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nettle extract: A potential ingredient for functional foods development
Journal of Functional Foods 57 (2019) 166–172
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Nanocomposite of montmorillonite/nettle extract: A potential ingredient for functional foods development
T
Abed Rutakhlia, Hossein Sabahia, , Gholam Hossein Riazib ⁎
a b
Department of Life Science Engineering, Faculty of New Sciences and Technologies, University of Tehran, P.O. Box 14395-1561, Tehran, Iran Department of Biochemistry, Institute of Biochemistry and Biophysics, University of Tehran, Tehran, Iran
ARTICLE INFO
ABSTRACT
Keywords: Nettle extract Encapsulation Functional food Diabetes Montmorillonite Antiglycation
The anti-diabetic effect of water extract of Urtica dioica as a green functional food ingredient has been reported to be very low. In this study, we prepared montmorillonite/nettle water extract (Mt/Ex) nanocomposite to enhance its therapeutic effects in vivo. The XRD, FTIR and TGA analyses showed the successful intercalation of phenolics into Mt. The oral delivery of Mt/Ex nanocomposite decreased serum HbA1c (glycated hemoglobin) significantly (p < 0.01) from 10.5% to 6%. In contrast, it did not decrease serum glucose, significantly. It appeared that Mt/ Ex contained a small amount of cyclic protein which is the major antidiabetic component of U. dioica but had the high level of polyphenols as antiglycation. No negative effect of Mt on blood alkaline phosphatase status was observed. These results suggest that intercalation of the plant extracts into Mt may be considered as a safe potential technique for developing of potent functional foods-antiglycation, for food and pharmaceutical industry.
1. Introduction The Urtica dioica leaves have been used as food and medical materials for centuries. The fresh leaves are added to the soup as a potherb or as a supplement to the salad, while herbal tea is made from its dried leaves (Orcic et al., 2014). In the traditional medicine, the aqueous and ethanolic extracts have been used to treat diseases such as gout, anemia, rheumatism, eczema and arthritis for thousands of years. Most importantly, they were used to treat kidney, urinary and bladder problems (Kavalali, 2003). New research also confirms these nettle-health benefits. Their anti-diabetic (Farzami, Ahmadvand, Vardasbi, Majin, & Khaghani, 2003; Ranjbari et al., 2016) and anti-inflammatory activities (Kavalali, 2003) have also been proven. Spinola, Pinto, and Castilho (2018) argued that the phenolic composition had strong correlation with their anti-diabetic effect. They introduced 5-O-caffeoylquinic as the most important hypoglycemic and anti-glycation compound in the extract of this plant. The presence of flavonoids and phenolic acids have also been reported as antioxidant compounds in nettle leaves (Zbikowska et al., 2018). However, among the green tea, blackcurrant seeds and nettles extracts, nettle has the least effect on reducing the oxidation of fats in oat flake cookies during 3 months storage. This was due to the lowest total phenolic content in nettle extract (Zbikowska et al., 2018). As a functional food, water extract of stinging nettle is safer than its ethanolic extract and its production is also more ⁎
economical and more environment-friendly, but unfortunately its therapeutic effects such as very little anti-hyperglycemic and antiglycation activity has been reported in streptozotocin or alloxan-induced diabetic rats (Bnouham et al., 2003). The extensive research have shown that intercalation of drugs into Mt can increase their therapeutic effects because of overcoming the low permeability, instability, extensive first pass metabolism and efflux of drug (Baek, Choy, & Choi, 2012; Dong & Feng, 2005; Kevadiya et al., 2012; Lin et al., 2002). Despite these extensive research on the intercalation of drugs into the Mt, there is no report on the development of the Mt/plant extract composites to improve their medicinal properties. Therefore, we suggest that Mt, as a safe oral drug carrier having FDA approval (Viseras, Cerezo, Sanchez, Salcedo, & Aguzzi, 2010) with large interlayer spacing and strong adsorption capacities for cations, hydrophobic and hydrophilic compounds (Yu et al., 2013) can be an excellent material for intercalation of phenolic compounds of stinging nettle leaf extract and therefore increasing their therapeutic effect as functional food. Therefore, our aim was to investigate the capacity of Mt for nettle water extract intercalation and study their antiglycation activity in vivo. This is despite the fact that most antiglycation experiments are performed in vitro (in processed foods) (Kuda et al., 2016; Wang et al., 2016; Xu et al., 2016) whose results cannot be generalized to the in vivo conditions. The results showed that Mt/Ex composite had no significant effect on glucose and lipid profiles of blood, however, it had a much
Corresponding author. E-mail address: [email protected] (H. Sabahi).
https://doi.org/10.1016/j.jff.2019.04.004 Received 4 January 2019; Received in revised form 1 April 2019; Accepted 1 April 2019 1756-4646/ © 2019 Elsevier Ltd. All rights reserved.
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stronger antiglycation effect in vivo than most functional foods reported so far.
Group IV
2. Materials and methods
Group VI
Group V
2.1. Materials
2.4.2. Induction of diabetes For inducing diabetes, streptozotocin (STZ) (55 mg/kg b.w) dissolved in citrate buffer (0.1 M, pH 4.5), were injected in 24 h fasted rats by a single intraperitoneal injection. Control rats were treated with the citrate buffer alone. After 96 h, glucose of blood plasma was measured and the rats with fasting blood glucose greater than 240 mg/dl were used in the next experiments.
Mt was purchased in mineral form (bentonite) from Poodrsazan, Tehran, Iran. The purification method and chemical composition of the local bentonite with mesh size of 200 µm and particle size less than 75 µm have been explained elsewhere (Rabiei, Sabahi, & Rezayan, 2016). 2.2. Preparation of Mt/Ex nanocomposite
2.4.3. Blood sampling The blood samples were prepared weekly for 30 days after 8 h fasting. Blood glucose was determined after and before fasting using diagnostics kits according to manufactured instructions. Moreover, the blood sample was also prepared at the end of 30 day and glycated hemoglobin (HbA1c), total cholesterol (TC), high density lipoprotein cholesterol (HDL) alkaline phosphatase (ALP) were measured using Hitachi 912 autoanalyser.
Mt/Ex nanocomposite was obtained through a previously reported method (Rabiei et al., 2016). Preparation of the impure nettle leaves was carried out based on a method by Li, Percival, Bonard, and Gu (2011) without performing partial purification. The air-dried nettle leaf (50 g) was milled in a kitchen mill, and then was mixed with water (250 mL). The water suspension was sonicated for 30 min at 60 °C. The sample was centrifuged (Model:MF20R, AWEL company, French) at 4500 rpm for 10 min at room temperature and was filtered. This extract was freeze-dried to obtain solid materials. 200 mg of purified Mt dissolved in 100 mL deionized water and stirred continuously for 2 h to achieve homogenized slurry. Then, the solid extract was solved in water to obtain a concentration of 200 mg/mL. Then 4 different volumes of this solute including 1, 2, 3 and 4 mL were added to 100 mL of mentioned Mt slurry to obtain 1:1, 1:2, 1:3, and 1:4 ratios of Mt/Ex. After 2 h stirring, the solution was centrifuged at 4000 rpm for 5 min. The sediment containing nanocomposite, was freeze-dried for 2 days.
2.5. Method validation Method validation is the process used to confirm that the analytical procedure employed for a specific test is suitable for its intended use. Results from method validation can be used to judge the quality, reliability and consistency of analytical results. Because we used readymade kits to measure blood parameters by the autoanalyzer, the validation of these kits has been reported by the manufacturer as Table 1. In this table the relative standard deviation (RSD) is the indicator of precision and was calculated using following equation. The value of RSD should not be more than 2.0% (Robbins, Gong, Wells, Greenspan, & Pegg, 2015).
2.3. Characterization of Mt/Ex nanocomposite Crystallization, chemical characterization and thermo gravimetric analysis of the nanocomposites were evaluated using XRD, FTIR and TG analyzer as described in our published article (Rabiei et al., 2016).
