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Thread: Terpens In Resin

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    Lightbulb Terpens In Resin

    TERPENS IN RESIN

    Cannabis is seen by many as a medicine or a recreational drug, but the more subtle aspect of it being an aromatic plant goes too often unnoticed. The herbs we use in our kitchen are considered aromatic plants because they contain a particular terpene profile that gives a distinctive flavor. Cannabis is so complex from this point of view that the possible combinations are endless, creating a broad spectrum of aromas and flavors that differ with the strains and the way they are cultivated.

    To give you an idea of what a terpene is, a quick look at Wikipedia reveals what follows:

    The word terpenes defines a large and varied class of hydrocarbons, produced primarily by plants (conifers and cannabis) and more rarely by insects. Terpens are the major components of resin, and of oils and extracts produced from resin. The name "terpene" is derived from the word "turpentine". Terpenes function as smell and taste molecules, and also as biosynthetic building blocks for the organism of almost all creatures. Terpenes are one of the most important components of the resin and essential oils of many types of plants and flowers.

    Enough with the science. Let’s see how these terpens are affecting cannabis in the process of breeding on flavor.

    The list of 16 terpens is a selection of the most carachteristic and recognizable ones, the ones involved in the distinctive flavor of any strain of cannabis. A few example:

    Alpha-Pinene is an organic compound, found in the oils of many species of many species of pine trees. It is also found in the essential oil of rosemary (Rosmarinus officinalis). 2 types of Alpha-pinene exist in nature, one more common in European pines, the other more common in North America. A mix of the two is present in the oil extracted from the eucalyptus tree. So if you like pine-tree smelling and tasting weed, think of growing this strain, or use it for crossings.

    Limonene is a hydrocarbon, classified as a cyclic terpene. It is a colourless liquid at room temperatures with an extremely strong smell of oranges. It takes its name from the lemon because lemon and other citrus fruits contain high quantities of this compound, which is responsible for much of their smell.

    Sabinene is a natural monoterpene and it is isolated from the essential oils of a variety of plants, mostly oak trees. Sabinene is one of the chemical compounds that contributes to the spiciness of black pepper and is a major constituent of carrot seed oil. It also occurs in tea tree oil at a low concentration.

    Myrcene, or β-myrcene,
    is also an organic compound. It is classified as a hydrocarbon and a monoterpene. It is obtained from the essential oil from various plants: bay, verbena, myrcia (from which is gets its name) and others. Myrcene is one of the most important chemicals used in the perfumery industry. Because of its pleasant odor, it is occasionally used directly. But it is also highly valued as an intermediate for the preparation of flavor and fragrance chemicals such as menthol, citronella, and geraniol.

    Many more terpenes are present in the resin and analyzed, and this represent a guide, a useful tool for a more mature type of smoker, conscious about flavor, and for all breeders interested in following a particular aroma in their lines.

    (This information is from GHSC)

    Aqua Lab Tech

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    Nice post aqua, a wealth of terpene knowledge, Rob' book hashish has some great info as well.
    I would only add that skunkman sam has told me in the past that he enjoy's his 60%THC melty dry sift more than the 100% he has gotten from the lab, because of the terpene content. It definitliy adds a syngery to the thc that is missed without it.

    I personaly havent ever hit a 100% thc, but i have hit 50 , 60 and 70%, and gotta say the volatile oils would be surely missed if they weren't present.



    Also here is a bit of information for those of you not familiar with some of the terms that are thrown around here.
    this is from cannabis-science.com

    Self-Titration
    Scientific Method - An in depth paper from Cambridge University (several pages). The Value of Science - Feynman

    Cannabinoids - A class of organic compounds (molecules) exclusive to the cannabis plant that are derived from natural terpene precursors. Some cannabinoids, such as CBD, have little to no psychoactive effect while others such as THC and CBN, are partially responsible for the psychoactive cannabis experience. Cannabinoids are terpene-phenolic compounds (derived from terpenes plus phenols).

    Pharmacology - The study of drugs, including their composition, biosynthesis, uses and effects in animals. Two important sub-disciplines are: pharmaco-dynamics and pharmaco-kinetics.

    Pharmacodynamics is the study of the molecular, biochemical, and physiological effects of drugs on cellular systems and their mechanisms of action. More simply stated, pharmacodynamics is the study of how drugs act on the body while pharmacokinetics is the study of how the body acts on drugs. You can't have one without the other.

    Pharmacokinetics - The study of the Absorption,
    Distribution, Metabolism and Excretion (ADME) of drugs. More simply stated, pharmacodynamics is the study of how drugs act on the body while pharmacokinetics is the study of how the body acts on
    drugs. You can't have one without the other.

    Medicinal Chemistry - The intersection of chemistry and pharmacy involving the design and development of therapeutic drugs. Medicinal chemistry involves the identification, synthesis and development of new chemical entities suitable for therapeutic use. It also includes the study of existing drugs, their biological properties, and their quantitative (measurement of) structure-activity relationships (QSAR). -WikiPedia

    It is through medicinal chemistry that the majority of man's modern pharmacopoeia is derived from natural products found plants. Say a plant-chemical shows some good effect in tests. A medicinal chemist modifies the molecule just a little, in different ways to see if they can get a better effect with fewer side effects. Looking at how the body reacts to all these little variations in the molecule tells scientists and doctors much more about the body and how diseases do what they do. These scientists continue keep building their understanding in this way. Liquid science.

