Phytochemistry xxx (2015) xxx–xxx
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Hypoglycemic activity of withanolides and elicitated Withania somnifera Gorelick Jonathan a,⇑, Rosenberg Rivka b, Smotrich Avinoam a, Hanuš Lumír c, Bernstein Nirit b,⇑⇑ a
Judea Regional Research and Development Center, Kiryat Arba, Israel Institute of Soil Water and Environmental Sciences, Volcani Center, POB 6, 50-250, Israel c Institute for Drug Research, Hebrew University Medical Faculty, Jerusalem 91120, Israel b
a r t i c l e
i n f o
Article history: Received 27 August 2014 Received in revised form 22 February 2015 Available online xxxx Keywords: Withania somnifera Withanolide Elicitation Glucose uptake Withaferin A
a b s t r a c t Withania somnifera, known in India as Asghawhanda, is used traditionally to treat many medical problems including diabetes and has demonstrated therapeutic activity in various animal models as well as in diabetic patients. While much of W. somnifera’s therapeutic activity is attributed to withanolides, their role in the anti-diabetic activity of W. somnifera has not been adequately studied. In the present study, we evaluated the anti-diabetic activity of W. somnifera extract and purified withanolides, as well as the effect of various elicitors on this activity. W. somnifera leaf and root extracts increased glucose uptake in myotubes and adipocytes in a dose dependent manner, with the leaf extract more active than the root extract. Leaf but not root extract increased insulin secretion in basal pancreatic beta cells but not in stimulated cells. Six withanolides isolated from W. somnifera were tested for anti-diabetic activity based on glucose uptake in skeletal myotubes. Withaferin A was found to increase glucose uptake, with 10 lM producing a 54% increase compared with control, suggesting that withaferin A is at least partially responsible for W. somnifera’s anti-diabetic activity. Elicitors applied to the root growing solutions affected the physiological state of the plants, altering membrane leakage or osmotic potential. Methyl salicylate and chitosan increased withaferin A content by 75% and 69% respectively, and extracts from elicited plants increased glucose uptake to a higher extent than non-elicited plants, demonstrating a correlation between increased content of withaferin A and anti-diabetic activity. Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction Known as ashwagandha in India, Withania somnifera (L.) Dunal, or Indian ginseng, is one of the most important plants in Ayurvedic medicine (Verma and Kumar, 2011). It is used traditionally to increase energy, strength and stamina and for the treatment of numerous ailments including hepatic, cardiovascular, immunological, neurological, and metabolic disorders such as diabetes (Mishra et al., 2000). Many of these usages have been confirmed in in vitro and in vivo models (Chengappa et al., 2013; Gupta and Singh, 2014; Mirjalili et al., 2009; Winters, 2006; Hosny Mansour and Farouk Hafez, 2012) One of W. somnifera’s more promising therapeutic properties is its anti-diabetic activity. It has been shown to increase insulin sensitivity in streptozotocin injected diabetic rats (Anwer et al., 2008) and lower blood glucose in alloxan induced diabetic rats (Udayakumar et al., 2009). In addition, oral
⇑ Corresponding author. Tel.: +972 526051332; fax: +972 29960460. ⇑⇑ Corresponding author. Tel.:+972 39683707. E-mail addresses: [email protected], [email protected] (J. Gorelick), [email protected] (L. Hanuš), [email protected] (N. Bernstein).
administration of W. somnifera root powder significantly lowered blood glucose in diabetic patients (Andallu and Radhika, 2000). However, the bioactive compounds responsible for the therapeutic activity of W. somnifera have not been adequately characterized. Many of W. somnifera’s therapeutic activities are attributed to withanolides, a family of steroidal lactones with a range of pharmacological activities (Mirjalili et al., 2009). Many of their biological activities have been documented (Chen et al., 2011), and a number of withanolides isolated from fruits of Withania coagulans have demonstrated anti-diabetic activity (Maurya et al., 2008). However, the anti-diabetic activity of withanolides from W. somnifera has not yet been adequately studied. Subsequent to bioactive chemical characterization, ideal growing conditions must be determined for optimization of bioactive compound production in the plant tissues. Although the physiological role of W. somnifera’s withanolides in the plant is not known, a widely held opinion for the role of secondary metabolites is associated with the plant stress response. When a plant is exposed to stressors, also called elicitors, enzymatic pathways are induced, which alter the content of bioactive secondary metabolites (Ebel and Cosio, 1994). This is especially true for compounds which are well known for pharmacological activity, such as terpenoids
http://dx.doi.org/10.1016/j.phytochem.2015.02.029 0031-9422/Ó 2015 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Gorelick, J., et al. Hypoglycemic activity of withanolides and elicitated Withania somnifera. Phytochemistry (2015), http:// dx.doi.org/10.1016/j.phytochem.2015.02.029
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J. Gorelick et al. / Phytochemistry xxx (2015) xxx–xxx
(Trapp and Croteau, 2001), alkaloids (Facchini, 2001), and phenylpropanoids (Dixon and Paiva, 1995). Elicitation, a technique in which elicitors are used to stimulate the plant defense response, has been utilized to increase the production of biologically active metabolites in medicinal plants (Gorelick and Bernstein, 2014). Elicitation may also be a valuable tool to increase the anti-diabetic activity of W. somnifera. In this paper, we demonstrate the antidiabetic activity of W. somnifera extract, and purified withanolides, as well as the effect of various elicitors on this activity.
increased glucose uptake by 35% and 22% in myotubes and adipocytes, respectively. In addition, leaf but not root extract increased insulin secretion in basal pancreatic beta cells (RIN5) by over 50%, but not in stimulated cells (Fig. 2). Improving basal insulin levels has been associated with lower fasting glucose and free fatty acid levels as well lower hepatic glucose production in type 2 diabetic patients (Del Prato et al., 2002). This effect did not increase with increasing concentrations of the extract, and further work is needed to better characterize this activity.
2. Results and discussion
2.2. Role of withanolides
2.1. W. somnifera’s anti-diabetic activity The anti-diabetic activity of W. somnifera leaf and root extracts was evaluated in cellular models of diabetes. Because diabetes is a complex disease involving multiple factors, different cellular models were used: glucose uptake in both skeletal muscle and adipocytes as well as insulin secretion in pancreatic beta cells. Both leaf and root extracts increased glucose uptake in rat myotubes (L6) and adipocytes (3T3-L1) (Fig. 1). The effect was dose dependent, with the greatest increase at a concentration of 100 lg * mL 1. The leaf extract was more active, producing a 50% increase in glucose uptake in myotubes and a 30% increase in adipocytes. Root extract
After confirming W. somnifera’s anti-diabetic activity, selected withanolides purified from W. somnifera were evaluated. Six isolated withanolides: withaferin A, withanone, withanolide A, withanolide B, and withanoside IV were tested for anti-diabetic activity based on glucose uptake in skeletal myotubes (Fig. 3A). While some of the withanolides slightly increased glucose uptake, withaferin A was the only compound that significantly increased glucose uptake and was selected for further study. Withaferin A increased glucose uptake in a dose dependent manner in myotubes (Fig. 3B) with 10 lM producing a 54% increase compared with control. These results suggest that withanolides, and specifically withaferin A are at least partially responsible for the anti-diabetic
Fig. 1. Effect of Withania somnifera shoot and root extracts on glucose uptake in L6 myotubes (A) or 3T3-L1 adipocytes (B). Differentiated myotubes or matured adipocytes were treated with increasing concentrations of Withania somnifera root and shoot extracts. After 4 h incubation with the glucose analog, 2-NBDG, fluorescence of the cell layer was measured to determine glucose uptake. Results represent the mean ± SE of 5 replicates. Results were tested using one-way ANOVA, and means were compared using Tukey-HSD. Different letters above the means represent significant differences (p < 0.05) within the leaf extract samples (lowercase letters) and the root extract samples (uppercase letters).
