**3. Ethnobotanical knowledge and modern science**

Out of the several plants biodiversity found on the earth surface, the plants which are used for the prevention and treatment of human and livestock disease are the significant ones due to the fact that those medicinal plants have secondary metabolites known as specialized metabolites [13]. Specialized metabolites with therapeutic possessions are dispersed throughout some plants genera, and these floras act as processing house for the natural products which are responsible for specialized metabolites [14]. The specialized metabolites have the potential biological activity that in turn used to protect the health and well-being of people and are the essential lead compounds for the modern medicines [15]. Study reported that medicinally important plants are the major source of treatment for up to 80% of the population until now, especially for underprivileged nations [16]. Also, the rest 20% of population living in higher-income

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countries still depends on complementary and alternative medicines which are especially plant origin and natural products [17]. Fascinatingly, out of the 25 dispensed drugs, about 12 of them are products which are plants origin [18, 19]. The role of ethnobotanists and researchers are incredible in the innovation of different present-day drugs, including artemisinin, aspirin, ephedrine, codeine and papaverine, colchicine, taxol, digoxin and digitoxin, capsaicin, tetrahydrocannabinol, and cannabidiol which are derived from Artemisia annua L., Filipendula ulmaria (L.) Maxim., Ephedra spp., Papaver somniferum L., Colchicum autumnale L., Taxus brevifolia Nutt., Digitalis purpurea L., Capsicum spp., and Cannabis sativa L., respectively [13, 20–22]. The uses of artemisinin in the modern medicine become acknowledged after a Chinese scientist (Tu Youyou) wins Nobel Prize in a year 2015 [23]. The results from the search of "medicinal plants" term on Google Scholar become more than 200,000 starting from the year 2000 until now.

Native people and ethnic groups use more than thousands of wild plants for the prevention and treatment of different human and livestock ailments, and even some of them are not identified and scientifically named still now, and hope several conventional medicines will be discovered from the plants and natural products in the future by the scientific community [24–26]. The well-known anthropologist David Maybury-Lewis had given emphasis to the role of indigenous people in supporting the invention of several plants which are medicinally important and used by this indigenous people for the treatment of different diseases [27]. Ethnobotanists can accelerate the proof of identity of plants which are medicinally important, and it is recommended that instead of conducting ethnobotanical assessment only, bioactive and lead compound can be extracted by mimicking the ethnobotanical information obtained from the indigenous people or traditional healers that can simplify the activity of bioprospecting of the plants [28]. Garnatje et al. [13, 29] advocated the term "ethnobotanical convergence" for the similar uses of plants included in the same node of a phylogeny. Although the term "ethnobotanical convergence" was condemned by Hawkins and Teixidor-Toneu [30], it is however assumed that connecting new technologies with the indigenous ethnobotanical information can accelerate the development of new modern drugs from the natural products and plants. Connecting ethnobotany with other fields such as phytochemistry, pharmacology, pharmacognosy, and molecular biology can support the botanical diversity identification and analysis of chemical constituents of medicinal plants which have the ability to prevent and treat human and livestock ailments [31]. Furthermore, approaches such as genomics and omics can also be employed to identify the genes underlying the (specialized) metabolites present in the plants characterized by high-throughput metabolomics approaches such as gas chromatography–mass spectrometry (GCMS), liquid chromatography-mass spectrometry (LCMS), and nuclear magnetic resonance (NMR) spectroscopy [32, 33]. The proper identification, utilization, and conservation of medicinal plants can assist in providing better alternative health care services in rural areas, especially in developing countries [34, 35]. Moreover, several medicinal plants collected from the traditional healers own garden or from the forest are majorly important, and high percent of individuals relay on these medicinal plants in order to get cure from their diseases [36]. The shortage of quality health care provisions that safeguard healthy lives and encourage safety of the individuals at all ages groups especially in the third-world nations of Asia and Africa strengthens the significance of medicinal plants used by the specific ethnic groups [34, 35]. In the past more than 10 years, the majority of population in the world depends on products derived from the plants which in turn shows the increasing demands of products derived from the

natural resources, and it may lead to the over-increment of the need of medicinal plants. So, the scientific community and ethnobotanists should fasten the investigation related with the development of products from the plants by working together with the indigenous people and traditional healers who have the ability to collect those plants which are medicinally useful by considering the emerging number of both communicable and noncommunicable and population growth and global climate change.

### **4. Plant collection and identification**

A **herbarium** is a process of collecting dried-out plant samples that used for study purposes. The key roles of a herbarium are to make available reference materials for botanical diversity identification of newly collected specimens, help as a supply for botanists and botany subjects, record the occurrence of a plant types in a specific area, decide taxonomy concerns, and store type and voucher samples. A **type specimen** is the exact specimen on which the name of a taxon is based. They are vastly valued and may be stored distinctly or attached onto different colored paper to escape loss or harm. A **voucher specimen** helps as the root for a scientific research. It is a consistent technique to authenticate the exact identity of the plant used for the research. Incase if questions are raised about the identity of the medicinal plants, the only way to answer this questions is by using the voucher numbers given for the specific plants. So, this shows whether the plants are correctly identified by the experts [37].

The person who collects the medicinal plant specimens are not allowed collecting endangered plant species or rare plants in a specific territory. And also, the individuals should respect the local regulations and need to obtain permission from the responsible bodies/officers. Incase if the medicinal plants were found only on individuals or traditional healers own gardens, verbal informed consent or permission should be obtained from the owner of plants to collect the specimens. After the plant specimens were collected from the traditional healers own garden or from the forest, the specimens should be put into the plastic bags, labeled, pressed, and transported to the national biodiversity centers. Then, the plant materials should be dried using the appropriate drying system and prepared for the botanical diversity identification using standard herbarium procedures. The identification mechanism is taken place by the botanists or taxonomists in the laboratory. But some botanical diversity identification processes can be taken place directly in the traditional healers own garden or in the forest if the owner or laws are not permitted to collect the plant specimens. The plant specimens should be deposited in the national herbarium or biodiversity laboratory by collecting them and immediately pressing between papers using a field press. Sticky specimens may be pressed between waxed paper. The pressing procedure in between newspaper should be carried out simultaneously by collecting the specimens due to the reason that once the specimens dried, they are difficult to press and arrange. The plant specimen should be arranged to show all the significant structures including lower and upper leaf, flower heads, and so on. Large specimens can be folded or cut into sections. Bulky fruit can be cut in half; large cones can be tagged to be stored in a box. Plant specimens should be dried as rapidly after it was collected and transported to the laboratory [38].

Sticks with good plants need to be gathered for the sample. The parts of the plants collected as sample should have to encompass pure phyllotaxy and the forking

#### *Ethnobotany DOI: http://dx.doi.org/10.5772/intechopen.104754*

scheme. For small herb, collection of more specimens as could fix on the herbarium sheet is desirable. In common, cutters are used to cut the stems, whereas for a while tallness, pruner is used, and for spiny samples, such as *Berberis mucrifolia*, leather glove is mandatory. For leaves and aromatic plants, digger is useful to dig out underground part. Particular plants stems lie horizontally under the ground. In such cases, the underground part should be washed from the mud elements, etc. If it is stem, then samples have been placed in a branching manner. Marine floras are light or to some extent filamentous and are hard to be organized on the page. A sheet of mounting paper is located below the moving or submerged tiny floras, and then the paper is slowly raised till the sample lies on the paper and out of the water. Then paper is raised to create a slope prudently so that it enables water runoff. These floras need to be shaken well before placing them in flimsies. Certain floras can be placed in plastic bags. The flowers that straightforwardly damage or can be lost among higher flowers from the same gathering place can be positioned in small bags within the larger bag. Unhealthy flowers, depauperate samples, diseased branches, etc., should be escaped [39].

Details about the plant specimens geographic area with the help of Geographic Information Systems (GIS), specimen collection date, and collection number should be noted down at the period of the specimen collection in a field notebook or electronically. This information is necessary to arrange the label for the herbarium specimen sheet. The collection number should be written on the edge of the sheet of paper. After the exact botanical diversity of the specimen becomes identified, the scientific name of their plant can be written together with the collection number. Herbarium specimen labels are organized in several means, but they usually hold a heading with state or region, province or district, country, and name of institution related with the sample, followed by the scientific name (genus, species, and author), details about the area such as geographic types or distance from adjacent town or landmark, and locale specifics (soil, altitude, humidity, etc.), Collection date, name of the specimen collector, and collection number are also significant information. The label is located at the bottommost-right-hand corner of a herbarium mounting sheet and attached immediately. Glue is also applied to the back of the plant sample, and it is organized on the mounting sheet. Lesser weight may be located on the top of the specimen till the glue is dry. Fragments of plant material such as seeds or pollen can be located in a small folded pocket, which is also glued immediately to the sheet. Once dried, the herbarium stamps the sheet with its name and assigns it an accession number. It is then filed in the herbarium cabinets that are usually ordered alphabetically as per plant family, genus, and species. Some herbaria use numerical arrangement as per the Adolf Engler method of arrangement [37].

