Saw Palmetto Extract Laboratory Guidance Document

Home , Iodine in biology, Saw palmetto extract, Serenoa

Keywords: Adulteration, animal fatty acids, canola oil, coconut oil, palm oil, saw palmetto, Serenoa repens, sunflower oil, vegetable oil

Citation (JAMA) style: Gafner S. Saw palmetto extract laboratory guidance document. Austin, TX: ABC-AHP-NCNPR Botanical Adulterants Prevention Program. 2019. 1. Purpose There is documented evidence of the adulteration of saw palmetto fruit extracts with a number of vegetable oils, such as canola (Brassica napus ssp. napus, Brassicaceae), coconut (Cocos nucifera, Arecaceae), olive (Olea europaea, Oleaceae), palm (Elaeis guineensis, Arecaceae), peanut (Arachis hypogaea, Fabaceae), and sunflower (Helianthus annuus, Asteraceae) oils. The partial or complete substitution of saw palmetto fruit extracts with mixtures of fatty acids of animal origin was first documented in 2018,1 and seems particularly common in materials sold as saw palmetto originating from China. This Laboratory Guidance Document (LGD) presents a review of the various analytical technologies used to differentiate between authentic saw palmetto extracts and ingredients containing adulterating materials. This document can be used in conjunction with the Saw Palmetto Botanical Adulterants Bulletin, rev. 3, published by the ABC-AHP-NCNPR Botanical Adulterants Prevention Program in 2018.2 2. Scope does not reduce or remove the responsibility of laboratory Various analytical methods are reviewed here with the personnel to demonstrate adequate method performance specific purpose of identifying their strengths and limita- in their own laboratories using accepted protocols. Such tions in differentiating saw palmetto fruit extracts from protocols are outlined in the United States Food and Drug potentially adulterating materials. Less emphasis is given to Administration’s Good Manufacturing Practices (GMPs) the authentication of whole, cut, or powdered saw palmetto rule (21 CFR Part 111) and those published by AOAC Inter- fruit and distinguishing it from potential confounding national, International Organization for Standardization materials, e.g., the Everglades palm (Acoelorrhaphe wrightii, (ISO), World Health Organization (WHO), and Interna- Arecaceae), by macroscopic, microscopic or genetic analy- tional Conference on Harmonisation (ICH), and national sis. Analysts can use this review to guide their selection pharmacopeial bodies, as may be applicable, depending on of appropriate analytical authentication techniques. The the regulatory requirements of the country in which the suggestion of a specific analytical method for testing saw saw palmetto extract is being offered for sale, re-sale, and/ palmetto materials in their particular matrix in this LGD or processing into finished consumer products.

Saw Palmetto Extract - Laboratory Guidance Document • 2019 • www.botanicaladulterants.org 1 3. Common and Scientific Names 4. Botanical Description and Geographical Range Saw palmetto grows as a small shrub, occasionally a small 3.1 Common name: Saw palmetto tree with creeping, horizontal, branched stems, usually to a height of 2-7 feet (0.6-2.1 m), although it may reach up to 3.2 Other common names for saw palmetto 25 feet (7.5 m). The stem systems run parallel to the soil English: Scrub-palmetto, sabal palm, saw palmetto berry surface, eventually branching beneath the substrate to form Chinese: Ju zonglu (锯棕榈) rhizomes. Saw palmetto leaves are fan-shaped, evergreen French: Sabal, palmier nain, palmier scie and about 3 feet (1 m) wide. The margins of the petioles German: Sabal, Sägepalme, Zwergpalme are lined with sharp spines that have given saw palmetto Italian: Palma nana, cavolo di palma its common name. The flowers are cream-colored and Russian: Сереноя ползучая (Serenoa repens), Сабаль fragrant, with three petals at the end of stalked panicles пильчатый (Sabal serrulata), карликовая пальма (karliko- that grow from the leaf axils. The fruit is a drupe, green or vaya palma, “dwarf palm”), пальма cереноа, co пальметто yellow at immature stages, and black when ripe (between Spanish: Sabal, palma enana americana3,4 August and October), resembling black olives in size and Swedish: sågpalmetto shape.6,7 The plant is endemic to the southeastern United States, growing from the coastal plains of Louisiana across 3.3 Latin binomial: Serenoa repens (W. Bartram) Small the Florida peninsula and up to South Carolina.6

