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Synergistic interactions between the antinociceptive effect of Rhodiola rosea extract and B vitamins in the mouse formalin test.


Article history:

Received 6 March 2013

Received in revised form 28 May 2013

Accepted 2 July 2013





Rhodiola rosea


Plant-drug interaction


Aim: In this study, the pharmacological interactions between a Rhodiola rosea ethanol extract and B-vitami ns such as thiamine ([B.sub.1]), riboflavine ([B.sub.2]), pyridoxine ([B.sub.6]), cyanocobalamin ([B.sub.12]) and a mixture of vitamins B1 + B6 + 812 was investigated in the mouse formalin test.

Methods: Individual dose response curves of the Rhodiola rosea ethanol extract, as well as B-vitamins alone or in a mixture were evaluated in mice in which nociception was induced with 2% formalin intraplantarly. The antinociceptive mechanisms of the Rhodiola rosea were investigated by exploring the role of the opioid and serotonin receptors and the nitric oxide pathway. Isobolographic analysis was used to evaluate the pharmacological interactions between the Rhodiola rosea ethanol extract and each B-vitamin individually or the mixture of vitamins [B.sub.1] + [B.sub.6] + [B.sub.12] by using the [ED.sub.30] and a fixed 1:1 ratio combination.

Results: Administration of the Rhodiola rosea extract alone or in combination with all of the vitamins produced a significant and dose-dependent antinociceptive response. The antinociceptive effect of the Rhodiola rosea extract ([ED.sub.50] = 81 mg/kg, p.o.) was significant and reverted in the presence of antagonists of the 5-1-[HT.sub.1A], GABA/BDZs and opioid receptors and by blocking mediators of the nitric oxide/cGMP/[K.sup.+]. channels pathway. Isobolograms demonstrate that all of the combinations investigated in this study produced a synergistic interaction experimental [ED.sub.30] values were significantly smaller than those calculated theoretically.

Conclusions: These results provide evidence that a Rhodiola rosea ethanol extract in combination with B-vitamins produces a significant diminution in the nociceptive response in a synergistic manner, which is controlled by various mechanisms. These findings could aid in the design of clinical studies and suggest that these combinations could be applied for pain therapy.

[c] 2013 Elsevier GmbH. All rights reserved.


Medicinal plants have been used throughout history as a source of new chemical substances with potential therapeutic effects. The presence of more than one active metabolite in plants supports the use of plant extracts in disease therapy. Rhodiola rosea L. (Crassulaceae) is an adaptogen plant also known as golden root, roseroot or arctic root. It is traditionally used in Eastern Europe and Asia to stimulate the nervous system (Kurkin and Zapesochnaya 1986; Kelly, 2001; Fintelmann and Gruenwald, 2007). It has also been reported that this species is involved in learning and memory (Petkov et al., 1986), an antidepressant (Darbinyan et al., 2007; van Diermen et al., 2009), an anxiolytic (Bystritsky et al., 2008) and an antioxidant (Pooja et al., 2006; Schriner et al., 2009). Approximately, 140 compounds have been isolated from the root and the rhizome of Rhodiola rosea, and these have been classified into different chemical groups including monoterpenes, phenylethanoids, phenylpropanoids, flavonoids, aryl glycosides, proanthocyanidins and gallic acid derivatives (Kurkin and Zapesochnaya 1986; Ganzera et al., 2001; Ma et al., 2006). The phenylpropanoid (e.g., rosavin) and phenylethanoid (e.g., salidroside) derivates are thought to be the most critical constituents needed for the numerous health benefits of Rhodiola rosea extracts (Kelly, 2001), and a standard dose for both rosavin and salidroside is used (Panossian and Wagner, 2005). Recent studies in models such as carrageenan and formaldehyde-induced arthritis or nystatin-induced edema in rats have shown that Rhodiola rosea possesses anti-inflammatory activity, possibly by inhibiting phospholipase A2 and cyclooxygenase-1 and 2 (Pooja et al., 2009). A preliminary study of Rhodiola rosea ethanol extracts demonstrated that this species also has antinociceptive effects also in the mouse formalin test (Montiel-Ruiz et al., 2012).