% RSD = standard deviation/mean × 100
2.4. In vivo studies
Linearity indicates the ability to produce results that are directly proportional to the concentration of the analyte in samples. A series of samples should be prepared in which the analyte concentrations span the claimed range of the procedure. If there is a linear relationship (R2), test results can be evaluated by appropriate statistical method. The limit of detection (LOD) was calculated based on the standard deviation (SD) of the data and the slop of the regression line as below equation: LOD = 3 ∗ SD/slope ((Robbins et al., 2015).
2.4.1. Animal preparation The animals tested were Wistar male rats (Rattus norvegicus) with an average weight of 200 ± 20 g. These animals were obtained from the animal center of the Institute of Biochemistry and Biophysics in University of Tehran and stored in the same center under controlled conditions: temperature 25 °C, 12 h light/dark cycles and free access to water and food. All rats left 1 week in animal house for acclimatization. At the time of blood sampling, the rats were anesthetized with ether. Then blood sampling was performed by cardiac punctur. The cages were cleaning daily. This study was approved by the Institutional Research Ethics Committee, Faculty of Physical Education and Sport Sciences, Tehran University under Ethics Number: IR.UT.SPORT.REC.1397.039. Thirty six rats were randomly assigned into six groups of six animals each. According to the guidelines of the American Association for Laboratory Animal Science (Fitts, 2011), the number of animals in each group was determined using the same number of animals used by other investigators in a published paper on the same topic (Opris et al., 2017). Treatments were applied in a randomized complete block design with six replications as below: Group I Group II Group III
Diabetic rats received 200 mg/kg b.w/day Mt (D + Mt) orally for 30 days. Diabetic rats received 80 mg/kg b.w/day extract (D + Ex) orally for 30 days. Diabetic rats received 80 mg/kg b.w/day extract in the form of Mt/Ex (D + Mt/Ex) orally for 30 days.
2.6. Statistical analysis Data of glucose, TC, HDL, ALP and HbA1c were presented as mean ± standard deviation (SD). Experimental data were analyzed by one way ANOVA test and the comparison of group for these biochemical parameters were done with Tukey test (HSD) set at P < 0.05 using the Social Package of Statistical System software (SPSS). Table 1 System precision (RSD%), system suitability (R2) and system limit (LOD) for blood biochemical parameters measurement.
Normal control (C). Normal rats received 200 mg/kg b.w/day Mt (C + Mt) orally for 30 days Diabetic control (D).
Parameters
HbA1C
ALP
Glucose
Cholestrol
HDL
RSD% (n = 20) R2 (n = 78) LOD
1.5 0.999 1%
0.9 0.999 5 ppm
1.28 0.996 5 mg/dl
0.62 0.995 5 mg/dl
0.9 0.999 2 mg/dl
RSD: relative standard deviation, R2: correlation coefficient, LOD: limit of detection. 167
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Fig. 1. X-ray diffraction patterns of Mt (red line), Mt/Ex nanocomposite prepared at two ratios of 1:2 (green line) and 1:4 (blue line). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 2. FTIR spectra of Mt (red line), nettle extract (blue line) and Mt/Ex nanocomposite (green line) at ratio of 1:4. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
3. Results and discussion 3.1. XRD analysis
and proteins (Cao et al., 2011; Jaeckels et al., 2017; Stankovic, Jovic, & Zivkovic, 2004; Yu et al., 2013) of extracts with outer and inner layers of Mt. The bands in 3405 cm−1 and 1632 cm−1 are related to the HeOeH stretching and bending-in-plan vibration of the interlayer water of Mt has been also broadened indicating that hydrogen bonding has been enhanced (Cao et al., 2011). Appearance of three shoulders at 1502 cm−1, 1098 cm−1 and 850 cm−1 also confirms extract intercalation between interlayers of Mt. The TGA analysis (Fig. 3) and DTA (Data not shown) also shows that interlayer water has been completely replaced by extract. In other words, hydrogen bonding between water and inner SieO has been changed to hydrogen bonding between extract mainly polyphenols (Gonzalez-Neves et al., 2014; Gutierrez et al., 2017; Turkyilmaz et al., 2012) and proteins (Cao et al., 2011; Yu et al., 2013) with outer and inner SieO, SieOH and AleOH. It has been proved that Mt mostly absorbs phenolic compounds, protein or alkaloids from plant extract and fruit juices. For instance, Mt adsorbed caffeine (a methylxanthine alkaloid) from tea (Shiono, Yamamoto, Yotsumoto, Kawai, et al., 2017) and coffee extracts, selectively (Shiono, Yamamoto, Yotsumoto, & Yoshida, 2017). In contrast, numerous workers found that natural and modified Mt absorbed phenolic compounds from plant extract or fruit juices (Gonzalez-Neves et al., 2014; Gutierrez et al., 2017; Turkyilmaz et al., 2012). On the other hand, many researchers have reported the selective absorption of protein from extracts (Cao et al., 2011) or fruit juices (Jaeckels et al., 2017; Stankovic et al., 2004). The phenolic compounds and protein content of wild-nettle leaf extract has been reported 1.5% and 23–24%, respectively. The caffeoylmalic acid (54%), chlorogenic acid (23%), rutin (12%) and caffeic acid derivative (7%) concentrations were 95% of total phenolic compounds (Orcic et al., 2014; Pinelli et al., 2008). According to the abovementioned research and FTIR analysis (Fig. 2), we can conclude that here, the Mt nanoparticles has mostly absorbed proteins and phenolic compounds from extract. Determining the exact ratio of absorbed proteins to phenolics and their types can be an attractive research subject for the future investigations. Balooch et al. (2018) also showed that Mt has high capacity for absorption of polyphenol compounds from plant extracts.
Mt/Ex nanocomposite was prepared in 1:1, 1:2, 1:3 and 1:4 ratios of Mt to extract. In two of the prepared nanocomposites, d-spacing was 13.67 Å (1:2 ratio) and 14.39 Å (1:4 ratio) (Fig. 1). The shift of peaks in the XRD spectra to left shows that increasing the extract content increased the amount of intercalation. By considering of real d-spacing of Mt, 11.93 Å, it can be concluded that d-spacing has been increased by 1.74 Å and 2.46 Å at 1:2 and 1:4 ratios, respectively. It is an evidence that extract has been intercalated into the interlayer of Mt (Balooch, Sabahia, Aminian, & Hosseini, 2018). It is interesting to note that there is a few report in the field of intercalation of the plant extract into Mt nanoparticles but there are many reports about other drug intercalation into Mt such as 5Fluorouracil (Lin et al., 2002), Paclitaxel (Dong & Feng, 2005), Timolol maleate (Joshi, Kevadiya, Patel, Bajaj, & Jasra, 2009), 5-Fluorouracil (Kevadiya et al., 2012), Glutathione (Baek et al., 2012), Gallic acid (Rabiei et al., 2016) and Insulin (Kamari, Ghiaci, & Ghiaci, 2017). 3.2. FTIR analysis The FTIR spectra of Mt showed characteristic absorption band at 3620 cm−1 which belonging to OeH stretching band in AleOH and SieOH in Mt (Fig. 2). The bands in 3405 cm−1 and 1632 cm−1 are related to the HeOeH stretching and bending-in-plan vibration of the interlayer water. SieO stretching in Mt create sharp band in 985 cm−1 and little band in 914 cm−1 and 795 cm−1 are related to AleOH and AleMg (Fe)eOH bending vibration in Mt molecule (Rabiei et al., 2016). The FTIR spectra of nettle extract (Fig. 2) shows existence of flavonoids, carbohydrate and protein. The band between 3270 and 3310 cm−1 probably corresponds to NH stretching vibration of protein (Barth, 2007). The 2934 cm−1 band are typically related to aldehydes (R-CHO) stretching vibration of polyphenol (Sabahi, Khorami, Rezayana, Jafaria, & Karami, 2018). The 1572 cm−1 band is maybe related to amid II (Barth, 2007). The 1393 cm−1 corresponds to the CH3 group. The 1257 cm−1 and 1046 cm−1 bands can be attributed to CeO and CeOeC stretching vibration in polyphenol, respectively (Jafari, Sabahi, & Rahaie, 2016). In the FTIR bands of Mt/Ex nanocomposite (Fig. 2), band at 3620 cm−1 which belongs to OeH stretching band in AleOH and SieOH in Mt has been broadened which shows increasing of hydrogen bonds between polyphenols (Gonzalez-Neves, Favre, & Gil, 2014; Gutierrez, Ponce, & Alvarez, 2017; Turkyilmaz, Yemis, & Ozkan, 2012)
3.3. TG analysis The TG analysis of pure Mt, extract and Mt/Ex nanocomposite have been shown in Fig. 3. The TGA curve of Mt represents two distinct steps. The major mass loss pattern observed at temperatures below 150 °C is due to the free water evaporation. The second step at 275 °C 168
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Fig. 3. TGA pattern of Mt (red line), and nettle extract (green line), Mt/Ex nanocomposite (blue line) at ratio of 1:4. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
corresponds to loss of structural water. Extract shows three steps mass losses at 147 °C, 245 °C and 403 °C (Fig. 3) related to thermal dehydration, boiling process and thermal decomposition (oxidation of organic matter), respectively. Mt/Ex composite boiling process and oxidation decomposition was shifted from 245 °C and 403 °C up to 341 °C and 500 °C (Data not shown) which shows increasing of thermal stability of extract after intercalation. This evidence also reflects the complete intercalation of extract into Mt interlayers. A more interesting event was elimination of the endothermic peak related to free water evaporation at Mt/Ex composite (Data not shown), indicating complete replacement of interlayer water by extract. Finally, the TGA analysis shows 24.7% loading for Mt/Ex composite (Fig. 3).
