostics Germany (Mannheim, Germany). 2.4. Plant extraction and isolation The air-dried root bark of Morus alba (1 kg) was successively extracted with 70% ethanol. The extract was concentrated in vacuo to give a residue (220 g). Fifty grams of the extract was fractionated over cellulose CC (800 g), eluted with H2 O (100%), 50% MeOH and finally with 100% MeOH to give three fractions (F-1, F-2 and F-3). Each fraction was evaporated in vacuo to give 14.8 g (F-1), 10.9 g (F-2) and 23.5 g (F-3) of residue, respectively. TLC analysis of these fractions indicated that the third fraction (MRBF-3) was the richest one of flavonoids (FeCl3 detection) and did not contain 1-deoxynojirimycin. Ten grams of MRBF-3 was subjected to silica gel CC and eluted with a mixture of n-hexane and acetone, increasing the amount of acetone, to yield 40 fractions. Betulinic acid (22 mg) and methyl ursolate (20 mg) were obtained from fractions 12 and 14, respectively, by crystallization from the mixture of methanol and acetone. Their identification was carried out by co-TLC with authentic specimens and by the EI-MS data. Methyl ursolate was isolated for the first time from family Moraceae. Fraction 15 showed a yellow precipitate after addition of benzene. This fraction (150 mg) was purified over silica gel CC eluted with benzene saturated with H2 O to give five fractions. Fraction 4 was crystallized from mixture of n-hexane and diethyl ether to yield 50 mg of morusin (1), and then the mother liquor of fraction 5 was recrystallized from n-hexane:diethyl ether mixture to give 6 mg of cyclomorusin (2). Fraction 16 showed a major spot on TLC using n-hexane:acetone (98:2), it was subjected to preparative TLC using n-hexane:diethyl ether (9:1) to yield 20 mg of a known compound (3); the compound was called tentatively as compound A in the previous paper (Nomura, 1988), and thus we name here as neocyclomorusin for this compound. Fractions 18 and 19 were collected and evaporated to give 100 mg of residues. These materials were subjected to preparative TLC, development systems; n-hexane:diethyl
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Fig. 1. Structures of flavonoids (1–3 and 5) and a 2-arylbenzofuran (4) from root bark of the Egyptian Morus alba.
ether (4:1), to yield 5 mg of moracin M (4). Fractions 20 and 21 were combined and purified with preparative TLC, using CHCl3 :ethyl acetate (5:1) to give 6 mg of (±)-kuwanon E (5). These known phenolic compounds (1–5, Fig. 1) (Nomura, 1988) were identified by the comparison with the authentic specimens.
blood was collected into heparinised chilled tubes containing sodium fluoride (to inhibit glycolysis). Serum was separated by centrifugation at 4 ◦ C and stored at −20 ◦ C until determination of serum glucose, lipid peroxides and serum insulin concentrations. 2.7. Measurements of the biochemical parameters
2.5. Animals and induction of diabetes Male Wister rats weighing 170–260 g were obtained from the experimental animal care centre of Faculty of Pharmacy, Al-Azhar University. The STZ was dissolved in 0.1 ml of citrate buffer (pH 4.5). Animals were made diabetic by injection of a single dose of STZ (60 mg/kg) intraperitoneally. STZ-treated rats were given 5% glucose in their drinking water for the first 24 h to counter any initial hypoglycemia. Control animals were similarly injected with vehicle only. On the third day, the animals were checked for the presence of glucose in the urine using enzymatic test strips. The animals were maintained under standard conditions of temperature 24 ± 5 ◦ C and 55 ± 5% relative humidity with a regular 12 h light:12 h dark cycle and allowed free access to standard laboratory food (Purina Chow) and water. All animals were treated humanely in accordance with the guideline for care of animals as set by WHO.
Blood samples were centrifuged at 4 ◦ C for 15 min at 3000 rpm and the sera obtained were used for biochemical analyses. Serum glucose was estimated by the glucose oxidase method (Trinder, 1969). Serum immunoreactive insulin was determined by the radioimmunoassay method using Amarsham insulin RIA kit using rat insulin as the standard (Gordon et al., 1985). Lipid peroxides (thiobarbituric acid reactive substance, TBARS production) were determined according to the previous method (Ohkawa et al., 1979). 2.8. Statistical analysis Data are expressed as means ± S.E.M. Statistical comparison between different groups were done using one-way analysis of variance (ANOVA) followed by the Tukey–Kramer multiple comparison test, to judge the difference between various groups. Significance was accepted at P < 0.05.