Knowledge of plant anatomy is essential for classifying plant species. Deprived of it, a plant may be not identified correctly, with possibly severe consequences. Botanical diversity identification keys are valuable tools if you have adequate information of plant anatomy to appropriately use them. A dichotomous key offers the user with two alternatives at each stage, while a polyclave key may offer quite a lot of choices at each stage. Electronic polyclaves usually let the user to effort some wellknown features of the specimen, thus removing various species in the key. It may, moreover, offer the possibility or chance that the left behind species are the right choice and may quick the user to say other features to remove further species. There are little elementary rules to make a dichotomous key. The entries should initiate with a couplet that has same first words (e.g. Leaves opposite or Leaves alternate) but are opposing statements. A leaf cannot be both alternate and opposite so that the decision is perfect. To avoid misunderstanding, they should not have several entries in a row that start with the identical word and should not use overlying ranges of measurement. Negative statements (e.g. Leaves not opposite) should also be avoided. Couplets can be numbered, lettered, or a combination of both. Occasionally indented keys use no numbers or letters at all [38].

Even though this key is only a rare line, it comprises terms that need specialized knowledge of plant anatomy. If you do not know what "samaroid wings" or "perianth" are, the key is unusable. Field guides frequently enclose keys and illustrations to support the plant identification. They usually emphasize a restricted geographic area and compressed adequate to carry in the field. A field guide lets the user to compare the unidentified plant with identified plants that cultivate in the area. Even with a good field guide and key, it is often hard to identify a plant down to the species level, specifically if it is not flowering or fruiting. If possible, a botanist will gather a specimen to take back to the laboratory for an additional systematic investigation [40].

### **5. Ethnobotany research and their applications**

Even though many new chemical constituents have been derived and identified from medicinal plants used by the multicultural ethnic group, there are no conventional drugs synthesized from these plants using ethnobotanical knowledge and regulated as pharmaceutical products in the United States in at least in the past 40 years [41]. This may look like astonishing, considering the amount of resources and materials invested during the investigation of ethnobotanical knowledge for the past 40 years. On the other hand, indigenous knowledge about the medicinal plant is still used by the scholars and researchers for the identification of new chemical constituents and structures [42] that can be used as the main points for the development of new chemicals that have biological activity. Nowadays, the jobs for scholars and researchers have become more difficult than the past. They did not focus on finding new cultures, rather they focused on previously invented more than 4000 cultures and knowledge. An essential problem challenged by researchers on medicinal plant is that the study on medicinal plant may not all the time result in perfect separation of mechanisms of action, rather they only show "in-vitro pharmacological activity" or "in-vivo pharmacological activity on different organ or "identification chemical constituents and suggesting that it may have such activity due to the presence of this functional groups," etc. A close relationship between ethnobotanists who conduct research for the assessment of medicinal plant use practices by the indigenous people and traditional healers and experimental pharmacologists who conduct study on the pharmacological activity of traditional medicinal plants claimed by the traditional healers is very important in order to add values on present health care system by discovering novel drugs from the natural products and plants [43].

Most of the scholars have related the current use of medicinal plants to their ancient civilization of ethnic groups by investigating the local names of the medicinal plants and uses in archival material and literature, but also more in recent times through past linguistic analysis of popular names [44, 45]. Investigation of the history of the use practices of *Rhododendron tomentosum* Harmaja in Norway by the indigenous people indicates continuity in vernacular names and the therapeutic indication of this medicinal plants from the first eighteenth to twentieth centuries, with only a loss in use as a salt substitute. The investigation of cognates to indigenous medicinal plant names offers us evidences to the past interaction between the medicinal plants

#### *Ethnobotany DOI: http://dx.doi.org/10.5772/intechopen.104754*

and the native society, whereas the specific name of medicinal plants indicates some striking characteristics of the plant, color, their native uses, and views of the indigenous people [46]. Sabine Nebel's studied that the evaluation of names for edible greens among *Grecanico* speakers in Italy and Greek findings shows astonishing continuity of language and customs. For instance, *Portulaca oleracea* L. (purslane) is called *andracla* in Gallicianò and *andrakla* in Greece. The uses of several of these medicinal plants are the most persistent form of traditional healing practices in ancient Greek civilization [38]. Spanish ethnobotanists and researchers also try to relate historicallinguistic method to the Spanish use practices of medicinal plants called *manzanilla* (chamomile) in drinking teas since the ages of Moorish practices in the twelfth and thirteenth centuries, and even further back to Dioscorides in ancient Greece [46]. Also, other researchers present an infrequent sight into Lithuanian botanical diversity classification by tracing and certainly separating the roots of cognate native names for sycamore maple (*Acer pseudoplatanus* L.), plane tree (*Platanus* spp.), black poplar (*Populus nigra* L.), guelder rose (*Viburnum opulus* L.), and blessed wreaths prepared from collected rye. They show the importance and risks of using traditional texts and ethnographic data, such as melodies, mysteries, and children's verses, as botanical indication for rebuilding the etymology and figurative history of ethnobotanical naming [47].

Many scholars give emphasis on the present uses of higher plants collected from the forest or wild not only due to the continuous historical use practices by the indigenous people or recurrence in new markets but also due to the their significance nutritional values. Generally, wild plants are nutritive because of their high content of minerals and vitamins [48, 49]. Study show that the consumption of common golden thistle (*Scolymus hispanicus* L.) among poor farmers in Portugal has a long history, also stretching back to ancient Roman times, and has now become popular among tourists. Underlying the use of this particular thistle, though, is the fact that it manages to maintain its high nutritional value regardless of the quality of the soils in which it grows. Native agriculturalists have acknowledged this distinctiveness and thus ignore all other thistles that cultivate in the area [49]. Also, other characteristics like sex are also essential concern in the collection and preparation of wild plants for healing and foods in Europe. Even though the depth knowledge of collection and preparation of wild fruits and plants is considered as the characteristic of females in various countries [50], but thistles collection and preparation is the activity of males in Portugal [49]. Researchers identified and presented about females who become males in the Albanian Alps: in this ancient form of transgenderism, there is convergence of the ethnomedicinal knowledge of "typical" males, regarding food and plants used for the treatment livestock ailments (ethnoveterinary), wild greens, and the ethnomedicinal knowledge of females, regarding weedy food and medicinal plants [51].

Around 679 ethnobotanical surveys were accompanied in Latin America until 2012. Out of these surveys, 41% of ethnobotanical studies were carried out in Brazil, 22% of these studies were conducted in Mexico, 9% of these surveys were carried out in Peru, 8% were conducted in Argentina, 6% were conducted in Bolivia, and 14% of the studies were conducted in the rest of the countries of Latin American continent [52]. Apart from the infamous development of ethnobiology as a field in Latin America, the amount of ethnobotanical research conveyed for the scientific community look like to delay in various countries. From the results of research conducted on ethnobatanical information in Latin America especially in Brazil, Mexico, and Argentina, it is true that there is rapid increment of scientific studies on the

ethnobotany fields in the continent. This condition could be improved by the integration of better funds for ethnobotanical studies by investing in human resources and economic capitals and through the formation of thematic areas and ethnobotanical cultures (as observed in Brazil and Mexico). The overall objective of these thematic areas are to promote and enlarge debates on the disciplines, build protocols, and create knowledge that may fulfill the existing gaps in various areas of ethnobotany. Ethnobotanical study may also be encouraged by an escalation in the number of scientific proceedings and journals to distribute the results obtained from the researches and by the design of undergraduate courses and master programs to teach scholars who will conduct quality researches on this field. These methods can fufill the existing gaps and intervals in ethnobotanical study and also lead to the alliance of ethnobotanical knowledge throughout the Latin America continent [52].

Ethnobotany seems to have appealed its correct place among the scientific field getting finance studies, but much task yet to be conducted. There is still a lot of ethnobotanical information yet to be recorded, especially in different parts of Africa like ethnobotanical knowledge of the Khoi, Ndebele, and Swazi, as well as the relationship of this ethnic group with traditional medicinal plants, wealth of unrecorded information, especially relating to the ethnobotany of the Khoi, Ndebele, and Swazi, as well as the interaction between plants and people, folk taxonomies, historical use practices of plants, uses of plants for the treatment of livestock ailments, and medicinal plant uses before the colonial period. In addition to these, medicinal plants used for the treatment of dental disease, plant parts used for the perfumes, cosmetics, insect repellents, colorant flowers, yeast plants, thatching plants, textile plants, musical instruments, as well as hunting, fishing, and other technologies. It is very important to isolate market-based research from indigenous knowledge-based research. Hence, market-based research may depend on the profit obtained from the study within reasonable time, but the former may be of more direct value in the short term, but indigenous knowledge-based research may be more urgent and important, as a consequence of their long period role on the way to a deeper identification of medicinal plants use practices by the traditional healers and native societies, especially in Africa. Study shows that urbanization and solid traditional effects from other parts of the globe are prominent to an extraordinary loss of indigenous knowledge in South Africa. Scholars and researchers should be stimulated to grasp the chances offered by ethnobotanical study and to document the aspects of their own historical-linguistic information and indigenous medicinal plant use practices by the different ethnic groups for the sake of future generations. It is obvious that once the traditional knowledge is documented well systematically, it is not adulterated for life time. It is truly accepted that each individuals and generations can understand and interpret indigenous knowledge in different angle. The sources of present-day technologies and discoveries by the scientists are the traditional and indigenous knowledge of our illiterate ancestors. However, the investigation of native knowledge concerning medicinal plants uses for the prevention and treatment of human and livestock diseases should be given emphasis especially in Africa. Hence, it is not feasible to give oral indigenous knowledge information for the scientific community without tangible and well-documented traditional knowledge about medicinal plants use practices in Africa [53].