degrading extract through gastrointestinal tract has not caused to obtain this result; it was also consumed it as injection. After injection, we did not observe antidiabetic effect for nettle extract (data not shown). These treatments also did not have significant effect on cholesterol and LDH level of serum on day 28 (Table 2). The reported anti-diabetic effect of leaf nettle (Urtica dioica) extract are different. Ethanolic nettle leaf extract has been reported to be antihyperglycemic (Ranjbari et al., 2016), in contrast, water nettle leaf extract has been reported to have no anti-diabetic properties (Bnouham et al., 2003; Roman Ramos, Alarcon-Aguilar, Lara-Lemus, & FloresSaenz, 1992). It has been shown that a cyclic hydrophilic protein in Urtica dioica is the major antidiabetic components of ethanolic nettle extract which facilitates glucose uptake through forming a permeable pore in the lipid bilayer of myoblasts cell (Shabani Domola, Christine, Doucette, Sweeney, & Wheeler, 2010). It appears that water nettle extract and Mt/Ex has contained a small amount of these proteins but the high level of polyphenols. Bnouham et al. (2003) also found no antihypoglycemic activity for nettle leaf extract. However, in hyperglycemia induced by oral glucose tolerance test (OGTT), water nettle
3.4. Antidiabetic effect It was observed that in type 2 diabetic rat, nettle extract and related composite means Mt/Ex did not decrease serum glucose (Fig. 4) on days 1, 14, 21 and 28 with respect to control group. To be sure that
Fig. 4. Serum glucose before (blue histogram) and after (red histogram) fasting in experiment groups. Data are average of 4 sampling; 1, 7, 14 and 28 days. Each value is mean ± S.D. for 6 rats in each group. C: normal control, D: diabetic control, Mt: montmorillonite, Ex: nettle extract, Mt/Ex: montmorillonite/nettle extract nanocomposite. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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glucose and serum lipid profile. Based on these results, they conclude that PFE could not completely reverse the hyperglycemia and PFEmediated reduction in HbA1c was probably due to the inhibition of the reaction between protein and sugar in the early stages of glycation. It should be noted that in this experiment diabetes was only induced by high-fat (28%) and high-sucrose (30%) diet which is not actual condition in diabetic rat type 2 (Abo-elmatty et al., 2013; Gheibi et al., 2017). An evidence for this claim is the similarity of HbA1c in normal and diabetics mice. Opris et al. (2017) also found that administration of sambucus nigra extract to animals with diabetes did not change the glucose concentrations in blood but it increased the antioxidant defense and reduced the inflammation in liver tissue. It has well been also documented that phenolic compounds, especially chlorogenic acid, caffeic acid and kaempferol-3-rutinoside that exit in the nettle leaf extract (Pinelli et al., 2008), are able to inhibit the glycation process (Justino et al., 2016). The mechanism by which plant polyphenols inhibit glycation can be attributed to antioxidant properties and the presence of their hydroxyl groups. Researchers have suggested that plant polyphenols might interact with glucose and prevent them from binding to proteins (Chao, Mong, Chan, & Yin, 2010; Justino et al., 2016). Mt/Ex composite showed to have a much stronger antiglycation effect than most extracts and nanoparticle/extracts that have been reported so far. For example, Daisy and Saipriya (2012) reported that the antiglycation activity of Cassia fistula extract and its gold nanoparticle were only 15.7% and 23.5%, respectively. Gold nanoparticle/Sambucus nigra extract also increase the antioxidant defense in diabetic rats only at p < 0.05 (Opris et al., 2017). Abo-elmatty et al. (2013) observed a decrease in %HbA1c from 10.5% to 8.3% in diabetic rate receiving 250 mg/kg purified extract of Urtica pilulifera having lectin; an antidiabetic compound which directly inhibited STZ activity. Caffeic acid (CA) and ellagic aid (EA) as two strong glycation inhibitor also reduced HbA1c in diabetic rate receiving 5% CA or EA, from 10.3% to 7.5%, at week 12 (Chao et al., 2010), While the antiglycation effect of the Mt/Ex composite in the present experiment was from 10.5% to 6.5% (40%) similar to pure asiatic acid and maslinic acid (Hung, Yang, & Yin, 2015) at high concentrations (0.2%). This strong effect can be related to (a) the enhanced effect of Mt on cellular uptake efficiency of the drugs through intestine endothelial cells (Dong & Feng, 2005), (b) polyphenol protection by Mt against harsh biological conditions such as intestinal enzymatic attack and gastric acid condition (Baek et al., 2012) and (c)
Table 2 The effect of different treatments on triglyceride (TG), high density lipid (HDL), cholesterol (Chol) and alkaline phosphatase (ALP) in serum on 28 days after treatments. Each value is mean ± S.D. for 6 rats in each group. Treatments
TG
HDL
C C + Mt D D + Mt D + Ex D + Mt/Ex
76 ± 22.4 34 ± 8.7 95 ± 27.6 105 ± 25.4 43 ± 10.8 84 ± 21.4
29 29 33 29 30 30
± ± ± ± ± ±
Chol 2.52 1.73 3.06 4.51 0.58 0.58
89 80 75 75 72 63
± ± ± ± ± ±
ALP 12.5 5.0 11.5 11.5 7.6 5.8
75 ± 10.8 71 ± 24.1 431 ± 76.4 490 ± 87.6 465 ± 71.6 434 ± 65.5
C: normal control, D: diabetic control, Mt: montmorillonite, Ex: nettle extract, Mt/Ex: montmorillonite/nettle extract nanocomposite.