Fig. 2. Basal (A) and glucose induced (B) insulin secretion in pancreatic RIN-5 beta cells after 1 h treatment with leaf and root Withania somnifera extract. Pancreatic beta cells were treated with increasing concentrations (50, 100, 500 lg * mL 1) of Withania somnifera root or shoot methanolic extract. Insulin secretion was measured before (A) and after stimulation of cells with high glucose + forskolin (B). Results are displayed as lg of secreted insulin per liter media. Results represent the mean ± SE of 3 replicates. * = p < 0.05 vs. control.
Please cite this article in press as: Gorelick, J., et al. Hypoglycemic activity of withanolides and elicitated Withania somnifera. Phytochemistry (2015), http:// dx.doi.org/10.1016/j.phytochem.2015.02.029
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were harvested and samples analyzed for physiological response, chemical composition, and bioactivity. 2.4. Physiological response The physiological response of elicited plants was compared to control plants by measuring membrane leakage, and osmotic potential of leaf sap (Bernstein et al., 2010). Evaluating changes in stress-related physiological parameters was aimed at identifying plant characteristics that correlate with stimulation of increased bioactivity. Elicitation with NaCl increased the osmotic potential of the cell sap by 56% and the leakage of electrolytes by 38% (Fig. 4), demonstrating that in spite of the short duration of exposure to the saline conditions (24 h), a stress condition was imposed, inducing a physiological response. Increased electrolyte leakage is an indication of reduced membrane stability resulting from direct injury to the membrane or reduced selectivity of the membrane transport system. Increased osmotic potential is an integrated result of reduced water content in the cells, accumulation of Na and Cl, and accumulation of osmolites as part of the osmotic adjustment mechanism. Surprisingly, under methyl salicylate and chitosan elicitation the membrane leakage reduced, demonstrating higher membrane stability compared to the control. Protective effects of chitosan on
Fig. 3. Glucose uptake in L6 myotubes treated with various withanolides (A) or increasing concentrations of withaferin A (B). Differentiated myotubes were treated with 1 lM of various withanolides (A) or increasing concentrations (0.1, 1, 10, or 100 lM) of withaferin A (B). After 4 h incubation with the glucose analog, 2-NBDG, fluorescence of the cell layer was measured to determine glucose uptake. Results are averages ± SE of 5 replicates. Samples were tested using one-way ANOVA, and means were compared using Tukey-HSD. Different letters above the means represent significant differences (p < 0.05).
effects observed in cellular models. Identification of at least one of the bioactive compounds will aid in correlating chemical composition and therapeutic activity, facilitating the optimization of growing conditions. 2.3. Elicitation Elicitors including methyl salicylate, and chitosan as well as NaCl were utilized to stimulate the plant stress response to increase the anti-diabetic activity of W. somnifera. Methyl salicylate and chitosan have been shown to be very effective at inducing the plant defense response (Ament et al., 2010; El Hadrami et al., 2010). Methyl salicylate is a natural plant derivative of salicylic acid implicated as an airborne signal involved with systemic acquired resistance (Shah et al., 2014). Chitosan is a polysaccharide produced from the breakdown of the fungal cell wall component chitin. Treatment with chitosan mimics an attack from a fungal pathogen which activates the plant defense response (Hadwiger, 1999). Rooted cuttings of W. somnifera were grown under hydroponic conditions with the various elicitors added to the hydroponic nutrient media. Selected elicitor concentrations were determined based on preliminary studies (results not shown). After 24 h, plants
Fig. 4. Physiological effects of elicitors on membrane leakage (A), and osmotic potential (B) in Withania somnifera. Leaf samples were taken from 30 day old plants following 24 h of elicitation (75 mM NaCl, 0.5 mM methyl salicylate, or 100 mg * mL 1 chitosan) and analyzed for membrane leakage (A) by comparative conductivity measurements or osmotic potential (B) using a cryoscopic microosmometer . Results are averages ± SE of 5 individual biological repeats. Samples were tested using one-way ANOVA, and means were compared using Tukey-HSD. Different letters above the means represent significant differences (p < 0.05).
Please cite this article in press as: Gorelick, J., et al. Hypoglycemic activity of withanolides and elicitated Withania somnifera. Phytochemistry (2015), http:// dx.doi.org/10.1016/j.phytochem.2015.02.029
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membrane stability have previously been demonstrated (Yang et al., 2009), and were suggested to result from a chitosan-induced reduction of oxidative damage by ROS scavenging. In accord with the increase in percent dry matter under the chitosan and the methyl salicylate elicitation treatments (results not shown), a small yet significant increase in osmotic potential of the plant sap occurred (Fig. 4) supporting the effect of these treatments on the water status of the plant cells. 2.5. Chemical response Chemical analysis of treated plants using HPLC revealed that elicitation for 24 h significantly increased the content of the bioactive compound, withaferin A, in W. somnifera leaves (Fig. 5). Methyl salicylate and chitosan increased withaferin A content by 75% and 69% respectively. NaCl treatment also slightly increased withaferin A content, but the high level of variability prevented any statistical significance. GC–MS analysis of leaf extracts from elicited plants revealed significant differences between treatments (results not shown). Most striking was the increase in fatty acid methyl esters found in the chitosan treated plants. Specifically, methyl 9,12-octadecadienoic acid, methyl 9,12,15-octadecatrienoic acid, methyl ester octadecanoic acid, and methyl ester hexadecanoic acid made up more than half of the compounds found in chitosan treated leaves, but were not present or present in only trace amounts in control leaves. The significance of these compounds and their role in the plant stress response remains to be determined. 2.6. Elicitation and bioactivity After demonstrating an increase in withanolide content, the correlation between increased chemical content and biological activity was analyzed. W. somnifera’s anti-diabetic activity was evaluated. While 50 lg * mL 1 of extracts from non-stimulated plants increased glucose uptake by 19%, extracts from plants treated with methyl salicylate or chitosan for 24 h increased glucose uptake by over 30% (Fig. 6).
Fig. 5. Elicitation of withaferin A in hydroponically grown Withania somnifera. 30 day old plants were exposed to 75 mM NaCl, 0.5 mM methyl salicylate or 100 mg * mL 1 chitosan in the growing solution for 24 h. Leaf samples were harvested and HPLC analysis performed to determine withaferin A content, Results are averages ± SE of 5 independent biological repeats. * = p < 0.05 vs. control.