Study conducted on ethnobotanical study in northern Angola showed that about 2390 medicinal plants were reported to be used by the traditional healers and community. Those medicinal plants were categorized under 358 species in 96 plant

#### *Ethnobotany DOI: http://dx.doi.org/10.5772/intechopen.104754*

families, while just 3 out of 358 stated species are endemic to Angola and about one-fifth are neophytes. As the distance from where the medicinal plants collected increased, the number of use citations also higher. According to this study, large proportion of women (83%) was involved in the collection and preparation of medicinal plants from the forest and garden. The authors of this study discover new medicinal plants used for the treatment of different ailments by the indigenous people in the study, including Gardenia ternifolia which is claimed to have anti-measles, and ethnobotanical knowledge and the chemical constituents of Annona stenophylla subsp. cuneata medicinal plant have never been identified and documented elsewhere especially in the study area. Regardless of the long-term fighting in Angola, indigenous use practices of medicinal plants for the prevention and treatment of human and livestock disease remain a crucial part of traditional heritage. For that reason, plants are critical components in all parts of livelihood, particularly in the health care system. This condition is compounded by the still low-quality medical sectors in the Angola, particularly in countryside of the countries [54].

Another study conducted on hierarchies of knowledge; ethnobotanical knowledge, practices, and beliefs of the Vhavenda in South Africa for biodiversity conservation showed that 84 medicinal plant species were stated by the respondents which are categorized under 44 families. The majority of the stated medicinal plants were categorized under the fabaceae. The authors were identified 6 new species which are not reported before in Vhavenda ethnobotanical documents, also 68 medicinal plants claimed to have new indications and another 14 species have the similar uses with previously record. In this survey, high percent of plants reported were consumed as dietary supplements (36.0%) and used for the treatment of different human and livestock diseases (26.1%) and comprised mostly indigenous plants (73.8%) paralleled to nonnative plants (26.2%). The Vhavenda takes a variety of activities for the management of plant diversity that can be endorsed to taboos avoiding the usage of endangered and rare species, advertisement of sustainable collecting activities, and the proliferation of plant species for environmental refurbishment. Also, the authors reported about 48.4% of indigenous plant knowledge was transferred to generations through family/relatives, followed by studying about plants cultivation, collecting information by individual itself, through traineeships with traditional practitioners, and through learning in the schools and clan gatherings which constituted 16.1%, 9.7% 6.4%, and 19.4%, respectively. The reasons behind the difficulty in transferring knowledge about traditional plants use practices of Vhavenda are alterations in traditional knowledge platforms for information exchange, destruction of traditional organizations, and shifting value structures. The Vhavenda ethnic groups preserve a complex "knowledge-practice-belief" structure nearby for the use, control, and protection of plant biodiversity. The documentations of indication of new medicinal plants for the different purpose in this research and the expansion of the previously invented plants for the treatment of different human and livestock disease are valuable for providing an additional complete understanding into the indigenous plants use practices by this ethic groups in South Africa. The indigenous knowledge of medicinal plants use practices of this ethnic group is widespread comprising a variety of indigenous, exotic, wild, and cultivated plants. The indigenous people and traditional healers cultivate and gather a diversity of plants from the own garden, and from the wild and deciduous woodlands representing an all-embracing knowledge base conceivably imitating the sociocultural perspective of comparative separation and long-term settlement of the Vhavenda in the area. The domination of native

plant diversity signified in ethnobotanical survey proposes that plant knowledge acclimatization may initiate with common and readily existing plants; conversely, the new popularity of nonnative plant diversity also reveals a form of adaptation to new acquired plant variety that has become integrated into the Vhavenda depository of valuable plant diversity. Native practices and organizations are also inserted in traditional settings and coded in cosmologies and belief systems that have safeguarded the sustainable utilization of plant biodiversity. Plant managing plans aim is to sustain consistent and continual supply of plant biodiversity for dietary purposes, medicinal use practices, and other uses through selective practices such as the prohibition of endangered and rare plants from use, the advertisement of sustainable collecting practices, and the proliferation of plant biodiversity [55].

A study review on medicinal plant use practices in Ethiopia showed that the country is rich in biodiversity and believed to have about 6000 higher plants diversity with about 10% of native higher plant species. This amount of biodiversity of plants species also includes most of the lower plant species. The genetic variety enclosed in the many biotic makeup is also great, consequently making the country a serious biodiversity homeland for plants. As one of the 12 Vavilovian midpoints of origin for home grown crops and their wild relatives, it is the country of various native crops and genetic stocks. Ethiopia is considered as the richest country in biodiversity since 5000 years ago when ancient Egyptians, Greeks, and Romans used it as a basis of exclusive merchandises like Frankincense, Myrrh, and other plant products, which are also used for the production of different drugs. Among the largest biodiversity of plant species found in Ethiopia, highest percent has medicinal purposes. With similar fashion with the rest of the world, most of the people of Ethiopia rely on medicinal plants for the treatment of human and livestock ailments. Accordingly, about 95% of traditional therapeutic synthesis are the products of plant source. It is not ambitious to say medicinal plants have been used as a basis for the traditional remedies in order to prevent and treat different human and livestock diseases in Ethiopia. Medicine preparation from the medicinal plants is an essential part of the tradition of Ethiopian people. Most Ethiopian indigenous healing knowledge is retained in stringent mystery; conversely, it is dynamic in that the traditional healers create every struggle to broaden their range by reciprocal transferring of traditional medicinal knowledge to each generations or by reading the traditional pharmacopeias. It is difficult to acquire indigenous healing information of the traditional practitioners for the reason that they claim that the knowledge is their own and only like to transfer their knowledge to their relatives, especially to the eldest son [56].

Traditional practitioners in Ethiopia use the medicinal plant existing in the biodiversity for the prevention and treatments of various human and livestock ailments. A study showed that around 800 plants diversity which is medicinally important in Ethiopia is used for treating around 300 diseases. As stated by several researchers, there are diverse kinds of plant diversity with their parts used for the treatment purposes, the place where they grow, and the type of conditions treated by using these medicinal plants. There are about 18 medicinal plant diversity with 63 genera, and they are used by the indigenous society for the prevention and treatment of different human diseases. According to the literature, the common medicinal plants used for the prevention and treatments of human conditions are *Aloe* species, *Eucalyptus globulus*, *Hagenia abyssinica*, *Cupressus macrocarpa*, *Buddleja polystachya*, *Acmella caulirhiza*, *Acacia* species, *Citrus* species, *Clematis* species, *Coffee Arabica*, *Croton macrostachyus*, *Euphorbia* species, *Ficus sycomorus*,

#### *Ethnobotany DOI: http://dx.doi.org/10.5772/intechopen.104754*

and *Moringa stenopetala*. According to other reviews, all plant life forms were not in the same way used as therapies, for the reason that there is the dissimilarity in the distribution among the life forms. Therefore, the growth forms of medicinal plants were trees, shrubs, herbs, climbers, and others. The most predominant plant parts used for the prevention and treatments of different human and livestock ailments are herbs, followed by shrubs and trees [57].

A study conducted on ethnobotanical assessment and physicochemical properties of Commonly Used Medicinal Plants in Southwest Ethiopia showed that a total of 72 plants species were stated by the respondents for the treatment of different human and livestock ailments and classified under 61 genera and 39 families. Herbs constituted the predominant plant growth parts followed by shrubs, tree, and climbers. Leaves were the most frequently used medicinal plant parts by the traditional healers in the study followed by roots and seeds. Regarding the method of preparation of the medicinal plants, crushing, powdering, pounding, and pressing were used sequentially by the traditional healers [58].


**Toxoplasmosis: Advances and Vaccine Perspectives**

Oscar Bruna-Romero1, Dulcilene Mayrink de Oliveira1 and Valter Ferreira de Andrade-Neto2,\*

*1Department of Microbiology, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais – UFMG, Belo Horizonte-MG 2Department of Microbiology and Parasitology, Centro de Biociências, Universidade Federal do Rio Grande do Norte – UFRN, Natal-RN Brazil*

### **1. Introduction**

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transmission du paludisme au hameau de Mahitsy, à Tananarive, Madagascar.