It is known that B-vitamins are widely used to treat acute and chronic pain (Sun et al., 2005). Several reports note that vitamins [B.sub.1], [B.sub.2], [B.sub.6] and [B.sub.12] have antinociceptive, anti-hyperalgesic and anti-allodynic properties in different experimental models (Fu et al., 1988; Jurna, 1998; Franca et al., 2001: Wang et al., 2005; Bertollo et al., 2006; Jolivalt et al., 2009; Song et al., 2009). The proposed mechanisms of action include the synthesis and secretion of serotonin and noradrenalin in the brain, suppression of nociceptive activity at the level of the spinal cord, the release of opioids and activation of opioid receptors, the nitric oxide cGMP pathway and a reduction in the current through [Na.sup.+] channels by tetrodotoxin receptor (Abacioglu et al., 2000; Franca et al., 2001; Fu et al., 1988; Song et al., 2009).

Because many mechanisms are involved in pain, it has been suggested that a combination of antinociceptive drugs that utilize different mechanisms of action could be more efficacious by working in a synergistic manner this strategy could also promote pharmacological effects at lower doses of each agent and thus reduces the intensity and incidence of unwanted effects. The aim of this study was to investigate the pharmacological interactions between Rhodiola rosea ethanol extracts and individual or a mixture of B-vitamins in the nociception induced by the formalin test in mice. Moreover, given that the mechanism of action underlying the antinociceptive properties of Rhodiola rosea ethanol extracts is unknown, this study also investigated the participation of the opioid and serotonin receptors and the nitric oxide pathway.

Materials and methods


Female ICR mice (body weight range, 25-30g) were used in this study. Animals were housed with a standard light/dark cycle (lights on 7:00 a.m.) and constant temperature (22-24 [degrees]C), and had free access to food and drinking water before the experiments. All experiments were performance between 8:00 a.m. and 2:00 p.m. All experimental procedures followed the Guidelines on Ethical Standards for Investigation of Experimental Pain in Animals (Zimmermann, 1983). The experimental protocol was approved by the local Institutional Animal Care and Use Committee in accordance with the Mexican federal regulations for the Care and Use of Laboratory Animals NOM-062-Z00-1999 (Mexican Ministry of Health). Each mouse was used only once during the protocol and was killed in a [CO.sub.2] chamber immediately after the experiment. For all experimental procedures, the groups were composed of at least six mice.

Plant material and drugs

A standardized ethanol extract (70%, w/v) prepared with the roots of Rhodiola rosea and vitamins [B.sub.1], [B.sub.2], [B.sub.6] and [B.sub.12] were acquired from Rectecma (Mexico City, Mexico). A standardized Rhodiola rosea extract contains 2.7% of rosavin and 2.5% of salidroside (Panossian and Wagner, 2005). Formaldehyde solution (37%), naltrexone, flumazenil, WAY 100635, L-[N.sup.G]-nitro arginine methyl ester (L-NAME), 1H-[1,2,4]0xidiazolo[4,2-[alpha]]quinoxalin-1-one (ODQ), linsidomine (SIN-1) and glibenclamide were purchased from Sigma Chemical Co (St. Louis, MO, USA). The extract was suspended in vehicle (0.5% tween-80 in saline solution (0.9% w/v). Naltrexone, flumazenil, WAY 100635, L-NAME, ODQ SIN-1 and glibenclamide were prepared in a saline solution (0.9% w/v). All substances and extract were freshly prepared for each use and administered orally in a volume of 0.2 m1/10 g body weight. Formaldehyde was dissolved in distilled water. Control animals received the same volume of vehicle [0.5% Tween-80 in saline solution (0.9% w/v) or saline solution alone].

