Gonick, Meghan. University of Bridgeport Acupuncture Institute, CT. generativehealth@gmail.com
(Presented at The Future of Ginseng and Forest Botanicals Symposium, July 12-14, 2017, Morgantown, WV)
Abstract
This presentation originated as a naturopathic doctorate thesis reviewing examining the research on the effects of growing conditions on qualities and quantities of constituent in medical herbs. Herb based medicines form a large portion of the materia medica and therapies currently used in Naturopathic practice and other alternative Medical practices. The international export value of pharmaceutical plants alone was estimated around $2.2 billion per year in 2011 (TRAFFIC). An estimated 50,000–70,000 medicinal and aromatic species are harvested from the wild (Schippmann, et al. 2006). This raises serious environmental and future supply concerns as the industry continues to grow.
Some herbs valued for their medical properties are quite difficult to produce agriculturally and are usually wildcrafted. Other plants are wildcrafted because practitioners of many traditional medicine system regard the natural plants as more potent. There appears to be little research comparing the effects of growing herbs in their natural environments versus otherwise. However, there may in many cases be validity to regarding wildcrafted plants as more potent. Specifically, many of the medically effective compounds are secondary metabolites, which fluctuate in response to predation and other environmental stressors. This suggest that further research comparing growing methods effects on herbal constituents may be helpful in determining, which methods of growing medical herbs
Keywords: naturopathy, herbs, secondary metabolites, constituents, wildcrafting
Introduction
One of the major modalities used for treating patients in Naturopathic medicine and other alternative medicine practices is through herbal medicine and phyto-pharmaceutical compounds. In recent years, increased consumer interest in plant-based medicine has increased demand for medical herbs. This has both increased opportunities in for those involved in the growth, collection, and processing of these plants as well as concerns about their availability and status in the wild. Additionally, with modern transportation, previously indigenous herbal medicines have become popularized worldwide, with 50,000-70,000 species used for medicine and cosmetics worldwide (Schippmann, et al. 2006). These plants can be threatened not only by over-collection, but by habitat destruction and disruption through deforestation, development, invasive plant species, overgrazing, and climate change. As early as 1930 the USDA commented on concerns that the increases in agriculture had led to a decrease in supply of medical herbs due to loss of habitat (USDA, 1930).
As medical herb growing methods change due to increased demand, it can alter the quality of the final product. From an economic point of view, research on growing methods for a plant may be concerned only with amount of final product, such as the study showing Calendula officinalis L. (Asteraceae) inflorescences size with changes in hydroponic nutrition (Stewart, Lovett-Doust, 2003). However, larger size doesn’t necessary indicate a better therapeutic effect. In fact, one study showed that Sanguinaria canadensis L. (Papaveraceae) rhizomes were larger in cultivated plants, but contained lower levels of active constituents: sanguinarine and chelerythrine (Graf et al. 2007). Similarly, some cultures believe that plants that appear more standardized are less useful in medicine, as shown by the demand in China for wild-appearing ginseng, Panax quinquefolius L. (Araliaceae) radix, due to the belief that it has more of the active constituents, ginsenosides (Teel, Buck, 1998).
It is estimated by the World Health Organization that 80% of people internationally rely on herbs for primary healthcare (Bodeker et al., 2005). While herbal medicine is a growth industry, it is very fragmented, uses a wide variety of plants from many regions, and doesn’t yet have the organization or resources to sponsor high levels of research. However, many researchers in fields such as botany, biochemistry, pharmacology, genetics, ethnobotany, and food chemistry have contributed individual studies.
Due to the scarcity of literature comparing wildcrafting vs cultivation, this paper reviews the chemical classes of medically active constituents and their biosynthesis, literature on any effects of growing conditions on these constituents, and finally different strategies for conservation.
Methods: Procedure of Literature Review
Databases searched included Google Scholar, Pubmed, and myEureka between March 10th and April 1st, 2014. Years searched were not limited. Search terms used included: (wildcrafting + constituents), (native + constituents), (agriculture, cultivation + native, wildcrafting). The first 20-30 hits for each were reviewed until the relevance seemed to have dropped. The initial search results were poor. A second set of searches using (secondary metabolite + wildcrafting, native, herb, cultivation) was more fruitful. The first 100 results on each database were checked for potential articles. Inclusion and exclusion criteria included whether or not full text was available, whether the article was a duplicate search result, whether there was comparison between different growing techniques or the article focused on environmental effects on secondary metabolites, with emphasis on any articles containing information about quantities of constituents. Articles that focused exclusively on molecular biology in model organisms to elucidate pathways were also excluded.
Using google scholar, 80 papers were deemed appropriate for review and 30 articles were downloaded and reviewed. Using myEureka, 63 articles were deemed appropriate for review and 36 articles were downloaded with 10 duplicates. In Pubmed, exclusion was easier; only 36 articles were reviewed with 27 downloaded.
