Various properties are attributed to “maca”, including immunomodulatory properties due to its secondary metabolites such as macamides, glucosinolates, isothiocyanates and flavonoids. Immunosuppression, hemolytic anemia, and thymic involution were induced with cyclophosphamide. Three concentrations of doses of dehydrated hydroalcoholic extract of purple maca (EHADM) were used for 30 days, the analysis of variance and Duncan’s multiple comparisons test the results are statistically significant (p<.05) which shows immunostimulatory activity in the marrow bone (monocytes, lymphocytes and white blood cells) and antianemic (hematocrit 31%) compared to the negative control group (G-1). At 84 mg/kg and at 167 mg/kg, it shows immunomodulatory activity on the humoral response in 66.70% of the experimental animals (G-3 and G-4). It is concluded that the dehydrated hydroalcoholic extract of purple maca presents immunostimulating and immunomodulatory activity on the humoral response in 66.7% of the Oryctolagus cuniculus induced to immunodeficiency with cyclophosphamide.

Ecotype, immunosuppression, macamides, purple maca

Lepidium meyenii Walp (maca), belongs to the genus Lepidium and family Brassicaceae (Granados et al. 2020) native to the highlands of the central Andes of Peru that grows at high altitudes (3500–4700) and severe environmental conditions (Chen et al. 2017; Granado et al. 2020); being cultivated in recent years in Tibet, Yunnan and Xinjiang of China (Chen et al. 2017). The hypocotyls of maca are roots of different phenotypic colors such as yellow, white, purple and black, which do not affect the composition of the primary metabolites such as fatty acids (2.2%: linolenic, oleic and palmitic), carbohydrates (59%), proteins (10%: alanine, arginine, aspartate, phenylamine, glycine, glutamate, isoleucine, lysine, leucine, serine, methionine and others) and fibers (8.5%), which are the primary nutritional source (Dini et al. 1994; Clément et al. 2010; Meissner et al. 2016); however, the phenotype influences the composition of secondary metabolites (McCollom et al. 2005; Zhao et al. 2012; Esparza et al. 2015; Meissner et al. 2016; Granados et al. 2020). Among the secondary metabolites, glucosinolates have been determined: isothiocyanates, p-methoxybenzylglucosinolate, m-methoxybenzylglucosinolate, 5-methyl-sulfinylpentyl (glucoalisin), p-hydroxybenzyl glucosinolate (glucosinalbin), glucoscobenantyl-4-enenzine glucosinolate (glucosinalbin), indolyl-3-methylglucosinolate (glucobrassicin) and 4-methoxyindolyl-3-methyl glucosinolate (4-methoxy-glucobrassicin) (Fahey et al. 2001; Dini et al. 2002; Lock and Rojas 2002); among the polyunsaturated fatty acids are macanes (0.15%) and macamides (0.84%) (Ganzera et al. 2002).

The reported macamides (N-benzylated alkamides) are acyclic keto acid, N-benzyloctadecanamide, N-benzyl-16-hydroxy-9-oxo10E, 12E, 14E-octadecatrienamide, N-benz1-9,16-dioxo-10E, 12E, 14E-octadecatrienamide, N-benzylhexadecanamide, N-benzyl-5-oxo-6E, 8E-octadecadienamide, 5-oxo-6E, 8E-octadecadienoic acid and macaridine (1,2-dehydro-N-hydroxypyridine) (Zheng et al. 2000; Lock and Rojas 2002; Zhao et al. 2005; Almukadi et al. 2013).

Hypocotyls have been studied according to the ecotype, reporting that red maca has an inhibitory effect on benign prostatic hyperplasia (BPH), black maca has aphrodisiac properties (Alzamora et al. 2007); in other studies, it is mentioned that macamides are responsible for antifatigue (Yang et al. 2016), antiosteoporotic (Zhang et al. 2006), analgesic, antidepressant and anxiolytic (Almukadi et al. 2013; Hajdu et al. 2014), and neuroprotective (Zhou et al. 2017). In traditional medicine it has been used for centuries to increase sexual activity and fertility (Gonzales et al. 2006; Uchiyama et al. 2014; Chen et al. 2017).