Pharmacological evaluation

Formalin test

This experimental model consists of placing each mouse in an open Plexiglass observation chamber with mirrors to allow them to become accustomed to their surroundings for 30 min. Each mouse was then removed for formalin administration; 20 pi of diluted 2% formalin was administered subcutaneously (s.c.) into the right hind paw of the mouse with a 30-gauge needle. The time of licking of the injected paw was defined as a nociceptive response, which was recorded during a 30-min period measured every 5 min after algesic injection (Hunskaar and Hole, 1987). The formalin-induced licking behavior was biphasic the initial acute phase (first phase, 0-10 min) is followed by a relatively short quiescent period that was then followed by a prolonged tonic response (second phase, 15-30 min) (Hunskaar and Hole, 1987; Shibata et al., 1989).

Antinociceptive properties of the Rhodiola rosea extract and B-vitamins

Groups of at least six mice each received increasing doses of either the Rhodiola rosea extract (10-177 mg/kg), vitamins [B.sub.1] (30-707 mg/kg), [B.sub.2] (1-100 mg/kg), [B.sub.6] (30-707 mg/kg), [B.sub.12] (1-177 mg/kg), a mixture of [B.sub.1] + [B.sub.6] + [B.sub.12] (30-562 mg/kg) or the Rhodiola rosea extract combined with each B-vitamin or the mixture (Table 1). All drugs were administered 15 min before formalin injection. The time schedule for drugs administration was determined in a pilot experiment in our laboratory (data not shown).

Table 1 Doses used in this study.

R.     [B.sub.1]   Total  [B.sub.6]  Total  [B.sub.12]  Total

0.84        8.75    9.59       3.86   4,70        2.09   2.93

1.69       17.51   19.20       7.72   9.41        4.19   5.88

3.38       85.03   38.41      15.45  18.83        8.38  11.76

6.74       70.06   76.80      30.90  37.64       16.76  23.50

13.54     140.12  153.66      61.80  75.34       33.50  47.05

R.      [B.sub.1]  Total  [B.sub.2]  Total
rosea   [B.sub.6]

0.84         4.62   5.46       0.16   1.00

1.69         9.24  10.93       0.33   2.02

3.38        18.48  21.86       0.67   4.05

6.74        36.96  43.70       1.34   8.08

13.54       73.92  87.46       2.68  16.22

Data are expressed in mg/kg.

Antinociceptive mechanism of action of the Rhodiola rosea extract

An [ED.sub.50] value of 81 mg/kg, p.o., calculated from the corresponding individual dose-response curve of the Rhodiola rosea extract, was used to investigate the possible mechanism of action. Fifteen min before the administration of Rhodiola rosea extract, animals were given 5 mg/kg flumazenil, s.c. (a [GABA.sub.A]/BDZ receptor antagonist); 0.16 mg/kg WAY100635, s.c. (a 5-[HT.sub.1A] serotonin receptor antagonist) or 1 mg/kg naltrexone, s.c. (an opioid receptor antagonist). To evaluate the involvement of the nitric oxide pathway in the antinociceptive effect of the Rhodiola rosea extract animals were pretreated with 10 mg/kg SIN-1, s.c. (a nitric oxide donor), 10 mg/kg L-NAME, s.c. (a nitric oxide synthase inhibitor), 1 mg/kg ODQ s.c. (an soluble guanylyl cyclase enzyme inhibitor) or 1 mg/kg glibenclamide, s.c. (an ATP-sensitive [K.sup.+]-channels blocker). Fifteen min later. the Rhodiola rosea extract ([ED.sub.30] =81 mg/kg, p.o.) or vehicle were administered for 15 min, and then each animal received an intraplantar injection of 20 [micro]L of 2% formalin to register a nociceptive behavior as described above.

