Evaluation of the antinociceptive effect of valerian and hops combination in experimental animal models: Involvement of the opioid system

Abstract

Pain is a common undertreated worldwide complaint. The need to explore the antinociceptive potential of alternative herbal products is essential. Although used as a mild sedative, limited evidence focused on the potential antinociceptive effect of valerian and hops combination. The present study was carried out to evaluate the in vivo anti-nociceptive effect of the valerian-hops combination to justify its use as an effective and safe analgesic agent. Anti-nociceptive effects of valerian-hops combination (50, 100, and 200 mg/kg) were assessed in swiss albino mice for performing the acetic acid-induced writhing test, the paw licking test using formalin, the paw licking test using glutamate, and the tail immersion test. The effects were compared to those of diclofenac or morphine in the presence or absence of the opioid receptor antagonist naloxone. Valerian-hops” extract of 100 and 200 mg/kg demonstrated a significant reduction in the number of writhing episodes induced by acetic acid compared to the control (p < 0.05), a significant reduction in the licking number at doses of 100 and 200 mg/kg in the late phase formalin-induced paw licking, significantly reduced the number of lickings after glutamate injection compared to control (p < 0.05). And significantly increased pain reaction after 60 and 90 min of tail immersion test, this effect was opposed by naloxone treatment. The valerian-hops combination produced a significant antinociceptive effect that involved the opioid system. Further studies are required to fully uncover the underlying active constituents and their mechanisms.

1. Introduction

Pain is an unpleasant sensory and emotional experience associated with actual or potential tissue damage or described in terms of such damage as defined by the International Association for the Study of Pain [1]. Pain is one of the foremost reasons for why individuals seek medical help in primary care [2].

According to the American Pain Foundation (APF), it is estimated that 100 million Americans suffer from pain, with approximately 25 million experiencing acute pain from injury or surgery and 75 million suffering from chronic pain conditions in 2007 reviewed in Ref. [2]. In Europe, 11.2 million reported severe pain, 29.4 million reported moderate pain and 9.0 million reported mild pain. The population prevalence of daily pain is 8.85% with 3.47% reporting severe daily pain and 4.70% moderate daily pain in 2011 [3]. Nociceptive pain is thought to be by far the most common human pain type, Nociception is the detection of tissue damage by specialized transducers attached to A-delta and C fibers, Nociceptors are usually activated by strong heat, intense cold, and harsh mechanical stimuli [4].

Nociceptive pain is managed by using Nonsteroidal Anti-inflammatory Drugs (NSAIDs) for mild to moderate pain severity and opioid analgesics for moderate to severe pain. Although NSAIDs are widely prescribed therapeutics, however, they are associated with several significant adverse effects with long-term use such as bleeding, peptic ulcer, and gastrointestinal lesion [5] Moreover, they are contraindicated in hypertensive patients and asthma patients [6]. Likewise, opioids are associated with many limitations such as constipation, respiratory depression, tolerance, dependence, and addiction [7,8].

Therefore, discovering and developing effective and safe alternative or adjuvant analgesics is a fundamental concern. The study of natural products is getting high attention [9]. A wide range of natural plants found their way from traditional folk remedies to modern medicine [10].

In the United States, valerian is regulated by the Food and Drug Administration (FDA) as a dietary supplement to alleviate anxiety (https://ods.od.nih.gov/factsheets/Valerian-HealthProfessional/).

Valerian root of (Valeriana -hops Valerianacea) is often combined with Hops strobile (Humulus lupulus L., Cannabaceae) to enhance its sedative and anxiolytic efficacy [11]. The valerian-hops combination is commercially available worldwide as an anxiolytic preparation [12]. Valerian is originally native to Asia and Europe [13] and hops plants are distributed throughout North America, Europe, and Asia [14].

The evidence of the potential analgesic effect of valerian is still emerging and controversial. Valerian demonstrated efficacy in dysmenorrhea as antispasmodic [15] most likely due to its calcium channel-blocking actions that inhibit intracellular calcium influx thereby decreasing muscle contractions [16]. While some studies an analgesic effect of valerian seen on acetic acid-induced writhing test and formalin-induced pain models in rodents [[17], [18], [19], [20]], however, according to Ref. [21] valerian infusion extract demonstrated anti-inflammatory activity as seen on the rat paw edema model compared to aspirin but lacked analgesic activity on the acetic acid-induced writhing test. Hops has analgesic and anti-inflammatory actions mediated by its’ active constituent Isohamolone [22,23].