M., Touré, Y. T. & Smith, T. (2007). Malaria transmission dynamics in Niono, Mali:

*Toxoplasma gondii* was first identified more than 100 years ago in the tissues of birds and mammals. In 1908 Nicolle and Manceoux described it for the first time in the gundi (*Ctenodactylus gundi*), a North African rodent, in tachyzoite forms. At the same time, Splendore in Brazil, identified the parasite in rabbit tissues. Due to its bow-like shape (Greek: *Toxo =* Arc) the genus was named *Toxoplasma*. However, only in the 1970's was the complete life cycle known and the parasite recognized as a coccidian parasite (member of the phylum Apicomplexa). It is ubiquitous throughout the world and estimated to infect approximately half of the world's population. It is characterized by a polarized cell structure and two unique apical secretory organelles called micronemes and rhoptries.

*Toxoplasma* has a complex life cycle consisting of a sexual cycle in its feline definitive hosts and an asexual cycle in its intermediate hosts. The latter, including humans, can be infected by ingestion of oocysts shed in cat feces. Unlike most other Apicomplexan parasites, *Toxoplasma* can be transmitted between intermediate hosts by either vertical (via placenta) or horizontal (carnivorism) transmission.

*Toxoplasma* parasite is found in intermediate hosts in two interconvertable stages: bradyzoites and tachyzoites. Bradyzoites, a dormant form, are slow-growing, transmissible and encysted. Infections with bradyzoite-containing cysts occur upon ingestion of undercooked meat. The wall of these cysts is digested inside the host stomach and the released bradyzoites, which are resistant to gastric peptidases, subsequently invade the small intestine. There, they convert into tachyzoites, the rapidly growing, disease-causing form that can infect most nucleated cells, replicate inside a parasitophorous vacuole, egress, and then infect neighboring cells. These tachyzoites activate a potent host immune response that eliminates most of the parasites. Some tachyzoites, however, escape destruction and convert back into bradyzoites. In the absence of an adequate immune response, tachyzoites

<sup>\*</sup> Corresponding Author

Toxoplasmosis: Advances and Vaccine Perspectives 171

cells become crucial to control parasite replication, not only by serving as additional sources of IFN-but also by developing cytotoxic activity against infected cells, eliminating parasite factories and thus preventing reactivation of infection (Denkers *et al*. 1993; Denkers and Gazzinelli 1998; Bhopale 2003). Whether B cells also play a role in protection against this parasite is not clear, but studies have generated indirect evidences that IgG antibodies may be important for protection (Kang *et al*. 2000). B cell-deficient mice have shown increased susceptibility to brain inflammatory pathology in chronic infections with the parasite, despite presenting similar levels of serum and tissue pro-inflammatory cytokines, such as IFN-γ. Furthermore, adoptive transfer of polyclonal anti- *T. gondii* IgG antibodies to these

To reproduce what the immune system does naturally to protect hosts against *T. gondii* infection (and re-infection), researchers have attempted several strategies for vaccination. These include the use of whole parasites (attenuated in different ways), soluble parasite antigens, recombinant purified proteins (subunit vaccines) or recombinant live vectors that express heterologous antigen(s) within host organisms (figure 1). Currently, some of these tools are also being used in combination, as part of prime-boost immunization protocols.

Sporulated oocysts (sporozoite-containing cysts) from the environment or tissue cysts (bradyzoite-containing cysts) from infected animals are the two major sources of infection with *T. gondii* (figure 2). However, vaccine candidates that include sporozoites or sporozoite antigens have traditionally been less studied because of the ease of access to bradyzoites and tachyzoites, e.g. using animal brain cysts or acutely infected animal peritoneal lavage/cell cultures, respectively. As a result, the first *T. gondii* whole-parasite experimental vaccines were mainly based on attenuated tachyzoites/bradyzoites, in particular those generated by inactivation or irradiation. Inactive parasites were used for immunization of experimental animals from 1956 (Cutchins and Warren 1956) to 1972 (Krahenbuhl *et al*. 1972) with not much success. In contrast, gamma-irradiated *T. gondii* tachyzoites were successfully tested as experimental vaccines in 1975 (Seah and Hucal 1975), in part after taking the idea from the pioneering irradiated-sporozoite malaria vaccines, which were initially tested in the 1960s and 70s (Nussenzweig *et al*. 1967; Gwadz *et al*. 1979). In the 1975 report, all animals inoculated with highly irradiated *T. gondii* parasites survived, were free of tissue cysts and were solidly protected against a subsequent rechallenge. Later, a few reports (Dubey *et al*. 1996; Omata *et al*. 1996; Dubey *et al*. 1998) have also used irradiated sporozoites (under the form of sporulated oocysts) to vaccinate mice, cats and pigs against toxoplasmosis, but in contrast to tachyzoites, results were not very

Other attempts to induce protection against toxoplasmosis with whole-parasite vaccines included the use of live attenuated parasites (tachyzoites) such as the S-48, the cps1-1, the temperature-sensitive TS-4, the MIC1-3 knock-out or the non-replicative Δrps13 strains (McLeod *et al*. 1988; Hakim *et al*. 1991; Buxton 1993; Gigley *et al*. 2009; Lu *et al*. 2009; Hutson

mice prevented both pathology and mortality.

**3.1 Whole-parasite attenuated vaccines**

encouraging, though some protection was also observed.

**3. Major toxoplasma vaccines and candidates studied to date**

Below is a review of current's state of the art of most of these technologies.

will grow unabated and cause tissue destruction, which can be severe and even fatal. However, the inflammatory immune response induced by tachyzoites can cause immunemediated tissue destruction. Therefore, a subtle balance between inducing and evading the immune response is crucial for *Toxoplasma* to establish a chronic infection.

The success of *Toxoplasma* as a widespread pathogen is due to the ease in which it can be transmitted between intermediate hosts. Humans do not play a major role in transmission; consequently, pathogenesis in humans is the indirect result of adaptations to infection in other hosts and treatment of human infections is unlikely to lead to the spread of drug resistance.

Once inside a host, the parasite develops powerful tools to modulate its host cell and develop into a chronic infection that can evade the host's immune system as well as all known anti-toxoplasmatic drugs. The ability of the parasite to replicate within a host cell, evade immune responses and undergo bradyzoite development requires the parasite to effectively modulate its host.

Toxoplasmosis remains a major health concern in pregnancy, where it causes severe birth defects or miscarriage, and in immunocompromised hosts. Thus, new toxoplasmosis control strategies are needed. The development of effective human and veterinary vaccines against toxoplasmosis is a relevant goal for Public Health (Gazzinelli *et al*. 1996; Pifer and Yarovinsky 2011). Even if new therapeutic drugs, with less hypersensitivity and toxicityrelated events, are developed, not only for acute *T. gondii* infection but also for the currently untreatable latent bradyzoite form of the parasite, a prophylactic vaccine against the disease would still be the best option from the financial, epidemiological, and social points of view. A vaccine would decrease the enormous costs of diagnosis/treatment, the premature loss of lives, the extensive rates of dissemination as well as the social impact of the disease. One major fact that suggests the possibility of vaccination against toxoplasmosis is that primary infection with the *T. gondii* parasite elicits protective immunity against re-infection in most individuals.


**5. Disabilities and Nervous System**

MeV can also cause damages to the nervous system.

#### *5.1. Hearing Loss*

MeV can induce hearing loss [69]. Before the introduction of mass vaccination, hearing loss was observed in 5% to 10% of measles cases in the USA. This remains highly frequent in under-developed countries where vaccination coverage is low [70]. One possible explanation is that otitis associated with measles in up to 25% of infected patients could cause hearing loss [71]. This pathology seems related to a super infection due to MeV-related transient immune-suppression.

Alternatively, hearing loss can occur immediately after the acute phase of the infection or later following measles acute encephalitis (described in paragraph 6.1) with typical bilateral and moderate to profound sensorineural hearing loss [69]. The mechanism associated with MeV-induced hearing loss remains unclear since neither viral antigen nor RNA have been detected in samples from the inner ear [57].

#### *5.2. Blindness*

Eye related symptoms such as conjunctivitis or corneal inflammation (keratitis) are commonly associated with measles [57]. Corneal complications are often more serious when superinfection (bacterial or viral) occurs during MeV-induced immune-suppression. However, there is a correlation between vitamin A deficiency and measles-induced blindness. Indeed, vitamin A deficiency is associated with severe keratitis and considerably increases the risk of xerophthalmia, corneal ulceration, and blindness [72]. This may explain why measles related blindness is more common in areas where children are already suffering of malnutrition.

Viral RNA can be detected in tear secretions [73]. In addition, human ex vivo cornea rim tissue is susceptible to MeV infection on its basolateral pole but neither syncytium formation nor released infectious particle have been found [74].

The relationship between MeV infection of ocular epithelial cells and the potential relationship with neural cell infection with cases of blindness is still unclear.