Fig. 6. Glucose uptake in L6 Myotubes treated with extracts from elicited Withania somnifera. Extracts were prepared from elicited Withania somnifera plants exposed to 75 mM NaCl, 0.5 mM methyl salicylate or 100 mg * mL 1 chitosan for 24 h in the growing solution. Differentiated myotubes were treated with extracts from elicited plants, non-elicited plants, or control. After 4 h incubation with the glucose analog, 2-NBDG, fluorescence of the cell layer was measured to determine glucose uptake. Results represent the mean ± SE of 5 replicates. * = p < 0.05 vs. control. # = p < 0.05 vs. non-elicited plants. Results are averages ± SE of 5 individual biological repeats.
Taken together, the physiological characterization of the plants suggests that exposure to each of the examined elicitors induced a response in the plant cells, likely eliciting a cascade of events leading to an increase in the concentrations of individual chemical components. Both methyl salicylate and chitosan, increased the chemical content of a bioactive compound (Fig. 5) as well as the related biological activity (Fig. 6).
3. Conclusions This study is the first to investigate elicitation in intact plants of W. somnifera. While elicitation in W. somnifera has been attempted in vitro (Sangwan et al., 2014) and in root culture (Sivanandhan et al., 2012), whole plant studies have not previously been performed. While the effectiveness of elicitors at increasing W. somnifera’s anti-diabetic activity has been supported, their practicality remains to be determined. Although in small quantities these elicitors may be prohibitively expensive, a survey of bulk suppliers showed a reasonable price range of $1–3/kg, especially considering the high value of medicinal plants. Many factors must be taken into consideration including cost of elicitors, changes in growing conditions, and regulatory issues to determine the cost effectiveness of elicitation. In any event, elicitation is a powerful tool in understanding the physiology, chemistry and ultimately the optimization of producing botanical therapeutics like W. somnifera. While studies have described anti-diabetic activity in withanolides including coagulanolide (Beg et al., 2014; Singh et al., 2012; Maurya et al., 2008), and 4b-hydroxywithanolide E (Takimoto et al., 2014), this is the first study documenting the anti-diabetic activity of withaferin A. Previously implicated as a possible treatment for cancer (Grover et al., 2010), inflammation (Maitra et al., 2009), and for inhibiting angiogenesis (Mohan et al., 2004), withaferin’s hypoglycemic activity had not previously been documented. Although withaferin A has been shown to aid in the treatment of diabetes by reducing inflammation in pancreatic beta
Please cite this article in press as: Gorelick, J., et al. Hypoglycemic activity of withanolides and elicitated Withania somnifera. Phytochemistry (2015), http:// dx.doi.org/10.1016/j.phytochem.2015.02.029
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cells and protecting against cytokine induced damage (Peng et al., 2010; SoRelle et al., 2013), the role of withaferin A as an insulin mimetic is quite novel. In addition, W. somnifera’s insulin secretagogue activity is also surprising. Because multiple factors are involved in the onset and development of diabetes, the most effective treatments usually entail a combination of an insulin sensitizer like metformin together with insulin or an insulin secretagogue (Lund et al., 2009). If indeed extracts of W. somnifera increase both insulin secretion as well as insulin sensitivity, it may possibly be a natural replacement for the current combinational therapy. However, more work is needed to better understand the role of W. somnifera and withaferin A for the treatment of diabetes. 4. Experimental 4.1. Plant material and growing conditions The study was conducted with W. somnifera plants that were developed from rooted cuttings of wild plants collected in the Har Hevron region of Israel near Moshav Carmel. Voucher specimens were submitted to the Israeli seed bank. For the elicitation experiments the plants were cultivated hydroponically. Rooted cuttings of W. somnifera were transferred to aerated one-quarter strength modified aerated Hoagland solution (Bernstein et al., 1995), five plants into each 13L container. At the time of transfer, the rooted cuttings were 10 cm high and had 5–7 leaves. The hydroponic solution was replaced weekly and solution pH was adjusted every three days to the value of 5.7 with the addition of KOH. The experiment was conducted in a temperature controlled greenhouse using a heating and cooling air conditioning system. The average maximum and minimum temperatures in the greenhouse were 35 °C and 22 °C day/night, respectively. The minimum day and maximum night relative humidities were 60% and 90%, respectively. The plants were cultivated for 30 days prior to the beginning of the elicitation treatments. Three elicitors (0.5 mM methyl salicylate; 100 mg * mL 1 chitosan and 75 mM NaCl) were evaluated for their effect on the chemical and the physiological response of the plants and bioactivity of the plant extract compared to a non-elicited control. The experiment had a random block design, with five replicated hydroponic containers of 5 plants each per treatment (25 plants per elicitation treatment). The elicitors were added to the hydroponic growing media. 24 h after exposure to the elicitors, roots and leaves from each plant were collected separately and freeze dried for further analysis. Leaf material was also collected for analysis of membrane leakage and osmotic potential. 4.2. Plant extracts Extracts were prepared from dried plant material. Lyophilized plant material was ground, extracted in methanol (10 mL * g 1), sonicated for 30 min, and incubated at room temperature. After 24 h, samples were centrifuged, filtered, and stored at 20 °C for further use. 4.3. Anti-diabetic activity The plant extracts were tested for anti-diabetic activity using cellular models of diabetes. The effects of W. somnifera extract on insulin secretion in pancreatic beta cells as well as glucose uptake in skeletal muscle and adipocytes were measured (Gorelick et al., 2011). Pancreatic RIN-5F (CRL-2058) cells were grown in RPMI media containing 10% fetal bovine serum (FBS) and 2 mM
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glutamine for measuring insulin secretion. RIN-5F cells were treated with increasing concentrations of extract for 1 h and subsequently incubated in Krebs–Ringer biocarbonate HEPES buffer (KRBH). After 1 h, the buffer was collected and replaced with KRBH buffer supplemented with 16.7 mM glucose and 10 lM forskolin to stimulate insulin secretion. After a 30 min incubation, the buffer was collected for analysis of insulin content using an insulin ELISA kit (Mercodia, Uppsala, Sweden). Glucose uptake was measured based on Yamada et al. (2000). L6 (CRL-1458) skeletal myoblasts were grown in Dulbecco’s Modified Eagle’s Medium (DMEM) containing 10% FBS. Differentiated myotubes were produced by replacing the FBS with 5% horse serum for 5 days. 3T3-LI (CL-173) fibroblasts were grown in DMEM containing 10% FCS. After cells approached confluency, they were grown in differentiation media containing 100 nM insulin, 500 nM dexamethasone, and 0.5 mM isobutylmethylxanthine. After 7–10 days, mature adipocytes were identified based on morphology and used for study. L6 or 3T3 cells were plated at a concentration of 105 cells * mL 1 on 96 well plates and differentiated. Differentiated L6 myotubes or 3T3 adipocytes were transferred to low glucose serum free media overnight before treatment with increasing concentrations of extract or insulin for 4 h. For withanolides assay, the 6 different purified withanolides (Natural Remedy, India) were each dissolved in methanol at a concentration of 1 mM and added to the cells for 4 h. For the withaferin assay, purified withaferin A (Sigma, St. Louis, USA) was dissolved in methanol and added to cells in increasing concentrations for 4 h. For elicited Withania assay, methanolic plant extracts were prepared from untreated or elicited plants as described in Section 4.2 and added at a concentration of 50 mg * mL 1 to cells for 4 h. After incubation, 200 lM of the fluorescent glucose analog 2-(N-(7-nitrobenz-2oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose (2-NBDG, Invitrogen, Carlsbad, USA) was added for 1 h. Cells were subsequently rinsed in cold PBS and fluorescence was measured with a fluorescence microplate reader (BMG Polarstar, Ortenberg, Germany). 