After conducting a review in the PubMed-NCBI database on the studies of the immunomodulatory activity of maca, it was found that the yellow ecotype has antitumor and immunomodulatory activity attributed to the stimulation of macrophages, increasing phagocytosis and production nitric oxide (NO), and other cells of the immune system (Alzamora et al. 2007). In another study it has been reported that the immunomodulatory activity of purple maca is due to alkaloids, macamides, glucosinolates and isothiocyanates (Alvarez and Alzamora-Gonzales 2013). This has prompted a preclinical phase 0 study of in vivo experimental pharmacology with purple maca ecotypes.

Our objective was to study the immunomodulatory activity of Lepidium meyenii Walp (purple maca) in Oryctolagus cuniculus (albino rabbits) induced to immunosuppression with cyclophosphamide.

Figure 1 shows the values ​​of monocytopenia, lymphopenia and leukopenia induced by cyclophosphamide and the immunomodulatory effect of Lepidium meyenii Walp (purple maca) in the samples of agranulocyte white blood cells (monocyte and lymphocyte) obtained from Oryctolagus cuniculus. By analysis of variance (ANOVA) and Duncan’s multiple comparisons test, the administered doses of EHADM for 30 days and after having induced immunosuppression with the alkylating agent are significant (p<.05), except in G-3 lymphocytes and G-5 compared to G-1 (φ not significant).

Figure 1. 

Immunomodulatory effect of Lepidium meyenii Walp (purple maca) on white blood cells agranulocytes of Oryctolagus cuniculus. *Monocyte; ^#lymphocyte; **white blood cell; Duncan’s multiple comparisons: ω significant and φ not significant.

Table 2 Shows the ANOVA and Duncan’s multiple comparisons test, it is observed that the means of agranulocytes (G-3, G-4 and G-5 with G-1) are greater than the critical values ​​to compare two means with corresponding distance in range p (Rp), so the difference is significant (ω), except in two groups of lymphocytes.

After treatment with EHADM for 30 days, the percentage of hematocrit (Fig. 2A) and hemoglobin (g/L) (Fig. 2B) were evaluated in a blood sample obtained from Oryctolagus cuniculus, after the induction of hemolytic anemia immune system generated by cyclophosphamide. The ANOVA showed that at the proposed doses of EHADM the percentage of hematocrit is significant (p <.05) and for the values ​​of g/L of hemoglobin the results were not statistically significant (p>.05). Duncan’s analysis shows that the differences in the hematocrit means (G-3, G-4 and G-5 with G-1) are less than the corresponding Rp, so the difference is not significant (φ).

Figure 2. 

Antianemic effect of Lepidium meyenii Walp (purple maca) determined by hematocrit percentage and hemoglobin weight (g/ L).

The hemagglutination of the rabbit serum was evaluated, after sensitization with GRC, observing serum anti-GRC antibodies (Ig M) in mesh form (positive reaction) in 66.70% in two groups of experimental animals (G-3 and G-4). In the control group (G-1) the presence of a button was observed (negative reaction).

The present study shows that cyclophosphamide caused monocytopenia, lymphopenia, a decrease in the number of white blood cells, a decrease in hematocrit and hemoglobin; involution of the thymus. After administering dehydrated hydroalcoholic extract of purple maca (EHADM) for 30 days, the immunostimulating and immunomodulatory effect was evaluated. At three EHADM dose concentrations, immunostimulatory activity is evidenced in the bone marrow, increasing the population of monocytes (cells of innate immunity), lymphocytes and total white blood cells; and an increase in hematocrit values, with respect to the control group (G-1). In previous studies, Alvarez (2008) demonstrated that the methanolic extract of purple maca induces a percentage increase in leukocytes in mice compared to the control group; Alvarez and Alzamora-Gonzales (2013) reported that yellow maca administered at a dose of 200 mg/kg in immunosuppressed mice favors the production of interleukin mRNA (IL-3 and IL-7 responsible for the stimulation of lymphocytes) and the granulocyte and monocyte colony stimulating factor (GM-SCF) in bone marrow, which supports what was reported in the present study. Lobatón (2003) reported an antianemic effect of maca in 50 people with iron deficiency anemia, as iron (Fe) increased significantly.

