Prunella vulgaris L. active components and their hypoglycemic and antinociceptive effects in alloxan-induced diabetic mice

Biomedicine & Pharmacotherapy 84 (2016) 1008–1018

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Original article

Prunella vulgaris L. active components and their hypoglycemic and antinociceptive effects in alloxan-induced diabetic mice K. Raafata,* , M. Wurglicsb , M. Schubert-Zsilaveczb a b

Department of Pharmaceutical Sciences, Faculty of Pharmacy, Beirut Arab University, 115020 Beirut, Lebanon Institute of Pharmaceutical Chemistry, Johann Wolfgang Goethe University Frankfurt, Marie-Curie-Straße 9, 60438 Frankfurt/Main, Germany

A R T I C L E I N F O

Article history: Received 30 July 2016 Received in revised form 6 September 2016 Accepted 24 September 2016 Keywords: Prunella vulgaris Diabetes mellitus Painful diabetic neuropathy HbA1c In vivo antioxidant Alpha-glucosidase Von frey filaments

A B S T R A C T

Prunella vulgaris L. (Lamiaceae) (PV) is a herbaceous plant traditionally utilized in management of diabetes and it has immunomodulatory activity. In this study, acute and subchronic antidiabetic, in-vivo antioxidant and antinociceptive effects of PV were evaluated in alloxan-induced type 1 diabetes (T1D) in a mouse model. Bio-guided fractionation, isolation, RP-HPLC, and 1H and 13C NMR identification of the active components responsible for PV effects were determined. RP-HPLC analysis showed that PV contained rosmarinic acid (RA) 4.5%, caffeic acid (CA) 9.8% and p-coumaric acid (pCA) 11.6%. Bio-guided fractionation showed that PV most active fraction was rich in caffeic acid, hence named, caffeic acid-rich fraction (CARF). RP-HPLC, and 1H and 13C NMR experiments showed that CARF contained CA (93.4%) and RA (6.6%). CARF reduced blood glucose levels and improved in-vivo oxidative-stress. It also inhibited the carbohydrate-hydrolyzing enzymes (alpha-amylase and alpha-glucosidase) and reduced HbA1c levels more significantly (p  0.05) than that of PV and equivalent amounts of CA or RA. For longer times, CARF had significantly (p  0.05) increased serum-insulin, ameliorated thermal hyperalgesia and tactile allodynia more significantly (p  0.05) than the effects of PV and equivalent amounts of CA or RA. Moreover, the tested compounds showed potential restoration of the lipid peroxide levels. Consequently, CARF and PV observed increase in serum-insulin, attenuation of alpha-amylase and alpha-glucosidase, and their antioxidant potentials might be responsible for their antidiabetogenic and antinociceptive properties. In conclusion, CARF isolated from PV could be a potential therapeutic agent to ameliorate T1D and related complications. ã 2016 Elsevier Masson SAS. All rights reserved.

1. Introduction Type 1 Diabetes mellitus (T1D) prevalence is rising worldwide, and has become a significant public health problem in all ages, with a high global prevalence of 10% [1,2]. Hyperglycemia and glycosylated hemoglobin (HbA1c) are widely used markers in diagnosing diabetes [3]. The proper management of diabetes includes blood glucose and HbA1c homeostasis, and improving antioxidant defenses by minimizing free radical adverse effects [4]. Primarily, the indigenous hemoprotein antioxidant enzyme, catalase (CAT), directly scavenges the reactive oxygen species (ROS), and catalyzes the lessening and detoxification of hydrogen peroxides [5–7]. As a result of non-enzymatic glycosylation and

oxidation, CAT has been found to be inhibited in diabetes [8]. Currently, oxidative stress associated with hyperglycemia is recognized as the driving force for the development of diabetic complications [9]. Therefore, natural herbs, and their isolates with potential antioxidant properties might be helpful in the management of T1D and its complications [10–14]. Diabetic somatic neuropathy (DN) is a chronic complication of diabetes which may provoke major conditions like foot amputation. The prevalence of DN was found to be 30.5% in men and 30.8% in women identified by clinical assessment [15]. DN is characterized by severe hyperalgesia and allodynia, which could be painful and disabling [16]. Nevertheless, as the mechanism of painful DN is incompletely understood, its conventional treatment remains

Abbreviations: DM, type 1 diabetes mellitus; DN, diabetic somatic neuropathy; CARF, caffeic acid rich fraction; ppm, parts per million; IP, intraperitoneal; RP, reversed phase; DMSO, dimethyl sulfoxide; LD50, lethality towards 50% of a population; ca., approximately; PV or P., vulgarisPrunella vulgaris; ROS, reactive oxygen species; CAT, catalase; BGL, blood glucose level; LPO, lipid peroxidation; TBRAS, thiobarbituric acid reactive substances. * Corresponding author. E-mail addresses: [email protected], [email protected] (K. Raafat). http://dx.doi.org/10.1016/j.biopha.2016.09.095 0753-3322/ã 2016 Elsevier Masson SAS. All rights reserved.

K. Raafat et al. / Biomedicine & Pharmacotherapy 84 (2016) 1008–1018

unsatisfactory [17]. Therefore, it is necessary to look for effective alternative drugs, especially with fewer side effects, for the management of painful DN. Self-heal or Prunella vulgaris L. (Lamiaceae) (P. vulgaris) is a perennial flowery herb normally cultivated in Asia and Europe. P. vulgaris inflorescence (spikes) has been used in the Asian folk medicine in blood glucose homeostasis and in the management of upper respiratory tract infections [18]. On the other hand, there are some evidence-based uses for P. vulgaris including immune modulation and antioxidant potential [19,20]. P. vulgaris is rich in triterpenoids. It comprises also many neuroactive phenolic compounds like p-coumaric acid, rosmarinic acid, caffeic acid, rutin and quercetin [18,21–23]. Furthermore, there are a number of attempts to understand the acute antihyperglycemic potentials of P. vulgaris and its triterpenoidal isolates [24–27]. Nevertheless, the literature survey showed that there are neither in-depth studies about the bio-guided fractionation of the most active compounds of P. vulgaris responsible for its hypoglycemic potential nor the subchronic effects of P. vulgaris use. Additionally, none of the researches intensely studied P. vulgaris effects on diabetes induced glycosylated hemoglobin, tactile allodynia and hyperalgesia. Moreover, alpha-amylase is known to degrade the dietary complex carbohydrates to di-saccharides and oligosaccharides, then alpha-glucosidase converts them into monosaccharides [28,29]. Consequently, the inhibition of these enzymes involved in the metabolism of carbohydrates and elevation of serum insulin levels are among the possible mechanisms for reducing hyperglycemia [30]. Therefore, the present work aims at assessing P. vulgaris utilizing bio-guided fractionation, isolation, RP-HPLC, and 1H and 13 C NMR identification of its active components. Moreover, this work also aims at investigating the acute and subchronic hypoglycemic activities of P. vulgaris in alloxan-provoked diabetic mice and the possible mechanism of glycemic homeostasis. The effect of P. vulgaris on glycosylated hemoglobin, T1D-inducedoxidative stress, mechanical allodynia and painful hyperalgesia were also assessed in this study. 2. Materials and methods 2.1. Preparation of extract Dried inflorescence (spikes) of P. vulgaris was a gift from Prof. M. Aboul-Ela (BAU, Lebanon), and authenticated by Prof. J. Habib (LU, Lebanon). After authentication with a reference sample, the spikes were given a voucher specimen number (PS-14-11) and were carefully stored in the Faculty herbarium. Two hundred grams of dried P. vulgaris spikes were sonicated for 1 h with 80% ethanol, filtered and concentrated in a rotating vacuum evaporator at 40  C with a yield of 22.58 g (yield ca. 11%, w/w) and stored at 40  C. 2.2. Standardization, bio-guided fractionation and identification P. vulgaris extract was fractionated using column chromatography (CC). A preparative CC, 50 mm diameter and 100 cm height, was used. Elution was done using reversed phase silica gel CC and developed in increasing polarity with EtAc-MeOH (100:0, 95:5, 90:10, 80:20, 70:30, 50:50,30:70, 20:80, 10:90, 0:100 V/V). During the whole chromatographic process the eluent was collected in a series of more than 200 fractions by time. Each fraction was tested the same way as the whole extract using in vivo alloxan-diabetic mice. The most active fraction [caffeic acid rich fraction (CARF), yield, ca. 9% w/w] was analyzed using1H NMR, 13C NMR and RPHPLC. All commercial solvents and chemicals were of analytical grade. The1H NMR and 13C NMR spectra were measured in MeOH-