Isobolographic analysis

To characterize the pharmacological interaction between the Rhodiola rosea extracts and B-vitamins, isobolograms were built using the values obtained at the dose that produces 30% of the possible maximum antinociceptive effect ([ED.sub.30]) from the drugs administered alone or in combination, and tested in the formalin test in mice. The theoretical additive dose (Zadd) with the S.E.M. for each combination at a 1:1 ratio was computed from the [ED.sub.30] of the single drugs, according to the method previously described by (Tallarida, 1992) to satisfy the following equation:

Zadd = fA + (1 - f)B

where A was the [ED.sub.30] of the Rhodiola rosea extract and B was the [ED.sub.30] of the B vitamins. For a 1:1 fixed ratio, f is 0.5 and (1 -f) is 0.5. Zadd represents the total additive dose of the drugs, theoretically providing a 30% reduction in the time of licking relative to the vehicle. The experimental dose (Zexp) is the total dose of the mixture that was experimentally determined by the two component drugs, which was administered at a 1:1 fixed-ratio combination sufficient to reduce the time of licking by 30% with respect to the vehicle. The Zexp values (with their 95% confidence limits) were determined from the dose-response curves of the combined drugs, by a standard linear regression analysis of the log dose--response curve (six animals in each group of at least five doses), and subsequently, the 95% confidence limits were transformed into the S.E.M. The experimentally determined Zexp was statistically compared to the theoretically calculated Zadd doses with by Student's t-test, according to the procedures previously described by Tallarida et al. (1989). The isobologram was constructed by connecting the [ED.sub.30] of the Rhodiola rosea extract on the abscissa with the [ED.sub.30] of the corresponding B-vitamins on the ordinate to obtain the additivity line (Tallarida, 1992). The amount of each component in the combination (Zexp and Zadd doses) was also plotted in the same graph. The theoretical additive point lies on a line connecting the [ED.sub.30] values of the individual drugs. Experimental values that lie below and to the left of this additive line are considered to be synergistic or super-additive, whereas values that lie above and to the right of the line demonstrate an attenuated or sub-additive interaction.

The interaction index indicates what fraction of the [ED.sub.30] value of the individual drugs accounts for the corresponding [ED.sub.30] values in the combination. The interaction index, denoted by [gamma], was calculated as follows:

[gamma] = a/A + b/B

where A and B are the [ED.sub.30] values when each drug acts alone and a and b are the values when each drug acts in the combination. A significant difference demonstration of 1 for the relation a/A + b/B was interpreted as a synergistic interaction if [gamma] was <1 and as an antagonistic interaction if [gamma] was >1; the absence of a significant difference was interpreted as an additive effect (Tallarida, 1992).

Statistical analysis

Curves were built by plotting the cumulative time of licking of the injected paw as a function of time. The area under the curve (AUC) of the licking time vs time was calculated by the trapezoidal rule. Dose-dependent curves for each compound tested were established by the percentage of antinociceptive effect which were calculated from the AUC in the reduction in time. The % of the antinociceptive effect was obtained from the AUC of the different treatments relative to the AUC of the vehicle. All results were presented as mean values [+ or -] S.E.M. for at least six animals per group. The statistical analysis was performed using the Student's t-test in Graphpad Prism version 4.0 for Windows (Graphpad Software, San Diego, CA, USA). Statistical significance between the isobolograms of theoretical and experimental points was determined if p< 0.05 by Student's t-test.


Antinociceptive properties of the Rhodiola rosea extract

In the vehicle group, a subcutaneous (s.c.) injection of 2% formalin into the right hind paw produced a typical pattern of licking behavior in mice, which is characterized by a biphasic time course. The first phase starts immediately after formalin injection and declines gradually for approximately 10 min. The second phase increases gradually up to 30 min (Fig. 1A). A nociceptive response in the vehicle group was compared to that observed in the group treated with 81 mg/kg of the Rhodiola rosea extract (Fig. 1A). The ED50 of the Rhodiola rosea extract was calculated from the time course curves obtained at the dosage tested from 10 to 177 mg/kg p.o. At these doses of the Rhodiola rosea extract, the licking time was inhibited in a dose-dependent manner in both phases of the mouse formalin test (Fig. 18). The maximal efficacy was reached at 35.7% and 50.2% antinociception in the neurogenic (first phase) and inflammatory (second phase) stages, respectively, with statistical significance at 177 mg/kg in the first phase and from 30 mg/kg for the second phase (Fig. 1B). It is important to mention that no side effects were observed in the animals that received the Rhodiola rosea extract during this study.