#### **6. Central Nervous System (CNS) Infection**

How the virus enters the CNS remains unclear since the known MeV receptors are not expressed. While its expression in the CNS seems to be only transient, nectin-4 has also been suggested to play a crucial role in MeV neuroinvasion based on observations made on closely related canine distemper virus whose neurotropism directly depends on nectin-4 specific patterns of expression [65,75–77]. Nectin-1 positive cells have recently been shown to be able to capture membranes and their cytoplasm from the surface of adjacent cells expressing nectin-4 at their surface via a trans-endocytosis mechanism [78]. In this context, viral RNP could transit from nectin-4 positive cells in nasal turbinate or meninges to neural cells expressing nectin-1 in olfactory bulb or brain parenchyma, respectively. The following key elements involved in the neural cell-to-cell dissemination and successful CNS invasion remain to be investigated (Figure 2A).

Three main CNS complications are associated with measles: The acute and the chronic forms, the latter being subdivided in two sub-types, the first as a measles inclusion-body encephalitis (MIBE) in immunocompromised patients and the second as a subacute sclerosing panencephalitis (SSPE) occurring in immunocompetent patients [79,80] (Figure 2B).


**The Switching of Trailing Limb Anticipatory Locomotor Adjustments is Uninfluenced by what the Leading Limb Does, but General Time Constraints Remain**

#### **Félix Fiset 1,2 and Bradford J. McFadyen 1,2,3,\***

Received: 24 February 2020; Accepted: 23 March 2020; Published: 26 March 2020

#### **Featured Application: The fundamental knowledge from this work has potential to eventually inform clinical interventions for gait and mobility training.**

**Abstract:** Research shows a blend of bilateral influence and independence between leading and trailing limbs during obstacle avoidance. Recent research also shows time constraints in switching leading limb strategies. The present study aimed to understand the ability to switch anticipatory locomotor adjustments (ALAs) in the trailing limb. Ten healthy young adults (24 ± 3 years) were immersed in a virtual environment requiring them to plan and step over an obstacle that, for the trailing limb, could change to a platform, requiring a switch in locomotor strategies to become a leading limb to step onto a new surface. Such perturbations were provoked at either late planning or early execution of the initial trailing limb obstacle avoidance. Sagittal plane trailing limb kinematics, joint kinetics and energetics were measured along with electromyographic activity of key lower limb muscles. Repeated measures ANOVAs compared dependent variables across conditions. To adjust to the new environment, knee flexor power around toe-off decreased (*p* < 0.001) and hip flexor power increased (*p* < 0.001) for late planning phase perturbations, while there was only an increase in mid-swing hip flexor power (*p* < 0.05) during perturbations at execution. Findings showed no influence of the leading limb function on the ability to switch trailing limb ALAs during late planning. However, the trailing limb was also constrained for modifying ALAs once execution began, but on-going limb control strategies could be exploited in a reactive mode.

**Keywords:** obstacle avoidance; anticipatory control; gait; locomotion; perturbation

#### **1. Introduction**

Navigating complex daily environments involves anticipatory control to safely avoid obstacles. There are robust and different lower limb strategies for stepping over an obstacle compared to stepping onto a platform. The anticipatory locomotor adjustment (ALA) to step over an obstacle involves a knee flexor generation strategy around toe-off (K5) [1,2] for both the leading and trailing limbs (respectively the first and second limbs to adapt) that puts energy in the lower limb to increase both knee and hip flexion. For the trailing limb, hip flexor generation power around toe-off (referred to as H3) for limb advancement can also be delayed until mid-swing (referred to as H3D) [3,4]. The higher the obstacle, the greater H3D [3]. For a level change accommodation, the leading limb instead increases the existing hip flexor generation at toe-off to help lift the limb to the new height [5].

In addition, there are differences in the modes of control between the leading and trailing limbs. Mohagheghi et al. [6] showed that the leading limb control is modifiable online by visual information during execution, whereas the trailing limb depends on visuomotor memory. The authors also concluded that there is independent control between the leading and the trailing limbs related to this difference in the use of visual information. The visual memory of the trailing limb for obstacle avoidance has been shown to be robust enough to be retained during cessation of the task up to two minutes after the leading limb has crossed in humans [7].

The hypothesis of independence between the two lower limbs was also reinforced by unexpected perturbations due to contact with the obstacle and the fact that only the limb that made contact was modified on subsequent obstacle crossings [8]. More recently, Howe et al. [9] found that lead limb toe trajectory was affected by the combined loss of the lower half of the visual field and ankle skin local anesthesia, whereas the trailing limb was only affected by loss of lower visual input. This emphasizes differences in motor control between limbs. Finally, such differences in adapted limb control have been highlighted during memory-guided obstacle crossing wherein the obstacle was removed and only its position replaced by a contrast tape [10]. The success rate of the trailing limb was four times lower than for the leading limb. The authors suggested that greater dependence on visual sampling during the approach phase for the trailing limb could explain this degraded performance.

However, Santos et al. [11] suggested that the hypothesis of independence between leading and trailing limbs could have been observed because of the predictability of the static environments used. By changing obstacle dimension one step before clearance, the authors showed that the trailing limb can use visual information from the leading limb's obstacle crossing. In studying foot–body balance geometries for adapted gait, Dugas et al. [12] perturbed obstacle placement both early and late in the approach to and beginning of obstacle avoidance. Secondary findings of differences between clearance and maximum foot heights for the leading and trailing limbs across perturbation conditions further supported limb independence in leading and trailing limb adaptations within non-predictable environments.

While limb independence seems well accepted, the above-noted studies using dynamic environments also point to the ability to modify obstacle avoidance once it has been planned. In an early example of this, Patla et al. [13] showed that relatively quick modifications of a planned obstacle avoidance can be made when a second obstacle is introduced unexpectedly. Yet, all of this work only supports the modification of an already planned strategy. Very recently, McFadyen et al. [14] used virtual reality (VR) to instantly change environmental demands, requiring one to switch between the respective knee and hip strategies described earlier either during the late planning stage at foot contact preceding the obstacle or at the beginning of step execution for the leading limb. Results showed that leading limb strategies could be switched within the late planning stage but appeared to be "locked-in" once execution began, at least to allow the planned strategy to be initiated uninterrupted, regardless of whether substituting a knee with a hip strategy or vice versa.

However, given the presumed independence between leading and trailing limb control for obstacle avoidance noted above and differences in the underlying motor control, it is not clear whether the same time-constrained control previously shown [14] exists for the trailing limb. Therefore, the purpose of the present work was to study the ability to switch from a trailing limb obstacle avoidance strategy to a level change accommodation strategy with the leading limb during either late planning or early execution when following obstacle avoidance by the contralateral leading limb. Knowing underlying locomotor control constraints could be relevant to clinicians for mobility training in pathologic populations.

#### **2. Materials and Methods**

#### *2.1. Participants*

Ten healthy young adults (24 ± 3 years; 5 males; height = 1.75 ± 0.79 m; mass = 67.5 ± 9.0 kg) with normal or corrected-to-normal vision (Snellen chart eye test) and without musculoskeletal or

neurological problems affecting their walking (self-reported) were recruited and provided their written consent to participate to the experiment. The study was approved by the ethics committee of the Centre intégré universitaire de santé et de services sociaux de la Capitale-Nationale (#2018-433).

#### *2.2. Experimental setup and protocol*

Non-collinear triads of markers were placed on the lateral aspects of the feet, lower legs and thighs and at the level of the sacrum for the pelvis. Markers were tracked by nine cameras (Vicon Motion Systems, Inc/MX T-series, Denver, USA; 100 Hz). Joint centers, segment centers of mass and radii of gyration were estimated in relation to anatomical landmarks (heels, toes, 5th metatarsal heads, medial/lateral malleoli, medial/lateral femoral condyles, left/right iliac spines and left/right anterior superior iliac spines) digitized with respect to the segment marker triads during a calibration set-up. It is unknown how limb dominance influences anticipatory locomotor adjustments (ALAs); and to control for any possible variability, the dominant limb, as determined from the Waterloo Footedness Questionnaire [15] (testing dominance during both movement and stabilization), was designated as the trailing limb for initial obstacle crossing.

Surface electromyography (EMG) was recorded (Noraxon, Scottsdale, USA; 1000 Hz) with electrodes on the mid-bellies of the rectus femoris (RF), vastus lateralis (VL), semitendinosus (ST), biceps femoris (BF), tibialis anterior (TA), medial gastrocnemius (GM) and soleus (SOL) muscles of the trailing limb. Signals were amplified (gain = 1000) and band-pass filtered at collection (15–500 Hz) and then offline (20–400 Hz). Signal strength and crosstalk were verified by isometric testing of each muscle, except for the GM and SOL, for which participants were asked to rise onto their toes while standing in order to solicit these muscles.

Participants donned a security harness used to prevent a complete fall. A starting line was determined at a point to allow the participant to make 3 steps at a self-selected natural speed with the trailing foot landing on a force plate (Bertec Corporation, Columbus, USA, 1000 Hz) corresponding to the final position before clearing an obstacle (1.6 cm2 wooden stick; Figure 1A). A second environment, involving the same starting position, introduced a 1.5 m deep platform (Figure 1B) positioned at 0.71 m (obstacle front to platform front) from where the obstacle was placed. Both surface changes were set to heights between 15% to 16% of participants' lower limb lengths. Participants were familiarized with both physical environments performing five trials of stepping over an obstacle and five trials to step onto the platform. Five additional trials of stepping over an obstacle with the leading limb only and then stepping onto the platform with both limbs (Figure 1C) were carried out to practice the experimental conditions requiring them to adjust for an obstacle with the leading limb first and a level change to the platform with the trailing limb immediately after. For all trials, participants were asked to walk at a natural pace.

