Localization of Salvinorin A and Related Compounds in Glandular Trichomes

Title: Localization of Salvinorin A and Related Compounds in Glandular Trichomes of the Psychoactive Sage, Salvia divinorum (Summary)
Authors: Daniel J. Siebert
Journal: Annals of Botany 93
Date: December 2003

Link to original paper: http://aob.oxfordjournals.org/cgi/reprint/93/6/763.pdf?ijkey=55zbQRbeCanlM&keytype=ref

Abstract

Background and Aims: Salvia divinorum produces several closely related neoclerodane diterpenes. The most
abundant of these, salvinorin A, is responsible for the psychoactive properties of the plant. To determine where
these compounds occur in the plant, various organs, tissues and glandular secretions were chemically analysed.
A microscopic survey of the S. divinorum plant was performed to examine the various types of trichomes
present and to determine their distribution.
Methods: Chemical analyses were performed using thin layer chromatographic and histochemical techniques.
Trichomes were examined using conventional light microscopy and scanning electron microscopy.
Key Results: It was found that neoclerodane diterpenes are secreted as components of a resin that accumulates
in peltate glandular trichomes, specifically in the subcuticular space that exists between the trichome head cells
and the cuticle that encloses them. Four main types of trichomes were observed: peltate glandular trichomes,
short-stalked capitate glandular trichomes, long-stalked capitate glandular trichomes and non-glandular
trichomes. Their morphology and distribution is described. Peltate glandular trichomes were only found on the
abaxial surfaces of the leaves, stems, rachises, bracts, pedicles and calyces. This was consistent with chemical
analyses, which showed the presence of neoclerodane diterpenes in these organs, but not in parts of the plant
where peltate glandular trichomes are absent.
Conclusions: Salvinorin A and related compounds are secreted as components of a complex resin that accumulates
in the subcuticular space of peltate glandular trichomes.

Introduction
Salvia divinorum has been used by the Mazatec Indians at sub-visionary doses to treat a variety of conditions. These include arthritis, headache and eliminatory complaints. It contains salvinorin A, which is the first naturally occurring, nonnitrogenous, opioid-receptor-subtype-selective agonist discovered.

The concentration of salvinorin A at the various stages of growth in an individual of the plant is surprisingly consistent. However, concentrations vary dramatically from plant to plant, even when they are genetically identical. Concentration in leaves was found to range from 0.089 to 0.37% (most around 0.245%). The stems contained roughly 4% of the concentration in the leaves. Salvinorin A is very stable under normal storage conditions; a 40-year-old specimen was found to contain a similar amount of salvinorin A as fresh leaves.

Closely related compounds also found in the plant include: salvinorin B, salvinorin C, salvinorin D, salvinorin E, salvinorin F, divinatorin A, divinatorin B, divinatorin C and (-)-hardwickiic acid. Of these compounds, salvinorin B, salvinorin C and salvinorin D are the most abundant and there are considerably lower concentrations of salvinorin E, salvinorin F, divinatorin A, divinatorin B,divinatorin C and (-)-hardwickiic acid. Hardwickiic acid is the only one of these compounds that is known to exist in anything besides S. divinorum.

Fig. 1. [VIEW] Salvia divinorum plant in bloom. This specimen was photographed at Cerro Camaron, in the Mazatec region of Oaxaca, Mexico.

Fig. 2. [VIEW] Structural diagrams of salvinorins A-F, divinatorins A-C and (-)-hardwickiic acid.

Most labiates (S. divinorum is in the Labiatae family) have glandular trichomes on the epidermal surface of their leaves, stems, and reproductive structures. These structures secret various types of compounds (many are terpenes) and evidence shows that terpene biosynthesis takes place within these trichomes. In the Labiatae family, glandular trichomes are generally classifed as either capitate (clavate) or peltate (subsessile), based on structural characteristics. Capitate trichomes are mostly excreted to the surrounding environment, apparently through pores in the cuticle (outer layer) of the head cell(s). In peltate trichomes, the secretions accumulate in a capacious subcuticular space that is formed by the separation of the head cell walls from the cuticular dome that encloses them, and they remain there until the cuticle is physically ruptured.

Methods
Plant material: Plants were asexually propagated.

Microscopy: The distribution and morphology of the glandular trichomes was examined using a microscope. Leaves were dipped in chloroform in order to make the trichomes more visible as they turn a milkey white. However, on very young leaves the peltate trichomes became iridescent. Scanning electron microscopy (SEM) was used to view trichomes in greater detail.

Sequencial extraction: The leaves were extracted by first dipping them into 3 beakers filled with chloroform, one after another. Then the dipped leaves were dried and powdered and then put into a 4th beaker for further extraction. Next, equal volume samples from each beaker were analyzed for salvinorin A concentration by spotting a TLC (Thin layer chromatography) plate.