Antinociceptive effect of the individual B-vitamins

Esophageal administration of thiamine ([B.sub.1]-vitamin; 30-707 mg/kg), riboflavin ([B.sub.2]-vitamin; 1-100 mg/kg), pyridoxine ([B.sub.6]-vitamin; 30-707 mg/kg), cyanocobalamin ([B.sub.12]-vitamin 1-177 mg/kg) and the mixture of thiamine, pyridoxine and cyanocobalamin ([B.sub.1] + [B.sub.6] + [B.sub.12] vitamins; 30-562 mg/kg) reduced the licking time both in the first and second phases of the formalin test in a dose- dependent manner. The maximal antinociceptive effect and the [ED.sub.30] produced by the B-vitamins were as follows: 85.2% and 173.5 [+ or -] 29.4mg/kg ([B.sub.1]-vitamin); 55.1% and 5.8 [+ or -] 1.8mg/kg ([B.sub.2]-vitamin); 81.2% and 102.8 [+ or -] 17.5mg/kg ([B.sub.6]-vitamin); 54.8 and 24 [+ or -] 17.5 mg/kg ([B.sub.12]-vitamin); 48.3% and 164.7 [+ or -] 17.5 mg/kg ([B.sub.1] + [B.sub.6] + [B.sub.12] vitamins) (Fig. 2). The ED30 of the B-vitamins and the Rhodiola rosea extract were used in subsequent studies using isobolographic analysis to evaluate the pharmacological interaction between the B vitamins and the extract.

Antinociceptive interaction between the Rhodiola rosea extract and B-vitamins

The individual [ED.sub.30] values for the Rhodiola rosea extract (27.5 [+ or -] 5.2 mg/kg) and the B-vitamins alone or in the mixture were used to determine the experimental values for the five closes of each combination at a 1:1 fixed ratio (Table 1). Dose-response curves of the antinociceptive effects of the Rhodiola rosea extract combined with B-vitamins demonstrated a dose-dependent response with the corresponding [ED.sub.30] as follows: [B.sub.1], 173.5 [+ or -] 29.4; [B.sub.2], 5.8 [+ or -] 1.8; [B.sub.6], 102.8 [+ or -] 17.5; [B.sub.12], 24 [+ or -] 17.5; and [B.sub.1] + [B.sub.6] + [B.sub.12] mixture, 164.7 [+ or -] 17.5 mg/kg (Fig. 3). Isobolograms were built taking these data into account the obtained experimental [ED.sub.30] values (Zexp) of the combinations were less than the calculated theoretic determined ED30 values (Zadd). Because the Zexp value was significantly different from Zadd, all of the combinations evaluated resulted in a synergistic interaction (Fig. 4A--E). The interaction index is the sum of the fraction of each [ED.sub.30] values for each drug alone to the [ED.sub.30] value measured when the same drug is given as part of a co-treatment. The interaction values measured were the following: Rhodiola rosea extract + [B.sub.1], 27.8 [+ or -] 3.8; Rhodiola rosea extract + [B.sub.2], 1.8 [+ or -] 0.2: Rhodiola rosea extract + [B.sub.6], 12.2 [+ or -] 2.3; Rhodiola rosea extract + [B.sub.12], 12.6 [+ or -] 1.7; and Rhodiola rosea extract + [B.sub.1] + [B.sub.6] + [B.sub.12] mix, 17.3 [+ or -] 1.9. As a result, the order of potency for the combinations was Rhodiola rosea extract + [B.sub.2]-vitamin > Rhodiola rosea extract + B-vitamin ([B.sub.1] + [B.sub.6] + [B.sub.12]) mixture > Rhodiola rosea extract + [B.sub.6-] vitamin > Rhodiola rosea extract + [B.sub.12]-vitamin (Table 2).