After these trials in the real world, participants were fitted with a head mounted display (HMD, Oculus Rift V1, Menlo Park, USA) tracked by three non-colinear markers. A virtual reality system similar to the one used previously [14] produced a virtual environment (VE) resembling the laboratory with the above described surface changes (Figure 1A,B). This VE was modeled in Blender and programmed with Vizard (WorldViz, Inc, Santa Barbara, USA) to be synchronized to the participant's movements. The lower limbs of a first-person avatar were also aligned with those of the participant's, using a skeletal template from the Vicon data to allow participants to also see their lower limbs in real time (refresh rate of 60 Hz). This was important for more realistic control of lower limbs during ALAs.

**Figure 1.** Virtual environment with the obstacle (**A**), platform (**B**) and the half obstacle and platform (**C**). The timing of instantaneous perturbations was triggered by a 20 N load at trailing limb heel contact (**D**; T1) or at 20 N of unloading at trailing limb toe-off (**E**; T2).

After participants were comfortably fitted in the HMD, both real obstacle and platform environments were also set in place and simultaneously presented in the VE, and participants were invited to step over the obstacle, step onto the platform and touch them both with a foot for as long as they needed to feel comfortable and to understand that the VE coincided spatially to the real environment. Participants then finally practiced both obstructed environments individually as the ALA tasks to be used in the protocol.

During data collection, participants were required to approach and step over the obstacle in the VE that could disappear and be instantaneously replaced with a platform appearing 71 cm further along the path requiring a new locomotor strategy by the trailing limb. Two experimental conditions, presented in blocks counterbalanced across participants, were related to the timing of this VE change during the trailing limb step preceding the obstacle at either foot contact (Figure 1D) or at the subsequent toe-off (Figure 1E). The environment changes were respectively triggered when the vertical ground reaction force under the trailing foot exceeded 20 N (T1 corresponding to late planning) and falling below 20 N (T2 corresponding to execution). Each block was comprised of a total of 15 trials, including 5 environment perturbations and 10 catch trials without environment perturbations. Participants were instructed to walk at a comfortable and natural rhythm and to adapt their gaits to the new environment if presented. Participants were assured that the physical environment always corresponded to the last environment that was presented in the HMD. To provide confidence to the participants, before collection of each experimental block, participants first observed the instantaneous change from obstacle to platform and then performed three practice trials for the environment, knowing it would change at the respective time. Between all data collection trials, the image in the HMD was blacked out and participants wore earphones that played pink noise at a volume determined to block all ambient noise within the laboratory to prevent them from anticipating the upcoming condition.

At the end of data collection, French versions of the simulator sickness questionnaire [16] were used to measure simulator sickness, and the Presence Questionnaire [17] and SUS questionnaire [18], both used to measure the degree of VE immersion, were administered.

#### *2.3. Data analysis*

Marker, force plate and full wave rectified EMG data were all passed through lowpass, zero lag, Butterworth filters with cut-off frequencies set respectively to 6, 50 and 100 Hz based on previous work [14,19]. Relative joint angles were calculated using Cardan rotation matrices prioritizing the sagittal plane. Maximum relative joint angles (MJRA) during the ALA swing phase, minimum foot clearance (MFC; the vertical distance between the heel and the obstacle or platform front edge) and mean COM velocity (COMv; as the mean velocity in sagittal plane of pelvis COM during the ALA stride) were also calculated. Newton–Euler inverse dynamics equations with a custom-made program were used to estimate lower limb kinetics and energetics respectively for net muscle moments of force (MMF) and muscle mechanical power (product of the MMF and relative joint angular velocities). These data were normalized to body mass, and muscle mechanical work of the trailing limb was calculated as the mathematical integral of targeted positive and negative power bursts corresponding to ankle plantar flexor generation (push-off; A2), knee extensor absorption at the end of stance (K3), the delayed knee extensor absorption around mid-swing (K3D), knee flexor generation at toe-off (K5) and hip flexor generation at both toe-off (H3) and mid-swing (H3D). Hip hiking work (HH) around toe-off was also calculated as the area under the positive power burst from the product of the vertical hip joint reaction force and vertical hip joint velocity with positive power corresponding to energy from the pelvis.

The areas under muscle bursts for BF and ST EMG activity that aligned with the K5 power burst for obstacle avoidance [1] were calculated. As in a previous study [14], we took electromechanical delays into consideration by choosing these muscle bursts to begin at the local minimum prior to the K5 power burst and continue for the duration found for the corresponding K5 power burst of that trial. This allowed EMG data to provide physiological confirmation of knee flexor strategies across conditions. To compare across participants, percentage changes in these EMG bursts from the mean unperturbed trials for each perturbation time within each environment block were also calculated. As there was no appropriate hip flexor muscle activity recorded (RF has not been shown to be a good measure of a hip flexor activity [20]), no corresponding EMG areas were presented for hip flexion.

For descriptive time series data, EMG, kinematic, kinetic and energetic (muscle mechanical power) data were normalized to 100% of stride, with toe-off fixed at 60% and ensemble averaged across trials for each condition.

#### *2.4. Statistical analysis*

Repeated measures ANOVAs for perturbation conditions were used (SPSS, IBM, New York, USA, v.24) to compare the three perturbation timings (unperturbed, early and late). Sphericity was verified for each variable with Mauchly's test of sphericity, and a Huynh–Feldt correction was applied when appropriate. When main effects were found, Bonferroni-corrected post-hoc comparisons were applied for further comparisons between conditions. A Wilcoxen signed-rank test was used to compare percentage changes in targeted knee flexor bursts between T1 and T2 perturbated conditions. Significance level was set to p = 0.05 (including after Bonferroni corrections) for all analyses. Effect sizes (partial eta squared, η2) with 95 % confidence intervals (CI; {lower endpoint, upper endpoint}) are presented with ANOVA data.

#### **3. Results**

It appears that all participants tolerated the VE well given average low group scores of 0.31/3 ± 0.19 on the Simulator Sickness Questionnaire. In addition, average group scores of 5.51/7 ± 0.77 were found on the Presence Questionnaire, and they were 4.54/7 ± 0.68 on the SUS Questionnaire, showing that participants were relatively well immersed.

Two participants contacted the platform without tripping when it appeared at trail foot toe-off (T2). One of them contacted it twice and the other only once. These trials were kept for analysis because the participants did not lose their balance and patterns prior to platform contact did not show any outlying behavior.

Before presenting the dependent variables, the general pattern changes within the time series patterns for angular displacement, net muscle moment of force and net muscle power plots are described. Across the three joints, locomotor adjustments for the new VE were mostly seen around toe-off and during the swing phase (Figure 2). At the ankle, a greater dorsiflexion occurred at mid-swing when the obstacle changed to the platform at trailing foot toe-off (T2). The related kinetic and energetic changes were less evident in the plots because of the relatively small mass of a foot to control during swing phase compared to the whole body during the stance phase.

**Figure 2.** Average plots of angular displacement (top), net muscle moment of force (middle) and net muscle power (bottom) at the ankle (left), knee (middle) and hip (right) joints of the trailing limb during unperturbed conditions (gray line), and perturbations of obstacle to platform at T1 (O-P-T1; dashed line) and T2 (O-P-T2; black line). Gait stride was normalized to 100% with toe-off fixed at 60%.

At the knee joint, there was a decrease in knee flexion combined with a decreased knee flexor moment associated with decreased generation for the early change (T1) at toe-off. For the late perturbation (T2), kinetic and energetic curves were similar to those for the obstacle condition.

Finally, at the hip joint, hip flexion was higher when the obstacle changed to a platform, regardless of the timing of perturbations (T1 or T2). The kinetic and energetic plots showed a larger hip flexor moment combined with energy generation around toe-off (H3) when the platform appeared early (T1). For the obstacle-only condition and late perturbation (T2), there was some hip flexor energy generation at toe-off that switched to a hip extensor moment with energy absorption before returning, at mid-swing, to hip flexor power generation (H3D). There did not appear to be any change in linear hip hiking power (HH) across conditions.

To better understand how an obstacle avoidance strategy can change to level accommodation strategy, specific kinematic variables were calculated and presented in Figure 3. First, MJRA of dorsiflexion, knee and hip flexion during the obstacle crossing swing phase were all significantly different across conditions with a main effect of time (*p* < 0.001 for the three joints; ankle η2 = 0.6811 CI{0.3308, 0.7906}; knee η2 = 0.8117 CI{0.5647, 0.8761}; hip η2 = 0.8340 CI{0.6107, 0.8907}). More precisely, at the ankle, dorsiflexion was greater for late perturbation than O (*p* < 0.001) and O-P-T1 (*p* = 0.007) conditions, while O-P-T1 was similar to obstacle condition (*p* = 0.715). At the knee, MJRA were only decreased for the early perturbation compared to the obstacle condition (*p* < 0.001) and to late perturbation (*p* < 0.001), but the late perturbation was not different from the obstacle condition (*p* = 1.000). At the hip, MJRA increased for both changes to Pl (*p* < 0.001 for T1 and T2), and no differences were observed between the two perturbation timings (*p* = 1.000).