Collection of glandular secretions: Collected from both the abaxial and adaxial surfaces of fresh leaves and from the abaxial stem surface by gently brushing chloroform-soaked cotton swabs over the epidermis.

Extraction of various parts: Individual extracts were made from the roots, external stem tissue, internal stem tissue, internal petiole tissue, leaves, cotyledons, rachises, bracts, pedicles, calyces and corollas by putting samples in chloroform and then spotting on TLC plate.

Isolation of peltate glandular trichomes from the abaxial (underside) of leaves: Adhesive tape was used to remove these trichomes (it removed approx 50% of them).

Removal of secretory products from the subcuticular reservoirs of individual peltate glandular trichomes: 1x3 cm section cut from leaf and dipped in chloroform. Then, under a microscope, a needle was used to pierce the cuticles of trichomes.
Extraction of leaves at six different stages of development: Six pairs of leaves from the uppermost six nodes were harvested from two plants. Leaves were dried and powdered, extracted with chloroform, and then spotted on a TLC plate.
Thin layer chromatography (TLC): Use Whatman silica gel plates developed in ethyl acetate/hexanes. The terpenes were visualized (they turn purple) by spraying the plates with vanillin reagent, and then heating it. Photos of the plates were then scanned onto a computer. To confirm that the compounds were correctly identified chromatography was also performed using acetone/dichloromethane and chloroform/methanol/water as solvents.

Histochemistry: Compounds were also visually observed under a microscope on whole dried leaves and stem segments by spraying the vanillin reagent directly onto plant tissue.

Results
The variety of trichome types in S. divinorum and their distribution on the plant: Two general types of trichomes: non-glandular trichomes and glandular (secretory) trichomes. Two types of glandular trichomes: peltate and capitate. Two types of capitate glandular trichomes: short-stalked and long-stalked.

Non-glandular trichomes are hair-like structures made up of a linear string of cells. They are present on stems, rachises, petioles, the abaxial veins of the leaves and bracts and the primary adaxial leaf veins. In seedling plants, the first few pairs of true leaves produce exceptionally long (8-12 mm), rigid, non-glandular trichomes that stand straight up from the adaxial leaf surface.

The peltate glandular trichomes consist of six cells: a basal epidermal cell, a short stalk cell and four head cells that are enclosed in a smooth cuticle. They secrete ricj terpene resin which accumulates in the large subcuticular space (the head). A fully developed head is quadrilobate and 35-45 µm in diameter. While absent on the adaxial side of the leaves they are abundant on the abaxial surfaces of the leaves, stems, rachises, bracts, pedicles and calyces. Young trichomes are milky-white or colorless, but as they age they turn honey-colored. The density of Peltate glandular trichomes on the abaxial surface of a fully developed leaf is typically about 1600 per cm².

Most capitate glandular trichomes are short-stalked and are present on the stems, leaves, cotyledons, rachises, pedicles, bracts and calyces. They seem to be short-lived because it is common for them to appear shrived and collapsed. Many short capitate trichomes have two head cells and long ones have a single head.

Sequential leaf extracts: First extract contained most salvinorins with no other pigments or chlorophylls present. Terepenes became progressively less concentrated after each extract.

Glandular secretions collected from the epidermal surfaces of leaves and stems: Salvinorins were found to be highly concentrated on the abaxial surface of the leaves while none was found on the adaxial leaf surfaces.

Extracts from various parts of the plant: Salvinorins were found in the external stem tissue, leaves, rachises, bracts, pedicles, and calyces but not in the roots, internal stem tissue, internal petiole tissue, cotyledons, or corollas.
Isolated peltate glandular trichomes and the leaf from which they had been removed: High salvinorin concentration in the peltate glandular trichomes that had been removed with adhesive tape (about 50% of total salvinorin in leaf was removed with tape).
Subcuticular contents of peltate glandular trichomes: Subcuticular cavities of peltate glandular trichomes had high concentrations of salvinorins.

Leaves at six stages of development: Salvinorin A was found in similar concentrations in all six leaf samples of each developmental stage. None of the samples contained salvinorin C, which apparently only occurs in leaves olders than those analysed. There were several unidentified compounds produced dark blue spots on the TLC plates. Concentrations of these compounds was greatest in the youngest leaves and in these young leaves its concentration was substantially greater than that of salvinorin A. These unknown compounds are also prominent in thecalyces, pedicles, and bracts.

Histochemical analysis: Salvinorin reacted with the reagent on glandular trichomes but not at all on the non-glandular trichomes.

Discussion
The chloroform dips showed that terpene compounds are secreted externally to the epidermis because the epidermis did not need to be penetrated to extract compounds. Swabbing the abaxial sides of leaves also proved this.