extract showed a strong anti-diabetic effect. It should be noted that Alloxan or Streptozotocin-induced diabetes is a well proven model of experimental diabetes even in type 2 (Abo-elmatty, Essawy, Badr, & Sterner, 2013; Daisy & Saipriya, 2012; Gheibi, Kashfi, & Ghasemi, 2017). These compounds produce oxidative stress similar to actual hyperglycemia in diabetic animal. Therefore, Alloxan or Streptozotocin are often used to stimulate diabetes in experimental animals because of their toxic effect on pancreatic β cells, while stimulating diabetes by oral glucose tolerance test (OGTT) is not like the actual condition of diabetes. According to these results, observing no anti-hypoglycemic effect of nettle extract and Mt/Ex composite is consistent with other studies (Bnouham et al., 2003; Roman Ramos et al., 1992). Interestingly, oral delivery of Mt/Ex composite in diabetic rat decreased HbA1c significantly (p < 0.01) and brought it down from 10.5% to 6.2% (normal level) on day 28 (Fig. 5). Nettle extract also decreased HbA1c significantly to 8% (p < 0.05) but it is higher than normal level. This result is also consistent with other studies. Spinola et al. (2018) also reported that the phenolic compound of Vaccinium cylindraceaeum plant extract effectively reduced glucosidases and glycation of proteins. In this experiment, the composition of polyphenols showed strong correlation with their bioactivity. They declared that 5O-caffeoylquinic acid (70%) was the most important anti-glycation compound in the extract of this plant. In nettle extract also, the major phenolic compound are 5-O-caffeoylquinic acid and 2-O-caffeoylmalic acid (70%) (Orcic et al., 2014; Pinelli et al., 2008). Kumagai et al. (2015) showed that pomegranate fruit extract (PFE) only had antiglycation effect on diabetic rats but with no effect on status of serum
Fig. 5. Serum HbA1c in experiment groups. Each value is mean ± S.D. for 6 rats in each group. C: normal control, D: diabetic control, Mt: montmorillonite, Ex: nettle extract, Mt/Ex: montmorillonite/nettle extract nanocomposite. *P < 0.05, **P < 0.01, ***P < 0.001. All treatments has been compared with diabetic control (D). 170
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increased circulation time and circulation amounts of polyphenols in the blood (Baek et al., 2012; Kevadiya et al., 2012). Insulin prevents degradation of blood protein through lowering serum glucose (Abo-elmatty et al., 2013). High serum glucose glycoside the various proteins of blood including hemoglobin (glycated hemoglobin or HbA1c), albumin, collagen, low-density lipoprotein and fibronectin at diabetic condition (Wang et al., 2016). In fact, glycation is a non-enzymatic reaction in processed food and in vivo condition. Advanced glycation end products (AGEs) are produced after subsequent intermediates steps (Xu et al., 2016). AGE formation is irreversible. It is believed that the AGEs induce many diseases including diabetes and other diabetes-related diseases and aging such as retinopathy, cataracts, arteriosclerosis, and renal dysfunction (Kuda et al., 2016). Therefore, the measurement of HbA1c is a trustworthy indicator in diagnosis and control of diabetes. To be sure that Mt nanoparticle and its related composites have not had toxic effect on liver enzymes, we measured alkaline phosphatase level of rat serum. No significant toxic effect was observed for Mt and their composites (Table 2). Alkaline phosphatase (ALP) is an enzyme which its increased activities in blood is mainly due to its leakage from the liver into the serum (Abo-elmatty et al., 2013), and it is also an indicator of the abnormal function of liver. In current study, the activity of ALP in diabetic rats was much higher than normal rats which is due to STZ application (Chao et al., 2010). In vivo test showed no negative effect of Mt on blood alkaline phosphatase status of normal and diabetic rats showing no toxicity for Mt (Table 2).
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(2016). Stability and loading properties of curcumin encapsulated in Chlorella vulgaris. Food Chemistry, 211, 700–706. Joshi, G. V., Kevadiya, B. D., Patel, H. A., Bajaj, H. C., & Jasra, R. V. (2009). Montmorillonite as a drug delivery system: Intercalation and in vitro release of timolol maleate. International Journal of Pharmaceutics, 374, 53–57. Justino, A. B., Pereira, M. N., Vilela, D. D., Peixoto, L. G., Martins, M. M., Teixeira, R. R., ... Espindol, F. S. (2016). Peel of araticum fruit (Annona crassiflora Mart.) as a source of antioxidant compounds with a-amylase, a-glucosidase and glycation inhibitory activities. Bioorganic Chemistry, 69, 167–182. Kamari, Y., Ghiaci, P., & Ghiaci, M. (2017). Study on montmorillonite/insulin/TiO2 hybrid nanocomposite as a new oral drug-delivery system. Materials Science and Engineering C, 75, 822–828. Kavalali, G. (2003). Urtica-therapeutic and nutritional aspects of stinging nettles (1st ed.). London: Taylor & Francis. Kevadiya, B. D., Patel, T. A., Jhala, D. D., Thumbar, R. P., Brahmbhatt, H., Pandya, M. P., ... Bajaj, H. C. (2012). Layered inorganic nanocomposites: A promising carrier for 5fluorouracil (5-FU). European Journal of Pharmaceutics and Biopharmaceutics, 81, 91–101. Kuda, T., Eda, M., Kataoka, M., Nemoto, M., Kawahara, M., Oshio, S., ... Kimura, B. (2016). Anti-glycation properties of the aqueous extract solutions of dried algae products and effect of lactic acid fermentation on the properties. Food Chemistry, 192, 1109–1115. Kumagai, Y., Nakatani, S., Onodera, H., Nagatomo, A., Nishida, N., Matsuura, Y., ... Wada, M. (2015). Anti-glycation effects of pomegranate (Punica granatum L.) fruit extract and its components in vivo and in vitro. Journal of Agricultural and Food Chemistry, 63, 7760–7764. Li, Z., Percival, S. S., Bonard, S., & Gu, L. (2011). Fabrication of nanoparticles using partially purified pomegranate ellagitannins and gelatin and their apoptotic effects. Molecular Nutrition & Food Research, 55, 1096–1103. Lin, F. H., Lee, Y. H., Jian, C. H., Wong, J. M., Shieh, M. J., & Wang, C. Y. (2002). A study of purified montmorillonite intercalated with 5-fluorouracil as drug carrier. Biomaterials, 23, 1981–1987. Opris, R., Tatomir, C., Olteanu, D., Moldovan, R., Moldovan, B., David, L., ... Filip, G. A. (2017). The effect of Sambucus nigra L. extract and phytosinthesized gold nanoparticles on diabetic rats. Colloids and Surfaces B: Biointerfaces, 150, 192–200. Orcic, D., Franciskovicc, M., Bekvalac, K., Svircev, E., Beara, I., Lesjak, M., & MimicaDukic, N. (2014). Quantitative determination of plant phenolics in Urtica dioica extracts by high-performance liquid chromatography coupled with tandem mass spectrometric detection. Food Chemistry, 143, 48–53. Pinelli, P., Ieri, F., Vignolina, P., Baccil, L., Bronti, S., & Romani, A. (2008). Extraction and HPLC analysis of phenolic compounds in leaves, stalks, and textile fibers of Urtica dioica L. Journal of Agricultural Food Chemistry, 56, 9127–9132. Rabiei, M., Sabahi, H., & Rezayan, A. H. (2016). Gallic acid-loaded montmorillonite nanocomposite as a new controlled release system. Applied Clay Science, 119, 236–242. Ranjbari, A., Azarbayjani, M. A., Yusof, A., Mokhtar, A. H., Akbarzadeh, S., Ibrahim, M. Y., ... Dehghan, F. (2016). In vivo and in vitro evaluation of the effects of Urtica dioica
4. Conclusion In this study, we tried to intercalate nettle leaf extract as an antidiabetic agent into the interlayer of Mt to prepare Mt/Ex nanocomposite. XRD, FTIR and TGA analysis showed the successful intercalation of extract between interlayers of Mt. Interestingly, intercalation of nettle extract in Mt turn it into a vigorous aniglycant product (p < 0.01) maybe due to increasing polyphenol bioavailability and higher amount and time in systemic circulation. Accordingly, we suggest to investigate anti-diabetic effects of Mt/Ex composite after incorporation into a food matrix. 5. Ethics statements All animal experiments complied with the ARRIVE guidelines and was carried out in accordance with the U.K. Animals (Scientific Procedures) Act, 1986 and associated guidelines, EU Directive 2010/ 63/EU for animal experiments, and the National Institutes of Health guide for the care and use of Laboratory animals (NIH Publications No. 8023, revised 1978). Conflict of interest The authors have no competing interest. Acknowledgements The authors thank the research Council of University of Tehran, Tehran, Iran, for financial support of this work. References Abo-elmatty, D. M., Essawy, S. S., Badr, J. M., & Sterner, O. (2013). Antioxidant and antiinflammatory effects of Urtica pilulifera extracts in type 2 diabetic rats. Journal of Ethnopharmacology, 145, 269–277. Baek, M., Choy, J. H., & Choi, S. J. (2012). Montmorillonite intercalated with glutathione for antioxidant delivery: Synthesis, characterization, and bioavailability evaluation. International Journal of Pharmaceutics, 425, 29–34. Balooch, M. R., Sabahia, H., Aminian, H., & Hosseini, M. (2018). Intercalation technique