3.4 Synonyms: Chamaerops serrulata Michx., Corypha 5. Adulterants and Confounding Materials obliqua W. Bartram, Corypha repens W. Bartram, Diglos- See Table 1 below. sophyllum serrulatum (Michx.) H. Wendl. ex Drude, Sabal serrulata (Michx.) Nutt. Ex Schult. & Schult. f., Serenoa 6. Identification and Distinction using serrulata (Michx.) G. Nicholson5 Macroanatomical Characteristics Botanical descriptions of saw palmetto fruit have been 3.5 Botanical family: Arecaceae published in a number of pharmacopeial monographs

Table 1. Scientific Names, Family, and Common Names of Plants Used as Sources of Vegetable Oils Known as Saw Palmetto Fruit Extract Adulterants*

Speciesa Synonym(s)a Family Common nameb Other common namesc Arachis hypogaea L. A. nambiquarae Hoehne Fabaceae Peanut Groundnut Brassica napus L. Brassica napus ssp. napus Brassicaceae Canolad Colza, rape, rapeseed Cocos nucifera L. Calappa nucifera (L.) Kuntze Arecaceae Coconut Coconut palm Cocos indica Royle C. nana Griff. Elaeis guineensis E. dybowskii Hua Arecaceae African oil palm Oil palm Jacq. E. madagascariensis (Jum. & H. Perrier) Becc. E. melanococca Gaertn. Helianthus annuus L. H. aridus Rydb. Asteraceae Sunflower H. jaegeri Heiser H. lenticularis Douglas H. macrocarpus DC H. ovatus Lehm. Olea europaea L. Oleaceae Olive

aThe Plant List and the Kew Medicinal Plant Names Services database.8,9 A comprehensive list of synonyms can be accessed through both websites. bAmerican Herbal Products Association’s Herbs of Commerce, 2nd ed.3 cAmerican Herbal Products Association’s Herbs of Commerce, 2nd ed.,3 and the USDA GRIN database.10 dAccording to the Canadian Food Inspection Agency,11 canola oil may be obtained from Brassica rapa and B. juncea as well.

*Note: Species other than those listed in Table 1 that are used for production of edible fatty oils are also known to be used as adulterants. In addition, admixture or substitution of saw palmetto extracts with “designer blends,” mimicking the saw palmetto fatty acid composition, which include fatty acids from animal sources have been described.1,12 The few reports of adulteration of saw palmetto berries with berries from related species, i.e., dwarf palmetto (Sabal minor),13,14 queen palm (Syagrus romanzoffiana),14 and Everglades palm (Acoelorrhaphe wrightii)15 of the palm family (Arecaceae) seem to suggest that such adulteration is rare. Fruits of dwarf palmetto (6-12 mm) and the Everglades palm (10-15 mm) are smaller and spherical compared to saw palmetto fruit, which is oval-shaped, of ca. 15 mm width and 12-25 mm length.4,13,14,16 Compared to saw palmetto, the fruit of queen palm is larger (20-25 mm) and heavier.14

