Effects of a water extract of Lepidium meyenii root in different models of persistent pain in rats

1 Introduction

Persistent pain is defined as pain lasting for a period of 6 months or more. Primary care settings in Asia, Africa, Europe, and in the Americas had patients reporting persistent pain in approximately a quarter of the population [1]. Arthritis-related diseases, nerve trauma or compression, and neuropathies induced by metabolic syndromes or neurotoxic drugs are among the most common causes of persistent pain. Clinical evidence of neuropathic or inflammatory pain are hyperalgesia, thermal allodynia and spontaneous pain [2]. The choice of therapy depends on the type of pain, its intensity, and how the patient responds to treatment. Drug therapy, however, continues to be inadequate to completely control persistent pain [3]. In this context, plant-derived products characterized by a safety profile (especially those used as food) emerge as interesting resources for individuating novel therapeutic possibilities to manage chronic pain.

Maca (Lepidium meyenii Walp.) is a traditional Andean crop belonging to the Brassicaceae family and it is traditionally used for its nutritional and medicinal properties [4]. Maca’s hypocotils are usually consumed fresh or dehydrated after boiling in water or milk, after being made into juices, maca coffee, or alcoholic beverages [5], [6]. The tubers of L. meyenii have been widely investigated during these last years, pointing out the copresence of two main groups of phytochemicals, the glucosinolates and the macamides and macaenes. The first group is typical of Brassicaceae; in maca, the main forms are aromatic glucosinolates [7], suggested by some authors as chemical markers to explain some biological effects of this plant [8] and denied by others. Over the last few years, different studies that have focused on the analysis and purification of macamides from this plant have been available in the literature. To this aim, lipophilic extracts of roots, such as those by petroleum ether [9] or by n-hexane [10], were selected. Nevertheless, Ganzera et al. [11] highlighted, using high performance liquid chromatography (HPLC), the presence of some macamides also in root powder methanol extract. Because the macamides have not been found in other Lepidium species, they have been proposed as phytochemical markers of maca [10]. Recently, it has been highlighted that some aromatic glucosinolates, abundant in fresh roots, consistently decrease in the dried material because they are involved in the ex novo biosynthesis of macamides, together with free fatty acids [12].

Maca extracts are used for the treatment of a wide range of pathological conditions such as sexual infertility, osteoporosis, and benign prostatic hyperplasia [13], [14]. On the contrary, limited evidence of efficacy against persistent pain has been described up to now [15]. However, in vitro studies confirmed macaenes family components’ involvement in enhancing cannabinoid transmission [16]. The cholinergic stimulation induced by L. meyenii extract (mainly by polyphenols) has been related to memory improvement [17] as well as antioxidant properties that may sustain the antidepressant-like effects of the plant extract [18]. Moreover, maca improves the antioxidant cell machinery [19]. Cannabinoid and cholinergic systems [20], [21], as well as redox unbalance [22], [23], [24], [25], play a relevant role in persistent pain processes. Maca consumption has been associated with lower serum levels of the proinflammatory and pro-algic cytokine IL-6 in populations living in the Peruvian Central Andes [8]. Starting from the theoretical approach that maca is able to modulate endogenous systems normally involved in pain control, the aim of this study was to evaluate the efficacy, after acute intake (oral administration), of a commercial water extract of L. meyenii, controlled in its composition by high performance liquid chromatography with diode array detector (HPLC-DAD) and high performance liquid chromatography-mass spectrometry (HPLC-MS). The study was carried out in rat or mouse models of persistent pain induced by the intra-articular injection of monoiodoacetate (MIA), chronic constriction injury (CCI) of the sciatic nerve and chemotherapy-induced neuropathy.

2 Materials and methods

3 Results

Several analytical methods were proposed to investigate macamides [11] and glucosinolates [7] recognized as the main bioactive phytochemicals of maca. According to these findings, the aim of this work was to check the presence of these two groups of molecules in the commercial extract selected for this in vivo study. The screening was done by HPLC-DAD-MS working on two extracts, at different polarities, obtained from the same commercial sample. The chromatographic profiles of the W extract on the Gemini^® column did not reveal the presence of analytes detectable in the 220–330 nm range. The same test on the HILIC column, specifically selected to highlight the glucosinolates, confirmed the absence of these compounds and the presence of several diglycosides and oligoglycosides, as expected. On the contrary, the AM extract after the HPLC-DAD-MS screening in positive and negative ionization mode, showed the best result in positive mode. By LC-MS several macamides were detected by their intense [M+H]⁺ and/or [M+Na]⁺ ions. Supplementary Figure S1A shows the base peak chromatogram in the 300–450 m/z range, selected in agreement with a recent article focused on the comparison of macamides in different batches of maca hypocotyls [9]. To summarize, some representative extract ion profiles at the selected masses are reported in Supplementary Figure S1B, whereas all the detected macamides are listed in Table 1.

The DAD profile also allowed us to point out a group of lipophilic metabolites with retention time values close to that of macamides and characterized by their UV-Vis spectra and ion molecular weight as reported in Supplementary Figure S2. To date, further information is not available on these compounds.