    Structure-Activity Relationship (SAR) - Process for measuring/predicting how small modifications to a molecule will change its effects. Example: Cannabinoids - The scientist measures the various effects of say THC. The chemist creates a few versions of THC by making slight changes to it, then each new analogue is tested. Based on the data, accurate assumptions can be made about what hundreds of other modifications will do. This helps folks decide which forms are worth putting more effort into.

    Drug Receptor - A cell component that combines with a drug, hormone, or chemical to alter the function of that cell. Drug receptors are conceptually like locks and chemical molecules are the keys. Many receptors will respond to multiple keys with each key having a different effect. Anandamide and other cannabinoids bond with the cannabinoid receptors to different degrees. Depending on the shape of the surface of the cannabinoid, it will affect the receptor in different ways. That is why some cannabinoids are psychoactive and some are not. It all comes down to how well and in what position that the key enters the lock.

    Receptors can regulate a biological process or it can turn processes on or off. An example might be depression meds called serotonin reuptake inhibitors (SSRI). The prescription drug inhibits the process of the body naturally turning serotonin into a byproduct and this elevates serotonin levels thus reducing depression.

    Receptors are classified by the biological systems or cycles that they regulate. Some regulate adrenaline and those receptors and drugs are said to be adrenergic. Other important receptor systems in your body include cholinergic, nicotinic and muscarinic to name a few.

    Learn more about receptors via PowerPoint: Intro to Pharmacology

    Anandamide - The first discovered endogenous (occurs in the body naturally) cannabinoid. It is a chemical derivative of a fatty acid type molecule known as arachidonic acid.

    Cannabinoid Receptors CB-1 & CB-2 - Click for Video

    Terpene - A type of strong-smelling chemical substance found in some plants, especially trees that have cones. Terpenes are found in essential oils. Terpenes are the major components of resin, and of turpentine produced from resin. The name "terpene" is derived from the word "turpentine". Cannabinoids are made using terpenes as building blocks. More: Wiki or Palomar.

    Trichome (Only ONE 'r') - Trichomes on plants are epidermal outgrowths of various kinds. Say What? There are two kinds of trichomes: glandular and non-glandular. Non-glandular trichomes are usually hair-like such as what covers a tomato plant versus the glandular type, which in cannabis contains the resins. Cannabis has both types of trichomes.

    Gas Chromatograph- A sensitive oven-like instrument that distills liquids/oils from a small sample. The hot gas mixture is passed through a thin tube that is lined with a stationary chemical, similar to an artery lined with cholesterol. Gas chromatography takes advantage of the fact that different components of the mixture are collected inside of the tube at different rates and this provides for isolation and measurement of each chemical in the mixture (e.g. THC, CBD, CBN). Sometimes unknown compounds can be identified.

    To the cannabis scientist the gas chromatograph (GC) is an extremely valuable tool. Because the GC allows you to measure most volatile compounds, it allows you to precisely determine the effects of your experiments, whether it be for THC content, THC/CBD ratio or to measure various flavor components for new floral strains (e.g. content % of a terpene called pinene).

    Here is an example of applied GC.

    HPLC is a related analytical tool.

    First-Pass Metabolism is a phenomenon of drug metabolism whereby the concentration of a drug is greatly reduced before it reaches the systemic circulation. -Wikipedia More

    Solvent - A substance that dissolves other substances, thus forming a solution (e.g. Table salt (NaCl) dissolved into water. Water is the solvent in this case).

    There are two classes of solvents: polar and non-polar. Polar solvents are usually alcohols or water, something with OH on the molecule (H2O=HOH). Non-polar solvents are usually long chain molecules like pentane or heptane or octane, or ring molecules like benzene.

    "Like dissolves like"
    With polar solvents one can dissolve polar solids such as sugars, other alcohols like menthol, or some salts. Cannabis extraction with polar solvents will extract the polar compounds including some of the smaller aroma molecules, chlorophyll and other small molecules. On the other end of the spectrum, non-polar solvents dissolve molecules like fats and oils such as cannabinoids and cholesterol.Think of polarity as a scale from 1-10 with 'polar' at 1 and 'non-polar' at 10. Mixing polar solvents with non-polar sometimes gives two layers such as with oil & vinegar because for example, vinegar is 2 and oil is 9. With numbers closer together, two solvents CAN be mixed to specific polarity in order to extract the hard-to-extract.p-value: On this principle, drug absorption in the body is estimated in the lab by adding the drug to a two-layer (biphasic) mixture of water (polar) and octanol (non-polar; the 8 carbons outweigh the OH by a little, so a little octanol dissolves in the water, and a little water dissolves in the octanol, but there are still two layers). The mixture is shaken distribute and dissolve the drug. The % ratio of how much dissolved into water and how much dissolved into water is the partition coefficient (p-value). Medicinal chemists design drugs with this number in mind.
    Soxhlet Extraction - A method of extracting certain components (e.g. oils) from a solid substrate such as plant biomass. Solvent is boiled, condensed and dripped through the plant matter to extract and collect the essential oils. The solvent is recycled making for a highly efficient and contained extraction provided appropriate solvent is used.
    Last edited by Bubbleman; 07-29-2008 at 08:39 PM.