Background
History of Herbal Medicine
It is important to understand the contexts of herbal medicine that has been adapted by current Naturopathic medicine. Naturopathic herbal medicine is primarily derived from American 19th century medicine’s use of medical herbs and modern phyto-therapeutic research, although other traditional herbal medicines such as Native American, Chinese herbal, Ayurvedic, and other cultural/ethnic groups’ herbal medical traditions have also contributed to the development of current Naturopathic materia medica. Advances in chemistry since the 1930’s and increased interest in the pharmaceutical qualities of individual constituents have increased the use of constituents either extracted from herbs or produced from cultures, leading to constituents based supplements, which does not represent the complete medical profile of the herb.
American 19th century medicine combined knowledge of herbs used by the European medical tradition and the knowledge shared by Native Americans available at the time through: education, oral traditions, writing, and clinical experience with patients. Even through the knowledge of medical herbs was not based on large standardized studies, it was not without scientific and empirical methods. Research was based on experiments and evaluation of individual cases/experiences to determine how well a medicine worked. Plant extracts were used for medicine teas, decoctions, tinctures, oils, gums, juices, and salts.
Many of the comprehensive texts written on medical herbs between the late 1800’s and the 1930’s are still in use today, and they describe not only herbs used, what they are used for and how they are processed, but also where they are obtained in nature. The 1892 Millspaugh’s Medical Plants describes 180 herbs with basic classes of chemical constituents, listings in the USP and a description of both processing and the final processed drug, while 1907 Potter’s New Cyclopaedia of Botanical Drugs and Preparations gives dosage and formulation used but less often a description of preparations (Millspaugh, 1974; Wren, 1975).
While these books provided physical descriptions of both the plant and its natural location, they did not describe methods of cultivation, since the majority of medical herbs were either widely available garden plants or collected from the wild (Millspaugh, 1974; Wren, 1975). In addition to descriptions and locations of plants in herbals, as late as 1966 the USDA published collection guides for the crude drug trade as a way of stimulating economic activity (Cavender, 2006), which provided locations, names, parts used, descriptions, and estimates of the demand for these herbs (USDA, 1930).
For over 150 years, one of the primary sources of medical herbs for pharmaceutical and other industries has been south central Appalachia, which includes western North Carolina, southeastern Virginia and eastern Tennessee and has around 1,100 species of plants with reported medical uses out of an estimated 2,500 total plant species, including native species such as, ginseng (P. quinquefolius), goldenseal (Hydrastis canadensis L.) (Ranunculaceae), bloodroot (S. canadensis), and black cohosh (Cimicifuga racemosa [L.] Nutt.) (Ranunculaceae) (Cavender, 2006). Interestingly, a study of the folk medicine of Appalachia shows that since at least the 1930’s, these peoples’ materia medica has focused on store-bought and gardened produce foods such as garlic and potatoes, suggesting that the collection of wildcrafted herbs was more for export profit than personal home use, and the author suggests this may be due to difficulty of identification, collection, and lack of seasonal availability (Cavender, 2006).
Modern Phytotherapies
In contrast to using traditional herbal medicine, phytopharmaceuticals use isolated plant constituents or herbal extracts standardized to one or two key constituent concentrations. These are usually based on modern research looking to confirm the activity described in traditional use or previous studies. These studies often focus on either showing the constituent can induce a specified activity in vivo or to elaborate the mechanisms of action in vitro. This research not only provides ideas for drug development, but evidence for the effectiveness of medical herbs and novel herbal treatments. The advantages of using isolated constituents or standardized products is that the dosage can be more standardized, growing conditions are not of therapeutic concern, and alternative plant sources (other species, or other parts) and culture-produced chemicals can be used.
The disadvantage of using isolated constituents or standardizing to only one or two constituents is that many medical herbs contain a multitude of active constituents, which may use multiple molecular pathways and reduce side effects. Sometimes, the traditional use of the whole herb or specific structures of the herb (ie. folium, flora, semen, radix) can be more clinically useful than isolated constituents. As in studies with Feverfew, Tanacetum parthenium L. (Asteraceae), attempts to use an isolated constituent responsible for its action can lead to poor effectiveness.
Phytochemistry of Medicinal Herbs
The constituents of an herb refer to all the chemical compounds normally found in the plant. This clearly varies by the solvent used for extraction (water, alcohol, oil, glycerin, CO2, vinegar) and part of the plant used (bark, root, flower, leaves, young shoots, seeds). Since plants are dynamic living organisms, concentrations and even presence of these constituents can also vary by age, time of year, soil minerals, and sun exposure. What may not be obvious for those not versed in plant sciences is that factors such as nearby plants, altitude, microbes, and nearby animals may in some cases be factors as well. When Chinese herbal medicine calls for the plant to be found on a specific mountain, or Cherokee medicine calls for the plant to have companions (other specific plants nearby), or European herbalism calls for the plant to be harvested in a specific manner, these can affect the chemical profile of the herb.
The reason that many medically active constituents are so easily affected by the environment is that the majority of them are what is known as secondary metabolites. Primary metabolites are chemicals found in plants used for structural, growth, and other metabolic purposes. These are necessary for the plant to maintain itself even under ideal conditions. These include sugars and carbohydrates used in structure, transportation and metabolism, lipids used in cell membranes or storage (in seeds), and nucleic acids and proteins uses in information storage and enzymes (Harborne, Baxter 1995). The distinction between primary and secondary metabolites is not always clear, for instance plant growth hormones are usually described as primary metabolites but they also belong to chemical classes usually classified as secondary metabolites (Harborne, Baxter 1995).