The pain reliever effect of L. meyenii extract was evaluated in several models of persistent pain by behavioral tests. In Figure 1, the effect of MIA administration in rats is shown. Fourteen days after MIA injection, the paw pressure test lead us to appreciate that the weight tolerated on the ipsilateral paw was significantly reduced compared with the contralateral (data not shown). A total of 10 g kg⁻¹ of L. meyenii extract time dependently reduced MIA-evoked mechanical hypersensitivity increasing the weight tolerated up to 70.8±2.2 g (Figure 1A). Figure 1B shows the difference between the weight burdened on the contralateral and the ipsilateral limb. ∆Weight was significantly increased in MIA+vehicle-treated rats (67.6±1.6 g) in comparison to vehicle+vehicle-treated rats (5.0±1.5 g). Lepidium meyenii (10 g kg⁻¹) was able to statistically reduce hind limb weight-bearing alterations peaking 15 min after treatment (41.1±6.0 g). Lepidium meyenii extract was also effective on neuropathic pain induced in rats by lesion to the peripheral nervous system. Rat paw pressure test revealed that 14 days after CCI, the weight tolerated on the ipsilateral paw significantly decreased (43.3±3.3 g) with respect to the contralateral (68.3±3.1 g) (data not shown). As depicted in Figure 2A, 10 g kg⁻¹ of L. meyenii completely reverted the CCI-induced hyperalgesia for 30 min after administration. Moreover, the effect of the extract was also evaluated by measuring spontaneous pain of CCI rats with the incapacitance test. As shown in Figure 2B, 10 g kg⁻¹ of L. meyenii extract reduced the difference between the weight burdened on the contralateral and the ipsilateral limb by approximately 45%. A different kind of neuropathic pain was induced in mice by the administration of the neurotoxic drugs oxaliplatin and paclitaxel. The platin-derivative anticancer compound was repeatedly administered and, on day 14, it induced a lowered threshold to cold stimuli that was measured by cold plate test (Figure 3A the licking latency of oxaliplatin+vehicle-treated mice is 11.6±0.7 s vs. 21.2±1.2 s of vehicle+vehicle-treated mice). Lepidium meyenii extract, acutely administered in a dose range of 0.5–10 g kg⁻¹, reverted oxaliplatin-induced neuropathic pain in a dose-dependent manner. A total of 10 g kg⁻¹ evoked the longest significant effect peaking 30 min after administration (20±0.9 s). Moreover, 3 and 1.5 g kg⁻¹ induced a statistically significant effect. In Figure 3B, the effect of repeated paclitaxel administrations in mice is shown. On day 14, the taxane derivative decreased the time that animals tolerate on a cold surface (11.5±0.9 s, paclitaxel+vehicle vs. 23.4±1.7 s, vehicle+vehicle). Lepidium meyenii extract was administered at a dose range of 0.5–10 g kg⁻¹. As depicted in Figure 3B, 0.5, 1.5, and 3 g kg⁻¹ doses induced a significant effect, whereas the highest dose significantly increased licking latency peaking (21.6±1.6 s) 30 min after the administration. Table 2 displays the effect of the maximum tested dose of L. meyenii (10 g kg⁻¹) on animals’ motor coordination as evaluated by rotarod test. Fifteen minutes after extract administration, the animals were placed on the rotating rod (10 rpm) for 10 min and the number of falls was counted. Administration of L. meyenii extract did not induce significant alterations of motor coordination in comparison with the control group.

Figure 1:

Articular pain.

On day 14 after MIA injection, L. meyenii extract (10 g kg⁻¹) was p.o. administered and pain threshold was evaluated over time. (A) Response to a noxious mechanical stimulus evaluated by paw pressure test on the ipsilateral paw. (B) Hind limb weight-bearing alterations were measured by incapacitance test. n=10; ^*P<0.01 versus vehicle+vehicle-treated rats; ^P<0.05 and ^^P<0.01 versus MIA+vehicle-treated rats.

Figure 2:

CCI-induced neuropathic pain.

On day 14 after nerve surgery, L. meyenii extract (10 g kg⁻¹) was p.o. administered and pain threshold was evaluated over time. (A) Response to a noxious mechanical stimulus evaluated by paw pressure test on the ipsilateral paw. (B) Hind limb weight-bearing alterations were measured by incapacitance test 14 days after surgery. n=10; ^**P<0.01 versus vehicle+vehicle-treated rats; ^P<0.05 and ^^P<0.01 versus CCI+vehicle-treated rats.

Figure 3:

Chemotherapy-induced neuropathic pain.