2.6. Experimental protocol for the biological study 3. Results The rats were divided at random into three groups of 10 animals each. The first group was the control (received citrate buffer alone), the second was STZ-diabetic control, while the third was the STZ-diabetic rats fed orally with MRBF-3 with a daily dose (600 mg/kg b.wt.) for 10 successive days (the administration of MRBF-3 to STZ-diabetic rats for 10 days in a dose of 200 and 400 mg/kg b.wt. showed no significant results). On the eleventh day, the rats were subjected to light ether anaesthesia and killed by cervical dislocation. Trunk
The i.p. injection of streptozotocin in a dose of 60 mg/kg b.wt. into rats resulted in loss of body weight from 230 to 207 g, P < 0.05 as compared with the normal control rats. The administration of MRBF-3 (600 mg kg−1 day−1 ) for 10 days to STZ-diabetic rats significantly restored the loss in body weight to reach a value of 226 g. Regarding the serum glucose level, the STZ-diabetic rat elicited a significant rise in serum glucose from 144 to 379 mg/dl, P < 0.05, as compared
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Table 1 Effect of Morus alba (MRBF-3) oral administration on body weight (g), serum glucose (mg/dl), serum insulin hormone (U/ml) and lipid peroxides production, described as nmol TBARS/ml serum in STZ-diabetic treated rats Control (untreated) Body weight (g) Serum glucose (mg/dl) Serum insulin (U/ml) Lipid peroxides
230 143.45 17.18 5.29
± ± ± ±
3.77 7.84 1.49 0.72
STZ-diabetic rats 207 378.77 10.75 6.26
± ± ± ±
2.49* 8.72* 0.28* 0.81*
STZ-diabetic rats fed on MRBF-3 225.98 155.07 15.58 5.13
± ± ± ±
2.06† 8.35† 0.25† 0.67†
Results are expressed as means ± S.E.M. (n = 10). * Significantly different from controls (P < 0.05). † Significantly different from diabetic control (P < 0.05).
to the normal control rats (Table 1). On the contrary, the STZdiabetic rats fed on MRBF-3 showed a significant decline in serum glucose level to a value of 155 mg/dl, P < 0.05 (which was nearly similar to those of normal control group) as compared to STZ-diabetic rats (Table 1). Consequently, serum insulin level had been significantly improved after administration of MRBF-3 to STZ-diabetic rats (15.6 U/ml; P < 0.05), as compared with STZ-induced diabetic rats (10.8 U/ml; P < 0.05) (Table 1). The production of lipid peroxides was significantly decreased in MRBF-3 treated STZ-diabetic rats from 6.3 to 5.1 nmol TBARS/ml serum; P < 0.05. The lipid peroxides was 5.3 nmol TBARS/ml serum for normal control rats, P < 0.05 (Table 1).
4. Discussion Asian and Egyptian mulberry tree (Morus alba, Morus australis, Morus bombycis, Morus mongolica and Morus lhou) is a candidate of promising drugs, because the plant has been widely cultivated in many countries and has been traditionally used in treatment of several diseases (Nomura, 1988; Chen et al., 1995). Fractionation of 70% alcohol extract of Morus alba root bark over cellulose column chromatography using H2 O, 50% MeOH and MeOH yielded three fractions, F-1, F-2 and F-3, respectively. TLC investigations of the three fractions indicated that F-3 was the richest one of flavonoids and was moranoline free. Therefore, this fraction (MRBF-3) was subjected to in vivo screening. It showed a promising hypoglycemic and inhibition of lipid peroxidation activities. So, it was subjected to chromatographic purification using different techniques in order to isolate its main constituents. Four main flavonoids with one or two isoprenoid groups (log P = 5–9)4 as well as a 2-arylbenzofuran (log P = 2.4) were isolated and identified. These compounds may be attributed to the fact that the phenolic contents of Morus alba root bark extract act as free radical scavengers (Fukai et al.,
4 1-Octanol-water partition coefficient (log P): data from database of Chemical Abstract Service (SciFinder Scholar). These log P were calculated using Advanced Chemistry Development (ACD) Software Solaris v.4.67.