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A. Rutakhli, et al. and swimming activity on diabetic factors and pancreatic beta cells. BMC Complementary Alternative Medicine, 16, 2–11. Robbins, K. S., Gong, Y., Wells, M. L., Greenspan, P., & Pegg, R. B. (2015). Investigation of the antioxidant capacity and phenolic constituents of U.S. pecans. Journal of Functional Foods, 15, 11–22. Roman Ramos, R., Alarcon-Aguilar, F., Lara-Lemus, A., & Flores-Saenz, J. L. (1992). Hypoglycemic effect of plants used in Mexico as antidiabetics. Archive of Medical Research, 23, 59–64. Sabahi, H., Khorami, M., Rezayana, A. H., Jafaria, Y., & Karami, M. H. (2018). Surface functionalization of halloysite nanotubes via curcumin inclusion. Colloids and Surfaces A: Physicochemical and Engineering Aspects, 538, 834–840. Shabani Domola, M., Christine, V., Doucette, R., Sweeney, G., & Wheeler, M. B. (2010). Insulin mimetics in Urtica dioica: Structural and computational analyses of Urtica dioica extracts. Phytotherapy Research, 24, 175–182. Shiono, T., Yamamoto, K., Yotsumoto, Y., Kawai, J., Imada, N., Hioki, J., ... Deuchi, K. (2017). Selective decaffeination of tea extracts by montmorillonite. Journal of Food Engineering, 200, 13–21. Shiono, T., Yamamoto, K., Yotsumoto, Y., & Yoshida, A. (2017). Caffeine adsorption of montmorillonite in coffee extracts. Bioscience, Biotechnology, and Biochemistry, 81, 1591–1597. Spinola, V., Pinto, J., & Castilho, P. C. (2018). Hypoglycemic, anti-glycation and
antioxidant in vitro properties of two Vaccinium species from Macaronesia: A relation to their phenolic composition. Journal of Functional Foods, 40, 595–605. Stankovic, S., Jovic, S., & Zivkovic, J. (2004). Bentonite and gelatin impact on the young red wine coloured matter. Food Technology and Biotechnology, 42, 183–188. Turkyilmaz, M., Yemis, O., & Ozkan, M. (2012). Clarification and pasteurization effects on monomeric anthocyanins and percent polymeric colour of black carrot (Daucus carota L.) juice. Food Chemistry, 134, 1052–1058. Viseras, C., Cerezo, P., Sanchez, R., Salcedo, I., & Aguzzi, C. (2010). Current challenges in clay minerals for drug delivery. Applied Clay Science, 48, 291–295. Wang, W., Liu, H., Wang, Z., Qi, J., Yuan, S., Zhang, W., ... Jia, A. Q. (2016). Phytochemicals from Camellia nitidissima Chi inhibited the formation of advanced glycation end-products by scavenging methylglyoxal. Food Chemistry, 205, 204–211. Xu, Y., Liu, G., Yu, Z., Song, X., Li, X., Yang, Y., ... Dai, J. (2016). Purification, characterization and antiglycation activity of a novel polysaccharide from black currant. Food Chemistry, 199, 694–701. Yu, W. H., Li, N., Tong, D. S., Zhou, C. H., Lin, C. X., & Xu, C. Y. (2013). Adsorption of proteins and nucleic acids on clay minerals and their interactions: A review. Applied Clay Science, 80–81, 443–452. Zbikowska, A., Kozlowska, M., Poltorak, A., Kowalska, M., Rutkowska, J., & Kupiec, M. (2018). Effect of addition of plant extracts on the durability and sensory properties of oat flake cookies. Journal of Thermal Analysis and Calorimetry, 134, 1101–1111.
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Journal of Functional Foods journal homepage: www.elsevier.com/locate/jff
Nanocomposite of montmorillonite/nettle extract: A potential ingredient for functional foods development
T
Abed Rutakhlia, Hossein Sabahia, , Gholam Hossein Riazib ⁎
a b
Department of Life Science Engineering, Faculty of New Sciences and Technologies, University of Tehran, P.O. Box 14395-1561, Tehran, Iran Department of Biochemistry, Institute of Biochemistry and Biophysics, University of Tehran, Tehran, Iran
ARTICLE INFO
ABSTRACT
Keywords: Nettle extract Encapsulation Functional food Diabetes Montmorillonite Antiglycation
The anti-diabetic effect of water extract of Urtica dioica as a green functional food ingredient has been reported to be very low. In this study, we prepared montmorillonite/nettle water extract (Mt/Ex) nanocomposite to enhance its therapeutic effects in vivo. The XRD, FTIR and TGA analyses showed the successful intercalation of phenolics into Mt. The oral delivery of Mt/Ex nanocomposite decreased serum HbA1c (glycated hemoglobin) significantly (p < 0.01) from 10.5% to 6%. In contrast, it did not decrease serum glucose, significantly. It appeared that Mt/ Ex contained a small amount of cyclic protein which is the major antidiabetic component of U. dioica but had the high level of polyphenols as antiglycation. No negative effect of Mt on blood alkaline phosphatase status was observed. These results suggest that intercalation of the plant extracts into Mt may be considered as a safe potential technique for developing of potent functional foods-antiglycation, for food and pharmaceutical industry.
1. Introduction The Urtica dioica leaves have been used as food and medical materials for centuries. The fresh leaves are added to the soup as a potherb or as a supplement to the salad, while herbal tea is made from its dried leaves (Orcic et al., 2014). In the traditional medicine, the aqueous and ethanolic extracts have been used to treat diseases such as gout, anemia, rheumatism, eczema and arthritis for thousands of years. Most importantly, they were used to treat kidney, urinary and bladder problems (Kavalali, 2003). New research also confirms these nettle-health benefits. Their anti-diabetic (Farzami, Ahmadvand, Vardasbi, Majin, & Khaghani, 2003; Ranjbari et al., 2016) and anti-inflammatory activities (Kavalali, 2003) have also been proven. Spinola, Pinto, and Castilho (2018) argued that the phenolic composition had strong correlation with their anti-diabetic effect. They introduced 5-O-caffeoylquinic as the most important hypoglycemic and anti-glycation compound in the extract of this plant. The presence of flavonoids and phenolic acids have also been reported as antioxidant compounds in nettle leaves (Zbikowska et al., 2018). However, among the green tea, blackcurrant seeds and nettles extracts, nettle has the least effect on reducing the oxidation of fats in oat flake cookies during 3 months storage. This was due to the lowest total phenolic content in nettle extract (Zbikowska et al., 2018). As a functional food, water extract of stinging nettle is safer than its ethanolic extract and its production is also more ⁎
economical and more environment-friendly, but unfortunately its therapeutic effects such as very little anti-hyperglycemic and antiglycation activity has been reported in streptozotocin or alloxan-induced diabetic rats (Bnouham et al., 2003). The extensive research have shown that intercalation of drugs into Mt can increase their therapeutic effects because of overcoming the low permeability, instability, extensive first pass metabolism and efflux of drug (Baek, Choy, & Choi, 2012; Dong & Feng, 2005; Kevadiya et al., 2012; Lin et al., 2002). Despite these extensive research on the intercalation of drugs into the Mt, there is no report on the development of the Mt/plant extract composites to improve their medicinal properties. Therefore, we suggest that Mt, as a safe oral drug carrier having FDA approval (Viseras, Cerezo, Sanchez, Salcedo, & Aguzzi, 2010) with large interlayer spacing and strong adsorption capacities for cations, hydrophobic and hydrophilic compounds (Yu et al., 2013) can be an excellent material for intercalation of phenolic compounds of stinging nettle leaf extract and therefore increasing their therapeutic effect as functional food. Therefore, our aim was to investigate the capacity of Mt for nettle water extract intercalation and study their antiglycation activity in vivo. This is despite the fact that most antiglycation experiments are performed in vitro (in processed foods) (Kuda et al., 2016; Wang et al., 2016; Xu et al., 2016) whose results cannot be generalized to the in vivo conditions. The results showed that Mt/Ex composite had no significant effect on glucose and lipid profiles of blood, however, it had a much
Corresponding author. E-mail address: [email protected] (H. Sabahi).