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d4 on a Bruker ARX 300 spectrometer (Bruker, Germany) of the Institute of Pharmaceutical Chemistry, Goethe-University, Frankfurt, Germany. Chemical shifts are measured in ppm using an internal standard, tetra-methylsilane (TMS) [31]. HPLC conditions were optimized using various columns with various stationary phases, wavelengths (200 nm- 400 nm) and solvent systems. The best chromatographic standardization for P. vulgaris and CARF were attained using RP-C18 endcapped Lichrospher column (250  4.6 mm I.D.; 5 mM particle size) (Merck, Germany), at 40  C and 1% formic acid in double distilled water (solvent A) and ACN (solvent B) as a mobile phase. Gradient elution conditions were 0–10 min 13% B, 10–20 min 41.5% B, 20–25 min 70% B, 25–35 min 10% B [32] at 280 nm and the flow rate was adjusted to 1 mL/min. 2.3. Animals and study design One week prior to the experimentation, male Swiss-Webster mice (22–30 g, aged 12–16 weeks) were housed (Faculty of Pharmacy, Beirut Arab University) with a free access to water and standard laboratory pellets (20% proteins, 5% fats, and 1% multivitamins) and 12-h light/dark cycle [23,33]. An adaptation period of one week, prior to the beginning of the experiment, was applied. Mice were fasted 16 h prior to the experiment, but had free water access. The experimental protocol (Table 1) was accomplished in accordance with the Ministry of Higher Education and legislation of animal experiments and with the approval (2015A008-P-R-0021) of BAU Institutional Review Board. Freshly prepared alloxan (Sigma-Aldrich, Germany) was dissolved in a vehicle [sterile cold saline (0.9%)] and IP injected in mice every 48-h for 3 times at a dose of 180 mg/kg to induce T1D. Seventy two hours after the last alloxan injection, blood glucose levels (BGL) and HbA1c were recorded for each mouse utilizing Accu-chek Active TM Test Meter and Accu-chek Active TM glucose strips (Roche, USA), and HbA1c micro column method (Analyticon, Germany), respectively. The mice with BGL  200 mg/dl and HbA1c > 8% were considered to be diabetic and were used in the experiment.BGL were then measured for each tested mouse, both acutely (6 h) and subchronically (8 days) as described in (Table 1). Also, HbA1c levels were tested at pre-dose and 8 weeks post-dose [12,34].On the day of the experiment, all test solutions were freshly prepared [35] and bacterially sterilized using UV and optimal in line bacterial filtration (ZHENFU) [13]. All doses were initially chosen equivalent to the amount of substance in P. vulgaris. This was followed by a pilot study, of ca. 10 fold adjustment in doses, to obtain the best doses compared to the original standardized extract. The adjusted doses, mentioned in Table 1, were applied in all experiments [36]. 2.4. Alpha-Glucosidase inhibition assay The inhibitory effects of PV, CARF, CA and RA on alphaglucosidase inhibitory activity were determined according to the method described before [28]. Briefly, PV (50, 100 and 150 mg/ml), CARF 7 mg/ml, CA (2.5, 5 and 10 mg/ml) or RA (5, 10, 20 mg/ml), and 100 mL of alpha-glucosidase (EC 3.2.1.20) solution (1 U/mL) was incubated at 25  C with (0.1 M) phosphate buffer for 10 min. After that, 50 mL of p-nitrophenyl-alpha-D-glucopyranoside solution (5 mM) in phosphate buffer (0.1 M) was added. Then, the mixtures have been incubated for 5 min at 25  C and absorbance were read at 405 nm in a JASCO spectrophotometer (JASCO, Japan). The inhibitory activity towards alpha-glucosidase was measured as (%) inhibition [28]. The inhibition percentage has been calculated utilizing the following equation: Inhibition (%) = [(Acontrol

Asample)/Acontrol]  100.

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Table 1 Protocol of the experimental design. Groups

n

Tested Substance(s)

Description

A. Acute (0, 0.5, 2 and 6 h) and subchronic (1, 3, 5, 8 days) effect of Prunella vulgaris ethanolic extract (P. vulgaris), caffeic acid rich fraction (CARF), rosmarinic acid (RA), caffeic acid (CA) and p-coumaric acid (pCA) and on blood glucose, body weights and CAT serum levels: I 7 Control Normal mice: Vehicle [sterile cold saline (0.9%)], IP II 7 Diabetic Control Diabetic mice: Vehicle, IP III 7 GB Diabetic mice: GB 5 mg/kg, IP 7 P. vulgaris Diabetic mice: P. vulgaris 50 mg/kg, IP IV V 7 P. vulgaris Diabetic mice: P. vulgaris 100 mg/kg, IP VI 7 P. vulgaris Diabetic mice: P. vulgaris 150 mg/kg, IP 7 CARF Diabetic mice: CARF 7 mg/kg, IP VII VIII 7 RA Diabetic mice: RA 5 mg/kg, IP IX 7 RA Diabetic mice: RA 10 mg/kg, IP X 7 RA Diabetic mice: RA 20 mg/kg, IP XI 7 CA Diabetic mice: CA 2.5 mg/kg, IP XII 7 CA Diabetic mice: CA 5 mg/kg, IP XIII 7 CA Diabetic mice: CA 10 mg/kg, IP XIV 7 pCA Diabetic mice: pCA 3 mg/kg, IP XV 7 pCA Diabetic mice: pCA 6 mg/kg, IP XVI 7 pCA Diabetic mice: pCA 12 mg/kg, IP B. Effect of P. vulgaris, CARF, RA, CA and pCA for longer times (0, 2, 4, 6 and 8 weeks) on hot plate and tail withdrawal latencies, and von Frey paw withdrawal thresholds: XVII 7 NORM Normal mice: Vehicle [sterile cold saline (0.9%)], IP XVIII 7 VEH Diabetic mice: Vehicle, IP XIX 7 P. vulgaris Diabetic mice: P. vulgaris 50 mg/kg, IP XX 7 P. vulgaris Diabetic mice: P. vulgaris 100 mg/kg, IP XXI 7 P. vulgaris Diabetic mice: P. vulgaris 150 mg/kg, IP XXII 7 CARF Diabetic mice: CARF 7 mg/kg, IP XXIII 7 RA Diabetic mice: RA 5 mg/kg, IP 7 RA Diabetic mice: RA 10 mg/kg, IP XXIV XXV 7 RA Diabetic mice: RA 20 mg/kg, IP XXVI 7 CA Diabetic mice: CA 2.5 mg/kg, IP XXVII 7 CA Diabetic mice: CA 5 mg/kg, IP XXVIII 7 CA Diabetic mice: CA 10 mg/kg, IP XXIX 7 pCA Diabetic mice: pCA 3 mg/kg, IP XXX 7 pCA Diabetic mice: pCA 6 mg/kg, IP XXXI 7 pCA Diabetic mice: pCA 12 mg/kg, IP

2.5. Alpha-Amylase inhibitory assay

2.7. Diabetic neuropathy management

PV (50, 100 and 150 mg/ml), CARF 7 mg/ml, CA (2.5, 5 and 10 mg/ ml) or RA (5, 10, 20 mg/ml) was mixed with alpha-amylase enzyme (EC3.2.11, 10 units/mL) (1%) and held for10 min incubation. One percent soluble starch (pH7.4) was added to the mixture and incubated at 25  C for 10 min. To stop the reaction, 1 mL dinitrosalicylic acid (1%) was added and boiled at 90  C for 15 min in a water-bath then cooled [29]. After that, the mixture was diluted with double-distilled water (1 mL) and the absorbance of the fractions/extract and control mixtures were measured at 540 nm at 20  C using a JASCO Spectrophotometer [29]. The inhibition percentage has been calculated utilizing the following equation:

After 8 weeks of inducing T1D in the animals, thermal hyperalgesia and tactile allodynia were measured at one week intervals for 8 weeks, using:

Inhibition (%) = [(Acontrol

Asample)/Acontrol]  100.

2.6. P. vulgaris, CARF, CA and RA effects on serum insulin Serum insulin was measured before and 8 weeks after P. vulgaris (150 mg/kg), CARF (7 mg/kg), CA (10 mg/kg) and RA (20 mg/kg) administration, utilizing an HPLC-RP method reported before [37]. Briefly, HPLC (Agilent, Japan) equipped with RP-C18 endcapped Lichrospher column (250  4.6 mm I.D.; 5 mM particle size) (Merck, Germany), at 40  C and ACN (solvent A) and 0.1% trifluoroacetic acid in double distilled water (solvent B) as a mobile phase. Gradient elution conditions used were 0–5 min 70% B, 5–15 min 60% B at 214 nm and the rate of flow was adjusted to 1 mL/min [37].