Often isolated to only a few families or even species of plants, secondary metabolites are usually the medically active or toxic constituents and are less ubiquitous and more varied than primary metabolites. They are usually not constitutive and have “no direct function in growth and development” (Buchanan et al. 2000). They are thought to be of use in plants to protect against herbivory and infection, act as attractants, and to serve in plant to plant interactions, called allelopathy (Buchanan et al. 2000). The three major groupings of phytochemicals that have medical benefits are mostly composed of secondary metabolites: nitrogen containing compounds; phenolics; and terpenes (Harborne, Baxter 1995; Buchanan et al. 2000).
Nitrogen Containing Compounds
There are over 15,000 know nitrogen-containing compounds synthesized in plants (Harborne, Baxter 1995). This includes the diverse group of over 10,000 alkaloids, the majority of nitrogen-containing compounds found in plants, which require nitrogen, but are not all formed via the same amino acid pathway (Harborne, Baxter 1995; Buchanan et al. 2000). Alkaloids are found in 20% of plant species making up 0.1-12% of dry weight of the plant and can be produced by fungal symbionts or the plant itself (Harborne, Baxter 1995; Buchanan et al. 2000). Alkaloids can act as herbivory deterrents, nitrogen storage, toxins to vertebrates, and have been shown to increase with initial damage to the plant (Buchanan et al. 2000).
Isoquinolines make up the largest group of alkaloids, are derived from Tyrosine and Phenylalanine precursors, and contain many medically active compounds such as morphine and papaverine (Harborne, Baxter 1995). Over 1,200 indole alkaloids have been identified including toxic species and medically interesting species such as reserpine and ergotamine (Harborne, Baxter 1995). Pyrrolizidine alkaloids are a diverse group of secondary compounds, thought to reduce herbivory through deterrent, repellent, or toxic effects on a wide range of generalist herbivores (Joosten, vanVeen 2011). Pyrrolidine and piperidine alkaloids are found mostly in the family Solanaceae and include nicotine (Harborne, Baxter 1995). Quinoline alkaloids are found in the family Rutaceae and a few others and several have shown pharmacological activity. Quinolizidine alkaloids found in the Fabaceae family have antiherbivory effects and potential medical uses (Harborne, Baxter 1995). Steroidal alkaloids are formed from triterpenoid (GDP) synthesis, found in the families Solanaceae, Apocynaceae and two other families, with members of this group having been found to have antihypertensive and other medical qualities (Harborne, Baxter 1995). Tropane alkaloids are found mostly in the families Solanaceae and Erythroxylaceae (Coca family) and to a lesser extent in 8 other families and have both toxic and medicinal compounds (Harborne, Baxter 1995).
Cyanogenic Glycosides and Glucosinolates are synthesized from amino acid precursors, often vary by plant population, and are bitter and/or toxic (Harborne, Baxter 1995). Both of these groups can serve a similar function; when plant tissue is crushed, glycosidase or thioglucosidase (respectively) cleaves the sugar from these compounds, releasing unpleasant aromatic cyanide or sulfur compounds that can be used as feeding deterrent (Buchanan et al. 2000). Cyanogenic glycosides are often bitter and toxic (Harborne, Baxter 1995). Glucosinolates compromise over 10,000 known compounds, have an acrid taste and are found mostly in the Brassicaceae family and others of the order Capparales (Harborne, Baxter 1995; Zenk, Juenger 2007).
Many of the other nitrogen-containing compounds are primary metabolites, such as, protein building amino acids, nucleic acids, and proteins used by the plants (Harborne, Baxter 1995). As deterrents, nonprotein amino acids, a group of 250 compounds, can be toxic in animals and are stored for protection in seeds (Harborne, Baxter 1995, Zenk, Juenger 2007).
Phenolic Compounds
There are over 10,000 known phenolic compounds found in plants, with nearly half of these being flavonoids (Harborne, Baxter 1995; Buchanan et al. 2000). Almost all phytochemicals classified as phenolics are derived from the Shikimate pathway via aromatic amino acids (Buchanan et al. 2000; Ribereau-Gayon 1972).
Flavonoid synthesis diverge from phenylpropanoids using chalcone synthase to form its precursors (Buchanan et al. 2000). Flavonoids have limited distribution throughout the plant kingdom and have been used medicinally due to their antioxidants, antiinflammatory, antimicrobial, free radical scavenging and metal chelating properties (Harborne, Baxter 1995; Perez et al. 2014). Flavones and flavonols are sometimes used as pigments and feeding attractants (Harborne, Baxter 1995). They include many medicinally active and antiinflammatory compounds such as: kaempferol, quercetin, myricetin, apigen and luteolin (Harborne, Baxter 1995). Almost exclusive to Fabaceae, isoflavonoids have over 600 recognized compounds, many of which are defensive compounds and beneficial to human health (Broeckling et al. 2005).