2 Saw Palmetto Extract - Laboratory Guidance Document • 2019 • www.botanicaladulterants.org and books.4,17-19 Criteria to distinguish saw palmetto fruits from other fruits in their whole form have been published by many authors.16,20,21 The macroscopic assessment is the method of choice to distinguish unripe (green) from semi-mature or mature (black) berries and is sufficient for identifying saw palmetto to species. For obvi- ous reasons, macroscopic identification is not applicable to saw palmetto extracts. 7. Identification and Distinction using Microanatomical Characteristics Microscopic descriptions of saw palmetto are found in the pharma- copeias of Europe and the United States, and the American Herbal Pharmacopoeia’s textbook on microscopic characterization of botanical medicines.17,18,22 Details of the fruit anatomy of saw palmetto, Everglades palm, and dwarf palmetto have been 21 published by Zona; however, no clear Figure 1: Color of authentic saw palmetto ethanol extracts (1,2) and CO2 differentiation criteria for the fruits of extracts (3,4); adulterated ingredients labeled as saw palmetto extract (5-8). these palms using botanical micros- Image provided by Euromed, SA (Mollet del Vallès, Spain). copy were provided. (18S, ITS, ms, prk, rpb2) were used to establish the rela- 8. Organoleptic Identification tionship among members of the palm family.25,26 Genome Saw palmetto berries are initially sweet, then pungent, skimming was applied to assemble the entire chloroplast acrid, and saponaceous. The aroma is strongly aromatic nucleotide sequence in leaf samples of 29 palm species, and foul, reminiscent of foul-smelling socks. Saw palmetto including saw palmetto and the Everglades palm.23 Genetic extracts also have a distinct aromatic and foul odor. Color data on commercial saw palmetto products are less abun- can provide an indication to detect adulterated ingredients dant. Nevertheless, Little and Jeanson15 investigated the authenticity of 37 commercial saw palmetto products (Figures 1 and 2). Ethanol, hexane, and high pressure CO2 extracts of saw palmetto from ripe berries typically have containing dried, cut and sifted plant material using mini- a dark green-brown color due to the extraction of chloro- barcodes from the matK and the rbcL regions. Amplifiable phyll with these solvents. Low pressure CO2 extracts have a yellow to orange- brown color. While organoleptic evaluation does not provide sufficient safeguard against adulteration, the extract color and the very characteristic odor are helpful in assessing the authenticity of a saw palmetto extract. 9. Genetic Identification and Distinction Several authors have looked into differ- ences among nucleotide sequences of various gene regions for saw palmetto and closely related palm species, including dwarf palm and Everglades palm. In most cases, the purpose was to determine phylogenetic relationships.23-26 Nucleo- Figure 2: UV/Vis spectrum of a 1% ethanolic solution of authentic saw tide sequences of the chloroplast (atpB, palmetto CO2 extract (green line) and adulterated ingredients labeled as matK, ndhF, rbcL, rps16 intron, trnD- saw palmetto extract. trnT, trnL-trnF, trnQ-rps16) and nucleus Image provided by Euromed, SA (Mollet del Vallès, Spain).