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    continued...


    Tincture - Usually an ethanolic solution containing some active ingredient(s). Often tinctures are simply 100% grain alcohol (ethanol) extracts of herbal biomass. Sometimes a percentage of water is used in order to optimize extraction of certain compounds.

    Self-Titration- Very Important Concept to understand when arguing with somone that claims increased potency is a bad thing. Any claim that more potent pot is significantly more physically harmful than the pot of yesteryear is absurd. Increased potency is a very positive trend and in the future should pharmaceutical companies need natural sources of cannabinoids, the cannabis strains that result will be unbelievably potent and genetically refined for maximum resin yield; Beyond comfort.

    Increased potency means that the user obtains the desired effect (via self-titration or self-thai-tration) using a lesser amount of the other components. That is to say, due to increased potency, a medical patient consumes less cannabis to reach the desired effect, thus less of the potentially harmful impurities and byproducts.. Higher potency cannabis consumed via inhalation is physically SAFER, in our opinion, than lower cannabinoid-content cannabis! And vaporization is much safer than combustion. Vaporization of superb quality cannabis is potentially one of the safest options for consumption.

    Caveat: One possibility is proposed in which the increased THC/CBD varieties of today (the genotype of tomorrow) are actually somewhat detrimental as medical grade cannabis, should it be that non-cannabinoid consituents of the resin synergize to give maximum medical benefit. That is to say, some papers pitch the liklihood that flavonoids, phytoestrogens and terpenes in cannabis can act either independently or together, with or without cannabinoids to offer therapetic benefit. If this is true, then breeders may be increasing cannabinoid content but actually sacrificing other chemicals and critical essences that are necessary for maximum synergy and maximum therapeutic index. Science needs to compare the effects of not only not only high cannabinoid ratios, but high terpene content (limonene), high flavonoid content, high-polypharmaceuticals content. Here is a paper suggesting the importance of Polypharmaceutical Cannabis as opposed to silver-bullet THC/CBD. This is an important argument for keeping synthetic cannabinoid pharmaceutical$ from overshadowing politics and the further induction/continuation of legal medicinal usage of whole cannabis.

    Therapeutic Index - The ratio of therapeutic benefit to side effects. The higher the therapeutic index, the greater the efficacy in relation to its safety.

    Glomerular Filtration - Links to an introductory video on kidney function. This a key concept in pharmacology.

    Spectroscopy - The study by which we utilize electromagnetic energy (e.g. light, UV, infrared, etc.) and expose readily willing molecules in order to perturb or excite them. How theses molecule react to being in the spotlight tells us much about them. With infrared spectroscopy (IR) we look at how the molecule uniquely dances about. With UV we might look at how much a molecule will absorb or throw back at us with vengence.

    Isomerization - Generally, the flipping of a molecule into another geometric shape, or the rearrangement of the positions of the atoms. With many drugs, one shape does one thing, the other shape does another, or sometimes nothing at all. Isomerization can be a natural process but is also often influenced by things like heat, light or pH. Some cannabinoids isomerize to more more potent forms. Others do the opposite.
    Decarboxylation - The loss of carbon dioxide (CO2) from a molecule. This usually happens when a carboxylic acid (R-COOH) loses CO2. THC acid (THCa) undergoes decarboxylation when it is heated and this transformation is a vital contribution to the conversion to the more psychoactive form, THC. Also CBDa -> CBD.

    Analytical Chemistry - The area of chemistry concerned with measuring the various aspects of chemicals in a sample. Analytical chemists use small samples of material and perform a variety of tests to determine the properties of the sample. The analytical chemist does not usually synthesize compounds, but instead only takes measurments to determine content and identity. Determining the cannabinoid content of cannabis requires requires application of analytical chemistry.



    THC - A cannabinoid; the most well known and often prolific psychoactive component of certain mature cannabis plants.
    THC content varies from 0-25%, based on dry weight. Strains containing an estimated 12-16% THC have become more more commonplace than the average strains of yesteryear. This is the result of a several factors including continued selective breeding, more experienced cultivators, easier technology access via internet, and the drastic shift in domestic cannabis production and decreased importation as a result of drug war dollars.