4.4. Plant physiology 4.4.1. Membrane leakage Membrane leakage measurements, indicators of membrane injury under stress (Lu et al., 2008), were measured as previously described with minor modifications (Shoresh et al. 2011). Leaf segments were shaken vigorously in 30 mL of double-distilled water for 24 h and the electrical conductivity (EC) was measured using a conductivity meter (CyberScan CON 1500; Eutech Instruments, Ayer Rajan Crescent, Singapore). The tubes with the leaf pieces were then autoclaved and shaken again. Conductivity was measured again and the membrane leakage was calculated as the ratio between the first and the second conductivity measurements. Results from 5 replicated leaves were averaged. 4.4.2. Osmotic potential For osmotic potential measurements, the sampled leaf tissue was frozen in 1.5 mL micro-test-tubes in liquid nitrogen and stored at 5 °C for further analyses. The frozen tissue was crushed inside the tubes with a glass rod, the bottom of the tubes was pin-pricked and the tubes, set inside another 1.5 mL tube, centrifuged for 4 min in a refrigerated centrifuge (Sigma Laboratory Centrifuges, Germany) at 5 °C at 10,000 rpm. One hundred microliters of the fluids collected in the lower micro test tube were used for measurement of osmotic potential using a cryoscopic microosmometer (lOsmette; Precision Systems, Natick, MA, USA) by measuring the freezing point of 100 lL of sap. Results are presented in mOsm * kg H2O 1. Five replicates from each treatment were analyzed.
Please cite this article in press as: Gorelick, J., et al. Hypoglycemic activity of withanolides and elicitated Withania somnifera. Phytochemistry (2015), http:// dx.doi.org/10.1016/j.phytochem.2015.02.029
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4.5. Chemical composition 4.5.1. HPLC HPLC analysis was performed to determine withanolide content in treated plants (Chaurasiya et al., 2008). 70% aqueous methanol extracts prepared from dried ground plant material were sequentially partitioned with hexane to remove non-polar impurities and then chloroform to extract the withanolides. The chloroform phase was evaporated and resuspended in methanol. 10 lL was injected using a Waters LC module equipped with a multi-wavelength UV detector set to 227 nm. A Gemini C-18 column (Phenomenex, Torrance, USA) was used with an isocratic mobile phase consisting of water and methanol (65:35). Retention times were determined using standard withanolides (Natural Remedies, India). For quantification, a standard curve was generated. For verification of withanolides, 10 lL of each sample was injected into a Thermo TSQ Quantum Access Max Triple Stage Quadrupole Mass Spectrometer using positive Electrospray Ionization. Identification of the molecular ion (M + H) of withaferin A as well as other characteristic ions was confirmed. 4.5.2. GC–MS For GC–MS analysis, 1 lL of each sample was injected into a Hewlett–Packard G1800A GCD system fitted with a 5% phenylmethylsiloxane (HP-5MS) capillary column (30 m 0.25 mm i.d. 0.25 mm), splitless injection with helium 1 mL * min 1 and a solvent delay of 4.8 min. The temperature of the detector was 280 °C and the injector was 250 °C. The column temperature was held for 4 min at 50 °C and then increased 8 °C * min 1 until 280 °C. 4.6. Statistical analysis Results are expressed as means ± standard errors. Statistical analysis was performed using JMP 5 software (SAS Institute Inc., 2002, Cary, NC, USA). Data were subjected to one-way ANOVA analysis (a < 0.05) and Tukey honestly significant difference for comparison of means. Acknowledgments This work has been carried out with support from the Israeli Ministry of Science, Technology, and Space as well as the Israeli Ministry of Agriculture project no. 3-8320. References Ament, K., Krasikov, V., Allmann, S., Rep, M., Takken, F.L., Schuurink, R.C., 2010. Methyl salicylate production in tomato affects biotic interactions. Plant J. Cell Mol. Biol. 62, 124–134. Andallu, B., Radhika, B., 2000. Hypoglycemic, diuretic and hypocholesterolemic effect of winter cherry (Withania somnifera, Dunal) root. Indian J. Exp. Biol. 38, 607–609. Anwer, T., Sharma, M., Pillai, K.K., Iqbal, M., 2008. Effect of Withania somnifera on insulin sensitivity in non-insulin-dependent diabetes mellitus rats. Basic Clin. Pharmacol. Toxicol. 102, 498–503. Beg, M., Chauhan, P., Varshney, S., Shankar, K., Rajan, S., Saini, D., Srivastava, M.N., Yadav, P.P., Gaikwad, A.N., 2014. A withanolide coagulin-L inhibits adipogenesis modulating Wnt/beta-catenin pathway and cell cycle in mitotic clonal expansion. Phytomed. Int. J. Phytother. Phytopharmacol. 21, 406–414. Bernstein, N., Silk, W.K., Läuchli, A., 1995. Growth and development of sorghum leaves under conditions of NaCl stress: possible role of some mineral elements in growth inhibition. Planta 196, 699–705. Bernstein, N., Shoresh, M., Xu, Y., Huang, B., 2010. Involvement of the plant antioxidative response in the differential growth sensitivity to salinity of leaves vs. roots during cell development. Free Radic. Biol. Med. 49, 1161–1171. Chaurasiya, N.D., Uniyal, G.C., Lal, P., Misra, L., Sangwan, N.S., Tuli, R., Sangwan, R.S., 2008. Analysis of withanolides in root and leaf of Withania somnifera by HPLC with photodiode array and evaporative light scattering detection. Phytochem. Anal. 19, 148–154. Chen, L.X., He, H., Qiu, F., 2011. Natural withanolides: an overview. Nat. Prod. Rep. 28, 705–740.
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Withaferin A inhibits pro-inflammatory cytokine-induced damage to islets in culture and following transplantation. Diabetologia 56, 814–824. Takimoto, T., Kanbayashi, Y., Toyoda, T., Adachi, Y., Furuta, C., Suzuki, K., Miwa, T., Bannai, M., 2014. 4beta-Hydroxywithanolide E isolated from Physalis pruinosa calyx decreases inflammatory responses by inhibiting the NF-kappaB signaling in diabetic mouse adipose tissue. Int. J. Obes. http://dx.doi.org/10.1038/ ijo.2014.33. Trapp, S., Croteau, R., 2001. Defensive resin biosynthesis in conifers. Ann. Rev. Plant Physiol. Plant Mol. Biol. 52, 689–724. Udayakumar, R., Kasthurirengan, S., Mariashibu, T.S., Rajesh, M., Anbazhagan, V.R., Kim, S.C., Ganapathi, A., Choi, C.W., 2009. Hypoglycaemic and hypolipidaemic effects of Withania somnifera root and leaf extracts on alloxan-induced diabetic rats. Int. J. Mol. Sci. 10, 2367–2382.