https://doi.org/10.1016/j.jff.2019.04.004 Received 4 January 2019; Received in revised form 1 April 2019; Accepted 1 April 2019 1756-4646/ © 2019 Elsevier Ltd. All rights reserved.
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stronger antiglycation effect in vivo than most functional foods reported so far.
Group IV
2. Materials and methods
Group VI
Group V
2.1. Materials
2.4.2. Induction of diabetes For inducing diabetes, streptozotocin (STZ) (55 mg/kg b.w) dissolved in citrate buffer (0.1 M, pH 4.5), were injected in 24 h fasted rats by a single intraperitoneal injection. Control rats were treated with the citrate buffer alone. After 96 h, glucose of blood plasma was measured and the rats with fasting blood glucose greater than 240 mg/dl were used in the next experiments.
Mt was purchased in mineral form (bentonite) from Poodrsazan, Tehran, Iran. The purification method and chemical composition of the local bentonite with mesh size of 200 µm and particle size less than 75 µm have been explained elsewhere (Rabiei, Sabahi, & Rezayan, 2016). 2.2. Preparation of Mt/Ex nanocomposite
2.4.3. Blood sampling The blood samples were prepared weekly for 30 days after 8 h fasting. Blood glucose was determined after and before fasting using diagnostics kits according to manufactured instructions. Moreover, the blood sample was also prepared at the end of 30 day and glycated hemoglobin (HbA1c), total cholesterol (TC), high density lipoprotein cholesterol (HDL) alkaline phosphatase (ALP) were measured using Hitachi 912 autoanalyser.
Mt/Ex nanocomposite was obtained through a previously reported method (Rabiei et al., 2016). Preparation of the impure nettle leaves was carried out based on a method by Li, Percival, Bonard, and Gu (2011) without performing partial purification. The air-dried nettle leaf (50 g) was milled in a kitchen mill, and then was mixed with water (250 mL). The water suspension was sonicated for 30 min at 60 °C. The sample was centrifuged (Model:MF20R, AWEL company, French) at 4500 rpm for 10 min at room temperature and was filtered. This extract was freeze-dried to obtain solid materials. 200 mg of purified Mt dissolved in 100 mL deionized water and stirred continuously for 2 h to achieve homogenized slurry. Then, the solid extract was solved in water to obtain a concentration of 200 mg/mL. Then 4 different volumes of this solute including 1, 2, 3 and 4 mL were added to 100 mL of mentioned Mt slurry to obtain 1:1, 1:2, 1:3, and 1:4 ratios of Mt/Ex. After 2 h stirring, the solution was centrifuged at 4000 rpm for 5 min. The sediment containing nanocomposite, was freeze-dried for 2 days.
2.5. Method validation Method validation is the process used to confirm that the analytical procedure employed for a specific test is suitable for its intended use. Results from method validation can be used to judge the quality, reliability and consistency of analytical results. Because we used readymade kits to measure blood parameters by the autoanalyzer, the validation of these kits has been reported by the manufacturer as Table 1. In this table the relative standard deviation (RSD) is the indicator of precision and was calculated using following equation. The value of RSD should not be more than 2.0% (Robbins, Gong, Wells, Greenspan, & Pegg, 2015).
2.3. Characterization of Mt/Ex nanocomposite Crystallization, chemical characterization and thermo gravimetric analysis of the nanocomposites were evaluated using XRD, FTIR and TG analyzer as described in our published article (Rabiei et al., 2016).
% RSD = standard deviation/mean × 100
2.4. In vivo studies
Linearity indicates the ability to produce results that are directly proportional to the concentration of the analyte in samples. A series of samples should be prepared in which the analyte concentrations span the claimed range of the procedure. If there is a linear relationship (R2), test results can be evaluated by appropriate statistical method. The limit of detection (LOD) was calculated based on the standard deviation (SD) of the data and the slop of the regression line as below equation: LOD = 3 ∗ SD/slope ((Robbins et al., 2015).
2.4.1. Animal preparation The animals tested were Wistar male rats (Rattus norvegicus) with an average weight of 200 ± 20 g. These animals were obtained from the animal center of the Institute of Biochemistry and Biophysics in University of Tehran and stored in the same center under controlled conditions: temperature 25 °C, 12 h light/dark cycles and free access to water and food. All rats left 1 week in animal house for acclimatization. At the time of blood sampling, the rats were anesthetized with ether. Then blood sampling was performed by cardiac punctur. The cages were cleaning daily. This study was approved by the Institutional Research Ethics Committee, Faculty of Physical Education and Sport Sciences, Tehran University under Ethics Number: IR.UT.SPORT.REC.1397.039. Thirty six rats were randomly assigned into six groups of six animals each. According to the guidelines of the American Association for Laboratory Animal Science (Fitts, 2011), the number of animals in each group was determined using the same number of animals used by other investigators in a published paper on the same topic (Opris et al., 2017). Treatments were applied in a randomized complete block design with six replications as below: Group I Group II Group III
Diabetic rats received 200 mg/kg b.w/day Mt (D + Mt) orally for 30 days. Diabetic rats received 80 mg/kg b.w/day extract (D + Ex) orally for 30 days. Diabetic rats received 80 mg/kg b.w/day extract in the form of Mt/Ex (D + Mt/Ex) orally for 30 days.
2.6. Statistical analysis Data of glucose, TC, HDL, ALP and HbA1c were presented as mean ± standard deviation (SD). Experimental data were analyzed by one way ANOVA test and the comparison of group for these biochemical parameters were done with Tukey test (HSD) set at P < 0.05 using the Social Package of Statistical System software (SPSS). Table 1 System precision (RSD%), system suitability (R2) and system limit (LOD) for blood biochemical parameters measurement.
Normal control (C). Normal rats received 200 mg/kg b.w/day Mt (C + Mt) orally for 30 days Diabetic control (D).