    Peace
    Bubble man

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    Default Diterpenes

    DITERPENES
    They have 20 carbon atoms and are derived from geranylgeraniol pyrophosphate.
    They are of fungal or plant origin and are found in resins, gummy exudates, and in the resinous high-boiling fractions remaining after distillation of essential oils. The rosin remaining after distilling pine turpentine, for instance, is rich in diterpenoids. In ancient times, conifer exudates were used for caulking boats and waterproofing ropes. Resin secretion is also recognized to be part of the resistance mechanism conifers employ against bark beetles and their associated pathogenic fungi. Diterpenoid groups that are physiologically active include: vitamin A activity (retinol), phytohormones that regulate plant growth and germination, e.g. gibberellin, fungal hormones that stimulate the switch from asexual to sexual reproduction, e.g. trisporic acid; disease resistance agents (phytoalexins), e.g. casbene and podocarpic acid and the anticancer drug, taxol, from the bark of the yew tree, and natural cannabinoids.
    The diterpenes have exceptionally open chain, as found in geranylgeraniol or phytol which forms a part of chlorophyll and the side chain of vitamin E and K, and crocetin which is a diacid diterpenoid and the lipid part of the crocins, glycosylated derivatives present in saffron.

    Examples of diterpene substances are given below :





    Abietic acid
    Abietic acid is an irritant compound present in pine wood and resin. It is the most abundant compound present in rosin, the solid fraction of the oleoresin of coniferous trees. It is mainly used to make lacquers and varnishes and metal resinates. These resinates are produced in reacting abietic acid (or a similar compound) with a metal salt (gold, indium, nickel, palladium, platinum, silver ...) and used in a wide variety of applications where high purity metals in organic solution form is needed (gravure printing inks, vitrifiable colors, antifouling agent, dryers for paints and varnishes ...).

    Steviol is the aglycone of stevia's sweet glycosides, one of them being formed by replacing one hydrogen atom (bottom) with glucose via an ester link, and another hydrogen atom (top) with a disaccharide (glucose and rhamnose). The steviol glycosides are responsible for the sweet taste of the leaves of the stevia plant (Stevia rebaudiana, Asteraceae). These compounds are 40 to 300 times sweeter than sucrose. They are developed to be used in sweet drinks.

    Steviol
    Retene is present in tars obtained by distillation of resinous wood, it is an important pollutant eliminated by the paper factories. This diterpene is present in geological sediment where it is formed by diagenesis from abietic acid, several intermediates having been recognized (Wakeham SG et al., Geochem Cosmochim Acta 1978, 42, 289). Thus, with cadalene (sesquiterpene), retene is used in paleobotanic to estimate the importance of ancient pine forests.


    Retene
    Gibberellins are a family of compounds, over 130 members exist whose structures and occurrence can be found on the web. The most important in plants is gibberellin A1 which is responsible for stem elongation. The most widely available compound is gibberellic acid (one double bond in the right cycle). Among the physiological properties, gibberellins are involved in stem growth, seed germination and fruit setting and growth.
    Dehydroleucodine was isolated from Artemisia douglasiana, a popular medicinein Argentina and was shown to have several physiological and therapeutic properties : anti-proliferative activity in G2 phase (Lopez ME et al., Protoplasma 2002, 219, 82), cytoprotective agent for gastric ulcers and a general antioxidant.
    Cafestol and kahweol are present in high concentrations (up to 18% diterpene esters) in the oil derived from coffee beans. The only difference between cafestol and kahweol is an extra double bond present in the second cycle of kahweol. These diterpenes are esterified with one fatty acid (C14 to C24), palmitic and linoleic acids being the major esterified fatty acids (Folstar P et al., Lebensm Wiss Technol 1975, 8, 286). Terpenes were also shown to induce an elevated plasma cholesterol content in human (Boekschoten MV et al Nutr J 2003, 2, 8).
    Cannabinoids are a group of diterpenes present in Cannabis (Cannabis sativa L). All these substances are structurally related to tetrahydrocannabinol (THC) and are able to bind to specific cannabinoid receptors.
    Tetrahydrocannabinol, also known as Δ9-THC or Δ9-tetrahydrocannabinol, is the main psychoactive substance found in the plant. It was isolated by Mechoulam R et al. in 1964 (review in
    Mechoulam Ret al., Chem Phys Lipids 2000, 108, 1).

    Δ9-Tetrahydrocannabinol (THC)

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    Lightbulb Cannabinoids and THC Part 1