**Figure 3.** Average (standard deviation) of maximum joint relative angles (MJRA) during trailing limb swing phase (**A**). Minimal trailing limb foot clearance (MFC) of the obstacle—negative values correspond to the foot passing below obstacle level (**B**). Average pelvis center of mass (COM) velocity during trailing limb swing phase (**C**) across unperturbed (blue), early perturbation of obstacle to platform (O-P-T1, orange) and late perturbation (O-P-T2, gray) conditions.

Finally, MFC of the trailing foot over the obstacle had a main effect of time (*p* < 0.001; η2 = 0.7874 CI{0.3939, 0.8743}) with decreased MFC for early VE change compared to obstacle only and to the T2 perturbation (*p* < 0.001 for both), and late perturbation was not different than obstacle only (*p* = 1.000). A main effect of time was also present for COMv (*p* = 0.002; η2 = 0.4987 CI{0.1064, 0.6677}). COMv was

significantly slower for O-P-T2 condition than for the obstacle condition (*p* = 0.008) and for O-P-T1 (*p* = 0.025), but these latter two conditions were not different from each other (*p* = 0.673). We can see in the plot of COMv (Figure 4) that participants appeared to slow down only during the swing phase.

**Figure 4.** Average plots of pelvis COM velocity (COMv) across unperturbed (gray), early perturbation of obstacle to platform (O-P-T1, dashed line) and late perturbation (O-P-T2, black line) conditions. Gait stride was normalized to 100% with toe-off fixed at 60%.

For the accommodation strategies related to the muscle mechanical work for the power bursts around toe-off and during swing (Figure 5), ankle push-off (A2) remained unchanged across conditions (*p* = 0.755; η2 = 0.028 CI{0, 0.1939}). Knee extensor absorption at the end of the stance phase (K3), however, showed a main effect of time (*p* = 0.014; η2 = 0.4357 CI{0.0216, 0.6544}). Post-hoc analyses showed a tendency of K3 to be augmented for early change (*p* = 0.053), but not for late one (*p* = 0.146), and the O-P-T1 (obstacle-platform at T1) condition was not different than O-P-T2 conditions (*p* = 0.141). For knee flexor generation (K5) and the subsequent delayed knee extensor absorption (K3D), there were main effects of time for both power bursts (*p* < 0.001; η2 = 0.7894 CI{0.3962, 0.8756}, *p* = 0.001; η2 = 0.6356 CI{0.1944, 0.7786} respectively). Post-hoc analyses showed that K5 significantly decreased when the obstacle switched to a platform early (O-P-T1; *p* < 0.001), but not late (O-P-T2; *p* = 1.000). Significant differences were also observed between the two perturbation timings (*p* < 0.001). The delayed knee extensor absorption right after K5 (K3D) was also significantly decreased for the early change compared to the obstacle only (*p* = 0.002) and late perturbation conditions (*p* = 0.019). There was no difference between late perturbated trials and obstacle only (*p* = 1.000). Hip flexor generation around toe-off (H3) showed a general effect of time (*p* < 0.001; η2 = 0.7530 CI{0.3192, 0.8555}) and was augmented in an early perturbation (O-P-T1; *p* < 0.001), but not for a late one (O-P-T2; *p* = 0.408). O-P-T1 and O-P-T2 conditions were also different from each other (*p* = 0.003). There was also a main effect of time for the hip flexor generation during mid-swing (H3D; *p* = 0.005; η2 = 0.4685 CI{0.0637, 0.6578}). Post-hoc analyses showed that it was increased for the late perturbation only (O-P-T2) compared to the obstacle condition (*p* = 0.037) and to the O-P-T1 condition (*p* = 0.034) with no significant difference observed between O-P-T1 and obstacle conditions (*p* = 0.379 ).The positive hip linear power burst at toe-off showed a main effect of time (*p* = 0.009; η2 = 0.4830 CI{0.0925, 0.6568}) with an increase of power burst for a late perturbation compared to the obstacle condition (*p* < 0.001). The obstacle condition was not different from O-P-T1 (*p* = 0.519). O-P-T1 was not different from O-P-T2 (*p* = 0.242).

**Figure 5.** Average (standard deviation) of net muscle work corresponding to ankle push-off (A2); knee extensor absorption around (K3); after toe-off (K3D); after knee flexor generation (K5); hip flexor generation around toe-off (H3); hip flexor generation at mid-swing (H3D); and hip hiking around at toe-off (HH) across the unperturbed (O; blue), early perturbation of obstacle to platform (O-P-T1, orange) and late perturbation (O-P-T2, gray) trials.

The related muscle activity is presented in Figure 6 for a participant with patterns representing what was observed across all participants, specifically for the areas of interest for statistical testing. EMG plots of tibialis anterior showed increased activity in swing during late perturbation (T2). Semitendinosus (ST) and the biceps femoris (BF) activity showed higher activity at toe-off, corresponding to the K5 muscle power burst, for the obstacle and late perturbation (T2) conditions. All other muscles appeared to be qualitatively similar across conditions. To confirm the qualitative observations for greater ST and BF activity at toe-off corresponding to the K5 power burst, a percentage of change relative to obstacle condition was calculated for each perturbation condition (Figure 7). For both targeted bursts of ST and BF, muscle activity for the early perturbation condition was lower than for the late one (*p* = 0.017 for ST; *p* = 0.007 for BF).

**Figure 6.** Surface EMG of seven lower limb muscles of one representative subject across conditions of unperturbed (O; gray), early perturbation of obstacle to platform (O-P-T1, dashed line) and late perturbation (O-P-T2, black line) conditions.

**Figure 7.** Percentage changes in the area under the curve of semitendinosus (ST) and biceps femoris (BF) EMG activity corresponding to the K5 power burst across the early perturbation of obstacle to platform (O-P-T1, orange) and late perturbation (O-P-T2, gray) conditions.

#### **4. Discussion**

ALAs are essential to maintaining equilibrium within the dynamic environments found daily. Earlier work has shown the capacity to add onto an already planned ALA online [13], while a more recent study has revealed that one can also switch from one ALA to another for the leading limb, but only before ALA execution begins [14]. Yet, there is ample research to suggest differences in sensorimotor control between leading and trailing limbs [6,7]. The present study involved a perturbation requiring participants to switch from an initially planned obstacle avoidance ALA in the trailing limb to a leading limb level change accommodation ALA. This strategy switch created a contrast between what the leading and trailing limb planned and executed. As previously observed by McFadyen et al. [14], it was also found here that a trailing limb ALA can be switched within the late planning stage, but not once execution begins. In addition, it was found that, differently than for the leading limb, the pre-existing hip flexor work at the transition to swing phase could be exploited after initial execution to accommodate the need to step up to a higher surface level.

The current observation of the ability to switch trailing limb ALAs during late planning (T1) at the same time as the leading limb executes obstacle avoidance, supports the fact that the leading limb obstacle avoidance function was of insignificant influence on the trailing limb successfully switching to a new ALA. Such an ALA switch must, therefore, be driven by visual information of the environmental change. This ability to use visual information for trailing limb control has already been suggested for a static obstacle avoidance situation when vision was manipulated during planning and approach [6]. In addition, Santos et al. [11] showed this in a more dynamic situation for changing obstacle heights at a trailing limb foot contact (similar timing to T1 in the present study). However, previous studies involved only modifying an already planned trailing limb strategy. In the present work, ALAs after switching were very different for both limbs, and the leading limb knee flexor strategy to step over the obstacle did not affect the capacity to switch ALAs within the trailing limb. This further supports independent control between limbs [6,8,12]. It appears that the previously suggested feedforward influence of leading limb function on trailing limb control [11] is thus context specific and likely only true when both limbs perform the same ALA (i.e., both stepping over an obstacle).

Despite this lack of influence of leading limb control at the early switch (T1) of ALAs in the trailing limb, it was observed that the initially planned obstacle avoidance strategy could not be canceled out at the beginning of its execution (T2). As noted above, this is probably not due to influence from the leading limb and is more likely for the same reason discussed by McFadyen et al. [14] for switching leading limb ALAs. Specifically, a locomotor strategy is not immediately modifiable once it is launched regardless of being a leading or trailing limb. This inability to switch as execution begins (T2) was clearly expressed in the lower limb kinematic, kinetic and EMG patterns with large effect sizes as shown by the partial eta squared values for most variables.