2003; Nomura et al., 1977, 1980; Sharma et al., 2001)5 that arise as a result of STZ-intoxication, resulting in alleviating the state of diabetes mellitus found in the STZ-induced diabetic rats (Furusho et al., 2002). Similar antihyperglycemic fraction containing 2-arylbenzofurans (moracin M and glycosides of 2-arylbenzofuran with an isoprenoid group) was prepared from the leaves of Argentine mulberry tree, Morus insignis (Basnet et al., 1993). Furthermore, some Morus flavones with a prenyl group at C-3 position have inhibitory potency against aldose reductase (Yamaguchi et al., 1991). It could be considered that hypoglycemic action of Morus root bark (Chen et al., 1989, 1995) is due to synergistic or additive action of moranoline (1-deoxynojirimycin), morans (glycopeptides), hydrophobic flavonoids (flavones and flavanones) and 2-arylbenzofurans. The i.p. injection of streptozotocin in a dose of 60 mg/kg b.wt. into rats resulted in a state of type 1 diabetes mellitus (Sharifi et al., 2004). Ten days after STZ treatment, the diabetic rats exhibited a state of severe hyperglycemia as compared with normal control group as shown in Table 1. Poor general conditions of the STZ-diabetic treated rats were observed. A significant decrease in body weight (↓−11%) was observed 10 days after STZ treatment when compared with intact normal control rats. It was observed that oral administration of MRBF-3 for 10 days normalized the body weight loss (↑+9%) elicited by STZ. The MRBF-3 treated STZ-diabetic rats showed significant rise in body weight to a value that was nearly similar to the intact control rats. This finding was in close agreement with that of other investigators (Junod et al., 1969; Craft and Failla, 1983; Failla and Kiser, 1981; Schwechter et al., 2003), who noticed a body weight gain upon improvement of the diabetes status. The metabolism of glucose, proteins and lipids is abnormal in diabetes due to insulin secretion defect, leading to various metabolic disorders (Genuth, 1973; Goldstein et al., 2004) and complications (Saudek and Eder, 1979; Kannel and McGee, 1979). The STZ-diabetic rats elicited a signifi5 The main flavonoid of MRBF-3, morusin (1), gives a minor pyranoflavone (±)-neocyclomorusin (3) by a radical reaction via a hydroperoxide with same skeletal structure of 3. On the other hand, (±)-cyclomorusin (2) is derived from 1 by the radical reaction under oxygen-free conditions. One of the roles of morusin (1) in the plants is presumably an antioxidant, a quencher of radical chain reactions.
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cant elevation of serum glucose by ↑+265% associated with a decrease in serum insulin by ↓−58%, indicating that their pancreatic  cells were irreversibly damaged. Our data indicated that the oral administration of MRBF3 for 10 days (600 mg kg−1 day−1 ) to the STZ-diabetic rats showed a significant reduction in serum glucose by ↓−41% and an increase in serum insulin level by +44% as compared to STZ-diabetic rats. Lipid peroxidation is a marker of cellular oxidative damage initiated by reactive oxygen species (Farber et al., 1990). It was reported that diabetics are highly sensitive to oxidative stress (Pritchard et al., 1986; Baynes, 1991; Urano et al., 1991). In STZ-diabetic animals, the STZ generates nitric oxide, which is a powerful free radical oxidant (Kwon et al., 1994) results in an increase in serum level of lipid peroxides due to oxidation of cells (Wakame, 1999). The STZ-diabetic rats exerted a significant elevation of lipid peroxides (expressed as nmol TBARS/ml serum) by ↑+15% as compared to the normal control. The production of lipid peroxides was significantly decreased in MRBF-3 treated STZ-diabetic rats by ↓−22% as compared to STZ-diabetic rats (Table 1). In this experiment, the oral administration of the extract of Morus alba root bark (MRBF-3) suppresses the elevation of lipid peroxides in STZ-diabetic rats compared with control. The data included in this work suggested that MRBF-3 prevents cellular damage induced by STZ via inhibition of lipid peroxidation possibly through antioxidant mechanisms due to its high flavonoids content, which is in accordance with the reported data on this kind of compounds (Oh et al., 2002), as well as it preserves the capability of insulin secretion. Also, this finding was in accordance with the work done by Coskun et al. (2005) who reported that a natural antioxidant flavonol (quercetin) has protective effect in diabetes by decreasing oxidative stress and preservation of pancreatic  cell integrity. In our study however, we demonstrated the presence of five phenolic compounds in the active extract; the data of these compounds as hypoglycemic agents are not reported here. So, further detail experiments are required to clarify the role of these flavonoids and 2-arylbenzofuran as hypoglycemic agents.
5. Conclusions The present study indicated that the flavonoids-rich fraction of 70% alcohol extract of the Morus alba root bark can recover from STZ-induced diabetes in rats. The fraction may protect pancreatic  cells from degeneration and diminish lipid peroxidation of cells. However, further merit investigations including clinical study are necessary in the future to confirm this hypothesis.