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J. Gorelick et al. / Phytochemistry xxx (2015) xxx–xxx Verma, S., Kumar, A., 2011. Therapeutic uses of Withania somnifera with a note on withanolides and its pharmacological actions. Asian J. Pharm. Clin. Res. 4 (S1), 1–4. Winters, M., 2006. Ancient medicine, modern use: Withania somnifera and its potential role in integrative oncology. Altern. Med. Rev. 11, 269–277.
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Phytochemistry journal homepage: www.elsevier.com/locate/phytochem
Hypoglycemic activity of withanolides and elicitated Withania somnifera Gorelick Jonathan a,⇑, Rosenberg Rivka b, Smotrich Avinoam a, Hanuš Lumír c, Bernstein Nirit b,⇑⇑ a
Judea Regional Research and Development Center, Kiryat Arba, Israel Institute of Soil Water and Environmental Sciences, Volcani Center, POB 6, 50-250, Israel c Institute for Drug Research, Hebrew University Medical Faculty, Jerusalem 91120, Israel b
a r t i c l e
i n f o
Article history: Received 27 August 2014 Received in revised form 22 February 2015 Available online xxxx Keywords: Withania somnifera Withanolide Elicitation Glucose uptake Withaferin A
a b s t r a c t Withania somnifera, known in India as Asghawhanda, is used traditionally to treat many medical problems including diabetes and has demonstrated therapeutic activity in various animal models as well as in diabetic patients. While much of W. somnifera’s therapeutic activity is attributed to withanolides, their role in the anti-diabetic activity of W. somnifera has not been adequately studied. In the present study, we evaluated the anti-diabetic activity of W. somnifera extract and purified withanolides, as well as the effect of various elicitors on this activity. W. somnifera leaf and root extracts increased glucose uptake in myotubes and adipocytes in a dose dependent manner, with the leaf extract more active than the root extract. Leaf but not root extract increased insulin secretion in basal pancreatic beta cells but not in stimulated cells. Six withanolides isolated from W. somnifera were tested for anti-diabetic activity based on glucose uptake in skeletal myotubes. Withaferin A was found to increase glucose uptake, with 10 lM producing a 54% increase compared with control, suggesting that withaferin A is at least partially responsible for W. somnifera’s anti-diabetic activity. Elicitors applied to the root growing solutions affected the physiological state of the plants, altering membrane leakage or osmotic potential. Methyl salicylate and chitosan increased withaferin A content by 75% and 69% respectively, and extracts from elicited plants increased glucose uptake to a higher extent than non-elicited plants, demonstrating a correlation between increased content of withaferin A and anti-diabetic activity. Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction Known as ashwagandha in India, Withania somnifera (L.) Dunal, or Indian ginseng, is one of the most important plants in Ayurvedic medicine (Verma and Kumar, 2011). It is used traditionally to increase energy, strength and stamina and for the treatment of numerous ailments including hepatic, cardiovascular, immunological, neurological, and metabolic disorders such as diabetes (Mishra et al., 2000). Many of these usages have been confirmed in in vitro and in vivo models (Chengappa et al., 2013; Gupta and Singh, 2014; Mirjalili et al., 2009; Winters, 2006; Hosny Mansour and Farouk Hafez, 2012) One of W. somnifera’s more promising therapeutic properties is its anti-diabetic activity. It has been shown to increase insulin sensitivity in streptozotocin injected diabetic rats (Anwer et al., 2008) and lower blood glucose in alloxan induced diabetic rats (Udayakumar et al., 2009). In addition, oral
⇑ Corresponding author. Tel.: +972 526051332; fax: +972 29960460. ⇑⇑ Corresponding author. Tel.:+972 39683707. E-mail addresses: [email protected], [email protected] (J. Gorelick), [email protected] (L. Hanuš), [email protected] (N. Bernstein).
administration of W. somnifera root powder significantly lowered blood glucose in diabetic patients (Andallu and Radhika, 2000). However, the bioactive compounds responsible for the therapeutic activity of W. somnifera have not been adequately characterized. Many of W. somnifera’s therapeutic activities are attributed to withanolides, a family of steroidal lactones with a range of pharmacological activities (Mirjalili et al., 2009). Many of their biological activities have been documented (Chen et al., 2011), and a number of withanolides isolated from fruits of Withania coagulans have demonstrated anti-diabetic activity (Maurya et al., 2008). However, the anti-diabetic activity of withanolides from W. somnifera has not yet been adequately studied. Subsequent to bioactive chemical characterization, ideal growing conditions must be determined for optimization of bioactive compound production in the plant tissues. Although the physiological role of W. somnifera’s withanolides in the plant is not known, a widely held opinion for the role of secondary metabolites is associated with the plant stress response. When a plant is exposed to stressors, also called elicitors, enzymatic pathways are induced, which alter the content of bioactive secondary metabolites (Ebel and Cosio, 1994). This is especially true for compounds which are well known for pharmacological activity, such as terpenoids
http://dx.doi.org/10.1016/j.phytochem.2015.02.029 0031-9422/Ó 2015 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Gorelick, J., et al. Hypoglycemic activity of withanolides and elicitated Withania somnifera. Phytochemistry (2015), http:// dx.doi.org/10.1016/j.phytochem.2015.02.029
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(Trapp and Croteau, 2001), alkaloids (Facchini, 2001), and phenylpropanoids (Dixon and Paiva, 1995). Elicitation, a technique in which elicitors are used to stimulate the plant defense response, has been utilized to increase the production of biologically active metabolites in medicinal plants (Gorelick and Bernstein, 2014). Elicitation may also be a valuable tool to increase the anti-diabetic activity of W. somnifera. In this paper, we demonstrate the antidiabetic activity of W. somnifera extract, and purified withanolides, as well as the effect of various elicitors on this activity.
increased glucose uptake by 35% and 22% in myotubes and adipocytes, respectively. In addition, leaf but not root extract increased insulin secretion in basal pancreatic beta cells (RIN5) by over 50%, but not in stimulated cells (Fig. 2). Improving basal insulin levels has been associated with lower fasting glucose and free fatty acid levels as well lower hepatic glucose production in type 2 diabetic patients (Del Prato et al., 2002). This effect did not increase with increasing concentrations of the extract, and further work is needed to better characterize this activity.