Table 2 Theoretical (Zadd) and experimental (Zexp) [ED.sub.30]
values for the combinations of the Rhodiolo rosea extract
with the B vitamins and the interaction index values.

Combination   Zadd (mg/kg)        Zexp  Interaction
                               (mg/kg)        index

R. rosea+    00.5 [+ or -]  27.8 [+ or         0.27
[B.sub.1]             14.9      -] 3.8

R. rosea +      16.6 [+ or   1.8 [+ or         0.11
[B.sub.2]            -]2.7      -] 0.2

R. rosea +   65.1 [+ or -]  12.2 [+ or         0.18
[B.sub.6]              9.1      -] 2.3

R. rosea +   25.8 [+ or -]  12.6 [+ or         0.48
[B.sub.12]             5.1      -] 1.7

R. rosea +      96.1 [+ or  17.3 [+ or         0.17
[B.sub.1] +          -]9.1      -] 1.9
[B.sub.6] +

Zadd, theoretical values: Zexp, experimental values.

The antinociceptive effect of the Rhodiola rosea extract and its mechanism of action

Pretreatment with 5 mg/kg flumazenil, s.c.; 0.16 mg/kg WAY 100635, s.c.; or 1 mg/kg naltrexone, s.c., did not alter the nociceptive responses observed in the vehicle group after injection of 2% formalin in any phase of the test (Fig. 5A). However, the antinociceptive effect of the Rhodiola rosea extract (81 mg/kg, p.o.) was partial and significantly (p <0.05) reverted in the presence of flumazenil and WAY100635 (Fig. 5A) but not in the presence of naltrexone (Fig. 5A).

Regarding the nitric oxide pathway, rats that received pretreatment of 10 mg/kg L-NAME or SIN-1, s.c.; 5 mg/kg ODQ i.p.; 10 mg/kg glibenclamide, i.p.; or the vehicle did show a change in the nociceptive response in the formalin test. In contrast, the antinociceptive effect of the Rhodiola rosea extract (81 mg/kg, p.o.) was partially reverted (p <0.05) in the presence of L-NAME, ODQ and glibenclamide but not in the presence of SIN-1 (Fig. 5B).


The use of herbal and natural products or plant extracts together with other medications is a common practice that is increasing in the general population. However, scientific studies that show whether these interactions are beneficial are lacking. In this study, the pharmacological interaction between the antinociceptive activity of Rhodiola rosea ethanol extracts and B-vitamins was investigated by using nociception induced by a formalin injection in mice. Furthermore, we found that the mechanism of action underlying the antinociceptive activity of this plant involved the GABA/BDZ and [5-HT.sub. IA] receptors but not the opioidergic system.

Relative to the large number of possible and common pharmacokinetic and pharmacodynamic herb-drug interactions between supplements and medication, currently only a small number of combinations have been examined (Yu et al., 2009; Li et al., 2011; Zhou et al., 2012). The distinction between "documented" interactions and "potential" interactions needs to be clearly drawn (Boullata, 2005). Preclinical and clinical studies have reported that efficacy of Rhodiola rosea extracts (Panossian and Wagner, 2005; Perfumi and Mattioli, 2007; Montiel-Ruiz et al., 2012) and B-vitamins alone or in a mixture, display anti-inflammatory and antinociceptive properties in experimental models (Abacioglu et al., 2000; Bertollo et al., 2006; Franca et al., 2001) or in humans (Aufiero et al., 2004; Bernstein, 1990; Jolivalt et al., 2009; Sun et al., 2005; Wyatt et al., 1999). Studying the combination of natural products or plant extract with B-vitamins is interesting and might be an advantageous and safe therapy, especially given that the pharmacological effects of each treatment can increase and that a reduction in side effects is probable. ADAPT-232 (Chisan[R]) is a traditional herbal medicinal product consisting of a fixed combination of extracts from Rhodiola rosea root, Schisan drachinensis berry, and Eleutherococcus senticosus root (Panossian et al., 2013). This combination significantly presents adaptogenic properties and it is use to increase tolerance to stress augmenting efficacy on cognitive functions and mental performance (Bogatova et al., 1997). As a afore mentioned, Rhodiola rosea extracts can act as a stimulant and an adaptogen in the central nervous system and B-vitamins are used in human to boost the immune system. As was demonstrated in this study, coadministration of a Rhodiola rosea extract with B-vitamins (vitamins [B.sub.1], [B.sub.2], [B.sub.6], [B.sub.12] and a [B.sub.1] +[B.sub.6]+ [B.sub.12] mixture) produced a synergistic a ntinociceptive effect with no observable side effects in mice.