2.7.1. Hot plate latency test The evaluation of thermal hyperalgesia management utilizing hot plate latency test, were assessed using the method described before [14]. Briefly, mice were located on the hot plate apparatus (UgoBasile, Italy) with accustomed temperature of 55  0.1  C (Table 1B). Time to first hind paw withdrawal or licking was set as an index of thermal nociceptive threshold, with 30 s cutoff time to avoid thermal paw damage. 2.7.2. Tail flick latency test Adjunct to hot plate test, amelioration assessment of thermal hyperalgesia were measured in mice using the method described before [38]. Concisely, mice were placed in a special cage of the tail-flick apparatus (Hugo Sachs Elektronik, Germany) and the time from the radiant heat onset to the withdrawal of the tail, was recorded using an electronic timer (Table 1B) with a 10 s cut-off time to prevent tail tissue damage. 2.7.3. Von frey filaments test By measuring the withdrawal response to mechanical stimuli with von Frey filaments (OptiHair TM, Marstock Nervtest TM, Germany) of different intensities (0.5 to 45.3 g), the level of

K. Raafat et al. / Biomedicine & Pharmacotherapy 84 (2016) 1008–1018

mechanical allodynia was measured. Mice were kept in an elevated inverted cage with a wire mesh floor and allowed to acclimatize for 10 min [39]. Afterwards, von Frey filaments were used in the method described before [11,12,39,40]. Briefly, flinching or sharply withdrawn paw immediately after filament removal was regarded as a positive response. By observing the mouse hind limb flinching before dosing (predose) and subsequently up to eight weeks following drug or vehicle administration, tactile allodynia withdrawal thresholds were measured (Table 1B). 2.8. Measurement of lipid peroxidation The effect of PV (50, 100 or 150 mg/kg), CARF 7 mg/ml, CA (2.5, 5 or 10 mg/kg) or RA (5, 10 or 20 mg/kg) on Lipid peroxidation (LPO), as demonstrated by thiobarbituric acid reactive substances (TBARS) formation, and measured by thiobarbituric acid test; modified from a method described before [41]. Briefly, on the 8 day post-administration, 0.2mlserumhas been added to thiobarbituric

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acid (0.8%), sodium dodecyl sulphate (8.1%) and acetic acid (20%) in double-distilled water. After that the mixture was heated for 1 h at 95  C in a water-bath, and cooled. The mixture was then extracted with (15:1, v/v) isopropyl alcohol/methanol and the absorbance of the product was measured at 532 nm by JASCO spectrophotometer [41]. 2.9. Estimation of in vivo antioxidant activity Subchronically, serum catalase (CAT) levels (kU/l) were measured utilizing the modified method explained in the literature before [36] (Table 1B). 2.10. Statistical analysis One-way analysis of variance followed by the Student– Newman–Keuls test were used in this study to statistically evaluate the results utilizing “OriginPro” statistic program. Data

Fig. 1. HPLC and 1H NMR analysis of caffeic acid (CA) standard and the most active fraction of P. vulgaris, caffeic acid rich fraction (CARF) (A) HPLC chromatogram utilizing RPC18 column and gradient mobile phase and flow rate 1.0 mL/min at 280 nm. Upper panel: (1) Standard CA (200 mg/ml). Lower panel: CARF; (1) CA (ca. 93.4%) (2) rosmarinic acid (RA) (ca. 6.6%) (B) 1H NMR spectrum. Upper panel: Standard CA. Lower panel: CARF; major peaks: CA (ca. 95%) and minor peaks: RA (ca. 5%).

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are explicated as means  S.E.M in each group. Statistical significance was taken at p  0.05.

Consequently, it is suggested that CARF is the most effective acute therapy in diabetic animals.

3. Results

3.3. Subchronic P. vulgaris, CARF, CA, RA and pCA effects on BGL, body weight, in vivo antioxidant and HbA1c activities

3.1. Standardization, bio-guided fractionation and identification The standardized P. vulgaris powder mainly contained caffeic acid (CA) (9.8%), rosmarinic acid (RA) (4.5%) and p-coumaric acid (pCA) (11.6%) using RP-HPLC UV method. Following bio-guided fractionation, the most active fraction of P. vulgaris extract was identified using RP-HPLC apparatus and NMR spectrometer. The major peak of the most active fraction was identified as CA, utilizing standard CA RP-HPLC calibration curves and hence this fraction was named “caffeic acid rich fraction” (CARF). RP-HPLC showed that CARF contained CA (ca. 93.4%) and RA (ca. 6.6%) compared to CA and RA standards (Fig. 1A). 1H NMR and 13C NMR analysis demonstrated that CARF contained CA (ca. 95%) and RA (ca. 5%) compared to CA and RA standards (Fig. 1B). 3.2. Acute effects of P. vulgaris, CARF, CA, RA and pCA on blood glucose A range of doses of P. vulgaris, CARF, CA, RA and pCA in alloxandiabetic mice were acutely studied (Table 2). Glibenclamide (GB) 5 mg/kg was used as a positive control. After 6 h of extract administration, P. vulgaris ethanolic extract (50, 100 and 150 mg/ kg) significantly reduced BGL by 24.7, 26.6 and 49.1% compared to control, respectively. The most active fraction, CARF (7 mg/kg) significantly reduced BGL by49.6% 6 h following CARF administration compared to the control group. Moreover, the active substance doses were chosen equivalent to their amounts in P. vulgaris and were tested the same way as the original extract. The CA (2.5, 5 and 10 mg/kg) showed a significant effect with BGL declining by 40.9, 42.9 and 44.5% compared to the control group after 6 h of CA administration, respectively. The RA (5, 10 and 20 mg/kg) showed a significant effect with BGL declining by 36.9, 38.0 and 42.9% compared to the control group after 6 h of RA administration, respectively. The pCA (3, 6 and 12 mg/kg) showed a significant effect with BGL declining by 27.6, 33.1 and 41.9% 6 h following pCA administration compared to control, respectively. The GB (positive control) prevented the increase of BGL 6 h (declining by 39.2%) following GB administration compared to the control group (Table 2).

Throughout 8 days, the subchronic effects of P. vulgaris, CARF, CA, RA and pCA were studied and GB (5 mg/kg) utilized as a positive control. After the administration of the test samples, the BGL of each mouse was observed on the 1st, 3rd, 5th and 8th day. The BGL of the diabetic control group were significantly higher than those of the control mice throughout the experimental period (Table 3). The highest decline in BGL was demonstrated with CARF (7 mg/kg). On the 8th day, CARF was more potent by 1.05, 1.09, 1.08, 1.44 and 2.72 folds than that of the highest doses of P. vulgaris (150 mg/kg), CA (10 mg/kg), RA (20 mg/kg), pCA (12 mg/kg) and GB (5 mg/kg), respectively (Table 3). Mice given P. vulgaris, CARF, CA, RA, pCA, and GB were also observed throughout the subchronic administration for changes in body weight (Table 4). On the 8th day, the test solutions showed 14.7, 15.1, 17.3, 2.7 and 13.7% increase in body weight with the highest doses of P. vulgaris (150 mg/kg), CARF (7 mg/kg), CA (10 mg/ kg), RA (20 mg/kg), pCA (12 mg/kg) and GB (5 mg/kg) compared to normal control mice, respectively (Table 4). In agreement with the measured acute and subchronic decline in BGL, significant decline in HbA1c levels has been observed in P. vulgaris (150 mg/kg), CARF (7 mg/kg), CA (10 mg/kg) and RA (20 mg/kg) (Fig. 2). Eight weeks post-administration, the decline in HbA1c was11.4, 19.3, 14.8 and 9.1% in P. vulgaris (PV) (150 mg/kg), CARF (7 mg/kg), CA (10 mg/kg) and RA (20 mg/kg) respectively, in comparison to vehicle control animals (Fig. 2). Acutely and subchronically, the antihyperglycemic, body weight improvement, in-vivo antioxidant actions and HbA1c levels of CARF are more potent and extended than those of other substances under investigation. 3.4. P. vulgaris, CARF, CA and RA effects on alpha-glucosidase and alpha-amylase The ability of PV (50, 100 or 150 mg/ml), CARF 7 mg/ml, CA (2.5, 5 or 10 mg/ml) or RA (5, 10 or 20 mg/ml) to inhibit alpha-glucosidase and alpha-amylase activities in vitro were investigated (Figs. 3 and 4).The results have shown that PV (50, 100 or 150 mg/ml), CARF

Table 2 Acute effect of Prunella vulgaris ethanolic extract (P. vulgaris), caffeic acid rich fraction (CARF), rosmarinic acid (RA), caffeic acid (CA) and p-coumaric acid (pCA) on blood glucose. Group

Control Diabetic control GB P. vulgaris P. vulgaris P. vulgaris CARF CA CA CA RA RA RA pCA pCA pCA

Dose (mg/kg)

– – 5 50 100 150 7 2.5 5 10 5 10 20 3 6 12

S.E.M.: mean standard error. * p < 0.05 significant from the control animals. ** p < 0.01 significant from the control animals.