(A) Oxaliplatin-induced neuropathic pain. Mice were treated i.p. daily with 2.4 mg kg⁻¹ oxaliplatin. (B) Paclitaxel-induced neuropathic pain. Mice were treated i.p. (on days 1, 3, 5, and 7) with 2 mg kg⁻¹ paclitaxel. The response to a thermal non-noxious stimulus was evaluated by cold plate test. Behavioral test was performed on day 14. Before and after L. meyenii extract (0.5–10 g kg⁻¹) p.o. administration. n=10; ^*P<0.05 versus vehicle+vehicle; ^P<0.05 and ^^P<0.01 versus oxaliplatin+vehicle; ^P<0.05 and ^^P<0.01 versus paclitaxel+vehicle.

4 Discussion

The present results describe the pain-relieving effects of an aqueous extract of L. meyenii, in three different models of persistent pain. In particular, the intra-articular injection of MIA provides a monolateral osteoarthritis characterized by a persistent inflammatory pain which, starting on day 14 after injection, possesses a neuropathic component [36]. Ten gram per kilogram of maca extract reduces pain induced by a mechanical noxious stimulus on the ipsilateral paw. Moreover, maca-dependent pain relief allows also to reduce postural unbalance, an outward of osteoarthritis progression, as measured by hind limb weight bearing alterations [34]. The anti-neuropathic properties of the L. meyenii extract are confirmed after the ligation of the sciatic nerve, a neuropathy model where the alterations of the nerve morphology are accompanied by remarkable inflammatory state emerged in terms of cellular infiltrate and edema [37], [38]. Maca extract, at maximum dosage, is able to revert mechanical hypersensitivity for 30 min, after injection, as shown by paw pressure test. Ten gram per kilogram of L. meyenii significantly reduces the postural unbalance induced by the nerve ligation as revealed by the incapacitance test. Our results also demonstrate that a single oral administration of maca extract (10 g kg⁻¹) is able to completely revert the thermal allodynia induced in mice after oxaliplatin or paclitaxel chronic treatment. The platinum and the taxane derivatives are used for treatment of several types of cancer as colorectal cancer, lung cancer or ovarian cancer [39], [40]. Their chronic administration causes a peripheral neuropathy characterized by hypersensitivity to cold stimuli [41], [42]. These models of neuropathic pain are free from inflammatory components and are characterized by specific molecular alteration of the peripheral and central nervous system [35]. The maintenance of efficacy in these different kinds of pain suggests an interesting pharmacodynamic profile. The dosages used are quite high but compatible with the use as food supplement. Furthermore, no signs of evident toxicity are highlighted in all treatment groups; in particular, as shown by the rotarod test performances, no motor or neurological alteration was detectable. This maca sample was obtained using an aqueous extraction of the root, as declared in the label, and consequently was not rich in lyphophilic components. Nevertheless, the high sensitivity of a mass spectrometer, differently from the DAD, allowed to detect the unexpected presence of several macamides tentatively identified according to their molecular weight [9].

Furthermore, by use of the HILIC column, suitable to analyzing very polar compounds, the absence of glucosinolates in our sample was confirmed. This finding is in agreement with a recent work that correlates the low content of glucosinolates in dried maca roots to the increase of macamides after the drying process. The hydrolytic phenomena associated with open-air drying are responsible of the formation of the precursors of macamides, which continue to be synthetized until a water content close to 13% is achieved [12]. Even if in low amounts, the macamides can contribute to the observed effect in rats.

At the end, it is worth noting the presence of a group of unknown metabolites with UV-Vis spectra indicating a chromophoric system that can indicate a possible antioxidant capacity that can contribute to the biological effects of this commercial maca sample. At the same time, the shape of these spectra did not confirm the presence of typical flavonoids or flavones, some of them previously found in maca. In vitro studies have demonstrated that macaenes and macamides are able to inhibit the FAAH enzyme, normally responsible for inactivation of anandamide, the main endogenous endocannabinoid. Anandamide is involved in several physiological processes such as painful sensitivity and neuroprotection [16]. Selective FAAH inhibitors enhance anandamide endogenous levels that cause antinociceptive effect by agonism on the CB₂ receptor [21]. Furthermore, many preclinical studies demonstrated that FAAH inhibitors reduce chronic pain without the development of tolerance after repeated administrations [43]. The absence of flavonoids in the present extract was confirmed by the investigation on the UV-vis and mass spectra registered, respectively, by HPLC-DAD and by MS at different fragmentation energies. Only a recent work cited two new flavolignans [44]. By the mass experiments, it was possible to exclude the presence of these compounds in our extract. At the same time, other known phenolic compounds were also reported in the same work, as pinoresinol and tricine were not detected. These results strongly suggest the exclusion of both cholinergic system modulation and antioxidant effects of phenolic compounds from the pharmacodynamic mechanisms. The absence of the glucosinolates, confirmed by mass spectrometric analyses, allowed us to exclude the contribution of these phytochemicals for the biological effect observed for the maca extract.

In conclusion, L. meyenii extract is effective as a pain reliever in different models of persistent pain, both in those with a predominant inflammatory component and in those with a predominant neuropathic aspect. The absence of evident toxicity, confirmed by its common use as a customary refreshment as well as a traditional remedy by Peruvian people, shows that L. meyenii extract is a possible treatment against osteoarticular and neuropathic pain.