Medicinal Plants from Mexico, Central America, and the Caribbean Used as Immunostimulants

A literature review was undertaken by analyzing distinguished books, undergraduate and postgraduate theses, and peer-reviewed scientific articles and by consulting worldwide accepted scientific databases, such as SCOPUS, Web of Science, SCIELO, Medline, and Google Scholar. Medicinal plants used as immunostimulants were classified into two categories: (1) plants with pharmacological studies and (2) plants without pharmacological research. Medicinal plants with pharmacological studies of their immunostimulatory properties were subclassified into four groups as follows: (a) plant extracts evaluated for in vitro effects, (b) plant extracts with documented in vivo effects, (c) active compounds tested on in vitro studies, and (d) active compounds assayed in animal models. Pharmacological studies have been conducted on 29 of the plants, including extracts and compounds, whereas 75 plants lack pharmacological studies regarding their immunostimulatory activity. Medicinal plants were experimentally studied in vitro (19 plants) and in vivo (8 plants). A total of 12 compounds isolated from medicinal plants used as immunostimulants have been tested using in vitro (11 compounds) and in vivo (2 compounds) assays. This review clearly indicates the need to perform scientific studies with medicinal flora from Mexico, Central America, and the Caribbean, to obtain new immunostimulatory agents.


Introduction
The immune system is a complex organization of leukocytes, antibodies, and blood factors that protect the body against pathogens [1]. Innate immunity consists of cells such as lymphocytes, macrophages, and natural killer (NK) cells, which are the first line of host defence [2,3]. The NK cells lyse pathogens and tumor cells without prior sensitization [4]. Activated macrophages defend the host by phagocytosis, releasing the enzyme lysosomal acid phosphatase, and through the synthesis and release of nitrous oxide (NO) and hydrogen peroxide (H 2 O 2 ) [5,6]. These two components inhibit the mitochondrial respiration and the DNA replication of pathogens and cancer cells [7]. When an infection occurs, macrophages and mast cells immediately release interleukins [2]. The interleukins link the communication between cells of the immune system, facilitating innate immune reactions. Among these cytokines, IL-2 and IL-6 induce the stimulation of cytotoxic T cells and enhance the cytolytic activity of NK cells [8,9]. Interferon gamma (IFN-), mainly produced by NK cells, exerts antitumor and antiviral effects, increases antigen presentation and lysosomal activity of macrophages, and promotes the cytotoxic effect of NK cells [10].
Immunodeficiency occurs when there is a loss in the number or function of the immune cells, which might lead to infections and diseases such as cancer [11,12]. Therefore, the discovery of agents which enhance the immune system represents an attractive alternative to the inhibition of tumor growth and the prevention and treatment of some infections. An immunostimulatory agent is responsible for strengthening the resistance of the body against pathogens. In preclinical 2 Evidence-Based Complementary and Alternative Medicine and clinical studies, some immunostimulatory medicinal plants (e.g., Viscum album and Echinacea purpurea) have increased the immune responsiveness by activating immune cells [3,11].
In ancient traditional medicine, the term immunostimulant was unknown. In some cases, medicinal plants species that "purify the blood," "strengthen the body," and "increase the body's defences" have been used as immunostimulant agents [13,14].
Some of the in vitro and in vivo tests used to evaluate the immunostimulatory effects of plant extracts and compounds include the following: (a) proliferation of splenocytes, macrophages, and lymphocytes, (b) phagocytosis, (c) pinocytosis, (d) production of NO and/or H 2 O 2 , (e) NK cell activity, (f) release of IFN-, IL-2, IL-6, and other interleukins, and (g) lysosomal enzyme activity. In vivo studies mainly consist in the induction of an immunosuppressed state in the animals by using (a) chemical agents such as 5-fluorouracil, cyclophosphamide, and methotrexate or (b) biological agents such as tumorigenic cells. All the abovementioned agents have been extensively studied on inducing immunosuppression [15,16].
This review provides ethnomedicinal, phytochemical, and pharmacological information about plants and their active compounds used as immunostimulants in Mexico, Central America, and the Caribbean. This information will be useful for developing preclinical and clinical studies with the plants cited in this review.