References Basnet, P., Kadota, S., Terashima, S., Shimizu, M., Namba, T., 1993. Two new 2-arylbenzofuran derivatives from hypoglycemic activity-bearing
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fractions of Morus insignis. Chemical & Pharmaceutical Bulletin 41, 1238–1243. Baynes, J.W., 1991. Role of oxidative stress in development of complications in diabetes. Diabetes 40, 405–412. Chen, F.-J., Nakashima, N., Kimura, I., Kimura, M., 1989. Hypoglycemic activity of mulberry leaves (Folium Mori) and Cortex Mori radicis in streptozotocin-induced diabetic mice fasted and none fasted. Wakan Iyakugaku Kaisshi 6, 374–375. Chen, F.-J., Nakashima, N., Kimura, I., Kimura, M., 1995. Hypoglycemic activity and mechanisms of extracts from mulberry leaves (Folium Mori) and Cortex Mori radicis in streptozotocin-induced diabetic mice. Yakugaku Zasshi 115, 476–482. Coskun, O., Kanter, M., Korkmaz, A., Oter, S., 2005. Quercetin, a flavonoid antioxidant, prevents and protects streptozotocin-induced oxidative stress and -cell damage in rat pancreas. Pharmacological Research 51, 117–123. Craft, N.E., Failla, M.L., 1983. Zinc, iron, and copper absorption in the streptozotocin-diabetic rat. American Journal of Physiology—Endocrinology and Metabolism 244, E122–E128. Failla, M.L., Kiser, R.A., 1981. Altered tissue content and cytosol distribution of trace metals in experimental diabetes. The Journal of Nutrition 111, 1900–1909. Farber, J.L., Kyle, M.E., Coleman, J.B., 1990. Biology of disease: mechanisms of cell injury by activated oxygen species. Laboratory Investigation 62, 670–679. Fukai, T., Nomura, T., 1998. Proof against 2 -hydroxy-3-prenylflavoneoxygen complex by laser desorption/ionization time-of-flight mass spectrometry. Rapid Communications in Mass Spectrometry 12, 1945–1951, and references cited therein. Fukai, T., Satoh, K., Nomura, T., Sakagami, H., 2003. Antinephritis and radical scavenging activities of prenylflavonoids. Fitoterapia 74, 720–724. Furusho, T., Kataoka, E., Yasuhara, T., Wada, M., Innami, S., 2002. Administration of -carotene suppresses lipid peroxidation in tissues and improves the glucose tolerance ability of streptozotocin-induced diabetic rats. International Journal for Vitamin and Nutrition Research 72, 71–76. Genuth, S.M., 1973. Plasma insulin and glucose profiles in normal, obese, and diabetic persons. Annals of Internal Medicine 79, 812– 822. Goldstein, D.E., Little, R.R., Lorenz, R.A., Malone, J.I., Nathan, D., Peterson, C.M., Sacks, D.B., 2004. Test of glycemia in diabetes. Diabetes Care 27, 1761–1773. Gordon, C., Yates, A.P., Davies, D., 1985. Evidence for a direct action of exogenous insulin on the pancreatic islets of diabetic mice: islet response to insulin pre-incubation. Diabetologia 28, 291–294. Halliwell, B., Gutteridge, J.M., 1990. Role of free radicals and catalytic metal ions in human disease: an overview. Methods in Enzymology 186, 1–85. Hikino, H., Mizuno, T., Oshima, Y., Konno, C., 1985. Validity of the oriental medicines. 80. Antidiabetes drugs. 4. Isolation and hypoglycemic activity of moran A, a glycoprotein of Morus alba root bark. Planta Medica, 159–160. Jin, W.Y., Na, M.K., An, R.B., Lee, H.Y., Bae, K.H., Kang, S.S., 2002. Antioxidant compounds from twig of Morus alba. Natural Product Sciences 8, 129–132 (Chemical abstracts 139, 106237.). Junod, A., Lambert, A.E., Stauffacher, W., Renold, A.E., 1969. Diabetogenic action of streptozotocin: relationship of dose to metabolic response. The Journal of Clinical Investigation 48, 2129–2139. Kannel, W.B., McGee, D.L., 1979. Diabetes and cardiovascular disease. The Framingham study. The Journal of the American Medical Association 241, 2035–2038. Kappus, H., 1987. Oxidative stress in chemical toxicity. Archives of Toxicology 60, 144–149. Kim, E.S., Park, S.J., Lee, E.J., Kim, B.K., Huh, H., Lee, B.J., 1999. Purification and characterization of Moran 20 K from Morus alba. Archives of Pharmacal Research 22, 9–12.