We hypothesize that the addition of hops could result in substantial antinociception in animal models.

Therefore, the present study was carried out to evaluate the in vivo anti-nociceptive effect of the valerian-hops combination to justify its use as an analgesic agent.

2. Methods

2.1. Extract preparation

Commercial Valerian root (Valeriana -hops)/hops strobile (Humulus lupulus) dried aqueous extract was generously donated from Roha Company (Germany). Valerian/hops dried extracts were prepared according to Roha's recommendation. Briefly, chopped valerian roots and hops strobile were extracted separately by drinking water at temperatures (temp for valerian = 30-40 °C and temp for hops = 70–85 °C) by exhausting percolation method. Ratios of the drug to extraction agents were 1:8–10 for valerian extract, and 1:12–18 for hops strobile extract. Ratios of drug-to-drug preparation carrier medium were 4:6–1 for valerian extract and 3:6–1 for hops strobile extract. Carrier medium composed of 2% highly dispersed silicon dioxide and Maltodextrin in a percentage of 13% for valerian extract and 28% for hops extract. Extracts were concentrated by vacuum evaporation at t = 45 ± 5 °C, reduction of microbial content was achieved by high-temperature short-time pasteurization −5 s at 130 °C for valerian extract, and 3–5 s at 140–143 °C for hops strobile extract. Extracts were separately dried by spray dryer at t = 50–60 °C then ground and sieved to be ready for use as 85% dry extract of valerian roots and 70% dry extract of hops strobile.

2.2. Experimental animals

24–30 g male 4 weeks old Swiss albino mice were used for performing the experiments. Mice were kept in the animal house under the proper conditions. One hour before applying the experiments, mice were moved to the testing area to adapt to the laboratory conditions to decrease stress. All experiments on animals were carried out in accordance with the U.K. Animals (Scientific Procedures) Act, 1986, and associated guidelines. The animals were maintained under a 12-h light/dark cycle, at relative humidity of 50–60% and room temperatuof re 21 ± 1 °C. The study protocol was approved by the Hashemite University IRB. No. 20/2/2018–2019.

2.3. Acetic acid-induced writhing test

As described before [24], in this experiment five groups of mice were treated intraperitoneally (i.p) as follows: the first group received normal saline as vehicle control; group II received 10 mg/kg diclofenac sodium (Sigma- Aldrich). Valerian-hops extract was given to groups two to five (50, 100, and 200 mg/kg, respectively). Acetic acid (0.6%, purity ≥99.0%, 10 ml/kg) (Sigma- Aldrich) was administered for each mouse 60 min after Valerian -hops extract, vehicle, or diclofenac sodium treatments. Complete writhing was recorded and counted for 30 min after acetic acid treatment. The occurrence of body elongation, abdomen contraction, pelvis ending twisting, and/or trunk twisting associated with limb extension was counted as writhing. Inhibition of writhing was considered as the results of this experiment which were represented as percent according to the following formula:

Percent of inhibition of writhing (PIW) = [(Writhing number (Control) – writhing number (treatment))/Writhing number (control)] × 100.

2.4. Paw licking test using formalin

As described in Ref. [25] six groups of mice were treated as follows: group one received 0.1 ml normal saline as vehicle control, group two received 10 mg/kg diclofenac sodium, groups three to five received 50, 100, 200 mg/kg Valerian -hops extract, respectively. An Intraperitoneal route of administration was performed for all treatments. 60 min after treatments, 20 μl of 2.5% formalin (purity ≥37%) (Sigma- Aldrich) were injected into the sub-plantar region of the right hind paw to induce pain. The nociceptive response was recorded by measuring the time for each mouse while licking the formalin injection site. Licking time was recorded at zero to 5 min after formalin injection, representing the early (neurogenic) phase, and 15 to 30 min after formalin injection, which represents the late (inflammatory) phase. Percent of licking inhibition was calculated according to the following formula:

Percent inhibition of licking (PIL) = [(Licking time (control)- Licking time (treatment))/Licking time (control)] × 100.