Many constituents among these species appear to be involved in the adaptogen activity of ADAPT-232, where salidroside, rosavine, and tyrosol belong to Rhodiola rosea (Panossian et al., 2007, 2012, 2013). It has been reported that the active constituent salidroside stimulates the expression of NPY and 72 kDa heat shock protein (Hsp72) in isolated human neuroglia cells (Panossian et al., 2012). As well described by this researching group, the activation of NPY initiates Hsp72 expression in human neuroglia cells, which are known to maintain homeostasis of neuronal cells, where NPY and Hsp72 are involved in an innate defense response to mild stressors (adaptogens), which increases tolerance and adaptation to stress (Panossian et al., 2012). Recently, the levels of phosphorylated MAK pJNIK, and pp38 were increased by L-glutamate treatment but decreased by the treatment with rosin and salidroside in murine microglial BV2 cells (Lee et al., 2013). The possible mechanism of action underlying the antinociceptive response of the B-vitamins has been reported, but that of the Rhodiola rosea remains unclear. It is known that vitamin [B.sub.1] and [B.sub.6], both exert their antinociceptive effects by activating guanylyl cyclase, which may involve the central and/or peripheral L-arginine/nitric oxide/cGMP pathway (Abacioglu et al., 2000, 2001). In injured DRG neurons vitamin B1 reduces hyperexcitability decreases alterations in Na+ and suppresses thermal hyperalgesia (Song et al., 2009). Vitamin B2 produces its antinociceptive and anti-inflammatory effects by activating IC+ channels or nitric oxide release, but not through the activation of the opioid system. The analgesic effect of the B-vitamin mixture is attributed to an increase in the availability and/or effectiveness of noradrenaline and 5-hydroxytryptamine, which are neurotransmitters that inhibit the nociceptive system (Dakshinamurti et al., 1990; Jurna 1998).

In this study, we found that the antinociceptive effects of the Rhodiola rosea extract were reverted in the presence of L-NAME, ODQ and glibenclamide, but an increase was observed in the presence of SIN-1. Our results are in agreement with the previous finding that nitric oxide donors, such as nitroglycerin and SIN-1, decrease hyperalgesia induced by carrageenans (Osborne and Coderre, 1999; Budzinski et al., 2000) and are able to potentiate the peripheral response of other analgesics (Alves et al., 2004). Inhibition of the antinociceptive response of the Rhodiola rosea extract by a blockage of the nitric oxide synthesis, cGMP synthesis or K+ channels suggests that the antinociceptive effect of the extract involves the nitric oxide-cGMP-K+ channel pathway. Our results are in agreement to previous studies reporting participation of nitric oxide and other mediators of nociception in the effects of Rhodiola rosea as stress-protector and adaptogen; it has been evaluated on the stress response markers stress-activated protein kinase/Jun N-terminal protein kinase (SAPK/JNK), the phosphorylated kinase p-SAPK/p-JNK, NO, cortisol, testosterone, prostaglandin E2, leukotriene B4 and thromboxane B2 in rabbits treated with Rhodiola rosea (Panossian et al., 2007; Panossian and Wikman, 2009). Similarly, pretreatment with the antagonist's flumazenil and WAY100635 prevented the antinociceptive effect of the Rhodiola rosea extract. Together, this results show that GABA/BDZs and [5-HT.sub.1A] receptors are involved in the activity of this plant. In vitro studies have reported that Rhodiola rosea extracts increase 5-HT levels in the hippocampus of depressed rats and that this induced neural stem cell proliferation in the cerebral area (Chen et al., 2009: Song et al., 2009). These effects are in concurrence to studies describing that Rhodiola rosea modulates biogenic monoamines at central nervous system, where norepinephrine and dopamine decreased in cerebral cortex and brain stem, but a substantial increase was observed in serotonin, most likely by modifying activity of monoamine oxidase and catechol-O-methyltransferase (Stancheva and Mosharrof, 1987). Moreover, it has been reported that Rhodiola rosea prevents both catecholamine release and subsequent cAMP elevation in the myocardium, and depletion of adrenal catecholamines induced by acute stress (Maslova et al., 1994). Recently profile gene expressions on the human neuroglial cell line showed that Rhodiola rosea reduce the serotonin level by down regulation of 5-1-113 receptors (Panossian et al., 2013).