    Cannabinoids and THC
    In this study we will examine two types of the several glands on Cannabis, the capitate sessile and capitate stalked forms. In addition, there are hairs on the surface of the plant. In our commentary we will refer to cannabinoids as the broad class of specialized compounds synthesized by Cannabis. Cannabinoids are dimers in that they are formed by condensation of terpene and phenol precursors (c).
    Diagram 1 (Above). Cannabinoid pathway. Cannabinoids represent a dimer consisting of a terpene and a phenol component. Cannabigerol (CBG) is the first component of the pathway. It undergoes chemical change to form either cannabichromene (CBC), or cannabidiol (CBD). Delta 9-tetrahydrocannabinol (THC) is derived from CBD.
    Cannabinoids include these more abundant forms:
    THC, delta 9-tetrahydrocannabinol
    CBD, cannabidiol
    CBC, cannabichromene
    CBG, cannabigerol
    Another cannabinoid, cannabinol (CBN), is formed from THC and can be detected in some plant strains. Typically, THC, CBD, CBC and CBG occur together in different ratios in the various plant strains. In fiber strains CBD/CBC are in high concentrations and THC is at a low concentration; in drug strains, THC is high and CBD/CBC are low. An important area to be studied relates to the occurrence in the plant of potential precursor for cannabinoid formation and where synthesis occurs in the cell.
    Distribution and types of glands
    Glands cover entire surface of the above ground portion of both pistillate (female) and staminate (male) plants, but are most abundant on bracts of female plants (Fig. 1).
    Figure 1. (Above) Stalked and sessile glands on underside of a bract. Hairs also are present on bract. x35.
    Figure 2. (Above). Stalked gland showing large head (star) and abscission zone at base of gland head (arrow). Sessile glands are evident in background along with a portion of a hair. X300.
    Legend for all figures: C, cuticle; D, disc cell; P, plastid; S, secretory material; T, secretory cavity; V, vesicle; W, disc cell wall.
    There are two types of glands active in cannabinoid secretion on female plants (Figs. 1, 2):
    a. Capitate sessile - most common in that it occurs on stems, leaves and bracts.
    b. Capitate stalked - develops only after flower formation, and occurs especially on the bracts subtending a flower and seed. Both types are present on the bract subtending the seed, but some factor(s) that stimulates flowering also stimulates development of the stalk related to this gland. Thus this gland has evolved from the sessile type.
    An abscission zone develops at the base of the head where the stipe cells attach to the disc cells resulting in abscission of glands upon attaining maturity (Fig. 2, arrow).
    Cannabinoid, including THC, composition of glands
    a. Effect of position and season on contents of gland.
    We examined both gland types for their cannabinoid composition. Whole glands (20 glands) were removed from bracts and leaves of the same plant to compare their contents. They were extracted for cannabinoids and analyzed (Table 1).
    We also separated them as to glands over a vein as contrasted to non-vein areas. Results showed that stalked glands over a vein on the bract contained more THC content, approximately 20 times more, than the sessile glands over a leaf vein. Similarly, stalked glands over a non-vein area contained much more THC than sessile glands over the leaf non-vein area.
    Surprisingly, even stalked glands on a bract varied for THC content between the vein and non-vein area. We also found that sessile glands varied for contents between vein and non-vein areas on the leaf.
    This table shows two intervals during the year, October and December, during which we compared levels of THC in glands. Comparison of October with December analyses showed again that stalked glands contained considerably more THC than sessile glands. For both types, however, the vein glands now showed less than the non-vein. Interestingly, the level of THC can decrease to a non-detectable level in the glands (leaf vein glands).
    In similar analyses of these glands on other strains we found the same pattern, but the cannabinoid levels in the stalked glands were not as high as for this strain. In this strain the sampling was repeated during the following March and, again, the stalked glands on the bract contained higher concentrations of THC than sessile glands on the leaf. Thus, a repeatable pattern appears to occur in the plant in which stalked glands usually contained greater quantities of THC than sessile glands.
    b. Effect of gland age on cannabinoid content
    We also examined cannabinoid content of stalked gland by age to measure the major cannabinoid components in both a fiber and drug strain (Table 2). Glands, viewed under a microscope, can be classified according to their secretory phases from the color of their contents. Glands most active in secretion (mature) are translucent in appearance, aged glands are yellow in appearance and senescent glands are brown in color. Mature glands possessed the highest content of their major cannabinoid in both the fiber and drug strains. Senescent glands possessed low levels of cannabinoids. The concentration of some components, as CBD in the drug strains, may be so low that is was not detectable in our analysis; similarly, for THC and CBN in the fiber strain. It is unknown where the cannabinoids go during the aging process, but we suggest that it is possible they volatilize into the atmosphere along with the terpenes in glands, as noted later in this report. Nevertheless, this phenomenon of altered content in glands during aging is one that should be studied to gain a more complete understanding of the secretory process of cannabinoids in the cell.
    Table 2. (Above) Cannabinoid content of capitate-stalked glands of different ages.
    Aqua Lab Tech

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    Default Cannabinoids and THC Part 2