Saw Palmetto Extract - Laboratory Guidance Document • 2019 • www.botanicaladulterants.org 3 DNA was obtained for 34 samples (92%), with 29 (85%) ing processes. Refining usually leads to a substantial loss samples containing saw palmetto. In three samples, only of phytosterols, tocopherols, and phospholipids. However, the matK mini-barcode sequence was obtained, which was there are numerous other vegetable oils that may be used as insufficient to distinguish between saw palmetto and its undeclared substituents of saw palmetto extracts. closest relative, the Everglades palm. Two products were made from fruit of other palm trees; one was made solely 11.1 Chemistry of Serenoa repens and potential adulter- from Everglades palm fruit, while for the other, the exact ants species could not be determined.15 Serenoa repens: Ripe saw palmetto fruit contains 15-20% lipids, primarily free fatty acids, fatty acid esters, Comments: Based on the report described above,15 the triglycerides, and sterols (Figure 3). The fruit is also rich use of DNA mini-barcoding is a suitable means for authen- in acidic polysaccharides. Additional compounds include tication of crude saw palmetto fruit materials.27 However, phenolic acids such as 4,5-di-O-caffeoylshikimic acid, the use of genetic techniques to determine the authenticity 4,5-di-O-caffeoylquinic acid, and gallic acid, as well as of saw palmetto extracts is not appropriate because fatty flavonoids (rutin, isoquercitrin, and astragalin), and carot- oils are generally devoid of DNA of appropriate quality to enoids.4,13,31 Based on Peng et al., immature berries contain permit reliable identification.28 lower concentrations of fatty acids than mature berries, and approximately the same amounts of lauric and oleic acids 10. Physicochemical Tests (compared to mature berries, where oleic acid concentrations The European Pharmacopoeia (Ph. Eur.) monograph for are higher than those of lauric acid).14 The main compounds saw palmetto extract includes specifications for the relative in saw palmetto oil are free fatty acids (70-95%), followed density, refractive index, acid value, iodine value, and perox- by glycerides (mono-, di-, and triglycerides [5-6%]), fatty ide value in saw palmetto extracts.29 Among these tests, acid methyl esters (ca. 2%), phytosterols (0.20-0.50%) and the determination of the acid value is the most important fatty alcohols (0.15-0.35%).29,32,33 Ethanol extracts are for the establishment of saw palmetto extract authentic- distinct from hexane and CO2 extracts by the relatively ity. Since saw palmetto extracts contain a large concentra- high concentration of phosphorylated glycerides. Hexane tion of free fatty acids, the acid value is much higher for extracts contain a higher proportion of free fatty acids, saw palmetto than for other vegetable oils.27,30 While this but lower amounts of mono-, di-, and triglycerides.32 The simple test is helpful in detecting saw palmetto adulteration, fatty acid composition (numbers in parentheses refer to the acid value assay must be used in combination with an the percentage of fatty acid compared to total [free and appropriate chemical test to rule out adulteration with some bound] fatty acids in a CO2 extract) is dominated by oleic of the materials mentioned in section 5. (30-35%) and lauric (26-32%) acids, followed by myris- The peroxide value is a function of fatty acid oxidation tic (10-12%), palmitic (8.5-9.2%), and linoleic (4.3-6.0%) (rancidity), and therefore does not provide any helpful infor- acids.34-36 Marti et al. reported the presence of hydroxyl- mation for authenticity determination. The relative density ated fatty acids (12-hydroxy-5,8,10,14-eicosatetraenoic acid, and refractive index of saw palmetto and common vegetable 10,11-dihydro-12-oxo-15-phytoenoic acid, corchorifatty oils do not differ substantially, and thus these analytical acid F) in commercial saw palmetto extracts.32 The fatty measurements are also not useful in establishing adultera- acid composition can be used to distinguish saw palmetto tion.27 With the exception of palm oil, the iodine value of extracts from most vegetable oils (Table 2), although some most vegetable oils is either above or below the value of saw oil blends are designed to mimic the authentic saw palmetto palmetto extract specified by the Ph. Eur.27 However, a fingerprint in order to make the detection of adultera- material that is compliant with the iodine value specifica- tion more difficult. The main phytosterols are β-sitosterol tions of saw palmetto oil in Ph. Eur. is easily obtained by (68-72% of total sterols), campesterol (20-23%), and stig- using a suitable mixture of vegetable oils. masterol (8-9%).36,37 Δ5-avenasterol, Δ7-avenasterol, cleros- terol, 24-methylenecholesterol, and Δ7-stigmasterol have 11. Chemical Identification and Distinction been reported present in minor amounts.1,38 Fatty alcohols Chemical authentication of saw palmetto extracts has include octacosanol, hexacosanol, tetracosanol, and triac- long been dominated by gas chromatographic (GC) meth- ontanol.33,39 ods, either analyzing the fatty acids directly, or after conversion into fatty acid esters. In addition to GC, thin-layer Arachis hypogaea oil: Peanut oil contains approximately chromatography (TLC) is an integral part for identity test- 96% triglycerides, with oleic, linoleic, and palmitic acids ing in pharmacopeial monographs. Other methods of chem- as the main fatty acids (Table 2).40 In addition, peanut oil ical authentication are less common, although a number contains approximately 0.50% phospholipids and 0.30% of additional techniques have been investigated and are phytosterols (β-sitosterol, campesterol, stigmasterol, and discussed below. Distinction based on the phytochemical Δ5-avenasterol).41 profile requires detailed knowledge of the constituents of saw palmetto and its likely adulterants. Some of the impor- Brassica napus oil: Canola oil contains 94.9-99.1% tant saw palmetto constituents and their significance in triglycerides, with oleic acid making up over 60% of the authentication are discussed below. The overview of poten- total fatty acids. Other major fatty acids in canola oil are tial adulterants is based on published literature. The chemi- palmitic and linoleic acids (Table 2).40,42 Commercial cal composition of vegetable oils is dependent on the refin- canola oil used in products on the market is usually low

4 Saw Palmetto Extract - Laboratory Guidance Document • 2019 • www.botanicaladulterants.org Figure 3: Major Fatty Acids and Phytosterols in Saw Palmetto