Phenylpropanoids are initially formed via the same pathway as flavonoids until they diverge at the enzyme, phenylalanine ammonia-lyase (PAL). PAL has been shown to be induced by UV light exposure, which is consistent with the functions of these compounds including: free radical scavenging, vascularization, pigmentation, phytoalexins, UV protectant, and signaling molecules (Broeckling et al. 2005; Hamberger, Bak 2013). Phenylpropanoids also contribute to flavor and aroma profiles, are insect deterrents, and have antimicrobial and antibiotic along with of medicinal uses (Harborne, Baxter 1995).
Phenolic acids can be used as primary metabolites in cell wall structures (lignin) or can be used as secondary metabolites such as THC, which is neuroactive in mammals or urushiol, which causes contact dermatitis (Harborne, Baxter 1995). Over 700 coumarins are known with members showing allergenic, insecticidal, blood thinning, antibacterial, brachycardic, and antitumor activities (Harborne, Baxter 1995).
Over 200 lignan compounds have been identified with most members found in the wood where they provide insecticidal properties (Harborne, Baxter 1995). Several extractable lignans have shown medical properties, such as, antiviral, antitumor, and antihepatotoxic properties. Stilbenoids can be fungal resistant and are often found in woody materials or glycosylated (Harborne, Baxter 1995). Tannins have astringent properties, which can be unpalatable but have beneficial properties in wound and burn healing (Harborne, Baxter 1995).
Terpenoid Compounds
Terpenes are the largest class of secondary metabolites with over 20,000 known compounds and have a more diverse range of uses in plants including: membranes components, defense compounds, phytohormones, and signaling molecules (Buchanan et al. 2000; Hamberger, Bak 2013). Terpenes are formed by geranylgeranyl diphosphate pathway using five carbon isoprene units. Terpenes are hydrophobic compounds forming important constituents of essential oils (Harborne, Baxter 1995; Buchanan et al. 2000). Most terpenoids are secondary metabolites, but the primary metabolites – gibberellins, sterols, and carotenoids – are used as phytohormones (gibberellins and abscisic acid), membrane components, and accessory photosynthetic pigments respectively (Buchanan et al. 2000; Zerbe et al. 2013).
There are over 600 identified monoterpenes (2 isoprene unit compounds) from plants with most being found in the essential oil and contributing to the aroma (Harborne, Baxter 1995). Many of these, like thymol, also have antiseptic and antiinflammatory properties (Harborne, Baxter 1995). Iridoids are a bitter tasting subgroup of monoterpenes, which are usually found in a glycosylated form acting as a feeding deterrent (Harborne, Baxter 1995). Iridoids also have medicinal uses as tonic bitters, are antimicrobial and anti-inflammatory, but some members are toxic (Harborne, Baxter 1995).
Over 10,000 different diterpenes (4 isoprene compounds) have been identified in plants; most are secondary metabolites but it also includes the primary metabolites gibberellins, a class of plant hormone, which consists of over 71 compounds (Harborne, Baxter 1995; Zerbe et al. 2013). These are normally hydrophobic and can be aromatic, so they are rarely glycosylated, although stevioside triglucoside is a noted exception (Harborne, Baxter 1995). Although many diterpenes are toxic, several are of great use to the pharmaceutical industry such as: taxol, a chemotherapeutic agent from Taxus sp.; forskolin, a vasodilator from Coleus forskohlii (Lamiaceae), and marrubiin, an analgesic and antidiabetic drug candidate from Marrubium sp. (Lamiaceae); to name just a few (Zerbe et al. 2013). The annual market value of diterpenes alone discovered from plants is in the billions of dollars from pharmaceutical, fragrance, herbal, and other industries (Zerbe et al. 2013).
Triterpenes derivatives include cardenolides, bufadienolides, and saponins. Cardenolides and bufadienolides, including digoxin, are known for their cardiac effects on vertebrates (Harborne, Baxter 1995; Buchanan et al. 2000). They are found in the families Apocynaceae, Asclepiadaceae, Moraceae, and Scrophulariaceae (Harborne, Baxter 1995).
Triterpene saponins are been found in over 100 families and have been shown to have defensive properties: antiherbivory, anti-nutritional, allelopathic effects, and are toxic to cold blooded animals, insects and mollusks (Harborne, Baxter 1995; Broeckling et al. 2005). They have pharmacological impact through their antimicrobial, antiinflammatory, hemolytic, anticholesterolemic, and cytotoxic activities (Harborne, Baxter 1995; Broeckling et al. 2005).
Steroidal saponins are found in the families Agavaceae, Dioscoreaceae, Scrophulariaceae, and Liliaceae (Harborne, Baxter 1995). Steroidal saponins have detergent properties, low human toxicity, can be used to stun fish, and include diosgenin, the precursor for synthetic progesterone (Harborne, Baxter 1995).
There are thousands of known sesquiterpenes, many of which are aromatic (Harborne, Baxter 1995). This class contains several biologically active secondary metabolites and the primary metabolite, abscisic acid, which acts to control plant growth (Harborne, Baxter 1995). Tetraterpenes, which are commonly referred to as carotenoids, are lipid soluble and used as accessory pigments in photosynthesis and as antioxidants and vitamin A precursors in animals (Harborne, Baxter 1995; Buchanan et al. 2000).