    c. Content of secretory cavity
    The gland consists of disc cells with their cytoplasm and a non-cellular intrawall secretory cavity. We examined the contents of this secretory cavity specifically to determine if cannabinoids occur in it. With microcapillary pipets we removed contents from only the secretory cavity without damaging the disc cells. Data show cannabinoids to be abundant in this cavity (Table 3). Each gland contained an average of 61 nanograms of cannabinoids. This strain was a hemp form with a CBD content. In these analyses each sample included the contents of approximately 100 or more glands. Some samples represent bracts of different lengths, ranging from 4 to 9 mm, the latter being the mature size. Those bract samples of small size, 4 mm., showed a range of variation in cannabinoid content per gland, but some showed the highest content (samples 12 and 16) while others showed a low content (sample 7). Similarly, samples from larger bracts showed relatively high content (sample 6) as well as low content (samples 1 and 2). Some samples also showed different ratios for CBD to THC even when total cannabinoid content was high (compare samples 12 and 16).
    Table 3. Cannabinoid contents in secretory cavities of capitate-stalked glands of a hemp strain showing average cannabinoid content in a gland.
    Although these difference perhaps should be expected in biological samples, it also emphasizes that the cellular component(s) which synthesize cannabinoids may not synthesize them in a constant ratio during development of an organ.
    Summary:
    a) Capitate stalked glands contained more THC (and total cannabinoids) than capitate sessile glands.
    b) THC, and total cannabinoid, quantity in both gland types can vary during the year, and can decrease to very low levels.
    c) Cannabinoids decrease with aging of glands.
    d) Cannabinoids occur in the secretory cavity of the gland.
    Location of cannabinoids in the gland
    a. Development of the gland.
    For both types of glands an epidermal cell enlarges and divides several times to form a tier of disc cells on a short stipe on the epidermis of the leaf or bract (Diagr. 2).
    Diagram 2. (Above). Representation of mature secretory gland. Disc cells, attached to leaf or bract by stipe cells and basal cells (below stipe), release fibrillar wall matrix into secretory cavity where it contributes to thickening of subcuticular wall (wall) during enlargement of secretory cavity. Plastids (P) in disc cells produce secretions which accumulate outside plasma membrane, pass through cell wall as hyaline areas to form secretory vesicles (L) in secretory cavity. Vesicles in contact with subcuticular wall release contents to contribute to growth of cuticle during enlargement of secretory cavity. THC occurs in walls, fibrillar matrix and other contents surrounding the vesicles, but not in the vesicles; little THC is present in the disc cells. Nucleus = black; vacuole = V; vesicles = L; plastids = P; endoplasmic reticulum = ER.
    The outer wall of the disc cells splits tangentially to initiate an intrawall cavity across the top of the entire gland surface. This cavity enlarges as secretions are accumulated in it (Fig. 3).
    Figure 3. Section where secretory cavity joins disc cell showing cuticle and subcuticular wall, vesicles in secretory cavity, secretions in disc cell just below the wall of the disc cell separating it from the secretory cavity. Bar = 0.5 µm.
    The outer portion of the wall remains associated with the cuticle to form the subcuticular wall; the inner portion remains associated with the disc cells. Both the cuticle and subcuticular wall increase in thickness as the secretory cavity enlarges and, therefore, precursors for their growth must be present in the secretory cavity. Secretions as vesicles are evident in the secretory cavity. Note the vesicles are similar in density (grayness) to gray secretions in the disc cell. The mechanism that controls the thickening of cuticle and subcuticular wall are as yet unknown.
    b. Secretion role of disc cells
    It is pertinent to examine the organization of the disc cells because all contents in this cavity must be derived from the disc cells. Cannabinoids, or their precursors, are secretions from these cells. Another major group of secreted compounds are the terpenes (monoterpenes and sesquiterpenes). Monoterpenes are the more abundant of the two. Terpenes compose the "essential oils"; they contribute to the odors of the plant, and are sticky in character, as evident when one touches the plant. Different combinations of terpenes in different strains contribute to odor differences among the strains. Cannabinoids, and THC, are odorless to most humans.
    Figure 4. Portion of gland showing several disc cells each with numerous lipoplasts. Portion of secretory cavity is evident. Fibrillar matrix has separated from the wall (arrow). Bar = 0.5µm.
    The tier of disc cells contains a typical cell complement including a large nucleus, plastids, mitochondria, endoplastic reticulum and abundant ribosomes, as well as vacuoles (Diagr. 2; Fig. 4). Plastids, however, represent the unique component of these cells. They are interpreted to be a source, perhaps the principal source, of secretions in the cavity. Plastids divide repeatedly and become very numerous in the disc cells. Plastids form a unusual central component, termed the reticulate body, derived from thylakoids (Figs. 4, 5). This body consists of thylakoids fused into a tubular array of light and dark areas in a hexagonal arrangement. This large body is somewhat like a prolamellar body that forms in chloroplasts when plants are grown in the dark because it has a lattice configuration similar to a prolamellar body. But it is unlike a prolamellar body in that it persists in both the light and the dark, and it does not contribute to formation of grana membranes as does the prolamellar body.
    Figure 5. Lipoplast showing presence of secretion on its surface and region where secretion joins the reticulate body (arrow). Bar = 0.2 µm.
    The reticulate body was associated with secretory activity. Enlarged quantities of secretions accumulated on the surface of the plastids, and were continuous with the light zone in the reticulate body (Fig. 5). The association of the secreted mass with the light zone on the surface of the plastid supported an interpretation that the reticulate body contributes to the synthesis of these secretions (Fig. 5, arrow). Such secretions were evident on nearly all plastids and may become so voluminous as to surround a plastid, as it appears in an electron micrograph. These secretions are more or less round in appearance in these sections, but undoubtedly spherical in three-dimensions; since they are oily in composition they form spherical masses in the aqueous medium of the cell. These secretions are interpreted to be terpenes; plastids in other plants are reported to produce terpenes.
    Figure 6. Lipoplast near plasma membrane (arrow). Portion of secretion in contact with and passing through the plasma membrane (arrow). It will accumulate between the membrane and cell wall. Bar = 0.1 µm.
    Aqua Lab Tech