2. Results and discussion
2.2. Role of withanolides
2.1. W. somnifera’s anti-diabetic activity The anti-diabetic activity of W. somnifera leaf and root extracts was evaluated in cellular models of diabetes. Because diabetes is a complex disease involving multiple factors, different cellular models were used: glucose uptake in both skeletal muscle and adipocytes as well as insulin secretion in pancreatic beta cells. Both leaf and root extracts increased glucose uptake in rat myotubes (L6) and adipocytes (3T3-L1) (Fig. 1). The effect was dose dependent, with the greatest increase at a concentration of 100 lg * mL 1. The leaf extract was more active, producing a 50% increase in glucose uptake in myotubes and a 30% increase in adipocytes. Root extract
After confirming W. somnifera’s anti-diabetic activity, selected withanolides purified from W. somnifera were evaluated. Six isolated withanolides: withaferin A, withanone, withanolide A, withanolide B, and withanoside IV were tested for anti-diabetic activity based on glucose uptake in skeletal myotubes (Fig. 3A). While some of the withanolides slightly increased glucose uptake, withaferin A was the only compound that significantly increased glucose uptake and was selected for further study. Withaferin A increased glucose uptake in a dose dependent manner in myotubes (Fig. 3B) with 10 lM producing a 54% increase compared with control. These results suggest that withanolides, and specifically withaferin A are at least partially responsible for the anti-diabetic
Fig. 1. Effect of Withania somnifera shoot and root extracts on glucose uptake in L6 myotubes (A) or 3T3-L1 adipocytes (B). Differentiated myotubes or matured adipocytes were treated with increasing concentrations of Withania somnifera root and shoot extracts. After 4 h incubation with the glucose analog, 2-NBDG, fluorescence of the cell layer was measured to determine glucose uptake. Results represent the mean ± SE of 5 replicates. Results were tested using one-way ANOVA, and means were compared using Tukey-HSD. Different letters above the means represent significant differences (p < 0.05) within the leaf extract samples (lowercase letters) and the root extract samples (uppercase letters).
Fig. 2. Basal (A) and glucose induced (B) insulin secretion in pancreatic RIN-5 beta cells after 1 h treatment with leaf and root Withania somnifera extract. Pancreatic beta cells were treated with increasing concentrations (50, 100, 500 lg * mL 1) of Withania somnifera root or shoot methanolic extract. Insulin secretion was measured before (A) and after stimulation of cells with high glucose + forskolin (B). Results are displayed as lg of secreted insulin per liter media. Results represent the mean ± SE of 3 replicates. * = p < 0.05 vs. control.
Please cite this article in press as: Gorelick, J., et al. Hypoglycemic activity of withanolides and elicitated Withania somnifera. Phytochemistry (2015), http:// dx.doi.org/10.1016/j.phytochem.2015.02.029
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were harvested and samples analyzed for physiological response, chemical composition, and bioactivity. 2.4. Physiological response The physiological response of elicited plants was compared to control plants by measuring membrane leakage, and osmotic potential of leaf sap (Bernstein et al., 2010). Evaluating changes in stress-related physiological parameters was aimed at identifying plant characteristics that correlate with stimulation of increased bioactivity. Elicitation with NaCl increased the osmotic potential of the cell sap by 56% and the leakage of electrolytes by 38% (Fig. 4), demonstrating that in spite of the short duration of exposure to the saline conditions (24 h), a stress condition was imposed, inducing a physiological response. Increased electrolyte leakage is an indication of reduced membrane stability resulting from direct injury to the membrane or reduced selectivity of the membrane transport system. Increased osmotic potential is an integrated result of reduced water content in the cells, accumulation of Na and Cl, and accumulation of osmolites as part of the osmotic adjustment mechanism. Surprisingly, under methyl salicylate and chitosan elicitation the membrane leakage reduced, demonstrating higher membrane stability compared to the control. Protective effects of chitosan on
Fig. 3. Glucose uptake in L6 myotubes treated with various withanolides (A) or increasing concentrations of withaferin A (B). Differentiated myotubes were treated with 1 lM of various withanolides (A) or increasing concentrations (0.1, 1, 10, or 100 lM) of withaferin A (B). After 4 h incubation with the glucose analog, 2-NBDG, fluorescence of the cell layer was measured to determine glucose uptake. Results are averages ± SE of 5 replicates. Samples were tested using one-way ANOVA, and means were compared using Tukey-HSD. Different letters above the means represent significant differences (p < 0.05).
effects observed in cellular models. Identification of at least one of the bioactive compounds will aid in correlating chemical composition and therapeutic activity, facilitating the optimization of growing conditions. 2.3. Elicitation Elicitors including methyl salicylate, and chitosan as well as NaCl were utilized to stimulate the plant stress response to increase the anti-diabetic activity of W. somnifera. Methyl salicylate and chitosan have been shown to be very effective at inducing the plant defense response (Ament et al., 2010; El Hadrami et al., 2010). Methyl salicylate is a natural plant derivative of salicylic acid implicated as an airborne signal involved with systemic acquired resistance (Shah et al., 2014). Chitosan is a polysaccharide produced from the breakdown of the fungal cell wall component chitin. Treatment with chitosan mimics an attack from a fungal pathogen which activates the plant defense response (Hadwiger, 1999). Rooted cuttings of W. somnifera were grown under hydroponic conditions with the various elicitors added to the hydroponic nutrient media. Selected elicitor concentrations were determined based on preliminary studies (results not shown). After 24 h, plants
Fig. 4. Physiological effects of elicitors on membrane leakage (A), and osmotic potential (B) in Withania somnifera. Leaf samples were taken from 30 day old plants following 24 h of elicitation (75 mM NaCl, 0.5 mM methyl salicylate, or 100 mg * mL 1 chitosan) and analyzed for membrane leakage (A) by comparative conductivity measurements or osmotic potential (B) using a cryoscopic microosmometer . Results are averages ± SE of 5 individual biological repeats. Samples were tested using one-way ANOVA, and means were compared using Tukey-HSD. Different letters above the means represent significant differences (p < 0.05).
Please cite this article in press as: Gorelick, J., et al. Hypoglycemic activity of withanolides and elicitated Withania somnifera. Phytochemistry (2015), http:// dx.doi.org/10.1016/j.phytochem.2015.02.029
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membrane stability have previously been demonstrated (Yang et al., 2009), and were suggested to result from a chitosan-induced reduction of oxidative damage by ROS scavenging. In accord with the increase in percent dry matter under the chitosan and the methyl salicylate elicitation treatments (results not shown), a small yet significant increase in osmotic potential of the plant sap occurred (Fig. 4) supporting the effect of these treatments on the water status of the plant cells. 2.5. Chemical response Chemical analysis of treated plants using HPLC revealed that elicitation for 24 h significantly increased the content of the bioactive compound, withaferin A, in W. somnifera leaves (Fig. 5). Methyl salicylate and chitosan increased withaferin A content by 75% and 69% respectively. NaCl treatment also slightly increased withaferin A content, but the high level of variability prevented any statistical significance. GC–MS analysis of leaf extracts from elicited plants revealed significant differences between treatments (results not shown). Most striking was the increase in fatty acid methyl esters found in the chitosan treated plants. Specifically, methyl 9,12-octadecadienoic acid, methyl 9,12,15-octadecatrienoic acid, methyl ester octadecanoic acid, and methyl ester hexadecanoic acid made up more than half of the compounds found in chitosan treated leaves, but were not present or present in only trace amounts in control leaves. The significance of these compounds and their role in the plant stress response remains to be determined. 2.6. Elicitation and bioactivity After demonstrating an increase in withanolide content, the correlation between increased chemical content and biological activity was analyzed. W. somnifera’s anti-diabetic activity was evaluated. While 50 lg * mL 1 of extracts from non-stimulated plants increased glucose uptake by 19%, extracts from plants treated with methyl salicylate or chitosan for 24 h increased glucose uptake by over 30% (Fig. 6).