Effects of Rhodiola rosea have been also associated to an ability to induce opioid peptide biosynthesis and through the activation of both central and peripheral opioid receptors (Lishmanov et al., 1993, 1997; Maimeskulova et al., 1997). However, in this study, the presence of naltrexone did not modify the effects of Rhodiola rosea suggesting that opioid system is not involved under our experimental conditions. It is possible that the synergistic antinociceptive responses obtained with the combination of the Rhodiola rosea extract and the B-vitamins might potentiate their individual anti nociceptive effects because of a multi-target mechanism of action. Finally, it is important to mention that not only pharmacodynamic, but also pharmacokinetic interactions are possible in presence of Rhodiola rosea, given that a significant increase in the bioavailability of losartan after concurrent oral administration to rabbits was reported because they are substrates of both CYPs and P-gp (Spanakis et al., 2013).


These results provide evidence that Rhodiola rosea ethanol extracts in combination with B-vitamins produce a significant diminution in the nociceptive response in a synergistic manner. This process involves several mechanisms of action and suggests that a combination therapy can be used in the design of clinical studies and as a pain therapy.


This work was supported by an SIP-IPN 20130857grant (MDC). This work is part of the PhD dissertation of Montiel-Ruiz Rosa Mariana. Montiel-Ruiz is a Consejo Nacional de Ciencia y Tecnologia (CONACYT) and Programa Integral de Fortalecimiento Institucional-Instituto Politecnico Nacional (PIFI-IPN) fellow.

* Corresponding author at: Seccion de Estudios de Posgrado e Investigacion. Plan de San Luis y Diaz Miron s/n, Col. Santo Tomas, Delegacion Miguel Hidalgo, C.P. 11340: Mexico DF, Mexico. Tel.: +52 57296300x5729.

E-mail addresses:, (M. Deciga-Campos).

0944-7113/$--see front matter [c] 2013 Elsevier GmbH. All rights reserved. http://dx.doi/org/10.1016/j.phymed.2013.07.006


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Rosa Mariana Montiel-Ruiz (a), Maria Eva Gonzalez-Trujano (b), Myrna Deciga-Campos (a), *

(a) Seccion de Estudios de Posgrado e Investigacion, Escuela Superior de Medicina, Instituto Politecnico Nacional, Mexico, DF 11340, Mexico

(b) Laboratorio de Neurofarmacologia de Productos Naturales de to Direccion de Investigaciones en Neurociencias. Instituto Nacional de Psiquiatria Raman de la Fuente Muniz. Mexico. DF 14370, Mexico
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Author:Montiel-Ruiz, Rosa Mariana; Gonzalez-Trujano, Maria Eva; Deciga-Campos, Myrna
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Geographic Code:1MEX
Date:Nov 15, 2013
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