Mean blood glucose concentration  S.E.M. (mg/dL) 0h

0.5 h

2h

6h

102.90  1.90 205.79  5.60 219.70  3.70 203.51  3.40 209.33  3.70 200.20  3.10 209.90  2.80 201.54  2.10 209.90  2.70 203.34  2.20 204.54  1.60 209.90 1.90 216.34  2.20 200.54  2.60 201.90  2.60 202.54  2.60

107.20  3.60 214.63  4.50 222.64  1.80 172.45  2.30 200.78  2.90 180.67  1.90 177.83  1.90 141.45  2.90 163.36  2.10 154.26  2.60 107.26  1.90 140.56  1.80 142.65  1.40 190.91  2.10 211.83  1.90 187.91  2.10

105.23  4.10 217.91  9.70 158.74  2.10 186.87  3.50 181.87  2.90 144.87  2.30 126.81  1.80 129.67  2.40 127.33  2.40 126.55  2.30 87.84  1.80 140.26  1.50 103.58  1.90 180.45  1.40 175.67  1.80 152.81  1.40

108.35  3.70 213.25  7.30 129.60  2.40** 160.59  2.40* 156.59  2.30* 108.59  2.70* 107.58  1.30* 126.12  1.50* 121.76  1.70* 118.44  1.90* 134.54  1.10* 132.26  1.60* 121.87  1.80* 154.35  1.90* 142.65  1.70* 124.00  1.90*

K. Raafat et al. / Biomedicine & Pharmacotherapy 84 (2016) 1008–1018

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Table 3 Subchronic effect of P. vulgaris, CARF, RA, CA and pCA on blood glucose. Group

Dose (mg/kg)

Mean blood glucose concentration  S.E.M. (mg/dL) 1 st day

3rd day

5th day

8th day

Control Diabetic controla GBb P. vulgarisa P. vulgarisa P. vulgarisa CARFa CAa CAa CAa RAa RAa RAa pCAa pCAa pCAa

– – 5 50 100 150 7 2.5 5 10 5 10 20 3 6 12

106.00  2.50 201.79  5.60*** 184.20  3.70 160.39  1.80 161.43  2.70 108.45  2.10 107.58  1.30 136.12  1.50 131.76  1.70 128.44  1.90 122.54  1.10 129.26  1.60 122.87  1.80 110.35  1.90 142.65  1.70 114.00  1.90

108.20  3.60 210.93  4.50*** 177.03  2.90 150.67  2.30 150.69  2.10 110.88  2.70 105.89  3.10 139.76  2.10 129.58  2.40 127.26  2.20 112.59  1.70 118.51  1.90 125.46  1.70 113.88  3.10 142.56  1.80 135.69  3.50

107.10  3.20 214.21  9.70*** 159.04  2.40** 144.33  2.30 141.45  1.90 108.73  2.40 104.32  2.50 119.76  2.10 125.76  2.10 118.26  2.10 107.66  2.20 114.35  1.90 105.54  1.80 131.48  3.60 118.66  1.90 134.98  3.60

115.50  4.70 209.50  7.30*** 169.80  3.10 139.63  2.40* 128.33  2.10* 106.34  2.90* 101.13  1.30* 117.76  2.50* 113.55  2.30* 108.98  2.10* 125.23  2.30* 112.56  1.10* 110.46  2.20* 136.55  2.40* 136.42  1.60* 134.33  2.30*

S.E.M.: mean standard error. * p < 0.05 significant from the control animals. ** p < 0.01 significant from the control animals. *** p < 0.001 significant from the control animals. a Compared to vehicle control. b Compared to diabetic control.

Table 4 Subchronic effect of P. vulgaris, CARF, RA, CA and pCA on body weights in alloxan-induced diabetic mice. Group

Dose (mg/kg)

Mean body weight  S.E.M. (gm) (% increase from the vehicle control) 1st day

3rd day

5th day

8th day

–

24.40  0.50

24.50  0.60

24.66  0.97

24.72  0.70

–

25.18  0.70

5

23.40  0.70

P. vulgarisa

50

28.00  1.80

P. vulgarisa

100

27.46  2.70

a

150

26.46  2.10

CARF

7

27.55  0.40

CAa

2.5

27.56  0.80

CAa

5

27.10  0.40

10

26.50  0.20

RAa

5

25.00  0.50

RAa

10

25.10  0.40

25.60  0.20 (4.2%) 28.67  1.70 (8.1%) 28.10  1.30 (0.4%) 28.30  1.10 (3.0%) 28.88  1.70 (9.1%) 28.85  0.80 (4.7%) 28.66  0.30 (4.0%) 28.50  0.40 (5.2%) 27.90  0.70 (5.3%) 26.10  0.50 (4.4%) 26.00  0.90 (3.6%) 28.50  0.60 (12.2%) 24.30  1.30 (0.4%) 24.80  0.60 (1.0%) 24.90  1.30 (1.6%)

25.65  0.80 (3.7%) 29.04  0.40 (8.8%) 28.50  1.50 (1.8%) 28.90  1.30 (5.2%) 29.77  1.40 (12.5%) 30.61  1.20 (11.1%) 28.99  0.90 (5.2%) 29.50  0.50 (8.9%) 30.05  0.30 (13.4%) 26.50  0.40 (6.0%) 26.20  0.70 (4.4%) 28.50  0.80 (12.2%) 24.40  1.10 (0.8%) 24.85  1.00 (1.2%) 24.90  1.60 (1.6%)

25.00  0.50 ( 0.4%) 30.87  1.10* (13.7%) 28.63  1.40* (2.25%) 29.20  1.10* (6.3%) 30.34  1.90* (14.7%) 31.71  1.40* (15.1%) 30.05  0.40* (9.0%) 31.05  0.80* (14.6%) 31.09  0.20* (17.3%) 27.50  0.50* (10.0%) 28.20  0.40* (12.4%) 29.70  0.60* (16.9%) 24.55  1.20* (1.4%) 25.10  1.60* (2.2%) 25.15  1.40* (2.7%)

Control a

Diabetic control GB

a

P. vulgaris a

CA

a

a

20

25.40  0.30

a

3

24.20  1.20

pCAa

6

24.56  1.10

pCAa

12

24.50  1.20

RA

pCA

S.E.M.: mean standard error. * p < 0.05 significant from the control animals. a Compared to vehicle control.

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7 mg/ml, CA (2.5, 5 or 10 mg/ml) or RA (5, 10 or 20 mg/ml) had significantly inhibited alpha-glucosidase and alpha-amylase activities. By comparing the inhibitory effects, CA had the highest inhibitory effect on alpha-glucosidase ca. (IC50 = 4.7 mg/ml) and alpha-amylase ca. (IC50 = 5.1 mg/ml), apparently more than that of CARF, and greater than that on RA alpha-glucosidase ca. (IC50 = 11.6 mg/ml) and alpha-amylase ca. (IC50 = 21.7 mg/ml), and that of PV on alpha-glucosidase ca. (IC50 = 90.9 mg/ml) and alpha-amylase ca. (IC50 = 47.2 mg/ml). 3.5. P. vulgaris, CARF, CA and RA effects on serum insulin

Fig. 2. Effects of P. vulgaris ethanolic extract (PV), caffeic acid rich fraction (CARF), caffeic acid (CA) and rosmarinic acid (RA) on HbA1c at pre-dose and 8 weeks postdose. Values represent the mean  SEM (n = 7). (NORM) normal non-diabetic untreated mice. * p < 0.05 vs. diabetic vehicle control (VEH).

In order to identify the mechanism of P. vulgaris, CARF, CA and RA hypoglycemic activity, serum insulin was monitored using RPHPLC analysis, before and 8 weeks post-administration. The highest concentration of P. vulgaris, CARF, CA and RA had shown significant increase in serum insulin levels when compared to vehicle treated groups, 8 weeks post-administration (Fig. 5). Eight weeks post-administration, the test compounds had shown10.7, 12.3, 7.3 and 1.9 folds increase in serum insulin in P. vulgaris (PV) (150 mg/kg), CARF (7 mg/kg), CA (10 mg/kg) and RA (20 mg/kg) respectively, in comparison to vehicle control animals (Fig. 5).

Alpha-Glucosidase Inhibition (%)

3.6. P. vulgaris, CARF, CA and RA ameliorated neurological function

80

c c

70

d

60

b

40

b

b

50

f a

a

30 e

20 10 5

1 RA 0 20

RA

RA

7

2. 5 CA 5 CA 10

CA

RF CA

PV 50 PV 10 PV 0 15 0

0

The effects of P. vulgaris (PV),CARF, CA and RA on nerve function were examined by measuring the thermal latency with the hot plate and tail flick tests using tramadol (TRA) 10 mg/kg as a positive control(Figs. 6 and 7). Moreover, mechanical allodynia was also tested using von Frey filaments latency test (Fig. 8). Management of the alloxan-treated diabetic mice with PV, CARF, CA and RA markedly improved the thermal latency (Figs. 6 and 7). After 8 weeks of alloxination, diabetic animals demonstrated a transitory hyperalgesic sensation in thermal tests. Upon treatment with highest doses of PV (150 mg/kg), CARF (7 mg/kg), CA (10 mg/kg) and RA (20 mg/kg), hot-plate latency markedly improved by 2.00, 2.04, 1.92 and 1.88 folds respectively, in the 8th week, in comparison to the vehicle treated animals (Figs. 6 A and 7 A).