in erucic acid (< 2%). There are some high erucic acid oils, and oleic acids (Table 2).27,43 The sterol content is 0.04- which are most often referred to as rapeseed oils (although 0.12%, dominated by β-sitosterol (32-51% of total sterols), the name sometimes is used interchangeably with canola Δ5-avenasterol (20-41%), and stigmasterol (11-16%).27,41 oil), with erucic acid content over 50% of total fatty acids. Unique to canola oil is the presence of sulfur-containing Elaeis guineensis oil: Fatty acids in palm oil exist mainly fatty acids (epithiostearic acids). The concentration of as triglycerides (92-96%) and diglycerides (4-7%). The latter sterols varies between 0.70-1.00%, mainly represented by are represented primarily by palmitoyloleoyl-, dioleoyl- and β-sitosterol, campesterol, and brassicasterol. Since brassi- dipalmitoyl-glycerols, and can be used to differentiate palm casterol is unique to Brassica oils, it can be used as a marker oil from other vegetable oils. In commercial palm oils, compound to detect adulteration with canola oil.41 The the 1,3-diacylglycerols are more abundant than the corre- content of phospholipids is between 0.10-2.50%, depending sponding 1,2-diacylglycerols.41,44 Palm oil has approxi- on processing, with water- or acid-degummed oils having mately equal amounts of unsaturated and saturated fatty lower contents (0.10-0.60%). acids. The fatty acid composition is dominated by palmitic and oleic acids, with lesser amounts of linoleic and stearic Cocos nucifera oil: The triglyceride content in coconut acids (Table 2).27,41 Minor components of palm oil include oil is reported to be approximately 97%. One of the features 0.03-0.07% sterols (consisting of 55-67% β-sitosterol and of coconut oil is that it contains mainly saturated fatty acids. 19-28% campesterol), 0.10-0.30% glycolipids, and 0.02- Lauric acid (45.1-53.2% of total fatty acids) represents the 0.10% tocopherols. The red color of crude palm oil is due most abundant fatty acid, followed by myristic, palmitic, to the presence of carotenoids (0.05-0.07%).27,41

Saw Palmetto Extract - Laboratory Guidance Document • 2019 • www.botanicaladulterants.org 5 Helianthus annuus oil: As with other vegetable oils, trations of α-tocopherol (0.04-0.11%) of all vegetable oils, sunflower oil is composed mainly of triglycerides (up to and contains 0.72-0.86% phospholipids (the latter are not 97%). Using selective breeding techniques, sunflower seeds found in refined sunflower oil).27 with different fatty acid compositions have been developed, with regular, high-oleic and mid-oleic types being Olea europaea oil: Olive oil is composed mainly of the most common. The fatty acid compositions of regular triglycerides (ca. 99%), with oleic, linoleic, and palmitic and high-oleic acid sunflower oils are presented in Table 2. acids as the most abundant fatty acids (Table 2).46-48 The The mid-oleic acid type is the most popular sunflower oil sterol content is between 0.1-0.2%, composed of a mainly in the US retail market, with approximately 55-75% oleic, β-sitosterol (75-90%), Δ5-avenasterol (5-20%), and campes- 15-35% linoleic, 5% stearic, and 4% palmitic acids.41,43,45 terol (up to 4%). Numerous unusual phytosterols (e.g., The sterol content in sunflower seed oil is between 0.17- Δ7-avenasterol, Δ7-stigmastenol) are present at low concen- 0.52%. Of this, 42-70% is represented by β-sitosterol, trations.48 The content of squalene, a phytosterol precursor, 5-13% by stigmasterol and campesterol, respectively, and is between 0.02-0.75%. Olive oil also contains pigments up to 9% Δ7-stigmastenol. The latter has not been reported such as pheophytin, α- and β-carotene, and lutein.48 While in saw palmetto and could be used as a marker for adul- the phenolics content is low, some of these molecules are teration with sunflower oil. While Δ7-stigmastenol can be rather unique and can be used as specific markers for olive eliminated by heat treatment or bleaching, these treatments oil. Of particular interest are the secoiridoids, with oleuro- result in a conversion of Δ7-stigmastenol into the corre- pein (≤ 0.035%), oleocanthal (0.004-0.021%), and oleacein sponding (Δ8,14)- and Δ14-sterols. Depending on the extent (0.002-0.48%) as the most abundant.49,50 of refinement, sunflower oil has one of the highest concen-

Table 2: Relative Fatty Acid (free and bound fatty acid) Composition (in %) of Saw Palmetto and Adulterat- ing Vegetable Oils27,34-36,43,46,47