Synthesis Pathways
In order to understand how the environment can affect these chemicals, it is important to review their biosynthetic pathways. The Shikimate pathway (Figure 1) is the most well understood, perhaps because it is ubiquitous throughout the kingdom and is the origin of most phenolic compounds (Ribereau-Gayon 1972; Taiz, Zeiger 2002; Tohge et al. 2013). It produces the aromatic amino acids tryptophan, phenylalanine, and tyrosine, which are the initial components for phenol synthesis (Ribereau-Gayon 1972). In higher vascular plants phenylalanine is used predominantly as the precursor for phenolic compounds (Harborne, Baxter 1995). This pathway also provides precursors for chlorogenic acid, alkaloids, glucosinolates, auxin, tannins, suberin, tocopherols, and betalains (Tohge et al. 2013).
From phenylalanine, the metabolism can directed towards the synthesis of phenylpropanoids (coumarins, lignans) or towards flavonoids (Ribereau-Gayon 1972). The phenylpropanoid pathway is first catalyzed by Phenylalanine ammonia-lyase (PAL), which produces cinnamic acid (Ribereau-Gayon 1972; Taiz, Zeiger 2002; Docimo et al. 2013). Low nutrient levels, low light levels, and fungal infections have all been shown to increase the activity of Phenylalanine ammonia lyase (PAL), thus increasing synthesis of phenolic compounds based on cinnamic acid (Ribereau-Gayon 1972; Taiz, Zeiger 2002; Docimo et al. 2013). Whereas the enzymes in the Shikimate pathway are fairly conserved, the genes for this pathway vary across taxa and there can be variation between different tissues in the same plant, allowing for a great specificity of elicitors and products (Taiz, Zeiger 2002; Tohge et al. 2013). Chalcone synthase (CHS) catalyzes the first specific step towards flavonoids, and this pathway has been well elucidated (Docimo et al. 2013).
Phenylalanine ammonia-lyase (PAL), Cinnamic acid 4-hydroxylase (C4H) and 4-Coumarate: CoA ligase (4CL) catalyzes the first three steps of the general phenylpropanoid pathway whereas chalcone synthase (CHS) catalyzes the first specific step towards flavonoids (Docimo et al. 2013).
Because alkaloids do not have a uniform classification and are such a diverse group of compounds, only sharing in common that most contain a Nitrogen in a heterocyclic ring and that most compounds are basic, they are synthesized from a variety of pathways. Many may originate from amino acids, including those from the Shikimate pathway, while others originate from terpene synthesis.
Some of the basics of terpene synthesis are well understood, while all the enzymes responsible for individual compounds have yet to be identified (Broeckling et al. 2005). This pathway combines isoprene, a 5-carbon branched molecules, to form terpenes and is alternately known as the Isoprenyl pyrophosphate (IPP), the Geranyl pyrophosphate (GPP), or the Geranyl diphosphate (GDP) pathway (Taiz, Zeiger 2002).
Since many secondary metabolites are unique to one or two families or species, much attention is directed at the evolution of the biosynthetic pathways for these molecules. One group of enzymes (P450s) has numerous isotypes and is thought to be a key in specialized phytochemicals since they have been found to be involved in the synthesis of several separate types of secondary metabolites: cyanogenic glucosides, glucosinolates, terpenes (mono-triterpenes), and phenylpropanoids (Hamberger, Bak 2013). In S. miltiorrhiza multiple P450s are thought to be involved in catalyzing the unknown steps in phenolic acid and tanshinone synthesis (Luo et al. 2014).
Genetic and transcription data available to researchers allows for comparisons of metabolic pathways through transcription profiles to compare transcription sequences across species and families to look for potential links in evolutionary pathways. In one study the transcript profile of curcumin, Curcuma longa L. rhizome, was compared with those of other important terpenes: taxol, vinblastine, artemisinin, and acridone alkaloids (Annadurai et al. 2013). The authors were able to show that 25% of each of the different terpenes biosynthesis transcripts overlapped with menthol, and that taxol shared 8.11% in common with other terpenes (Annadurai et al. 2013).
Evolution, Physiology and Native Plants
Plants lack mobility and have evolved strategies to deal with stressors such as light, poor nutrition, microbes, and herbivores (general and specific). Most of the pathways used to react to these stressors may overlap with each other and growth and development pathways. For instance, light signaling pathways have been shown to overlap with pathways associated with wounding, pathogen attack, ozone exposure, and oxidative stress involving the use of reactive oxygen species, salicylic acid, jasmonate, and ethylene (Nawkar et al. 2013).
Some defenses of plants can be intrinsic or structural, such as pigments that absorb excess light, lignification, epidermal thickness, hairs, and thorns, while others are chemical, such as antioxidants, antimicrobials, and deterrents to herbivores (Pankoke, Müller 2013). Secondary metabolites are variable to reduce resource use, and the mechanisms for their evolution are theorized to be highly adaptable (Sønderbyet al. 2010). Secondary metabolite concentrations differ between tissues and with metabolic levels of the tissue. Mature leaves, which act as a carbon store and are much less metabolically active than growing leaves, have shown less induced resistance to herbivory than growing leaves (Pankoke, Müller 2013; Tallamy, Raupp, 1991).