Methodology
A literature search was conducted from December 2014 to July 2015 by analyzing the published scientific material on native medicinal flora from Mexico, Central America, and the Caribbean. Academic information from the last five decades that describes the ethnobotanical, pharmacological, and chemical characterization of medicinal plants used as immunostimulants was gathered. The following keywords were used to search for the academic information: plant extract, plant compound, immune system, immunostimulant, immunostimulatory, Mexico, Central America, and the Caribbean. No restrictions regarding the language of publication were imposed, but the most relevant studies were published in Spanish and English. The criteria for the selection of reports in this review were as follows: (i) plants native to Mexico, Central America, and the Caribbean, (ii) plants used in traditional medicine as immunostimulants with or without pharmacological evidence, and (iii) plants and their active compounds with information obtained from a clear source. The immunostimulatory activity of plant extracts or compounds in combination with a known immunostimulant agent (such as lipopolysaccharide, CD3) was omitted in this review.
Medicinal plants used as immunostimulants were classified into two categories: (1) plants with pharmacological studies and (2) plants without pharmacological research. The information on medicinal plants with pharmacological studies was obtained from peer-reviewed articles by consulting the academic databases SCOPUS, Web of Science, SCIELO, Medline, and Google Scholar. Medicinal plants with pharmacological studies of their immunostimulatory properties were subclassified into four groups: (a) plant extracts that have been evaluated for in vitro effects, (b) plant extracts with documented in vivo effects, (c) active compounds tested using in vitro studies, and (d) active compounds that have been assayed in animal models. The information for medicinal plants without pharmacological research was obtained from both undergraduate and postgraduate theses, in addition to peer-reviewed articles, and scientific books.

Medicinal Plants from Mexico, Central America, and the Caribbean Used as Immunostimulants
We documented 104 plant species belonging to 55 families that have been used as immunostimulants. Of these plants, 28 have pharmacological studies (Table 1), and 76 plants lacked pharmacological research regarding their immunostimulatory activity (  (Tables 1 and 6). We found that 46% of plants used as immunostimulants, with or without pharmacological studies, are also used for the empirical treatment of cancer. This was confirmed, for many plant species, by consulting our previous work [94]. Therefore, we highly recommend evaluating the immunostimulatory effects of medicinal plants used for cancer treatment. Medicinal plants used as immunostimulants are also used for the treatment of diarrhea (23%), cough (18%), and inflammation (18%). Diarrhea and cough are two symptoms associated with gastrointestinal and respiratory infections, respectively. We may therefore infer that immunostimulatory plants may also be used for the treatment and prevention of infections. Medicinal plants used as an antiparasitic agent may treat diseases such as malaria, whereas plants used as antivirals may treat diseases such as measles, smallpox, and others (Tables 1 and 6). A total of 20 plants, belonging to 15 botanical families, have in vitro studies regarding their immunostimulatory effects (Table 2). Furthermore, 8 plant species from 8 botanical families were assessed using in vivo assays (Table 3). A total of 11 compounds, isolated from 7 plants, have been tested using in vitro assays (Table 4). Only two compounds, isolated from two plants, were studied using in vivo models (Table 5).
Among the in vitro studies, Lophophora williamsii was one of the plant species that showed good immunostimulatory effects. This plant tested at 0.18 g/mL showed a similar activity (2.4-fold, compared to untreated cells) on the proliferation of human primary lymphocytes, compared to the positive control 0.6 g/mL concanavalin A [26]. Further studies with Lophophora williamsii, as well as the isolation and purification of its active compounds, are highly recommended. Among the in vivo studies, an ethanol extract from Evidence-Based Complementary and Alternative Medicine 3 Phoradendron serotinum leaves, tested from 1 to 10 mg/kg i.p., showed immunostimulatory effects, in a dose-dependent manner, by increasing the levels of IFN-, IL-2, and IL-6 in serum from C57BL/6 mice bearing TC-1 tumor [41]. The immunostimulatory effects obtained using in vitro studies were confirmed in in vivo studies for some plant species such as Mollugo verticillata, Phoradendron serotinum, and Petiveria alliacea and compounds such as maturin acetate ( Figure 1). This indicates that these plants and the compound can be metabolized, and their immunostimulatory effects are also shown in animals.
On the other hand, in many works cited in this review, only one concentration or dose was tested. Further studies will be required to obtain the EC 50 or ED 50 values, if possible, and analyze whether the plant extracts or compounds induce a concentration/dose-dependent effect. In many studies, a single immunostimulant test is used (e.g., the NO production). Authors are encouraged to perform more than one immunostimulatory test in further studies to provide more information on the immunostimulant effects of plant extracts or compounds. In some cases, the initial screening of the in vivo immunostimulatory effects is carried out using immunocompetent mice. Further studies are necessary to be performed on plant extracts and compounds using models of immunosuppressed mice, induced with chemical or biological agents.