Concentration µg/mL Fig. 3. Alpha-glucosidase inhibitory effect of P. vulgaris ethanolic extract (PV), caffeic acid rich fraction (CARF), caffeic acid (CA) and rosmarinic acid (RA). Values are shown as mean  S.E.M. (n = 3). Values with different letters are significantly different (p  0.05).

Alpha-Amylase Inhibition (%)

80 70

c

60

b

b b

a

a

50

d

d f

40

e

30 20 10 RA 5 RA 1 RA 0 20

2. 5 CA 5 CA 10

CA

7 F CA R

PV 5 PV 0 10 PV 0 15 0

0 Concentration µg/mL Fig. 4. Alpha-amylase inhibitory effect of PV, CARF, CA and RA. Values are shown as mean  S.E.M. (n = 3). Values with different letters are significantly different (p  0.05).

Fig. 5. Effects of P. vulgaris ethanolic extract (PV), caffeic acid rich fraction (CARF), caffeic acid (CA) and rosmarinic acid (RA) on serum insulin at pre-dose and 8 weeks post-dose. Values represent the mean  SEM (n = 7). (NORM) normal non-diabetic untreated mice. *p < 0.05 vs. diabetic vehicle control (VEH).

K. Raafat et al. / Biomedicine & Pharmacotherapy 84 (2016) 1008–1018

10 9

* ***

7

*

* *

*

*

*

*

5 0

2

4 Weeks

NORM DIA+VEH DIA+TRA 10 mg/kg DIA+PV 50 mg/kg DIA+PV 100 mg/kg

7 6

* ** *

DIA+PV 50 mg/kg DIA+PV 100 mg/kg DIA+PV 150 mg/kg

6

B Tail Withdrwal Latency (Seconds)

*

* * *

*

*

8

4

DIA+ CARF 7 mg/kg

5

*

*

6

8

*

3

* *

* * * *

*

* *

*

2 1 0

2

4

9 8

* ** * * *

7

* * * * * *

* * * * * *

* * * * * *

DIA+RA 5 mg/kg DIA+RA 10 mg/kg DIA+RA 20 mg/kg

6 5 4

* *

DIA+VEH DIA+CA 2.5 mg/kg DIA+CA 5 mg/kg DIA+CA 10 mg/kg

10

0

DIA + CARF 7 mg/kg DIA+PV 150 mg/kg

*

4

A Hot Plate Latency (Seconds)

NORM DIA+VEH DIA+TRA 10 mg/kg

6

8

Weeks Fig. 6. Effect of P. vulgaris ethanolic extract (PV) and caffeic acid rich fraction (CARF) on the hot plate and tail withdrawal latencies in alloxan treated mice. (A) Hot plate latency: (Closed-Circle, straight line) NORM: normal control mice. (Closed-squares, straight-line) DIA + VEH: diabetic animals treated with vehicle as control. (Starred, dotted-line) DIA + TRA 10 mg/kg: diabetic animals treated with TRA 10 mg/kg as a positive control. (Open-circles, straight-line) DIA + PV 50 mg/kg: diabetic animals treated with PV 50 mg/kg. (Up-triangles, dashed-line) DIA + PV 100 mg/kg: diabetic animals treated with PV 100 mg/kg. (Right-triangles, dashed-dotted-line) DIA + PV 150 mg/kg: diabetic animals treated with combination of PV 150 mg/kg. (Crossedtriangles, straight-line) DIA + CARF 7 mg/kg: diabetic animals treated with CARF 7 mg/kg. (B) Tail withdrawal latency: (Closed-Circle, straight line) NORM: normal control mice. (Closed-squares, straight-line) DIA + VEH: diabetic animals treated with vehicle as control. (Starred, dotted-line) DIA + TRA 10 mg/kg: diabetic animals treated with TRA 10 mg/kg as a positive control. (Open-circles, straight-line) DIA + PV 50 mg/kg: diabetic animals treated with PV 50 mg/kg. (Up-triangles, dashed-line) DIA + PV 100 mg/kg: diabetic animals treated with PV 100 mg/kg. (Right-triangles, dashed-dotted-line) DIA + PV 150 mg/kg: diabetic animals treated with combination of PV 150 mg/kg. (Crossed-triangles, straight-line) DIA + CARF 7 mg/kg: diabetic animals treated with CARF 7 mg/kg. Data (n = 7) are expressed in mean  S.E.M. "*" means P < 0.05 compared with control.

Furthermore treatment with PV (150 mg/kg), CARF (7 mg/kg), CA (10 mg/kg) and RA (20 mg/kg), tail-flick latency has been markedly improved by 2.90, 3.04, 2.80 and 1.48 folds respectively, in the 8th week in comparison to the vehicle treated animals (Figs. 6 B and 7 B). Treatment of mice with highest doses of PV (150 mg/kg), CARF (7 mg/kg), CA (10 mg/kg) and RA (20 mg/kg) has significantly ameliorated mechanical allodynia utilizing von Frey filaments by 7.11, 7.69, 6.96 and 5.81 folds respectively, in the 8th week in comparison to the vehicle treated animals (Fig. 8). 3.7. Measurement of lipid peroxidation The LPO (TBARS) and CAT antioxidant enzyme levels were measured as markers of diabetes-induced oxidative stress. Administration of alloxan provoked a significant elevation in LPO as a free radicals generation marker, as compared with normal non-diabetic mice (Fig. 9). This elevation were significantly restored in mice treated with PV (50, 100 and 150 mg/kg) respectively by 17.7, 50.6 and 69.1%, CARF (7 mg/kg) by 66.2%, CA (5 and 10 mg/kg) respectively by 46.5 and 63.0%, and RA (10 and

B Tail Withdrwal Latency (Seconds)

Hot Plate Latency (Seconds)

A

1015

2

4 Weeks

6

DIA+VEH DIA+CA 2.5 mg/kg DIA+CA 5 mg/kg DIA+CA 10 mg/kg DIA+RA 5 mg/kg DIA+RA 10 mg/kg DIA+RA 20 mg/kg

7 6 5

*

4

* * * * *

3

* * *

8

*

* * * * * *

* * * * *

2 1 0

2

4 Weeks

6

8

Fig. 7. Effect of caffeic acid (CA) and rosmarinic acid (RA) on the hot plate and tail withdrawal latencies in alloxan treated mice. (A) Hot plate latency: (Closed-squares, straight-line) DIA + VEH: diabetic animals treated with vehicle as control. (Closedleft-triangles, dotted-line) DIA + CA 2.5 mg/kg: diabetic animals treated with CA 2.5 mg/kg. (Closed-Up-triangles, straight-line) DIA + CA 5 mg/kg: diabetic animals treated with CA 5 mg/kg. (Closed-Right-triangles, straight-line) DIA + CA 10 mg/kg: diabetic animals treated with combination of CA 10 mg/kg. (Open-left-triangles, straight-line) DIA + RA 5 mg/kg: diabetic animals treated with RA 5 mg/kg. (OpenUp-triangles, dash-dotted-line) DIA + RA 10 mg/kg: diabetic animals treated with RA 10 mg/kg. (Open-right-triangles, dotted-line) DIA+ RA 20 mg/kg: diabetic animals treated with combination of RA 20 mg/kg. (B) Tail withdrawal latency: (Closed-squares, straight-line) DIA + VEH: diabetic animals treated with vehicle as control. (Closed-left-triangles, dotted-line) DIA + CA 2.5 mg/kg: diabetic animals treated with CA 2.5 mg/kg. (Closed-Up-triangles, straight-line) DIA + CA 5 mg/kg: diabetic animals treated with CA 5 mg/kg. (Closed-Right-triangles, straight-line) DIA + CA 10 mg/kg: diabetic animals treated with combination of CA 10 mg/kg. (Open-left-triangles, straight-line) DIA + RA 5 mg/kg: diabetic animals treated with RA 5 mg/kg. (Open-Up-triangles, dash-dotted-line) DIA + RA 10 mg/kg: diabetic animals treated with RA 10 mg/kg. (Open-right-triangles, dotted-line) DIA + RA 20 mg/kg: diabetic animals treated with combination of RA 20 mg/kg. Data (n = 7) are expressed in mean  S.E.M. "*" means P < 0.05 compared with control.