The two most widely discussed and researched signals to recognize potential stressors and pathogens in plants are jasmonate and salicylic acid (and their methylated forms). Jasmonate has been shown to act as a downstream signaling molecule in NO and H2O2 mediated stress induced by fungi (Ren, Dai, 2012). Salicylic acid occurs constitutively at low concentrations and is also involved in stomatal closure, transpiration, photosynthesis, nutrient uptake, chlorophyll and protein synthesis (Perez et al. 2014). Methyl salicylate has been shown to be transmitted through the air to different parts of the plant and nearby plants and has been shown to directly affect many insect species (Taiz, Zeiger 2002; Pickett et al. 2007).
There are four basic types of plants in most environments: native, naturalized, invasive, and cultivated. Cultivated species or subspecies may simply be clones or offspring of wild plants or show little resemblance to their wild ancestors. When a plant is introduced to a new environment, it can either: survive only with assistance; become naturalized; or become invasive. Invasive species are those that spread broadly within their newly occupied regions, while naturalized species survive at a moderate level (Bezemer et al. 2014).
Factors Shown to Alter Constituents
Abiotic factors, such as moisture, sunlight, soil nutrients, and toxins can alter the constituent profile of a plant. Usually there is a well-established set of ideal growing conditions for a given species or variety. This ideal is usually based on increasing the rate of growth for the commercially useful part of the plant.
Nitrogen deficiency conditions show increased assimilation of ammonium into the GS/GOGAT system and increased PAL activity, stimulating the recycling of nitrogen containing phenolics and antioxidants (Kováčik, Klejdus 2014). In nitrogen-deficient Nicotiana tabacum (Solanaceae) there was an observable increase in phenolic acids and in Matricaria chamomilla (Asteraceae) an increased lignification, elevations in chlorogenic acid, umbelliferone, flavones, and growth, but decreased flavonols (Kováčik, Klejdus 2014).
Light can be a potent stimulus for induction of secondary metabolites. PAL activity increases with reductions in light, while P450s can be activated by light (esp 450nm). UV-B radiation can be severely damaging to plant growth and development and can induce defenses including: increased salicylic acid (SA) and increased responsiveness to jasmonate (Nawkar et al. 2013). A study of UV-B induction of glycosyl flavonoids orientin, isoorientin, vitexin, and isovitexin in Passiflora quadrangularis (Passifloraceae) showed an increase of all four flavonoids, with a 40 times greater increase in isovitexin by UV-B exposure than by induction with methyl jasmonate (Antognoni et al. 2007). Overall the flavonoid production antioxidant activity increased from 28-76% in UV-B treated callus versus untreated (Antognoni et al. 2007).
Another study found UV-B added to herbivory increased glucosinolates in Brassica oleracea, broccoli (Mewis et al. 2012). Yet another study showed SA and using transgenic comparisons based on the model organism Arabidopsis metacaspase (Brassicaceae) was able to show that salicylic acid and jasmonate’s involvement in the UV signaling pathways (UVR8-COP1-HY5) increases reactive oxygen species and sunscreen pigments (secondary metabolites) in response to UV levels (Nawkar et al. 2013).
Biotic Factors
Biotic factors such as pollinators, herbivores, soil microbes, infections, and nearby plants can also alter constituents. Many plants have specialized pollinators, which have evolved with them. Some may also be capable of being fertilized by foreign pollinators when introduced to a new environment, but this is not always the case. The pollinator must notice the plant through scent or visual cues; it must be capable of extracting the pollen and depositing it on the next plant and it must do this during the flowering stage, which can change in a new climate. Forsythia suspense (Oleaceae) fructus is an example of a useful herbal medicine, which is limited by its inability to produce seed capsules. As a medical herb used for its activities as a broad spectrum antibiotic, an antifungal, an antipyretic, antinausea, diuretic and hepatoprotective properties, it is grown in Shanxi, Henan, and Shandong in China, where the fruit production is greatest (Foster, Yue 1992). While forsythia grows well (to the point of being classified sometimes as invasive in northeastern America, possibly due to the lack of insect pests), it does not produce fruit (Foster, Yue 1992).
Insects also affect plants as herbivores, but the effects can differ due to the presence of specific oral enzymes and the different patterns of damage during their feeding. For example, in V.vinifera folium culture, saliva Manduca sexta larva (Lepidoptera), was able to induce seven times the production of 3-O-glucosyl- resveratrol in 24 hours (Cai et al. 2012). A study with Plantago lanceolata L. (Plantaginaceae) showed clipping reduced sugars; only with feeding by generalist, Grammia incorrupta (Lepidoptera) was leaf iridoid content increased (Pankoke, Müller 2013). In contrast, for N. tabacum exposed to tobacco hornworm, M. sexta, cutting of equivalent amounts of leaf tissue, or cutting of identical patterns of leaf tissue, it was found that all treatments significantly increased alkaloids concentrations but both cutting treatments were significantly more effective than feeding, suggesting the specialist saliva may be reducing the response (Tallamy, Raupp, 1991).