2.5. Paw licking test using glutamate

This test was performed to assess the antinociceptive role of valerian-hops extract through the glutamatergic system. As described in Ref. [26], five groups of mice were treated as follows: 0.1 ml normal saline to the first group as vehicle control, group two received 10 mg/kg diclofenac sodium, whilst groups three to five received Valerian -hops extract (50, 100, 200 mg/kg, i.p. respectively). 20 μl of glutamate (Sigma-Aldrich, purity ≥98%, 10 μmol/paw) (Sigma- Aldrich) were injected via intraplantar route, and the animals were observed immediately for 15 min to measure the time each mouse took a licking and/or biting of the glutamate injection site.

2.6. Tail immersion test

The method described by Uma-Devi was used for this experiment. 66 albino mice were randomly divided into 11 groups with six mice each. Group (1) was treated with normal saline and served as control; group (2) was with morphine (5 mg/kg) (Sigma- Aldrich); groups (3–5) received extract (50, 100, 200 mg/kg respectively); group (6) was treated with Naloxone (Sigma- Aldrich); group (7) was treated with normal saline and Naloxone. Group (8) received Naloxone and morphine; Groups (9-11) were treated with Naloxone and extract (50, 1and 00, 200 mg/kg respectively). Then about 2–3 cm of the tail of each of the mice was dipped into a water bath containing warm water maintained at a temperature of 50 ± 1 °C and the time taken for the mice to flick its tail or withdraw it from the warm water known as the pain reaction time was recorded for all the micut-off cut off time was put at 15 s.

2.7. Statistical analysis

IBM SPSS statistics software (IBM, USA) was used for the analysis of the data. Data were represented as mean ± SD. The data were compared using one-way ANOVA followed by Tukey post-hoc test. P < 0.05 was taken as the cut-of value for statistical significance.

3. Results

3.1. Acetic acid-induced writhing

The intraperitoneal administration of “Valerian-hops” extract of 50 mg/kg has not shown a significant reduction in the number of writhing episodes induced by acetic acid (Fig. 1, n = 6), however, “Valerian-hops” extract of 100 and 200 mg/kg demonstrated a significant reduction in the number of writhing episodes induced by acetic acid compared to the control (n = 6, P < 0.05) and the highest reduction was observed at a dose of 200 mg/kg. The reference drug, diclofenac sodium 10 mg/kg, also reduced the number of writhing episodes induced by acetic acid (Fig. 1, n = 6, p < 0.05).

Fig. 1.

Effect of aqueous extract of Valerian -hops (50, 100, 200 mg/kg) alone and diclofenac sodium (10 mg/kg) on writhing response induced by acetic acid in mice. Treatments were given 30min before i.p. injection of acetic acid (0.6%). A number of animals per group (n) = 6. *p < 0.05 Significantly different compared to respective control.

3.2. Formalin-induced paw-licking test

The number of lickings after formalin injection was not reduced with the administration of “Valerian-hops” extract with all doses in the early phase (Fig. 2A, n = 6). However, the “Valerian-hops” extract has demonstrated a significant reduction in the licking number at doses of 100 and 200 mg/kg in the late phase (Fig. 2B, n = 6, p < 0.05). Diclofenac sodium, the reference drug, has demonstrated a significant reduction in the licking number in both early and late phases (Fig. 2A&B, n = 6, p < 0.05).

Fig. 2.

Effect of aqueous extract of Valerian -hops (50, 100, 200 mg/kg) and diclofenac sodium (10 mg/kg) on a number of licking induced by intraplantar injection of formalin ((2.5%, 20 μl/paw) in mice. Treatments were given 60min before injection of formalin. A number of animals per group (n) = 6. *p < 0.05 Significantly different compared to respective control.

3.3. Glutamate-induced paw-licking response

Fig. 3 shows that “Valerian-hops” extract treatments (50, 100, 200 mg/kg) significantly reduced the number of lickings after glutamate injection in a dose-dependent manner compared to the control (Fig. 3, n = 6, p < 0.05). Diclofenac sodium has also shown a significant reduction in the number of lickings after glutamate injection compared to control (Fig. 3, n = 6, p < 0.05).

Fig. 3.

Effect of aqueous extract of Valerian-hops (50, 100, 200 mg/kg) and diclofenac sodium (10 mg/kg) on a number of licking induced by intraplantar injection of glutamate (10μMol/paw) in mice. Treatments were given 30min before injection of glutamate. Several animals per group (n) = 6. *p < 0.05 Significantly different compared to respective control.