Immunomodulatory activity on the humoral response was observed in the present study at two dose concentrations of EHADM (89 mg/kg G-3 and 167 mg/kg G-4), after sensitizing with 10% GRC and inducing immunodeficiency with cyclophosphamide to experimental animals. In an experimental study carried out in mice immunosuppressed with cyclophosphamide, by Alvarez (2008) it is concluded that the administration of the purple maca extract increases the weight and cellularity of the thymus; while Torres (2008) observed that the yellow maca ecotype in immunosuppressed mice induces a high concentration of antibodies against GRC, this is possible, since the yellow ecotype stimulates type 2 helper T lymphocytes (Th2 or CD4+) produce IL-4, IL-5, IL-10, and IL-13, which are responsible for generating antibodies, as indicated by Alzamora et al. (2007). Quispe (2010) promotes the consumption of 3 g/day of the spray of the yellow ecotype of Lepidium peruvianum Chacón, to increase the production of surface antibodies to the hepatitis B virus (anti-HBs), the same that is generated between 1.2 and 6 months after starting the consumption of maca; while the count of Th2 lymphocytes (CD4 + involved in humoral immunity) and CD8+ cytotoxic lymphocytes (involved in cellular immunity) increased 2 months after the start of treatment compared to the control group.

Zhang et al. (2016), characterized a new polysaccharide of 11.3 kDa (MC-1: made up of arabinose, mannose, glucose and galactose) from Lepidium meyenii Walp, evaluating its activity in murine macrophages (Raw 264.7) and its immunomodulatory effect, concluding that MC- 1 stimulates macrophages (phagocytic and pinocytic capacity) and induces the expression of NO, IL-6 and TNF-α. Huayhua (2018) indicates that the methanolic extract of the purple ecotype of Lepidium meyenii Walp (maca) has an activating and immunomodulatory effect on peripheral blood mononuclear cells (PBMC) including T, B, NK lymphocytes, dendritic cells and monocytes, increasing the nitric oxide (NO) production and mRNA expression for TNF-α.

Through in vitro methods, the antioxidant activity of the aqueous extract of maca has been demonstrated, being capable of sequestering DPPH (1,1-diphenyl-2-picryl-hydrazyl) radicals at a concentration of 0.61 mg/mL, and at 0.43 mg/mL sequestering peroxynitrile radicals, the latter being a compound that occurs in chronic inflammations and oxidizes DNA (Sandoval et al. 2002); In a study carried out on macrophages RAW 264.7 it has been shown that at a concentration of 1 mg/mL of aqueous extract of maca it inhibits apoptosis induced by peroxynitrile and maintains the synthesis of intracellular ATP under conditions of oxidative stress caused by hydrogen peroxide (H₂O₂) (Lock and Rojas 2002); However, when making a comparison of the catechin content in maca, it has been shown that it is low (2.5 mg/g) compared to green tea (145 mg/g), so that the antioxidant activity in vitro is higher in tea (Lock and Rojas 2002); while the anthocyanin content in purple corn is 0.86–2.87 mg/g, with a higher antioxidant activity (Ramos et al. 2012). Tang et al. 2017 reported that maca polysaccharides at high doses of 100 mg/kg bw/day of mice could significantly improve the activities of glutathione peroxidase and creatine kinase (p < 0.05), decrease the activity of lactate dehydrogenase (p < 0.01).

Liu (2019) postulates that purple maca contains thymic phytohormones that increase the size and cellular activity of the thymus. Alvarez and Alzamora-Gonzales (2013), have reported that the immunomodulatory activity of purple maca is due to alkaloids, macamides, glucosinolates and isothiocyanates, which stimulate the production of IFN-γ by human T lymphocytes cultured in vitro. Recently Batiha et al. (2020), have described that the anthocyanins and flavonoids (quercetin and catechins) in maca are responsible for the antioxidant and anti-inflammatory activity.

The main limitations of the study lie in the small size of the sample (n = 18), not having extracted the main secondary metabolites of maca and not having individually tested the immunological activity of glucosinolates, macaenes, macamides, flavonoids and anthocyanins. However, the main strength of this preclinical study (phase 0) is to generate scientific evidence of the dehydrated hydroalcoholic extract of purple maca in the immune system, which will allow the initiation of phase 1 clinical studies. In this sense, our research team does not promote the consumption of maca as a treatment, but as a functional food that could help maintain immune capacity.

In conclusion, our results suggest that the dehydrated hydroalcoholic extract of purple maca at the concentrations studied shows significant immunostimulatory and immunomodulatory activity on the humoral response in 66.7% of Oryctolagus cuniculus induced to immunodeficiency with cyclophosphamide.

Peruvian Association of Immunogenomics and Personalized Medicine; Molecular Pharmacology Society of Peru.