20 mg/kg) respectively by 17.7 and 38.2%, when compared with vehicle treated control mice, after 8 day post-administration (Fig. 9). Therefore, the maximum restoration of LPO levels was shown with PV (150 mg/kg) and CARF (7 mg/kg), after 8 day postadministration, respectively (Fig. 9). 3.8. Estimation of in vivo antioxidant activity The serum CAT of each mouse was observed to facilitate the evaluation of the in vivo antioxidant effect of P. vulgaris, CARF, CA, RA and pCA. As shown in Table 5, diabetic mice taking the uppermost concentration of P. vulgaris (150 mg/kg), CARF (7 mg/ kg), CA (10 mg/kg), RA (20 mg/kg), pCA (12 mg/kg) and GB (5 mg/

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K. Raafat et al. / Biomedicine & Pharmacotherapy 84 (2016) 1008–1018

Fig. 8. The effect of P. vulgaris ethanolic extract (PV), caffeic acid rich fraction (CARF), caffeic acid (CA), rosmarinic acid (RA) and tramadol (TRA) 10 mg/kg on tactile allodynia in neuropathic model in alloxan-induced diabetic mice. Paw withdrawal thresholds to von Frey filaments were determined on hind paw prior to (Predose) and up to 8 weeks following i.p. injection of PV 150 mg, CARF 7 mg/kg, CA 10 mg/kg and RA 20 mg/kg. (NORM) normal non-diabetic untreated mice. *P  0.05 and **P  0.01 compared to vehicle (VEH) (n = 7 animals/group).

kg), had a potential rise in CAT serum activity to achieving a significant difference on the 5th (20.4, 37.8, 30.6, 22.3, 24.6 and 35.0% respectively) and 8th day (18.6, 38.3, 33.7,21.5, 16.7 and 29.6% respectively) in comparison with diabetic control animals (Table 5). Following Wagner model of synergy, the results have shown that CARF had more prominent amelioration of painful hyperalgesia and mechanical allodynia than other individual substances, suggesting an apparent synergism between CA and RA [42]. 4. Discussion The amelioration of DM and DN continues to be challenging, with significant consequences on patients' morbidity and quality of life [43]. To prevent the progression of DN, glycemic homeostasis is essential, however, DN mechanisms stay unclear, and as a result,

TBRAS Level (LPO) nM /100g

3.0 2.5 *

2.0

* *

1.5

*

*

1.0

*

*

*

0.5

RA 5 RA RA 10 20

7

2. 5 CA 5 CA 10

CA

CA RF

PV 5 PV 0 1 PV 00 15 0

No r

m al VE H

0.0 Concentration mg/kg Fig. 9. The subchronicallevel of LPO (TBARS) in P. vulgaris ethanolic extract (PV), caffeic acid rich fraction (CARF), caffeic acid (CA) and rosmarinic acid (RA) in (8 days post-administration) treated mice.Data (n = 4) are expressed in mean  S.E.M. "*" means P < 0 0.05 compared with vehicle control (VEH).

usually its management remains unsatisfactory [44]. Tricyclic antidepressants, anticonvulsants, 5-HT-NA reuptake inhibitors (SNRIs), opiates and topical medications are agents used to palliate DN symptoms. Nevertheless, these conventional medications in many cases provide partial pain alleviation and possess unpleasant side effects [44–47]. Consequently, alternative therapies with higher efficacy, tolerability and safety should be investigated for an effective DN pain management [43,44]. Moreover, several lines of evidence indicated that the susceptibility of diabetes and its related complications have been ameliorated by natural compounds [11–14]. A number of conducted studies have well established the safety of P. vulgaris in several indications [19,20]. Furthermore, the most active antidiabetic compounds of P. vulgaris and their effects on hyperglycemia, diabetes induced glycosylated hemoglobin, tactile allodynia and hyperalgesia are not clearly described in the literature. Currently, we evaluated the acute and subchronic effects of P. vulgaris administration in alloxan-induced diabetic mice. Moreover, we performed a bio-guided fractionation to identify the active components of P. vulgaris and their effects on glycosylated hemoglobin, T1D-induced oxidative stress, tactile allodynia and hyperalgesia. The bio-guided fractionation utilizing column chromatography, RP-HPLC, 1H NMR and 13C NMR (Fig. 1) indicated that the most active fraction of P. vulgaris contains caffeic acid (major peaks ca. 93.4%) and rosmarinic acid (minor peaks ca. 6.6%), and is hence named caffeic acid rich fraction (CARF). The acute and subchronic hypoglycemic activity of CARF was 1.27 and 2.72 folds more effective than that of glibenclamide, respectively. Subchronically, CARF has shown significant improvement in body weights, indicating its long-term efficacy in T1D amelioration (Table 4). One of the essential biomarkers of hyperglycemia severity is glycosylated hemoglobin. HbA1c levels are very helpful in the measurement of overall glycemic control, since HbA1c levels reflect the cumulative glycation throughout the erythrocytes lifetime [34]. The current results show that CARF significantly decreases HbA1c following an 8 week administration. Moreover, CARF has shown the highest HbA1c reduction indicating that CARF provides continuing glycemic restoration in diabetic animals. In order to recognize the mechanism by which the tested compounds restore the glycemic homeostasis, the inhibition of alpha-glucosidase and alpha-amylase, and the elevation of serum insulin levels have been studied. Alpha-amylase degrades dietary complex carbohydrates to di-saccharides and oligosaccharides, then alpha-glucosidase converts them into monosaccharides [28,29]. Consequently, the inhibition of these enzymes involved in the metabolism of carbohydrates is one of the possible mechanisms for reducing hyperglycemia [30]. In the current study, CARF and PV have shown significant decrease in alphaamylase and alpha-glucosidase activity. Nevertheless, CARF has shown more significant inhibitory effects than PV, suggesting that CARF had more potent hypoglycemic potential than PV. Moreover, with the aim of identifying the mechanism of the antidiabetogenic properties of PV and CARF, serum insulin levels were monitored before and 8 weeks post-administration. Currently, CARF and PV have shown significant increase in serum insulin levels. However, CARF has shown more significant elevation in serum insulin levels more than PV, suggesting that CARF had more potent insulin secretagogue potential than PV. Therefore, the potential hypoglycemic effects of CARF and PV may be attributed to their insulin secretagogue potentials, and to their high inhibitory effect on alpha-glucosidase and alpha-amylase activities. The effect of PV and CARF to inhibit both alpha-amylase and alphaglucosidase activities and the insulin secretagogue potentials were witnessed before with earlier studies on polyphenolic compounds [48,49].

K. Raafat et al. / Biomedicine & Pharmacotherapy 84 (2016) 1008–1018

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Table 5 in vivo assessment of the antioxidant activity of P. vulgaris, CARF, RA, CA and pCA using CAT levels in serum of alloxan-induced diabetic mice. Group

Dose (mg/kg)

Catalase level  S.E.M. (kU/I) 1st day

3rd day

5th day

8th day

Control Diabetic controla GBb P. vulgarisa P. vulgarisa P. vulgarisa CARFa CAa CAa CAa RAa RAa RAa pCAa pCAa pCAa

– – 5 50 100 150 7 2.5 5 10 5 10 20 3 6 12

38.50  1.50 19.17  1.60*** 20.10  1.70 20.12  1.90 21.89  2.10 20.22  2.20 21.44  1.60 20.61  1.20 21.13  1.70 19.94  2.10 19.42  1.80 20.37  1.70 20.45  1.50 20.81  1.40 19.97  1.70 20.22  1.60

39.1  1.60 18.43  1.30*** 22.5  1.70 20.47  2.70 21.44  2.50 22.44  2.80 27.73  1.40 21.90  1.60 23.92  1.20 24.16  1.70 22.47  2.50 24.93  1.90 25.88  1.50 23.98  1.10 24.15  1.10 25.67  1.30

40.36  1.20 21.51  1.90*** 29.04  1.40 20.45  2.65 21.80  1.90 25.89  2.10 29.64  1.80 25.16  2.40 24.10  2.70 28.10  2.50 23.64  1.80* 26.16  2.40* 26.31  1.80* 24.50  2.50 24.70  2.30 26.80  2.60

39.12  1.70 23.05  1.40*** 29.87  1.00** 21.55  2.60** 22.65  2.40** 27.34  1.90** 31.87  1.30** 27.28  3.10* 27.38  2.90* 30.82  1.60* 25.37  1.10* 27.93  2.60* 28.01  1.10* 25.60  1.60* 25.70  1.20* 26.90  1.30*

S.E.M.: mean standard error. * p < 0.05 significant from the control animals. ** p < 0.01 significant from the control animals. *** p < 0.001 significant from the control animals. a Compared to vehicle control. b Compared to diabetic control.