3.4. Tail immersion test

“Valerian-hops” extract has not demonstrated an increase in pain reaction time after 30 min of treatment compared to control at all doses (Table 1, n = 6). The same result was observed after 30 min of morphine treatment. On contrary, after 60 min of “Valerian-hops” extract treatments (50, 100, and 200 mg/kg) significantly increased pain reaction time at doses of 100 and 200 mg/kg, the pain reaction time was significantly increased compared to the control (Table 1, n = 6, p < 0.05). The reference drug, morphine, also demonstrated a significant increase in pain reaction time compared to contthe rol (Table 1, n = 6, p < 0.05). Interestingly, after 90 min of “Valerian-hops” extract treatments (50, 100, 200 mg/kg) a significant increase in pain response time was observed only with a dose of 200 mg/kg comparedthe to control which was comparable to the morphine effect (Table 1, n = 6, p < 0.05). Naloxone, the non-selective opioid receptor antagonist, was administered to assess the involvement of the opioidergic system in the antinociceptive effect of Valerian-hops extract. As expected, pain reaction time was significantly reduced after administration of naloxone with morphine compared to morphine alone after 30 and 60 min of treatment (Table 1, n = 6, p < 0.05). Naloxone administration with 100 mg/kg “Valerian-hops” extract treatments (50, 100, 200 mg/kg) significantly reduced the number of lickings after glutamate injection extract has reduced the pain reaction time compared to 10Valerian-hopsan- hops extract alone after 90 min of treatment (Table 1, n = 6, p < 0.05). Moreover, pain reaction time was significantly reduced after administration of naloxone with 200 mg/kg “Valerian-hops” extract treatments (50, 100, 200 mg/kg) significantly reduced the number of lickings after glutamate injection extract after 30 and 90 min of treatment (Table 1, n = 6, p < 0.05), suggesting that Valerian -hops extract anti-nociceptive effect.

Table 1.

Antinociceptive effect of Valerian -hops extract, morphine, and naloxone in tail immersion test.

Treatment (mg/kg)	Response time (s)
	Pretreatment	30min	60min	90min
Control (0.1ml/mouse)	2.04 ± 0.12	2.51 ± 0.11	2.62 ± 0.15	3.1 ± 0.15
Morphine (5)	1.87 ± 0.08	3.23 ± 0.09	3.65 ± 0.21*	4.52 ± 0.24*
Extract 50 mg	2.31 ± 0.12	2.51 ± 0.12	3.12 ± 0.24	3.41 ± 0.11
Extract 100 mg	2.23 ± 0.10	2.82 ± 0.15	3.74 ± 0.23*	3.99 ± 0.18
Extract 200 mg	2.25 ± 0.06	3.21 ± 0.07	3.95 ± 0.15*	4.35 ± 0.16*
NLX (2)	2.21 ± 0.13	2.41 ± 0.14	2.93 ± 0.21	2.62 ± 0.09
Control (0.1ml/mouse) + NLX (2)	1.95 ± 0.08	2.23 ± 0.09	2.28 ± 0.14	2.24 ± 0.10
NLX (2)+ Morphine (5)	2.10 ± 0.07	2.45 ± 0.14	2.84 ± 0.11a	3.15 ± 0.14a
NLX (2)+ Extract 50 mg	2.12 ± 0.15	2.38 ± 0.09	2.65 ± 0.22	2.89 ± 0.19
NLX (2)+ Extract 100 mg	2.31 ± 0.11	2.47 ± 0.10	2.99 ± 0.18	3.15 ± 0.14b
NLX (2)+ Extract 200 mg	2.23 ± 0.09	2.55 ± 0.11	3.31 ± 0.12c	3.47 ± 0.13c

Each value is presented as the Mean ± SEM (n = 6); NLX=Naloxone.

*p < 0.001 compared with control group.

^ap < 0.001 compared with morphine group.

^bp < 0.05 compared with the extract 100 group.

^cp < 0.05 compared with the extract 200 group.

4. Discussion

The current study aimed at evaluating the anti-nociceptive effect of valerian/hops combination in an attempt to justify its future possible role as an analgesic. Our findings showed a significant anti-nociceptive effect of the valerian-hops combination in the acetic-acid-induced writhing test, the formalin-induced paw test, the glutamate-induced paw licking test, and the tail immersion test. Our study adds up to the existing literature about the role of valerian-hops in central and peripheral effects in pain management possibly via a synergy between the active constituents in the two herbs such as valerenic acid and isohamolone.