Fig. 5. Elicitation of withaferin A in hydroponically grown Withania somnifera. 30 day old plants were exposed to 75 mM NaCl, 0.5 mM methyl salicylate or 100 mg * mL 1 chitosan in the growing solution for 24 h. Leaf samples were harvested and HPLC analysis performed to determine withaferin A content, Results are averages ± SE of 5 independent biological repeats. * = p < 0.05 vs. control.
Fig. 6. Glucose uptake in L6 Myotubes treated with extracts from elicited Withania somnifera. Extracts were prepared from elicited Withania somnifera plants exposed to 75 mM NaCl, 0.5 mM methyl salicylate or 100 mg * mL 1 chitosan for 24 h in the growing solution. Differentiated myotubes were treated with extracts from elicited plants, non-elicited plants, or control. After 4 h incubation with the glucose analog, 2-NBDG, fluorescence of the cell layer was measured to determine glucose uptake. Results represent the mean ± SE of 5 replicates. * = p < 0.05 vs. control. # = p < 0.05 vs. non-elicited plants. Results are averages ± SE of 5 individual biological repeats.
Taken together, the physiological characterization of the plants suggests that exposure to each of the examined elicitors induced a response in the plant cells, likely eliciting a cascade of events leading to an increase in the concentrations of individual chemical components. Both methyl salicylate and chitosan, increased the chemical content of a bioactive compound (Fig. 5) as well as the related biological activity (Fig. 6).
3. Conclusions This study is the first to investigate elicitation in intact plants of W. somnifera. While elicitation in W. somnifera has been attempted in vitro (Sangwan et al., 2014) and in root culture (Sivanandhan et al., 2012), whole plant studies have not previously been performed. While the effectiveness of elicitors at increasing W. somnifera’s anti-diabetic activity has been supported, their practicality remains to be determined. Although in small quantities these elicitors may be prohibitively expensive, a survey of bulk suppliers showed a reasonable price range of $1–3/kg, especially considering the high value of medicinal plants. Many factors must be taken into consideration including cost of elicitors, changes in growing conditions, and regulatory issues to determine the cost effectiveness of elicitation. In any event, elicitation is a powerful tool in understanding the physiology, chemistry and ultimately the optimization of producing botanical therapeutics like W. somnifera. While studies have described anti-diabetic activity in withanolides including coagulanolide (Beg et al., 2014; Singh et al., 2012; Maurya et al., 2008), and 4b-hydroxywithanolide E (Takimoto et al., 2014), this is the first study documenting the anti-diabetic activity of withaferin A. Previously implicated as a possible treatment for cancer (Grover et al., 2010), inflammation (Maitra et al., 2009), and for inhibiting angiogenesis (Mohan et al., 2004), withaferin’s hypoglycemic activity had not previously been documented. Although withaferin A has been shown to aid in the treatment of diabetes by reducing inflammation in pancreatic beta
Please cite this article in press as: Gorelick, J., et al. Hypoglycemic activity of withanolides and elicitated Withania somnifera. Phytochemistry (2015), http:// dx.doi.org/10.1016/j.phytochem.2015.02.029
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cells and protecting against cytokine induced damage (Peng et al., 2010; SoRelle et al., 2013), the role of withaferin A as an insulin mimetic is quite novel. In addition, W. somnifera’s insulin secretagogue activity is also surprising. Because multiple factors are involved in the onset and development of diabetes, the most effective treatments usually entail a combination of an insulin sensitizer like metformin together with insulin or an insulin secretagogue (Lund et al., 2009). If indeed extracts of W. somnifera increase both insulin secretion as well as insulin sensitivity, it may possibly be a natural replacement for the current combinational therapy. However, more work is needed to better understand the role of W. somnifera and withaferin A for the treatment of diabetes. 4. Experimental 4.1. Plant material and growing conditions The study was conducted with W. somnifera plants that were developed from rooted cuttings of wild plants collected in the Har Hevron region of Israel near Moshav Carmel. Voucher specimens were submitted to the Israeli seed bank. For the elicitation experiments the plants were cultivated hydroponically. Rooted cuttings of W. somnifera were transferred to aerated one-quarter strength modified aerated Hoagland solution (Bernstein et al., 1995), five plants into each 13L container. At the time of transfer, the rooted cuttings were 10 cm high and had 5–7 leaves. The hydroponic solution was replaced weekly and solution pH was adjusted every three days to the value of 5.7 with the addition of KOH. The experiment was conducted in a temperature controlled greenhouse using a heating and cooling air conditioning system. The average maximum and minimum temperatures in the greenhouse were 35 °C and 22 °C day/night, respectively. The minimum day and maximum night relative humidities were 60% and 90%, respectively. The plants were cultivated for 30 days prior to the beginning of the elicitation treatments. Three elicitors (0.5 mM methyl salicylate; 100 mg * mL 1 chitosan and 75 mM NaCl) were evaluated for their effect on the chemical and the physiological response of the plants and bioactivity of the plant extract compared to a non-elicited control. The experiment had a random block design, with five replicated hydroponic containers of 5 plants each per treatment (25 plants per elicitation treatment). The elicitors were added to the hydroponic growing media. 24 h after exposure to the elicitors, roots and leaves from each plant were collected separately and freeze dried for further analysis. Leaf material was also collected for analysis of membrane leakage and osmotic potential. 4.2. Plant extracts Extracts were prepared from dried plant material. Lyophilized plant material was ground, extracted in methanol (10 mL * g 1), sonicated for 30 min, and incubated at room temperature. After 24 h, samples were centrifuged, filtered, and stored at 20 °C for further use. 4.3. Anti-diabetic activity The plant extracts were tested for anti-diabetic activity using cellular models of diabetes. The effects of W. somnifera extract on insulin secretion in pancreatic beta cells as well as glucose uptake in skeletal muscle and adipocytes were measured (Gorelick et al., 2011). Pancreatic RIN-5F (CRL-2058) cells were grown in RPMI media containing 10% fetal bovine serum (FBS) and 2 mM