As reported before, CAT activity declined in alloxan-provoked diabetic animals [8,10,13]. This decline might be attributed to alloxan-generated ROS deactivation. Subchronically, the elevation in CAT levels have evidenced that T1D treatment with tested compounds especially as CARF had reversed the T1D generated oxidative stress (Table 5). Mechanical allodynia and painful hyperalgesia are distinguishing symptoms of DN [50]. Alloxan-induced diabetic animals have shown similar symptoms [13,51,52]. In this study, elevated HbA1c levels in alloxinated mice induced DN characterized by mechanical allodynia and painful hyperalgesia. DN success rate (loss of sensory of thermal sensitivity with latency significantly below 10S) [14,53] after 6 weeks of T1D induction in mice, was above 85%. Those mice with satisfactory DN success rate were used in DN experiments. Using hot plate and tail flick latency tests, P. vulgaris, CARF, CA and RA ameliorated alloxaninduced thermal hyperalgesia. CARF lowered painful thresholds more significantly than P. vulgaris, CA and RA. P. vulgaris, CARF, CA and RA had significantly recovered the decrease in latency response for tactile withdrawal thresholds. Nevertheless, CARF has shown the best antinociceptive properties of the tested substances. Although CARF is rich in CA, it reversed T1D and T1D-induced tactile allodynia more significantly than CA. These results suggested that there is an apparent synergistic effect between CA and RA in CARF producing more significant amelioration of T1D and nociceptive thresholds. In addition, the results indicated that CARF exhibited marked antinociceptive properties in alloxan-provoked diabetic mice which might be due to long term blood glucose control, declining in the HbA1c levels and the improvement of CAT levels. These effects might be contributed in the mechanism by which CARF possesses its antidiabetogenic and hypoalgesic properties. Moreover, it is obvious that CARF treatment provides more pronounced anti-hyperglycemic effects, which resulted in the reversal of painful hyperalgesia and tactile allodynia associated with DN. A marked elevation in TBARS level in diabetic-animals was observed in a previous and in the present study, indicating the association of oxido-nitrosative stress in DN pain [41]. We detected

that the highest doses of PV, CARF, CA and RA attenuated oxidative stress, measured as TBARS level (ROS marker) and elevated the antioxidant enzyme (CAT) level in alloxan-induced diabetic mice that exhibited thermal hyperalgesia and tactile allodynia. Consequently, the mechanism by which PV and CARF has ameliorated the thermal hyperalgesia and mechanical allodynia is likely attributed to the antioxidant effect of PV and CARF. Our findings are in-line with previously reported studies that suggested a neuroprotective effect of other natural compounds, like G. biloba polyphenolics, against neurotoxicity [54,55]. Therefore, the observed antioxidant and hypoglycemic effects of PV, CARF, CA and RA may be involved in the mechanism of their antinociceptive and neuroprotective effects. CARF (ca. 93.4% CA and 6.6% RA, RP-HPLC, 1H NMR and 13C NMR) administration in diabetic mice not only ameliorated the diabetogenic condition but has also reversed DN painful hyperalgesia and tactile allodynia. CARF has shown potent anti-hyperglycemic, as well as the ability to inhibit TBRAS and elevate the antioxidant enzyme (CAT) in T1D. Therefore, the observed CARF attenuation of alpha-amylase and alpha-glucosidase, its elevation in serum insulin levels, and its antioxidant potential might be responsible for its antidiabetogenic and antinociceptive properties. In conclusion, the data of the present study suggests a potential effective role of P. vulgaris against alloxan-induced T1D and DN in mice. These findings further validate the traditional use of P. vulgaris against T1D and its complications. Conflicts of interest The authors declare no conflicts of interest. References [1] Y. Chiba, Y. Kimbara, R. Kodera, Y. Tsuboi, K. Sato, Y. Tamura, S. Mori, H. Ito, A. Araki, Risk factors associated with falls in elderly patients with type 2 diabetes, J. Diabetes Complicat. (2015). [2] WHO, Global guideline for type 2 diabetes, Diabetes Res. Clin. Pract. 104 (1) (2014) 1–52. [3] S.S. Kwon, J.-Y. Kwon, Y.-W. Park, Y.-H. Kim, J.-B. Lim, HbA1c for diagnosis and prognosis of gestational diabetes mellitus, Diabetes Res. Clin. Pract. (2015). [4] M.G. Rajanandh, S. Kosey, G. Prathiksha, Assessment of antioxidant supplementation on the neuropathic pain score and quality of life in

1018

[5]

[6]

[7]

[8] [9]

[10]

[11]

[12]

[13]

[14]

[15]

[16]

[17]

[18]

[19] [20] [21]

[22]

[23]

[24]

[25]

[26]

[27] [28]

[29]

[30]

K. Raafat et al. / Biomedicine & Pharmacotherapy 84 (2016) 1008–1018 diabetic neuropathy patients – a randomized controlled study, Pharmacol. Rep. 66 (1) (2014) 44–48. P. Arulselvan, S.P. Subramanian, Beneficial effects of Murraya koenigii leaves on antioxidant defense system and ultra structural changes of pancreatic betacells in experimental diabetes in rats, Chem. Biol. Interact. 165 (2) (2007) 155–164. I.S. Punitha, K. Rajendran, A. Shirwaikar, Alcoholic stem extract of Coscinium fenestratum regulates carbohydrate metabolism and improves antioxidant status in streptozotocin-nicotinamide induced diabetic rats, Evid. Based Complement Altern. Med. 2 (3) (2005) 375–381. G. Manonmani, V. Bhavapriya, S. Kalpana, S. Govindasamy, T. Apparanantham, Antioxidant activity of Cassia fistula (Linn.) flowers in alloxan induced diabetic rats, J. Ethnopharmacol. 97 (1) (2005) 39–42. H.F. Al-Azzawie, M.S. Alhamdani, Hypoglycemic and antioxidant effect of oleuropein in alloxan-diabetic rabbits, Life Sci. 78 (12) (2006) 1371–1377. S. Kawahito, H. Kitahata, S. Oshita, Problems associated with glucose toxicity: role of hyperglycemia-induced oxidative stress, World J. Gastroenterol. 15 (33) (2009) 4137–4142. A. Sepici-Dincel, S. Acikgoz, C. Cevik, M. Sengelen, E. Yesilada, Effects of in vivo antioxidant enzyme activities of myrtle oil in normoglycaemic and alloxan diabetic rabbits, J. Ethnopharmacol. 110 (3) (2007) 498–503. K.M. Raafat, A.G. Omar, Phytotherapeutic activity of curcumol: isolation, GC– MS identification, and assessing potentials against acute and subchronic hyperglycemia, tactile allodynia, and hyperalgesia, Pharm. Biol. (2015) 1–11. K. Raafat, A. El-Lakany, Acute and subchronic in-vivo effects of Ferula hermonis L. and Sambucus nigra L. and their potential active isolates in a diabetic mouse model of neuropathic pain, BMC Complement. Altern. Med. 15 (2015) 257. K. Raafat, M. Aboul-Ela, A. El-Lakany, Alloxan-induced diabetic thermal hyperalgesia, prophylaxis and phytotherapeutic effects of Rheum ribes L. in mouse model, Arch. Pharm. Res. (2014). K. Raafat, W. Samy, Amelioration of diabetes and painful diabetic neuropathy by Punica granatum L. extract and its spray dried biopolymeric dispersions, Evid. Based Complement Altern. Med. 2014 (2014) 180495. L. Salvotelli, V. Stoico, F. Perrone, V. Cacciatori, C. Negri, C. Brangani, I. Pichiri, G. Targher, E. Bonora, G. Zoppini, Prevalence of neuropathy in type 2 diabetic patients and its association with other diabetes complications: the Verona Diabetic Foot Screening Program, J. Diabetes Complicat. (2015). J. Ma, H. Yu, J. Liu, Y. Chen, Q. Wang, L. Xiang, Metformin attenuates hyperalgesia and allodynia in rats with painful diabetic neuropathy induced by streptozotocin, Eur. J. Pharmacol. 764 (2015) 599–606. M. Greig, S. Tesfaye, D. Selvarajah, I.D. Wilkinson, Insights into the pathogenesis and treatment of painful diabetic neuropathy, Handb. Clin. Neurol. 126 (2014) 559–578. H.Y. Cheung, Q.F. Zhang, Enhanced analysis of triterpenes, flavonoids and phenolic compounds in Prunella vulgaris L. by capillary zone electrophoresis with the addition of running buffer modifiers, J. Chromatogr. A 1213 (2) (2008) 231–238. X. Fang, R.C. Chang, W.H. Yuen, S.Y. Zee, Immune modulatory effects of Prunella vulgaris L, Int. J. Mol. Med. 15 (3) (2005) 491–496. J. Psotova, M. Kolar, J. Sousek, Z. Svagera, J. Vicar, J. Ulrichova, Biological activities of Prunella vulgaris extract, Phytother. Res. 17 (9) (2003) 1082–1087. Z.J. Wang, Y.Y. Zhao, Y.Y. Chen, B.N. Ma, Triterpenoid compounds of Prunella genus and their features of 13C NMR spectroscopy, Zhongguo Zhong Yao Za Zhi 25 (10) (2000) 583–588. Y. Wang, J. Yin, Q. Guo, Y. Xiao, Dynamic change of active component content in different parts of Prunella vulgaris, Zhongguo Zhong Yao Za Zhi 36 (6) (2011) 741–745. K. Raafat, U. Breitinger, L. Mahran, N. Ayoub, H.-G. Breitinger, Synergistic inhibition of glycinergic transmission in vitro and in vivo by flavonoids and strychnine, Toxicol. Sci. 118 (2010) 171–182. J. Zheng, J. He, B. Ji, Y. Li, X. Zhang, Antihyperglycemic activity of Prunella vulgaris L. in streptozotocin-induced diabetic mice, Asia Pac. J. Clin. Nutr. 16 (Suppl. 1) (2007) 427–431. Q.X. Zhou, F. Liu, J.S. Zhang, J.G. Lu, Z.L. Gu, G.X. Gu, Effects of triterpenic acid from Prunella vulgaris L. on glycemia and pancreas in rat model of streptozotozin diabetes, Chin. Med. J. (Engl.) 126 (9) (2013) 1647–1653. Q. Cheng, X. Zhang, O. Wang, J. Liu, S. Cai, R. Wang, F. Zhou, B. Ji, Anti-diabetic effects of the ethanol extract of a functional formula diet in mice fed with a fructose/fat-rich combination diet, J. Sci. Food Agric. 95 (2) (2015) 401–408. P.K. Perera, Y. Li, Functional herbal food ingredients used in type 2 diabetes mellitus, Pharmacogn. Rev. 6 (11) (2012) 37–45. Y.I. Kwon, E. Apostolidis, K. Shetty, In vitro studies of eggplant (Solanum melongena) phenolics as inhibitors of key enzymes relevant for type 2 diabetes and hypertension, Bioresour. Technol. 99 (8) (2008) 2981–2988. K.A. Abo, A.A. Fred-Jaiyesimi, A.E. Jaiyesimi, Ethnobotanical studies of medicinal plants used in the management of diabetes mellitus in South Western Nigeria, J. Ethnopharmacol. 115 (1) (2008) 67–71. Y.J. Shim, H.K. Doo, S.Y. Ahn, Y.S. Kim, J.K. Seong, I.S. Park, B.H. Min, Inhibitory effect of aqueous extract from the gall of Rhus chinensis on alpha-glucosidase