Parameters
HbA1C
ALP
Glucose
Cholestrol
HDL
RSD% (n = 20) R2 (n = 78) LOD
1.5 0.999 1%
0.9 0.999 5 ppm
1.28 0.996 5 mg/dl
0.62 0.995 5 mg/dl
0.9 0.999 2 mg/dl
RSD: relative standard deviation, R2: correlation coefficient, LOD: limit of detection. 167
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Fig. 1. X-ray diffraction patterns of Mt (red line), Mt/Ex nanocomposite prepared at two ratios of 1:2 (green line) and 1:4 (blue line). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 2. FTIR spectra of Mt (red line), nettle extract (blue line) and Mt/Ex nanocomposite (green line) at ratio of 1:4. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
3. Results and discussion 3.1. XRD analysis
and proteins (Cao et al., 2011; Jaeckels et al., 2017; Stankovic, Jovic, & Zivkovic, 2004; Yu et al., 2013) of extracts with outer and inner layers of Mt. The bands in 3405 cm−1 and 1632 cm−1 are related to the HeOeH stretching and bending-in-plan vibration of the interlayer water of Mt has been also broadened indicating that hydrogen bonding has been enhanced (Cao et al., 2011). Appearance of three shoulders at 1502 cm−1, 1098 cm−1 and 850 cm−1 also confirms extract intercalation between interlayers of Mt. The TGA analysis (Fig. 3) and DTA (Data not shown) also shows that interlayer water has been completely replaced by extract. In other words, hydrogen bonding between water and inner SieO has been changed to hydrogen bonding between extract mainly polyphenols (Gonzalez-Neves et al., 2014; Gutierrez et al., 2017; Turkyilmaz et al., 2012) and proteins (Cao et al., 2011; Yu et al., 2013) with outer and inner SieO, SieOH and AleOH. It has been proved that Mt mostly absorbs phenolic compounds, protein or alkaloids from plant extract and fruit juices. For instance, Mt adsorbed caffeine (a methylxanthine alkaloid) from tea (Shiono, Yamamoto, Yotsumoto, Kawai, et al., 2017) and coffee extracts, selectively (Shiono, Yamamoto, Yotsumoto, & Yoshida, 2017). In contrast, numerous workers found that natural and modified Mt absorbed phenolic compounds from plant extract or fruit juices (Gonzalez-Neves et al., 2014; Gutierrez et al., 2017; Turkyilmaz et al., 2012). On the other hand, many researchers have reported the selective absorption of protein from extracts (Cao et al., 2011) or fruit juices (Jaeckels et al., 2017; Stankovic et al., 2004). The phenolic compounds and protein content of wild-nettle leaf extract has been reported 1.5% and 23–24%, respectively. The caffeoylmalic acid (54%), chlorogenic acid (23%), rutin (12%) and caffeic acid derivative (7%) concentrations were 95% of total phenolic compounds (Orcic et al., 2014; Pinelli et al., 2008). According to the abovementioned research and FTIR analysis (Fig. 2), we can conclude that here, the Mt nanoparticles has mostly absorbed proteins and phenolic compounds from extract. Determining the exact ratio of absorbed proteins to phenolics and their types can be an attractive research subject for the future investigations. Balooch et al. (2018) also showed that Mt has high capacity for absorption of polyphenol compounds from plant extracts.
Mt/Ex nanocomposite was prepared in 1:1, 1:2, 1:3 and 1:4 ratios of Mt to extract. In two of the prepared nanocomposites, d-spacing was 13.67 Å (1:2 ratio) and 14.39 Å (1:4 ratio) (Fig. 1). The shift of peaks in the XRD spectra to left shows that increasing the extract content increased the amount of intercalation. By considering of real d-spacing of Mt, 11.93 Å, it can be concluded that d-spacing has been increased by 1.74 Å and 2.46 Å at 1:2 and 1:4 ratios, respectively. It is an evidence that extract has been intercalated into the interlayer of Mt (Balooch, Sabahia, Aminian, & Hosseini, 2018). It is interesting to note that there is a few report in the field of intercalation of the plant extract into Mt nanoparticles but there are many reports about other drug intercalation into Mt such as 5Fluorouracil (Lin et al., 2002), Paclitaxel (Dong & Feng, 2005), Timolol maleate (Joshi, Kevadiya, Patel, Bajaj, & Jasra, 2009), 5-Fluorouracil (Kevadiya et al., 2012), Glutathione (Baek et al., 2012), Gallic acid (Rabiei et al., 2016) and Insulin (Kamari, Ghiaci, & Ghiaci, 2017). 3.2. FTIR analysis The FTIR spectra of Mt showed characteristic absorption band at 3620 cm−1 which belonging to OeH stretching band in AleOH and SieOH in Mt (Fig. 2). The bands in 3405 cm−1 and 1632 cm−1 are related to the HeOeH stretching and bending-in-plan vibration of the interlayer water. SieO stretching in Mt create sharp band in 985 cm−1 and little band in 914 cm−1 and 795 cm−1 are related to AleOH and AleMg (Fe)eOH bending vibration in Mt molecule (Rabiei et al., 2016). The FTIR spectra of nettle extract (Fig. 2) shows existence of flavonoids, carbohydrate and protein. The band between 3270 and 3310 cm−1 probably corresponds to NH stretching vibration of protein (Barth, 2007). The 2934 cm−1 band are typically related to aldehydes (R-CHO) stretching vibration of polyphenol (Sabahi, Khorami, Rezayana, Jafaria, & Karami, 2018). The 1572 cm−1 band is maybe related to amid II (Barth, 2007). The 1393 cm−1 corresponds to the CH3 group. The 1257 cm−1 and 1046 cm−1 bands can be attributed to CeO and CeOeC stretching vibration in polyphenol, respectively (Jafari, Sabahi, & Rahaie, 2016). In the FTIR bands of Mt/Ex nanocomposite (Fig. 2), band at 3620 cm−1 which belongs to OeH stretching band in AleOH and SieOH in Mt has been broadened which shows increasing of hydrogen bonds between polyphenols (Gonzalez-Neves, Favre, & Gil, 2014; Gutierrez, Ponce, & Alvarez, 2017; Turkyilmaz, Yemis, & Ozkan, 2012)
3.3. TG analysis The TG analysis of pure Mt, extract and Mt/Ex nanocomposite have been shown in Fig. 3. The TGA curve of Mt represents two distinct steps. The major mass loss pattern observed at temperatures below 150 °C is due to the free water evaporation. The second step at 275 °C 168
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Fig. 3. TGA pattern of Mt (red line), and nettle extract (green line), Mt/Ex nanocomposite (blue line) at ratio of 1:4. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
corresponds to loss of structural water. Extract shows three steps mass losses at 147 °C, 245 °C and 403 °C (Fig. 3) related to thermal dehydration, boiling process and thermal decomposition (oxidation of organic matter), respectively. Mt/Ex composite boiling process and oxidation decomposition was shifted from 245 °C and 403 °C up to 341 °C and 500 °C (Data not shown) which shows increasing of thermal stability of extract after intercalation. This evidence also reflects the complete intercalation of extract into Mt interlayers. A more interesting event was elimination of the endothermic peak related to free water evaporation at Mt/Ex composite (Data not shown), indicating complete replacement of interlayer water by extract. Finally, the TGA analysis shows 24.7% loading for Mt/Ex composite (Fig. 3).