Microbes, bacteria and fungi that bind to roots can also affect constituents. Joosten and van Veen found that soil microorganisms had a significant effect on pyrrolizidine alkaloid content in both the roots and shoots of Jacobaea vulgaris (Asteraceae). (Joosten, vanVeen 2011). Since microbial infections can stimulate defense pathways and predated herbivores as a stimulus in plant evolution, they theorize that pyrrolizidine alkaloids might have evolved initially as a pathogen defense (Joosten, vanVeen 2011).
A study found that infection with Xylella fastidiosa, pathogenic bacteria responsible for Pierce’s disease, resulted in induction of phenolic compounds in V. vinifera ‘Thompson Seedless’ (Wallis, Jianchi 2012). The full effect was reached by 2 months, but after 6 months, levels had dropped below control, likely due to loss of defensive capabilities after resources had declined, photosynthesis had declined (Wallis, Jianchi 2012).
Similarly, induction of phenolic compounds occurs with Colletotrichum lupini spores applied to Lupinus angustifolius L. (Fabaceae) (Wojakowska et al. 2013). Metabolites (20-hydroxygenistein and phytoalexins: wighteone and luteone) increased within 24 hours with a maximum concentration at 7 days, after that genistein and the previous compounds were reached levels 50 times greater than in controls (Wojakowska et al. 2013).
Yeast has shown increased M. truncatula cell cultures isoflavonoid production benzophenanthridine alkaloids induction in Eschscholzia californica Cham. (Papaveraceae) suspension cell cultures (Broeckling et al. 2005; Cho et al. 2008). Derckel et al. found that the less virulent strain of a pathogenic gray mold, Botrytis cinerea, led to an increase in secondary metabolites, chitinase, b-1,3-glucanases and defensive proteins, while the more virulent strain showed no increase in secondary metabolites and delayed weaker induction of chitinases and b- 1,3-glucanases in infected V.vinifera tissues (Derckel et al. 1999). They reported similar findings with several agricultural plants: french bean (Phaseolus vulgaris), apple (Malus domestica), strawberry (Fragaria × ananassa), carrot (Daucus carota subsp. Sativus), and potato (S. tuberosum) (Derckel et al. 1999).
Many microbes help to form the complex interaction of plants with their environment, helping to fix nitrogen in the soil, and in some cases aiding in production of defensive chemicals either directly or through inducing the plants’ defenses. For some medicinal plants, bacteria and fungi have been shown to be necessary for the production of active constituents, such as microbial endophytes used in TCM (Schmidt et al. 2014). A co-culture of the fungus, F. mairei, with Taxus chinensis L. (Taxaceae) showed a 38-fold increase in taxol over the plant culture alone (Soliman et al. 2013). Similarly taxol-producing fungi, Paraconiothyrium SSM001, does not function in the absence of Taxus spp. tissue, but with the addition of the wood and bark material has been shown to yield a 10-30 fold increase in taxol (Soliman et al. 2013).
Studies have shown rhizobacteria can affect aroma profiles in strawberries and grapes (Schmidt et al. 2014). Biosynthesis of all major classes of secondary compounds (alkaloids, phenolics, and terpenes) have been shown to be stimulated by Arbuscular mycorrhizal fungi (Schmidt et al. 2014). Strains of rhizobacteria have been shown to secrete salicylic acid in beans, Phaseolus spp., to increase resistance to the mold B. cinerea (Derckel et al. 1999).
Signaling Molecules
Nearby plants can affect each other through both damage signals such as methyl jasmonate and allelopathic compounds, where the plant secrete chemicals to inhibit competition. Secondary metabolites found to be active in allelopathy come from all major groups: phenolics (flavonoids), terpenes, and alkaloids (Macías et al. 2007). An example of negative allelopathic effects (plants using phytochemical against other plants) comes from the invasive Centaurea maculosa Lam. (Asteraceae) which secretes catechin, adversely affecting the growth of nearby natives in North America (Bezemer et al. 2014). The antimalarial compound, artemisinin from Artemisia annua L. (Asteraceae) has been shown to inhibit seedling growth in a number of plants (Bharati et al. 2012).
Methyl jasmonate has been shown to induce defense genes and secondary metabolites inducing GDP synthesis and taxadiene synthase enhancing taxol production from T. canadensis and taxane from T. chinensis var. mairei (Sun et al. 2013). M. truncatula cell cultures showed an increase in triterpene saponins and the primary metabolites (b-Ala, GABA, and succinic acid) levels following elicitation with methyl jasmonate (Broeckling et al. 2005). The authors were uncertain as to the function of the primary metabolites, but GABA is highly neurotoxic in many insects.
Salvia miltiorrhiza radix (Lamiaceae), Danshen, is used in TCM to treat cardiovascular, cerebrovascular, and menstrual disorders and methyl jasmonate has been shown to stimulate both genes responsible for and the synthesis of tanshinones (terpenes) and phenolic acids (Luo et al. 2014). Similarly, Atractylodes lancea (Asteraceae) incubated with the fungus endophytic Gilmaniella sp. AL12 used in TCM for its antimicrobial volatile oil, were shown to have increased oil production and increased sesquiterpene components when exposed to jasmonate (Ren, Dai, 2012). Methyl jasmonate also increased benzophenanthridine alkaloids in E. californica suspension cell cultures (Cho et al. 2008).