[31]

[32]

[33]

[34]

[35] [36]

[37]

[38]

[39]

[40]

[41] [42] [43]

[44]

[45] [46] [47] [48]

[49]

[50]

[51]

[52] [53]

[54]

[55]

activity and postprandial blood glucose, J. Ethnopharmacol. 85 (2–3) (2003) 283–287. M. Hieke, J. Ness, R. Steri, C. Greiner, O. Werz, M. Schubert-Zsilavecz, S. Weggen, H. Zettl, SAR studies of acidic dual gamma-secretase/PPARgamma modulators, Bioorg. Med. Chem. 19 (18) (2011) 5372–5382. S. Sahin, C. Demir, H. Malyer, Determination of phenolic compounds in Prunella L. by liquid chromatography-diode array detection, J. Pharm. Biomed. Anal. 55 (5) (2011) 1227–1230. K.M. Raafat, H. Jassar, M. Aboul-Ela, A. El-Lakany, Protective effects of Origanum majorana L. against neurodegeneration: fingerprinting, isolation and in vivo glycine receptors behavioral model, Int. J. Phytomed. 5 (1) (2013) 46–57. W. Zhang, A. Welihinda, J. Mechanic, H. Ding, L. Zhu, Y. Lu, Z. Deng, Z. Sheng, B. Lv, Y. Chen, J.Y. Roberge, B. Seed, Y.-X. Wang, EGT1442, a potent and selective SGLT2 inhibitor, attenuates blood glucose and HbA1c levels in db/db mice and prolongs the survival of stroke-prone rats, Pharmacol. Res. 63 (4) (2011) 284–293. U. Breitinger, K.M. Raafat, H.G. Breitinger, Glucose is a positive modulator for the activation of human recombinant glycine receptors, J. Neurochem. (2015). W.G. Yasmineh, T.P. Kaur, B.R. Blazar, A. Theologides, Serum catalase as marker of graft-vs-host disease in allogeneic bone marrow transplant recipients: pilot study, Clin. Chem. 41 (11) (1995) 1574–1580. B. Sarmento, A. Ribeiro, F. Veiga, D. Ferreira, Development and validation of a rapid reversed-phase HPLC method for the determination of insulin from nanoparticulate systems, Biomed. Chromatogr. 20 (9) (2006) 898–903. A. Ulugol, C. Oltulu, O. Gunduz, C. Citak, R. Carrara, M.R. Shaqaqi, A.M. Sanchez, A. Dogrul, 5-HT7 receptor activation attenuates thermal hyperalgesia in streptozocin-induced diabetic mice, Pharmacol. Biochem. Behav. 102 (2) (2012) 344–348. H. Smits, C. Ultenius, R. Deumens, G.C. Koopmans, W.M.M. Honig, M. van Kleef, B. Linderoth, E.A.J. Joosten, Effect of spinal cord stimulation in an animal model of neuropathic pain relates to degree of tactile allodynia, Neuroscience 143 (2) (2006) 541–546. A. Fox, C. Gentry, S. Patel, A. Kesingland, S. Bevan, Comparative activity of the anti-convulsants oxcarbazepine, carbamazepine, lamotrigine and gabapentin in a model of neuropathic pain in the rat and guinea-pig, Pain 105 (1–2) (2003) 355–362. H. Ohkawa, N. Ohishi, K. Yagi, Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction, Anal. Biochem. 95 (2) (1979) 351–358. H. Wagner, G. Ulrich-Merzenich, Synergy research: approaching a new generation of phytopharmaceuticals, Phytomedicine 16 (2–3) (2009) 97–110. M. Jaiswal, C.L. Martin, M.B. Brown, B. Callaghan, J.W. Albers, E.L. Feldman, R. Pop-Busui, Effects of exenatide on measures of diabetic neuropathy in subjects with type 2 diabetes: results from an 18-month proof-of-concept open-label randomized study, J. Diabetes Complicat. (2015). N. Galeotti, A. Maidecchi, L. Mattoli, M. Burico, C. Ghelardini, St John's Wort seed and feverfew flower extracts relieve painful diabetic neuropathy in a rat model of diabetes, Fitoterapia 92 (2014) 23–33. M.C. Wong, J.W. Chung, T.K. Wong, Effects of treatments for symptoms of painful diabetic neuropathy: systematic review, BMJ 335 (7610) (2007) 87. A.J. Boulton, Pharmacologic management of painful diabetic neuropathy, Curr. Diabetes Rep. 8 (6) (2008) 429–430. T.J. Lindsay, B.C. Rodgers, V. Savath, K. Hettinger, Treating diabetic peripheral neuropathic pain, Am. Fam. Phys. 82 (2) (2010) 151–158. T. Matsui, T. Tanaka, S. Tamura, A. Toshima, K. Tamaya, Y. Miyata, K. Tanaka, K. Matsumoto, alpha-Glucosidase inhibitory profile of catechins and theaflavins, J. Agric. Food Chem. 55 (1) (2007) 99–105. L.G. Ranilla, Y.I. Kwon, E. Apostolidis, K. Shetty, Phenolic compounds, antioxidant activity and in vitro inhibitory potential against key enzymes relevant for hyperglycemia and hypertension of commonly used medicinal plants, herbs and spices in Latin America, Bioresour. Technol. 101 (12) (2010) 4676–4689. H. Gul, O. Yildiz, A. Dogrul, O. Yesilyurt, A. Isimer, The interaction between IL1b and morphine: possible mechanism of the deficiency of morphine-induced analgesia in diabetic mice, Pain 89 (1) (2000) 39–45. M. Paro, G. Italiano, R.A. Travagli, L. Petrelli, R. Zanoni, M. Prosdocimi, M.G. Fiori, Cystometric changes in alloxan diabetic rats: evidence for functional and structural correlates of diabetic autonomic neuropathy, J. Auton. Nerv. Syst. 30 (1) (1990) 1–11. N.A. Calcutt, S.R. Chaplan, Spinal pharmacology of tactile allodynia in diabetic rats, Br. J. Pharmacol. 122 (7) (1997) 1478–1482. K.A. Sullivan, J.M. Hayes, T.D. Wiggin, C. Backus, S. Su Oh, S.I. Lentz, F. Brosius 3rd, E.L. Feldman, Mouse models of diabetic neuropathy, Neurobiol. Dis. 28 (3) (2007) 276–285. J. Hibatallah, C. Carduner, M.C. Poelman, In-vivo and in-vitro assessment of the free-radical-scavenger activity of Ginkgo flavone glycosides at high concentration, J. Pharm. Pharmacol. 51 (12) (1999) 1435–1440. C. Shi, J. Liu, F. Wu, D.T. Yew, Ginkgo biloba extract in Alzheimer's disease: from action mechanisms to medical practice, Int. J. Mol. Sci. 11 (1) (2010) 107–123.