Fatty acid Saw Canolaa Coconut Olive Palm Peanut Sunflowerb Sunflowerc palmetto Caprylic C8:0 2.0-2.8 n.d. 4.6-10 n.d. n.d. n.d. n.d. n.d. Capric 2.7-3.2 n.d. 5.0-8.0 n.d. n.d. n.d. n.d. n.d. C10:0 Lauric 26-32 n.d. 45-53 n.d. ≤ 0.2 ≤ 0.1 ≤ 0.1 n.d. C12:0 Myristic 10-12 ≤ 0.2 17-21 < 0.1 0.5-2.0 ≤ 0.1 ≤ 0.2 ≤ 0.1 C14:0 Palmitic 8.5-9.2 2.5-7.0 7.5-10 7.5-20 39-48 8.0-14 5.0-7.6 2.6-5.0 C16:0 Stearic 1.7-2.1 0.8-3.0 2.0-4.0 0.5-5.0 3.5-6.0 1.0-4.5 2.7-6.5 2.9-6.2 C18:0 Oleic 30-35 51-70 5.0-10 55-83 36-44 35-69 14-39 75-90 C18:1 Linoleic 4.3-6.0 15-30 1.0-2.5 3.5-21 9.0-12 12-43 48-74 2.1-17 C18:2 Linolenic 0.7-1.2 5.0-14 ≤ 0.2 ≤ 1.0 ≤ 0.5 ≤ 0.3 ≤ 0.3 ≤ 0.3 C18:3 Arachidic < 0.1 0.2-1.2 ≤ 0.2 ≤ 0.6 ≤ 1.0 1.0-2.0 0.1-0.5 0.2-0.5 C20:0 Eicosenoic 0.2 0.1-4.3 ≤ 0.2 n.d. ≤ 0.4 0.7-1.7 ≤ 0.5 0.1-0.5 C20:1 Behenic < 0.1 ≤ 0.6 n.d. ≤ 0.2 ≤ 0.2 1.4-4.5 ≤ 0.7 0.5-1.6 C22:0 Lignoceric < 0.1 ≤ 0.3 n.d. ≤ 0.2 n.d. 0.5-2.5 ≤ 0.5 ≤ 0.5 C24:0

aLow erucic acid canola oil bRegular (= linoleic-type) sunflower oil cHigh-oleic acid sunflower oil

6 Saw Palmetto Extract - Laboratory Guidance Document • 2019 • www.botanicaladulterants.org 11.2 Laboratory methods Comments: Hanson et al. evaluated the authenticity Table 4, which appears at the end of this section, provides of 16 retail samples of saw palmetto products by infrared a summary comparison of different methods of analysis of (IR) spectroscopy and subsequent chemometric analysis saw palmetto oil. using principal component analysis (PCA). The addition of vegetable oils was readily detected due to the higher content 11.2.1 High-Performance Thin-Layer Chromatography of triglycerides.53 Similarly, Villar and Mulà presented Methods from the following sources were evaluated in the results of an analysis of 28 saw palmetto samples this review: Ph. Eur. 9.1,17,29 the HPTLC Association,51 using FT-IR (Figure 4) followed by PCA. The method and Halkina and Sherma.52 clearly distinguished between authentic and adulterated extracts.53,54 Based on these investigations, IR spectros- Comments: The HPTLC conditions in both documents copy combined with appropriate statistical methods may be include a relatively non-polar mobile phase combination suitable for detection of palmetto extract adulteration with with 1% acetic acid to prevent peak tailing on silica gel vegetable oils. However, adulterants with low triglyceride plates. The detection is carried out with anisaldehyde17,29 or content may be missed. 52 phosphomolybdic acid reagent. The conditions employed 11.2.3 High-performance liquid chromatography by Halkina and Sherma provide a rough separation into compound categories: triglycerides, free fatty acids, and Methods described in the following articles were evalu- 55 56 phytosterols.52 Identification of target analytes in the Ph. ated in this review: Bedner et al., Fibigr et al., Marti et 32 57 Eur. is not provided, although Melzig et al.4 suggest that al., and Al-Achi et al. the method identifies the presence of lauric acid, oleic acid, and β-sitosterol, which is not sufficient for the detection of Comments: High-performance liquid chromatography adulteration. Images of representative saw palmetto extract (HPLC) is rarely used for the analysis of saw palmetto due HPTLC fingerprints using the Ph. Eur. conditions can be to the challenges in separating the analytes of interest, and viewed on the website of the HPTLC Association,51 since their lack of a chromophore. the conditions are the same as in the Ph. Eur. Based on the paper by Halkina and Sherma, total substitution with Phytosterol analysis: Three HPLC methods evaluated as vegetable oils can be determined by the larger concentra- part of this laboratory guidance document, were devel- tions of triglycerides. However, HPTLC is not the method oped for the analysis of phytosterols using either a RP-18 of choice to detect admixture of vegetable oils, or the pres- or a phenyl column, with mass spectrometric (MS) detec- ence of designer blends with a similar fatty acid profile to tion. Bedner et al. developed two isocratic HPLC methods saw palmetto extract. (comparing RP-18 and phenyl columns) for the separation of campesterol, cycloartenol, lupenone, lupeol, β-sitosterol, 11.2.2 Infrared spectroscopy and stigmasterol. The peak shapes and resolution were Two methods, Hanson et al.53 and Villar and Mulà,54 better with the phenyl column, but despite the 80-minute to detect saw palmetto extract adulteration using infrared run time, campesterol and stigmasterol co-eluted. Quanti- spectroscopy were identified for this review. tative results with the APCI MS detector were comparable to gas chromatography with flame-ionization detection