Peltate glandular trichomes were rich in salvinorins, and more specifically rich in the extracellular subcuticular space of these trichomes. Chemical analysis showed that salvinorins are only present in parts of the plant that contain peltate glandular trichomes. Therefore, it is suggested that production of salvinorins is specific to these kinds of trichromes. Histochemical analyses of various parts of the plant show that salvinorins are absent from the non-glandular tissues.
The ratio of salvinorin C to salvinorin A is greater in the mature leaves, stems and rachises, than in the young leaves, bracts, pedicles and calyces.

Fig. 3. [VIEW] Scanning electron micrographs showing S. divinorum trichomes. (A) An intervenous area of the abaxial leaf surface with several peltate glandular trichomes clearly visible. Many of these are slightly sunken in the epidermis. Although difficult to see because of their much smaller size, several short-stalked capitate glandular trichomes are also present in this frame; many of these have withered. Scale bar = 100 mm. (B) A peltate glandular trichome on the abaxial leaf surface. The cuticle is broken, revealing the salvinorin-rich resinous secretion that accumulates in the subcuticular cavity. The spongy appearance of the secretion suggests that it accumulates as a heterogeneous emulsion. Scale bar = 10 mm. (C) Non-glandular trichomes on a vein of the abaxial leaf surface. A peltate glandular trichome is visible in the upper left quadrant of the frame. Scale bar = 50 mm. (D) Adaxial surface of a young leaf with non-glandular trichomes and short-stalked capitate glandular trichomes. A large percentage of the capitate glandular trichomes here have two head cells. The non-glandular trichomes are particularly stout on this surface, except for those on the veins, where they are slender. The trichomes would be scattered much further apart on a fully developed leaf. Scale bar = 200 mm. (E) Peltate
glandular trichomes and short-stalked capitate glandular trichomes on the abaxial calyx surface. The capitate glandular trichomes shown in this frame all have two head cells, some of which have collapsed and shrivelled. Scale bar = 20 mm. (F) The abaxial surface of a young calyx showing a dense covering of non-glandular trichomes, peltate glandular trichomes, and both short-stalked and long-stalked capitate glandular trichomes. Scale bar = 100 mm.

Unknown compounds were detected in young leaves, bracts, pedicles, and calyces, but their reaction on the TLC plates suggest that they are not likely to be neoclerodane diterpenes. Concentrations of one of these compounds was especially high in very young leaves as well as in the bracts, pedicles, and calyces. Since the calyces protect reproductive organs and that young leaves are especially vulnerable, this compound may have a protective function.
The inflorescence emits a pleasant fragrance that is not present in other parts of the plant which suggests there might be volatile terpenes. The long large-stalked capitate glandular trichomes might secrete compounds responsible for the aroma.

The density peltate glandular trichomes on the abaxial stem surface is about the same as that on the abaxial leaf surface. They occur at higher densities on the young leaves compared older leaves; however, salvinorin A content stays constant which is probably because it accumulates as the plant develops.

Fig. 4. [VIEW] Long, rigid, upright non-glandular trichomes on the adaxial leaf surface of a S. divinorum seedling (present only on first few sets of leaves).

Fig. 5. [VIEW] Thin layer chromatograms. Arrows: A = salvinorin A, B/D = salvinorin B and salvinorin D together as an unresolved single spot, C = salvinorin C. (A) Chromatogram of sequential chloroform extracts of fresh leaves. Lane 1, first 30-s extract; lane 2, second 30-s extract; lane 3, third 30-s extract; lane 4, 2-h chloroform extract prepared from the previously extracted leaves after they had been dried and powdered. (B) Chromatogram of chloroform extracts prepared from six organs of S. divinorum. The stems and rachises both contain a high percentage of pith and vascular tissue. The low concentration of salvinorins in the rachis extract rejects this. The stem extract shown here was prepared from the external stem tissue only, and therefore the concentration of salvinorins is much higher than it would be if whole stems had been used. Of the other plant parts analysed, but not pictured here, only the pedicles contained salvinorins. (C) Comparative analysis of leaves at six progressive stages of development. Lane 1, leaves from the uppermost node of the stalk (the youngest leaves); lane 2, leaves from the second node down; lane 3, leaves from the third node down; lane 4, leaves from the fourth node down; lane 5, leaves from the fifth node down; lane 6, leaves from the sixth node down. The concentration of salvinorin C in these leaves is too low to detect in this chromatogram. This compound is more concentrated in leaves that are older than those analyzed here.

Although Salvia is susceptible to many pests, since salvinorin-secreting trichomes cover plant surfaces they may have an insect antifeeding property. It could also deter deer or other mammals that are likely to be susceptible to its psychoactive effects.

Further Research Questions
What are the unidentified compounds that occur at high concentrations in young leaves?
Are salvinorins biosynthesized in the glandular trichomes?
Are the other various neoclerodane diterpenes also localized in peltate glandular trichomes?

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