338
A.N.B. Singab et al. / Journal of Ethnopharmacology 100 (2005) 333–338
Kwon, N.S., Lee, S.H., Choi, C.S., Kho, T., Lee, H.S., 1994. Nitric oxide generation from streptozotocin. The FASEB Journal 8, 529–533. Nomura, T., 1988. Phenolic compounds of the mulberry tree and related plants. In: Herz, W., Grisebach, H., Kirby, G.W., Tamm, Ch. (Eds.), Progress in the Chemistry of Organic Natural Products, vol. 53. Springer, Vienna, pp. 87–201, and references cited therein. Nomura, T., Fukai, T., Hano, Y., 2003. Chemistry and biological activities of isoprenylated flavonoids from medicinal plants (moraceous plants and Glycyrrhiza species). In: Atta-ur-Rahman (Ed.), Studies in Natural Product Chemistry, vol. 28: Bioactive Natural Products (Part I). Elsevier, Amsterdam, pp. 199–256, and references cited therein. Nomura, T., Fukai, T., Katayanagi, M., 1977. Oxidative cyclization of morusin with manganese dioxide. Heterocycles 6, 1847–1854. Nomura, T., Fukai, T., Matsumoto, J., 1980. Studies on the constituents of the cultivated mulberry tree. 6. Oxidative cyclization of morusin. Journal of Heterocyclic Chemistry 17, 641–646. O’Brien, B.A., Harmon, B.V., Cameron, D.P., Allan, D.J., 1997. Apoptosis is the mode of beta-cell death responsible for the development of IDDM in the nonobese diabetic (NOD) mouse. Diabetes 46, 750– 757. Oh, H., Ko, E.-K., Jun, J.-Y., Oh, M.-H., Park, S.-U., Kang, K.-H., Lee, H.-S., Kim, Y.-C., 2002. Hepatoprotective and free radical scavenging activities of prenylflavonoids, coumarin, and stilbene from Morus alba. Planta Medica 68, 932–934. Ohkawa, H., Ohishi, N., Yagi, K., 1979. Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Analytical Biochemistry 95, 351–358. Pritchard Jr., K.A., Patel, S.T., Karpen, C.W., Newman, H.A., Panganamala, R.V., 1986. Triglyceride-lowering effect of dietary vitamin E in streptozotocin-induced diabetic rats. Increased lipoprotein lipase activity in livers of diabetic rats fed high dietary vitamin E. Diabetes 35, 278–281.
Saudek, C.D., Eder, H.A., 1979. Lipid metabolism in diabetes mellitus. The American Journal of Medicine 66, 843–852. Schwechter, E.M., Vel´ısˇkov´a, J., Vel´ısˇek, L., 2003. Correlation between extracellular glucose and seizure susceptibility in adult rats. Annals of Neurology 53, 91–101. Sharifi, A.M., Mousavi, S.H., Larijani, B., 2004. Study of interaction between nitric oxide and ACE activity in STZ-induced diabetic rats: role of insulin. Pharmacological Research 50, 261–266. Sharma, R., Sharma, A., Shono, T., Takasugi, M., Shirata, A., Fujimura, T., Machii, H., 2001. Mulberry moracins: scavengers of UV stressgenerated free radicals. Bioscience, Biotechnology, and Biochemistry 65, 1402–1405. Trinder, P., 1969. Determination of blood glucose using an oxidaseperoxidase system with a non-carcinogenic chromogen. Journal of Clinical Pathology 22, 158–161. Urano, S., Hoshi-Hashizume, M., Tochigi, N., Matsuo, M., Shiraki, M., Ito, H., 1991. Vitamin E and the susceptibility of erythrocytes and reconstituted liposomes to oxidative stress in aged diabetics. Lipids 26, 58–61. Wakame, K., 1999. Protective effects of active hexose correlated compound (AHCC) on the onset of diabetes induced by streptozotocin in the rat. Biomedical Research 20, 145–152. WHO Expert Committee on Diabetes Mellitus, Diabetes Mellitus Second Report. Technical Report Series 646, World Health Organization, Geneva, 1980. Yagi, M., Kouno, T., Aoyagi, Y., Murai, H., 1976. The structure of moranoline, a piperidine alkaloid from Morus species. Nippon Nogei Kagaku Kaishi 50, 571–572. Yamaguchi, T., Sato, S., Mitsuhashi, H., Nomura, T., 1991. Aldose reductase inhibitors containing benzopyrans for treatment of diseases associated with diabetes. Japan Kokai Tokkyo Koho 1991. 19910325 (Chemical abstracts 116, 46283).