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    Default Cannabinoids and THC Part 3

    The secretions on the surface of plastids can be observed in contact with the plasma membrane (Figs. 5, 6). Similarly, other quantities of secretions of similar density, but not in contact with a plastid in a thin section, were observed in contact with the plasma membrane (Figs. 3, 7).
    Figure 7. Secretions (small light areas in wall) emerging as a vesicle (V) in the secretory cavity (T). Fibrillar matrix (arrow) appears to be released from the wall into the secretory cavity. In disc cell, secretions are evident in the cytoplasm adjacent to the plasma membrane and on the surface of lipoplasts. Bar = 0.2 µm.
    Secretions in the secretory cavity
    Secretions somehow passed through the plasma membrane and accumulated in the space between the plasma membrane and cell wall (Fig. 6, arrow and S). Secretions subsequently passed through the cell wall, often appearing as small light areas in the wall (Figs. 3, 5, 6).
    Secretions emerged into the secretory cavity as small accumulations on the wall surface facing the cavity (Figs. 3). Small gray vesicles, partly embedded in the wall, at W and to the lower left of the large vesicle (V) near the "corner" of the secretory cavity. They have the same gray density as secretions in disc cell (Fig. 6). It appeared that the material passing through the wall accumulated in enlarged vesicles on the wall surface facing the secretory cavity. As vesicles emerge from the cavity they became surrounded with a surface feature about Ω the thickness of a typical membrane (Figs. 3, 7).
    Vesicles are released from the wall surface to aggregate in the secretory cavity (Fig. 8). These vesicles are transported to the subcuticular wall surface where some of them released their contents into the wall (Fig. 8, curved arrow). The contents also moved through the subcuticular wall to the cuticle where the contents aided thickening of the cuticle (Figs. 3, 8). The irregular contour of the inner surface of the cuticle with dark fiber-like extensions in the cuticle, is derived from the fusion of quantities of vesicular material along with its surrounding surface feature.
    Figure 8. Outer sheath of secretory cavity consists of cuticle and subcuticular wall. Vesicles (light areas) are evident in the subcuticular wall (curved arrow). Vesicles and fibrillar matrix are evident in the cavity. Bar = 0.1 µm.

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    Wow guys, thats a lotta info, thanks for sharing.
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    Default Cannabinoids and THC Part 4