While no previously published data addressed the antinociceptive effects of the valerian-hops combination, some findings from very limited publications highlighted the antinociceptive properties of valerian and hop separately support our results.

In the acetic acid test, valerian hops inhibited acetic acid-induced nociception. Our results are in line with previous reports that showed similar results [[27], [28], [29], [30]]. Although the mechanism is not well understood it could be related to the suppression of prostaglandin synthesis by several anti-inflammatory constituents of valerian-hops [17].

In the formalin-induced paw-licking test, valerian hops significantly decreased the pain score in the late phase. One similar study reported [19] that valerian root resulted in a significant reduction in the pain score in the formalin model. This can be explained by the effect of flavonoids and phenolic compounds of valerian-hops to inhibit the synthesis of nitric oxide, a hyperalgesia mediator [19]. Our previous studies (data unpublished) showed that valerian-hops decreased nitric oxide in stressed mice, furthermore, data showed that flavonoids suppress nitric oxide after formalin injection leading to an analgesic effect [31].

To assess the ability of valerian hops to interfere with glutamate-mediated nociceptive transmission, a glutamate-induced paw-licking test was performed. Valerian-hops extract treatments (50, 100, 200 mg/kg) significantly reduced the number of lickings after glutamate injection in a dose-dependent manner compared to the control (Fig. 3, n = 6, p < 0.05).

Glutamate, an important excitatory amino acid neurotransmitter, is most widely distributed in the central nervous system and induces nociceptive transmission at the peripheral, spinal and supra-spinal sites [32]. Additionally, the release of nitric oxide (NO) or some NO-derived substances, as well as activation of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors and N-methyl-d-aspartate (NMDA) receptors can mediate the nociceptive response induced by glutamate including pro-inflammatory mediators such as cytokines, which enhance the inflammatory reaction [26,33].

Our findings can be explained through an interaction of the valerian-hops extract with the glutamatergic system. The interaction between valerian extract and valerenic acid in precise with the glutamate receptor is documented [34] It was evident that the valerian anxiolytic effect is partially attained via interacting as a modest NMDA antagonist [35,36].

The tail immersion method indicated the central analgesic effect of the valerian-hops combination extract was revealed by the increased reaction time after giving thermal stimulus to the mice. Also, the involvement of the opioid system is evident due to the naloxone antagonizing effect of the extract. The valerian-hops combination is known for its central effects as anxiolytic and sleep aid [[37], [38], [39]], additionally was beneficial in alleviating heroin withdrawal symptoms in addicts [40] suggesting a possible interaction with the opioid system, in addition, the analgesic effect of hops might involve the opioidergic system [29], however more studies are needed to clarify its interaction with the opioidergic system.

In contrast, the valerian south African extract failed to suppress the acetic acid-induced writhing [21]. This can be explained by the differences in species, subspecies, and constituents. There are more than 350 species and several more subspecies of valerian worldwide [41] with over 150 constituents reported to have different biological activities. The climatic conditions, processing, and storage conditions play a role in the constituents’ concentrations and hence their effects [42,43].

Pain is still considered an undertreated medical condition worldwide, despite the availability of numerous analgesics. Exploring the antinociceptive effects of a well-established effective and safe herbal combination such as valerian hops provides an incremental advancement in this therapeutic area.

This is the first study that explores the antinociceptive properties of the valerian-hops combination on animal pain models. The valerian-hops combination is already an over-the-counter anxiolytic supplement, findings of this research can pave the road to broaden its indication as an adjuvant analgesic providing its high safety and tolerability profile compared to the NSAIDs and opioids. The study limitations include the small sample size and the use of the whole extract rather than the active constituents. Next step tis o isolate and characterize the active constituents of the extract and uncover their antinociceptive mechanisms.

Author contribution statement

Omar Gammoh: Conceived and designed the experiments; Analyzed and interpreted the data; Wrote the paper.

Esam Anais: Conceived and designed the experiments; Analyzed and interpreted the data; Contributed reagents, materials, analysis tools or data.

Yousra Bseiso, Khaled Alrosan: Performed the experiments; Contributed reagents, materials, analysis tools or data.

Abdelrahim Alqudah: Performed the experiments; Analyzed and interpreted the data; Wrote the paper.

Funding statement

Dr Omar Gammoh was supported by Hashemite University.

Dr Omar Gammoh was supported by Yarmouk University.

Data availability statement

Data will be made available on request.

Declaration of interest’s statement

The authors declare no conflict of interest.