degrading extract through gastrointestinal tract has not caused to obtain this result; it was also consumed it as injection. After injection, we did not observe antidiabetic effect for nettle extract (data not shown). These treatments also did not have significant effect on cholesterol and LDH level of serum on day 28 (Table 2). The reported anti-diabetic effect of leaf nettle (Urtica dioica) extract are different. Ethanolic nettle leaf extract has been reported to be antihyperglycemic (Ranjbari et al., 2016), in contrast, water nettle leaf extract has been reported to have no anti-diabetic properties (Bnouham et al., 2003; Roman Ramos, Alarcon-Aguilar, Lara-Lemus, & FloresSaenz, 1992). It has been shown that a cyclic hydrophilic protein in Urtica dioica is the major antidiabetic components of ethanolic nettle extract which facilitates glucose uptake through forming a permeable pore in the lipid bilayer of myoblasts cell (Shabani Domola, Christine, Doucette, Sweeney, & Wheeler, 2010). It appears that water nettle extract and Mt/Ex has contained a small amount of these proteins but the high level of polyphenols. Bnouham et al. (2003) also found no antihypoglycemic activity for nettle leaf extract. However, in hyperglycemia induced by oral glucose tolerance test (OGTT), water nettle
3.4. Antidiabetic effect It was observed that in type 2 diabetic rat, nettle extract and related composite means Mt/Ex did not decrease serum glucose (Fig. 4) on days 1, 14, 21 and 28 with respect to control group. To be sure that
Fig. 4. Serum glucose before (blue histogram) and after (red histogram) fasting in experiment groups. Data are average of 4 sampling; 1, 7, 14 and 28 days. Each value is mean ± S.D. for 6 rats in each group. C: normal control, D: diabetic control, Mt: montmorillonite, Ex: nettle extract, Mt/Ex: montmorillonite/nettle extract nanocomposite. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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glucose and serum lipid profile. Based on these results, they conclude that PFE could not completely reverse the hyperglycemia and PFEmediated reduction in HbA1c was probably due to the inhibition of the reaction between protein and sugar in the early stages of glycation. It should be noted that in this experiment diabetes was only induced by high-fat (28%) and high-sucrose (30%) diet which is not actual condition in diabetic rat type 2 (Abo-elmatty et al., 2013; Gheibi et al., 2017). An evidence for this claim is the similarity of HbA1c in normal and diabetics mice. Opris et al. (2017) also found that administration of sambucus nigra extract to animals with diabetes did not change the glucose concentrations in blood but it increased the antioxidant defense and reduced the inflammation in liver tissue. It has well been also documented that phenolic compounds, especially chlorogenic acid, caffeic acid and kaempferol-3-rutinoside that exit in the nettle leaf extract (Pinelli et al., 2008), are able to inhibit the glycation process (Justino et al., 2016). The mechanism by which plant polyphenols inhibit glycation can be attributed to antioxidant properties and the presence of their hydroxyl groups. Researchers have suggested that plant polyphenols might interact with glucose and prevent them from binding to proteins (Chao, Mong, Chan, & Yin, 2010; Justino et al., 2016). Mt/Ex composite showed to have a much stronger antiglycation effect than most extracts and nanoparticle/extracts that have been reported so far. For example, Daisy and Saipriya (2012) reported that the antiglycation activity of Cassia fistula extract and its gold nanoparticle were only 15.7% and 23.5%, respectively. Gold nanoparticle/Sambucus nigra extract also increase the antioxidant defense in diabetic rats only at p < 0.05 (Opris et al., 2017). Abo-elmatty et al. (2013) observed a decrease in %HbA1c from 10.5% to 8.3% in diabetic rate receiving 250 mg/kg purified extract of Urtica pilulifera having lectin; an antidiabetic compound which directly inhibited STZ activity. Caffeic acid (CA) and ellagic aid (EA) as two strong glycation inhibitor also reduced HbA1c in diabetic rate receiving 5% CA or EA, from 10.3% to 7.5%, at week 12 (Chao et al., 2010), While the antiglycation effect of the Mt/Ex composite in the present experiment was from 10.5% to 6.5% (40%) similar to pure asiatic acid and maslinic acid (Hung, Yang, & Yin, 2015) at high concentrations (0.2%). This strong effect can be related to (a) the enhanced effect of Mt on cellular uptake efficiency of the drugs through intestine endothelial cells (Dong & Feng, 2005), (b) polyphenol protection by Mt against harsh biological conditions such as intestinal enzymatic attack and gastric acid condition (Baek et al., 2012) and (c)
Table 2 The effect of different treatments on triglyceride (TG), high density lipid (HDL), cholesterol (Chol) and alkaline phosphatase (ALP) in serum on 28 days after treatments. Each value is mean ± S.D. for 6 rats in each group. Treatments
TG
HDL
C C + Mt D D + Mt D + Ex D + Mt/Ex
76 ± 22.4 34 ± 8.7 95 ± 27.6 105 ± 25.4 43 ± 10.8 84 ± 21.4
29 29 33 29 30 30
± ± ± ± ± ±
Chol 2.52 1.73 3.06 4.51 0.58 0.58
89 80 75 75 72 63
± ± ± ± ± ±
ALP 12.5 5.0 11.5 11.5 7.6 5.8
75 ± 10.8 71 ± 24.1 431 ± 76.4 490 ± 87.6 465 ± 71.6 434 ± 65.5
C: normal control, D: diabetic control, Mt: montmorillonite, Ex: nettle extract, Mt/Ex: montmorillonite/nettle extract nanocomposite.
extract showed a strong anti-diabetic effect. It should be noted that Alloxan or Streptozotocin-induced diabetes is a well proven model of experimental diabetes even in type 2 (Abo-elmatty, Essawy, Badr, & Sterner, 2013; Daisy & Saipriya, 2012; Gheibi, Kashfi, & Ghasemi, 2017). These compounds produce oxidative stress similar to actual hyperglycemia in diabetic animal. Therefore, Alloxan or Streptozotocin are often used to stimulate diabetes in experimental animals because of their toxic effect on pancreatic β cells, while stimulating diabetes by oral glucose tolerance test (OGTT) is not like the actual condition of diabetes. According to these results, observing no anti-hypoglycemic effect of nettle extract and Mt/Ex composite is consistent with other studies (Bnouham et al., 2003; Roman Ramos et al., 1992). Interestingly, oral delivery of Mt/Ex composite in diabetic rat decreased HbA1c significantly (p < 0.01) and brought it down from 10.5% to 6.2% (normal level) on day 28 (Fig. 5). Nettle extract also decreased HbA1c significantly to 8% (p < 0.05) but it is higher than normal level. This result is also consistent with other studies. Spinola et al. (2018) also reported that the phenolic compound of Vaccinium cylindraceaeum plant extract effectively reduced glucosidases and glycation of proteins. In this experiment, the composition of polyphenols showed strong correlation with their bioactivity. They declared that 5O-caffeoylquinic acid (70%) was the most important anti-glycation compound in the extract of this plant. In nettle extract also, the major phenolic compound are 5-O-caffeoylquinic acid and 2-O-caffeoylmalic acid (70%) (Orcic et al., 2014; Pinelli et al., 2008). Kumagai et al. (2015) showed that pomegranate fruit extract (PFE) only had antiglycation effect on diabetic rats but with no effect on status of serum
Fig. 5. Serum HbA1c in experiment groups. Each value is mean ± S.D. for 6 rats in each group. C: normal control, D: diabetic control, Mt: montmorillonite, Ex: nettle extract, Mt/Ex: montmorillonite/nettle extract nanocomposite. *P < 0.05, **P < 0.01, ***P < 0.001. All treatments has been compared with diabetic control (D). 170
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increased circulation time and circulation amounts of polyphenols in the blood (Baek et al., 2012; Kevadiya et al., 2012). Insulin prevents degradation of blood protein through lowering serum glucose (Abo-elmatty et al., 2013). High serum glucose glycoside the various proteins of blood including hemoglobin (glycated hemoglobin or HbA1c), albumin, collagen, low-density lipoprotein and fibronectin at diabetic condition (Wang et al., 2016). In fact, glycation is a non-enzymatic reaction in processed food and in vivo condition. Advanced glycation end products (AGEs) are produced after subsequent intermediates steps (Xu et al., 2016). AGE formation is irreversible. It is believed that the AGEs induce many diseases including diabetes and other diabetes-related diseases and aging such as retinopathy, cataracts, arteriosclerosis, and renal dysfunction (Kuda et al., 2016). Therefore, the measurement of HbA1c is a trustworthy indicator in diagnosis and control of diabetes. To be sure that Mt nanoparticle and its related composites have not had toxic effect on liver enzymes, we measured alkaline phosphatase level of rat serum. No significant toxic effect was observed for Mt and their composites (Table 2). Alkaline phosphatase (ALP) is an enzyme which its increased activities in blood is mainly due to its leakage from the liver into the serum (Abo-elmatty et al., 2013), and it is also an indicator of the abnormal function of liver. In current study, the activity of ALP in diabetic rats was much higher than normal rats which is due to STZ application (Chao et al., 2010). In vivo test showed no negative effect of Mt on blood alkaline phosphatase status of normal and diabetic rats showing no toxicity for Mt (Table 2).
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