The other major defensive signaling molecule in plants is salicylic acid, which in its methylated form, methyl salicylate, is aromatic. It has been shown to control aphids on cereal crops in fields (Pickett et al. 2007). Its production is increased with UV-B exposure (Nawkar et al. 2013). Salicylic acid has been shown to double taxol production in fungi and increase it in Taxus spp. cultures (Soliman et al. 2013). It has been shown to increase growth (height, branching, and number of leaves) in several members of Lamiaceae: Mentha piperita, Ocimum basilicum and O. majorana (Perez et al. 2014).
Under normal conditions, M. piperita folium contains 19-23% phenolics per dry weight, rutinoside making up much of the flavor and medicinal properties of the plant, with 12% as flavonoids composed of rosmarinic acid, hesperidin, eriocitrin, luteolin, and 7-O-rutinoside (Perez et al. 2014). Salicylic acid has been shown to increase growth in M. piperita at 2mM concentration, but only increase phenolic compounds at 0.5mM and 1mM concentrations, and to produce phenolics (sinapic acid, rutin and naringin) not seen in the controls (Perez et al. 2014). Similarly in Zingiber officinale (Zingiberaceae) folium, salicylic acid increased total phenolics by 20% and presented phenolics (ferulic and vanillic acid) not found in controls (Perez et al. 2014).
Comparisons of Growing Methods Effect On Constituents
Few comparisons of overall growing conditions were found. One showed hydroponically grown C. officinalis would increase inflorescence size with supplementation of phosphorus, but it neglected to look into the constituent profile of the inflorescences or compare them to other plants (Stewart, Lovett-Doust, 2003). Another study compared conventional (synthetic urea CO(NH2)2 followed by NH4NO3) versus organic (organic urea) fertilizers in cultivation of Olea europaea L. (Oleaceae), olive trees, finding no difference in yield of fruits and only a slight increased bitterness in organic fruits (Rosati, 2014). However the NMR spectrometry showed increased polyphenols in the organic fruits and significant variations in many primary compounds (nucleotides, some amino acids, fatty acids, and glucose) between the two groups (Rosati, 2014).
One of the few studies comparing growing methods and medical constituent quantities looked at bloodroot, S. canadensis, which is predominantly wildcrafted, although some is now produced via cultivation (Graf et al. 2007). The researchers found that the some of the most medically interesting compounds, the benzophenanthridine alkaloids, sanguinarine and chelerythrine, were consistently higher but more variable in the roots of wildcrafted plants, while the root mass was larger in cultivated rhizomes (Graf et al. 2007).
Difficulty with germination is one of the largest obstacles to cultivating many native medical herbs. A study of germination of Collinsonia canadensis L. (Lamiaceae) and Dioscorea villosa L. (Dioscoreaceae), which are often wildcrafted or grown from rootstock due to the difficulty with seed germination, indicated that both species require a period of cold temperatures prior to cool temperatures to germinate, similar to the winter period after the seed dispersal (Albrecht, McCarthy 2016). Twelve weeks of cold treatment provided good results for both species; however, they found that neither species will overcome dormancy with a 6-month dry storage treatment and it would take any seeds planted until the following spring to germinate (Albrecht, McCarthy 2016).
In Sardinia the endemic population of Helichrysum italicum ssp. italicum (Asteraceae), which is valued for anti-inflammatory, antioxidant, and antimicrobial activity (against Staphylococcus aureus and Candida albicans) was examined. Its effects are thought to be due to secondary metabolites – flavonoids, sesquiterpene lactones and essential oils – which depended on both the site of collection and the stage of plant growth (Melito et al. 2013). Of the 50 populations examined, analysis of their genes showed two distinct clades varied by elevation, with cluster A at lowland sites, while cluster B was at mid to high altitudes (Melito et al. 2013). In each cluster the essential oil contents did not vary significantly, but between the populations it varied significantly based on population. The authors expressed regret that they did not compare growing conditions with secondary metabolites, so it is unclear whether the changes were genetic drift or environmental (Melito et al. 2013).
Tims’ thesis on H. canadensis chemical ecology also looked at microecologies of subpopulations. He found elevation was inversely related to quantity of alkaloids present, toxicity of alkaloids, and combined herbivore and pathogen pressures (Tims, 2006). He found wild populations demonstrate increased alkaloid content with increased rhizome size and increased seed number and theorized that this may be an adaptation to protect seed, which are disturbed by ants that collect them for food (Tims, 2006). Increased growth (increased aerial mass and density of population) was noted in disturbed areas, but fertilization did not increase these parameters (Tims, 2006). He also found that proliferation of rhizome size and leaf biomass were greatest under 70% shade, providing valuable information for propagation (Tims, 2006).
Summary and Conclusions
Because of the complexity of environmental plant interactions and their effects on secondary metabolites, there is much opportunity for future research. Understanding of the complex ecological dynamics of secondary metabolites can create opportunities for conservation and more potent herbal medicines, which may provide research and job opportunities. Those who use plant-based medicines in their practice should be aware of threatened and endangered species included in the materia medica. They may be interested in becoming more educated or involved in cultivation or wildcrafting of herbs. Some may even be interested in investigating opportunities in small-scale alleycropping for their own herbal needs.
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