Yet, despite the fact that ALAs could not switch at the beginning of the execution, some modifications in locomotor patterns were seen at mid-swing. More precisely, for the T2 perturbation, the trailing limb increased the already existing mid-swing delayed hip flexor generation (H3D) seen for trailing limb obstacle avoidance. This was likely to compensate for the inability to increase the pre-planned hip flexor generation at toe-off (H3) for obstacle avoidance. This may be similar to what Patla et al. [13] proposed as an ability to exploit an on-going plan (in their case for knee flexion of the leading limb) and modulate it online at a later latency. That is, the delayed hip flexor generation at mid-swing would was already planned for trailing limb obstacle avoidance, but then further exploited to replace lost H3 strategy for a platform ALA. In a previous study [14], when the leading limb switched at execution, a hip extensor burst appeared during swing phase 300 ms after the environment perturbation. This timing is similar to the fine-tuning stage of gait adaptations [6] and was considered to be more a reactive response rather than a strategy substitution. The present study shows a similar reaction time for the trailing limb for the increased H3D in mid-swing. This reactive strategy would also be assisted by a slight increase in hip hiking power (HH) as well representing a frontal plane contribution. Yet, this latter change on its own was at the same time as when the initially planned obstacle ALA was first carried out. Thus, it would appear that HH is autonomous to the lower limb ALA and can be added on at early latency, even when the newly erroneous obstacle ALA must be initiated for the late perturbation at execution. The observed significant changes in H3D and HH power bursts are supported by good effect sizes with approximately half the variance explained by the conditions. However, with the larger confidence intervals, some caution is required.

Results at the ankle joint also showed greater mid-swing dorsiflexion and TA activity for the late perturbation condition (T2) but without changes immediately following toe-off. This again supports no immediate means to switch an ALA once executed, but an ability to adapt online at a later latency as a reactive strategy. Why the ankle creates more dorsiflexion than the other conditions even if it appears to finish the stride on the new surface level with the same relative joint angles between T1 and T2 perturbations is unclear. It could be a preventative overshoot to avoid tripping in this reaction to the new environment.

It is difficult from the present study to deduce the underlying neural control. With respect to the same observations for lead limb ALA switching, McFadyen et al. [14] offered some conjecture from the literature. They noted that perhaps motor plans involving posterior parietal cortex (PPC) [21] could lead to muscle synergies set in the motor cortex through pyramidal tract neurons [22]. These synergies may be initially locked-in upon execution, but can also be modified at a later point, perhaps through reticular spinal neuron pathways suggested to be involved in pattern modifications [23]. In the present study, wherein participants had to switch from a trailing limb obstacle crossing strategy to a leading limb level accommodation strategy, it is highly likely that similar neural control is involved. However, while PPC would likely be involved in trailing limb motor planning, it appears to also be important for the visual memory of the obstacle for this limb [24,25]. However, this visual memory would be useless in the current ALA switch. PPC cell discharge is related to bilateral function rather than any specific limb. Thus, it could perhaps reorganize motor planning such that cells related to trailing limb function would be inhibited while other cells related to the new leading limb function would need to newly discharge in relation to the new position of the limb relative to the platform that appeared. The premotor cortex (PMC) might also be involved in this transition from trailing to leading limb roles given recent literature suggesting some limb-dependent cells of PMC in cats that contribute the selection of the first limb to step over and the related interlimb coordination [26]. How such neural

pathways are be related to the constraints in anticipatory and reactive control of trailing and leading limb function for ALAs discussed above will require more study.

Although more research is required to understand any clinical relevance of these present findings, given the differences in the underlying control of the leading and trailing limbs, it could be clinically relevant to train each side in leading and trailing functions to improve dynamic balance. As to the inability to switch ALAs once execution begins, but with online adaptation of hip flexors to assure final and safe accommodation, perhaps reactive training could be another point of focus.

The present study is not without limitations. Virtual reality is a great way to control environmental factors and produce protocols not possible in the physical environment. However, it is important participants see it as being as real as possible. The results showed acceptable presence levels, but as discussed in previous similar work [14], VR did also result in some behavior (higher foot clearance and maintaining some knee flexor generation power (K5) for a platform accommodation) that is different from what might be expected in an only physical environment. However, in this within-subject design, we are confident that we were able to show general control changes between the ALA switching conditions. The present study was also limited in number of participants and to healthy young adults only. Further work is needed to understand such adaptability constraints and their consequences for safe ALAs in other populations (e.g., older adults, populations with neurological impairments).

#### **5. Conclusions**

The present study involved switching from a planned trailing limb obstacle avoidance involving a knee flexor strategy to a leading limb surface level accommodation involving a hip flexor strategy. The findings showed that, despite presumed feedforward control involving the leading limb during the original plan, such leading limb influence appears context specific, and the trailing limb can benefit from ongoing visual information. However, there also appears to be a limb independent rule that anticipatory locomotor adjustments can be switched during the planning phase, but at execution, the planned pattern must be initiated. Yet, the control system appears able to exploit other aspects of the ongoing lower limb control strategies, such as the hip flexor power at mid-swing, to accommodate to the new locomotor demands of the environment. Underlying neural mechanisms for such flexibility and constraints in anticipatory locomotor adjustments are only speculative for now and require further study. More research is also needed to better understand the implications of such control for locomotor control changes with age and impairments.

**Author Contributions:** Conceptualization, methodology, data curation: B.J.M. and F.F.; formal analysis, writing—original draft preparation: F.F.; writing—review and editing, supervision, project administration, funding acquisition: B.J.M. All authors have read and agreed to the published version of the manuscript.

**Funding:** Research was funded Natural Sciences and Engineering Research Council of Canada (RGPIN/191782-2017).

**Acknowledgments:** The authors thank N. Robitaille (programming), S. Forest (infrastructure construction), G. St-Vincent and F. Dumont (motion capture) for valuable technical assistance and Dr. Jean Leblond for statistical consulting. We also thank Dr. Philippe Jackson for the use of the HMD.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

#### *Article*


**Preface to "Metabolomics Data Processing and Data Analysis—Current Best Practices"**

Metabolomics analysis has taken its place as a staple tool in all research areas across the bioscience and medical scientific fields where chemical matter is involved. While technical developments on the instruments used for metabolomics analytics allow for deeper than ever chemical exploration of biological samples, the importance of appropriate data-analytical approaches to treat, analyze, and interpret the vast metabolomics data is increasingly highlighted. The data-analytical workflow required for metabolomics study is a multi-step procedure necessitating different software and algorithm approaches for different steps that include but are not limited to peak picking, data preprocessing, and metabolite annotation and identification as well as visualization. In this book, we present a collection of papers focusing on practices and resources for various aspects of the metabolomics data-analytical workflows, starting from data collection all the way to the presentation of publication-ready metabolomics results, including both reviews on the current best practices as well as reports describing novel, innovative approaches for aspects such as in silico prediction of metabolite structures. Any metabolomics study is a multidisciplinary effort necessitating expertise across areas of, e.g., analytical chemistry, biochemistry, bioinformatics, and data-analytics. Therefore, fluent combination of the various steps involved in the workflow involves various challenges and factors to be taken into consideration. Here, current practices are reviewed, from samples to biochemical interpretation (Ivanisevic and Want, 2019), advances in metabolic modeling on a genomic scale (Sen and Oresiˇ c, 2019), as well as possibilities for open-source ˇ algorithms for data-analysis (Klavus et al. 2020). In addition, is the presentation of a web-based ˚ interface that fosters many parts of the metabolomics statistical workflow (Chong et al. 2019). As described by Ivanisevic and Want, metabolite identification remains as one of the pitfalls of metabolomics analysis, and it is therefore essential that advanced procedures are developed for both data acquisition (DIA and DDA) as well as MSMS annotation and metabolite identification. In this book, novel strategies for the computational prediction of mass fragmentation spectra (Djoumbou-Feunang et al. 2019), the integration of computational predictions provided by various algorithms to foster the in silico prediction of metabolite structures (Ernst et al. 2019), handling of DIA MS/MS spectra (Peris-D´ıaz et al. 2019), as well as an in silico framework to optimize the acquisition of mass fragmentation data (Wandy et al. 2019) are presented. Databases and data repositories are an inevitable part of an efficient metabolomics analysis workflow. Curated databases of spectral libraries encompassing both experimental and in silico predicted fragmentation spectra are a prerequisite for efficient metabolite identification. This book contains a section where databases were reviewed and gaps in coverage in terms of different metabolite classes were identified (Oberacher et al. 2018; Frainay et al. 2018). Repositories holding raw metabolomics data enable further utilization of data collections and enable efficient collaborative attempts as described in Tsugawa et al. 2019. Within metabolomics data-analysis an essential element is the efficient strategy for feature selection benefitting especially from the multivariate nature of metabolomics data. Therefore, both advanced methods for the chemometric processing of the data, as well as visualization of the results with ease of interpretation of the biological significance are focus areas requiring further development, as described in the final three papers of this book (Schillemans et al. 2019; Gao et al. 2019; del Castillo et al. 2019). We hope the book will serve as useful resource for anyone entering the field of metabolomics and, especially, the data-analytical part of the technology. Likewise, the book presents various novel algorithms and combined pipelines that may well be utilized by experienced researchers in the field, as well as be further developed owing to the open-source nature of all the presented resources.

#### **Justin J.J. van der Hooft, Kati Hanhineva**

*Editors*

*Review*