    Fibrillar matrix is released from the surface of the wall into the secretory cavity. This matrix is evident at this wall as well as in the secretory cavity (Figs. 4 arrow, 7 arrow). The fibrillar matrix is transported to the subcuticular wall where it becomes incorporated thereby contributing to the thickening of this wall. The mechanisms controlling deposition of fibrillar matrix in the subcuticular wall, as well as the deposition of vesicular materials into the subcuticular wall and cuticle, remain to be studied. Since the sites of deposition are very distant from the origin of these materials, we speculate that the control mechanism somehow resides in the non-cellular secretory cavity.
    Summary:
    a) Plastids produce a major quantity of secretions that are released from their surface to pass through the plasma membrane and outer wall into the secretory cavity.
    b) Secretions accumulate in the secretory cavity as secretory vesicles whose contents contribute to thickening of the cuticle.
    c) Volatile components of secretory vesicles may volatilize into atmosphere and contribute to plant odor.
    d) Fibrillar matrix and other material released from the wall surface surround the secretory vesicles with a surface feature of yet unknown composition.
    Fibrillar matrix contributes to thickening of the subcuticular wall.
    Localization of THC in the gland
    Although cannabinoids were detected in the contents isolated from the secretory cavity, we do not yet know whether they occur in the disc cells. Further, we do not yet know where they are in the secretory cavity: in or around the vesicles, or elsewhere in the cavity.
    For this phase of study fresh glands were frozen by high pressure cryofixation and then fixed (killed) by cryosubstitution to prevent movement of THC in the gland during the fixation process. We prepared a monoclonal antibody for THC as a probe by attaching gold particles to it so that it would be visible under the electron microscope. Then thin sections of glands were treated with the antibody probe; the antibody will attach to any THC in the tissue. Under the electron microscope we will see the electron dense gold particles, as dense black dots, where the antibody has attached to THC.
    Upon examining the tissues we find that the THC, as indicated by the location of gold particles, is present in the cell wall facing the secretory cavity (W), and in the subcuticular wall (large arrowhead) under the cuticle (Fig. 9). It is also present in the fibrillar matrix being released into the cavity from the disc cell wall (open arrow). It was along the surface feature (small arrowhead) surrounding the large secretory vesicles in the cavity. It was also in the cuticle (arrow).
    Figure 9. (Above) Sites of THC accumulation are evident as black dots representing the gold attached to the THC antibody. THC is present in the disc cell wall (W), subcuticular wall (large arrowhead), along the surface features around vesicles in the cavity (small arrowhead), in fibrillar matrix being released from the disc cell wall (open arrow) and in the cuticle (arrow). No particles were evident outside the gland. Bar = 0.2 µm. Figure 10. THC is abundant along the surface features (arrow) of the numerous vesicles in the secretory cavity, but absent from the content in the vesicles. Where the surface feature of a vesicle is sectioned in surface view, THC appears over the entire surface. Bar = 0.1 µm.
    Deep within the cavity we note, again, that THC was associated with the surface feature around the numerous secretory vesicles, but it was not inside the vesicles (Fig. 10 arrow). Some vesicles were cut with their surface feature in planar view (slightly gray appearance), and gold grains are associated with the area of the surface feature. The contents of these vesicles (clear area), presumably, are the monoterpenes (lipoidal materials).
    Surprisingly, it is not in the cytoplasm of the disc cells (Fig. 9). We detected it only along the plasma membrane and in the wall proper of the disc cell. Only an occasional grain was detected in the cytoplasm, either over a plastid, or mitochondrion or among ribosomes. If cannabinoids are synthesized in the cytoplasm of disc cells, there should be abundant gold particles at sites of THC synthesis and accumulation.
    In other cells, as epidermal cells, THC was present in the wall, but in smaller quantities than in walls of the disc cell. Few gold particles were in the cytoplasm or vacuoles of other cells.
    Controls included sections treated with antibody alone, or treated with protein A-gold alone. Controls showed no antibody in the disc cell wall, around vesicles, in the subcuticular wall or in cuticle. No antibody was detected outside of the cells.
    Possible site of cannabinoid synthesis
    Our studies contribute several pieces to the puzzle on where cannabinoids are synthesized, yet we lack definitive information on the precise site of their formation. Data from the antibody probe showing that they are not evident in the cytoplasm of the disc cells suggest that they may be formed at or outside the plasma membrane surface. A working model for continued study on their synthesis is embodied in diagram 3.
    Diagram 3. (Left) Representation of gland illustrating the possible process in cannabinoid localization in secretory cavity. A phenol glucoside is transported into a disc cell and stored as free phenol in the vacuole. Terpene is synthesized by the specialized plastid, lipoplast, in disc cells. These precursors, terpene and phenol, react to form cannabinoids at the plasma membrane surface or in the wall whereupon they appear in the secretory cavity.
    As described in the cannabinoid pathway, these dimeric compounds consist of terpene and phenolic components. The abundant secretory activity of the disc cell plastids, and knowledge that this organelle does synthesize terpenes, suggests that they contribute the terpene component. Our detection, in previous studies, of abundant phenol in whole glands, and knowledge that phenols accumulate in vacuoles of cells, suggests that this cell feature may contribute the phenol component. Phenols are transported in the plant as glycosides and, when becoming localized in a cell vacuole, they accumulate there upon dissociation of the sugar moiety which returns to the cell cytoplasm.
    We hypothesize that terpenes and phenols, when released from their respective sources, accumulate at the plasma membrane and cell wall interphase where enzymes dimerize these compounds into cannabinoids. It is necessary to determine enzymes involved in cannabinoid synthesis. Such an enzyme, when available, can be prepared as an antibody probe that can be used to identify more precisely the locus of cannabinoid, and THC, synthesis. Glands represent unique structures, and can be utilized to broaden our understanding of cannabinoid synthesis and aid in our effort to reduce the cannabinoid content of Cannabis strains for production of industrial hemp.
    CONCLUSIONS
    1. THC accumulated in abundance in the secretory cavity where it was associated with the: a) cell walls, b) surface feature of secretory vesicles, c) fibrillar material released from disc cell wall, and d) cuticle. It was not associated with the content of the secretory vesicles. The association of THC with structural components, particularly the wall, fibrillar matrix and surface feature of vesicles, suggests that it may be chemically bound to them rather than being free in the cavity. If THC and other cannabinoids are bound to components in the cavity, their presence and movement may require a source of energy in the cavity. Additional studies are necessary to determine their bound or free status.
    2. Little or no THC was detected in the cytoplasm of the disc cells. This suggests that the terpene and phenol precursors, which must occur in the disc cells at some interval, may form the cannabinoids at the surface of the plasma membrane, or in the cell wall facing the secretory cavity.
    3. Some THC was present in the cell walls of other cells. Genes for cannabinoid synthesis are present in all cells of the plant, but tissues other than glands produce low levels of these compounds.
    4. Reduction or elimination of glands by mutation procedures will reduce significantly the quantity of THC in the plant. However, the pathway for cannabinoid synthesis is controlled genetically: glands are specialized to synthesize high levels of cannabinoids. Thus, a glandless plant can be expected to synthesize very low levels of cannabinoids. We do not know the roles of cannabinoids in the glands. They may be involved in some way with "protection", or other role. The absence of glands may or may not alter the functional role. Since other cells also synthesize these compounds, at very low levels, the quantity may be sufficient to perform the functional role. Therefore, a glandless mutant(s) would serve our purpose to reduce the THC concentration for utilization of such strains in the hemp industry.

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    Thumbs up Trichome University

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