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glutamine for measuring insulin secretion. RIN-5F cells were treated with increasing concentrations of extract for 1 h and subsequently incubated in Krebs–Ringer biocarbonate HEPES buffer (KRBH). After 1 h, the buffer was collected and replaced with KRBH buffer supplemented with 16.7 mM glucose and 10 lM forskolin to stimulate insulin secretion. After a 30 min incubation, the buffer was collected for analysis of insulin content using an insulin ELISA kit (Mercodia, Uppsala, Sweden). Glucose uptake was measured based on Yamada et al. (2000). L6 (CRL-1458) skeletal myoblasts were grown in Dulbecco’s Modified Eagle’s Medium (DMEM) containing 10% FBS. Differentiated myotubes were produced by replacing the FBS with 5% horse serum for 5 days. 3T3-LI (CL-173) fibroblasts were grown in DMEM containing 10% FCS. After cells approached confluency, they were grown in differentiation media containing 100 nM insulin, 500 nM dexamethasone, and 0.5 mM isobutylmethylxanthine. After 7–10 days, mature adipocytes were identified based on morphology and used for study. L6 or 3T3 cells were plated at a concentration of 105 cells * mL 1 on 96 well plates and differentiated. Differentiated L6 myotubes or 3T3 adipocytes were transferred to low glucose serum free media overnight before treatment with increasing concentrations of extract or insulin for 4 h. For withanolides assay, the 6 different purified withanolides (Natural Remedy, India) were each dissolved in methanol at a concentration of 1 mM and added to the cells for 4 h. For the withaferin assay, purified withaferin A (Sigma, St. Louis, USA) was dissolved in methanol and added to cells in increasing concentrations for 4 h. For elicited Withania assay, methanolic plant extracts were prepared from untreated or elicited plants as described in Section 4.2 and added at a concentration of 50 mg * mL 1 to cells for 4 h. After incubation, 200 lM of the fluorescent glucose analog 2-(N-(7-nitrobenz-2oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose (2-NBDG, Invitrogen, Carlsbad, USA) was added for 1 h. Cells were subsequently rinsed in cold PBS and fluorescence was measured with a fluorescence microplate reader (BMG Polarstar, Ortenberg, Germany). 4.4. Plant physiology 4.4.1. Membrane leakage Membrane leakage measurements, indicators of membrane injury under stress (Lu et al., 2008), were measured as previously described with minor modifications (Shoresh et al. 2011). Leaf segments were shaken vigorously in 30 mL of double-distilled water for 24 h and the electrical conductivity (EC) was measured using a conductivity meter (CyberScan CON 1500; Eutech Instruments, Ayer Rajan Crescent, Singapore). The tubes with the leaf pieces were then autoclaved and shaken again. Conductivity was measured again and the membrane leakage was calculated as the ratio between the first and the second conductivity measurements. Results from 5 replicated leaves were averaged. 4.4.2. Osmotic potential For osmotic potential measurements, the sampled leaf tissue was frozen in 1.5 mL micro-test-tubes in liquid nitrogen and stored at 5 °C for further analyses. The frozen tissue was crushed inside the tubes with a glass rod, the bottom of the tubes was pin-pricked and the tubes, set inside another 1.5 mL tube, centrifuged for 4 min in a refrigerated centrifuge (Sigma Laboratory Centrifuges, Germany) at 5 °C at 10,000 rpm. One hundred microliters of the fluids collected in the lower micro test tube were used for measurement of osmotic potential using a cryoscopic microosmometer (lOsmette; Precision Systems, Natick, MA, USA) by measuring the freezing point of 100 lL of sap. Results are presented in mOsm * kg H2O 1. Five replicates from each treatment were analyzed.
Please cite this article in press as: Gorelick, J., et al. Hypoglycemic activity of withanolides and elicitated Withania somnifera. Phytochemistry (2015), http:// dx.doi.org/10.1016/j.phytochem.2015.02.029
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4.5. Chemical composition 4.5.1. HPLC HPLC analysis was performed to determine withanolide content in treated plants (Chaurasiya et al., 2008). 70% aqueous methanol extracts prepared from dried ground plant material were sequentially partitioned with hexane to remove non-polar impurities and then chloroform to extract the withanolides. The chloroform phase was evaporated and resuspended in methanol. 10 lL was injected using a Waters LC module equipped with a multi-wavelength UV detector set to 227 nm. A Gemini C-18 column (Phenomenex, Torrance, USA) was used with an isocratic mobile phase consisting of water and methanol (65:35). Retention times were determined using standard withanolides (Natural Remedies, India). For quantification, a standard curve was generated. For verification of withanolides, 10 lL of each sample was injected into a Thermo TSQ Quantum Access Max Triple Stage Quadrupole Mass Spectrometer using positive Electrospray Ionization. Identification of the molecular ion (M + H) of withaferin A as well as other characteristic ions was confirmed. 4.5.2. GC–MS For GC–MS analysis, 1 lL of each sample was injected into a Hewlett–Packard G1800A GCD system fitted with a 5% phenylmethylsiloxane (HP-5MS) capillary column (30 m 0.25 mm i.d. 0.25 mm), splitless injection with helium 1 mL * min 1 and a solvent delay of 4.8 min. The temperature of the detector was 280 °C and the injector was 250 °C. The column temperature was held for 4 min at 50 °C and then increased 8 °C * min 1 until 280 °C. 4.6. Statistical analysis Results are expressed as means ± standard errors. Statistical analysis was performed using JMP 5 software (SAS Institute Inc., 2002, Cary, NC, USA). Data were subjected to one-way ANOVA analysis (a < 0.05) and Tukey honestly significant difference for comparison of means. Acknowledgments This work has been carried out with support from the Israeli Ministry of Science, Technology, and Space as well as the Israeli Ministry of Agriculture project no. 3-8320. References Ament, K., Krasikov, V., Allmann, S., Rep, M., Takken, F.L., Schuurink, R.C., 2010. Methyl salicylate production in tomato affects biotic interactions. Plant J. Cell Mol. Biol. 62, 124–134. Andallu, B., Radhika, B., 2000. Hypoglycemic, diuretic and hypocholesterolemic effect of winter cherry (Withania somnifera, Dunal) root. Indian J. Exp. Biol. 38, 607–609. Anwer, T., Sharma, M., Pillai, K.K., Iqbal, M., 2008. Effect of Withania somnifera on insulin sensitivity in non-insulin-dependent diabetes mellitus rats. Basic Clin. Pharmacol. Toxicol. 102, 498–503. Beg, M., Chauhan, P., Varshney, S., Shankar, K., Rajan, S., Saini, D., Srivastava, M.N., Yadav, P.P., Gaikwad, A.N., 2014. A withanolide coagulin-L inhibits adipogenesis modulating Wnt/beta-catenin pathway and cell cycle in mitotic clonal expansion. Phytomed. Int. J. Phytother. Phytopharmacol. 21, 406–414. Bernstein, N., Silk, W.K., Läuchli, A., 1995. Growth and development of sorghum leaves under conditions of NaCl stress: possible role of some mineral elements in growth inhibition. Planta 196, 699–705. Bernstein, N., Shoresh, M., Xu, Y., Huang, B., 2010. Involvement of the plant antioxidative response in the differential growth sensitivity to salinity of leaves vs. roots during cell development. Free Radic. Biol. Med. 49, 1161–1171. Chaurasiya, N.D., Uniyal, G.C., Lal, P., Misra, L., Sangwan, N.S., Tuli, R., Sangwan, R.S., 2008. Analysis of withanolides in root and leaf of Withania somnifera by HPLC with photodiode array and evaporative light scattering detection. Phytochem. Anal. 19, 148–154. Chen, L.X., He, H., Qiu, F., 2011. Natural withanolides: an overview. Nat. Prod. Rep. 28, 705–740.
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