Medicinal Plants Used as Immunostimulants without Pharmacological Studies
We documented 75 medicinal plants used as immunostimulants that lack pharmacological studies (  Evidence-Based Complementary and Alternative Medicine 5    Lymphocyte proliferation (1.6-fold) at 600 mg/kg [59] Evidence-Based Complementary and Alternative Medicine 9   [41]. However, the toxicity of the mistletoe species should be assessed.

Further Considerations
More ethnobotanical studies are necessary to provide information on medicinal plants used as immunostimulants in Mexico, Central America, and the Caribbean. The ethnomedicinal information of plant species will be updated with these studies. The toxicity of plant species cited in this review should also be assessed. For instance, Xanthium strumarium is considered a toxic plant. Recently, it was described that this plant induces hepatotoxicity [ courbaril was shown to lack genotoxic and mutagenic effects [98]. Toxicological studies are necessary to provide safety in the use of plant extracts and their compounds in clinical trials. To our knowledge, there are no pharmacokinetic studies carried out with plant compounds cited in this review. This might be due to (a) the lack of established methodologies for their quantitation, (b) the quantity of the obtained compound being not enough to carry out a pharmacokinetic study, and (c) many plants extracts not being chemically characterized, and there is no main metabolite for its quantification using HPLC. Further pharmacokinetic studies will provide additional pharmacological information prior to carrying out clinical trials. The isolation and elucidation of the structure of bioactive principles should also be encouraged.
Eight percent of medicinal plants listed in this review are classified as endangered. In the order of most endangered, Juglans jamaicensis, Cedrela odorata, and Lophophora williamsii are cataloged as vulnerable, whereas Taxodium mucronatum, Rhizophora mangle, Eysenhardtia polystachya, Cordia alliodora, and Hymenaea courbaril are cataloged as of least concern [99]. For instance, Lophophora williamsii (peyote) is a species that has been overexploited because of its high content of hallucinogenic alkaloids. The conservation of these species, as well as their habitats, should be encouraged by national and international programs to preserve biodiversity.
There is null or limited information regarding the trade of medicinal plants used as immunostimulants. Therefore, we performed direct interviews ( = 45) with local sellers of medicinal plants in Mexico, called "hierberos" or "yerbateros" in 7 different markets (Portales, Sonora, Xochimilco, Milpa Alta, Tlahuac, and Ozumba) located in Mexico City and the metropolitan area ( Figure 2). Two of the markets are located in Xochimilco. In order of importance, the most recommended plant species used as immunostimulants are Justicia spicigera, Polygonum aviculare, Carlowrightia cordifolia, Amphipterygium adstringens, Uncaria tomentosa, and others. It was interesting to find that 85% of yerbateros recommended the use of Justicia spicigera as immunostimulant (Figure 2(a)). Its way of preparation consists of the following: four or five branches and leaves are boiled with 1 L of water during 30 min. The recommended administration is 3 times daily. The rest of plant species were cited by less than 10% of yerbateros.
The demand for medicinal plants used as immunostimulants clearly indicates that these plant species are a current topic of interest. This indicates that ethnobotanical knowledge is a valuable tool, which supports the selection of plants to carry out pharmacological studies. Some of the medicinal plants cited in our survey have been pharmacologically investigated. Carlowrightia cordifolia showed poor immunostimulatory effects [17]. Amphipterygium adstringens showed in vivo immunostimulatory effects [51], whereas masticadienonic acid (Figure 1), its active compound at 0.001 M, increased the NO production (1.8 fold) with higher activity compared to 0.001 M ursolic acid (1.4 fold) [55]. Uncaria tomentosa showed in vitro immunostimulatory effects [50], whereas pteridine (Figure 1), its active compound, tested at 600 mg/kg i.p., increased the lymphocyte proliferation in immunocompetent mice [59]. Justicia spicigera and kaempferitrin (Figure 1), its active compound, showed in vitro immunostimulatory effects [14,54]. Nevertheless, the in vivo immunostimulatory effects remain to be performed with Justicia spicigera, kaempferitrin, and masticadienonic acid. The molecular mechanism by which this plant and the compounds exert their immunostimulatory effects should also be assessed. Finally, this review highlights the need to perform pharmacological, phytochemical, toxicological, and ethnobotanical studies with medicinal flora, from Mexico, Central America, and the Caribbean, to obtain new immunostimulatory agents.