Figure 4: Saw palmetto extract analysis by FT-IR. One adulterated sample (turquoise line) has a distinct spectrum different from authentic saw palmetto. Spectra were acquired by direct application of the samples (without any dilution) onto an ATR probe (ATR-FTIR Spectrum Two™, Perkin Elmer). Image provided by Euromed, SA (Mollet del Vallès, Spain).

Saw Palmetto Extract - Laboratory Guidance Document • 2019 • www.botanicaladulterants.org 7 (GC-FID).55 Fibigr et al. achieved acceptable separation as catalyst, allowing the use of an ultraviolet (UV) detector of eight phytosterols, including campesterol, β-sitosterol, for quantification. Gradient conditions suggest a normal and stigmasterol, on a narrow-bore RP-18 column in 8.5 phase separation, but neither the column packing nor the minutes. However, the test method was not applied to a detection wavelength was indicated. The method allowed saw palmetto extract.56 Establishing the presence of ubiq- for quantification of eight fatty acids in commercial saw uitous phytosterols such as campesterol, β-sitosterol, and palmetto products.57 The omission of important method stigmasterol does not provide a definitive means to detect information, lack of a chromatogram to assess the resolu- adulteration. However, the assessment of the phytosterol tion and peak shape, and absence of validation data means fingerprint as an approach for the detection of saw palmetto that the method cannot be evaluated for its fitness to detect adulteration could be useful as a complementary method adulteration. Since validated GC methods are available for in evaluation of the extract authenticity. While none of the fatty acid analysis (see below) with comparable time and above methods have measured phytosterols in adulterating complexity requirements regarding sample preparation, vegetable oils, the qualitative and quantitative sterol compo- these validated methods are considered a better option for sition of these adulterating materials is well known. Some use in evaluating the authenticity of saw palmetto extracts. of the “saw palmetto” samples containing animal fats have In 2019, Marti et al. analyzed 35 samples of saw palmetto low (< 0.2%) amounts of total sterols, but unusually high extract by ultra high-performance liquid chromatography content of Δ5,24-stigmastadienol or Δ7-avenasterol. In addi- (UHPLC)-high-resolution MS. In addition to the fatty tion, these samples tend to have a low campesterol/stigmas- acids, the authors determined the amounts of mono-, di-, terol ratio (0.78 – 1.67) compared to authentic saw palmetto and triglycerides, and phosphorylated glycerides. Etha- extracts (2.22-2.35).1 Compared to most GC-FID methods, nol, hexane, and CO2-extracts were readily distinguished HPLC-MS has the advantage of a faster sample preparation using multivariate statistics (PCA, orthogonal partial least since there is no need to silylate the phytosterols after hydro- squares discriminant analysis [OPLS-DA]).32 No adulter- lysis in potassium hydroxide. However, the resolution of the ated samples were included in the analysis, but based on the sterols is generally better using GC-FID. inclusion of a large number of saw palmetto constituents, and the discriminatory power of the assay, this approach Fatty acid analysis: Al-Achi et al. analyzed fatty acids after may be very useful in the determination of saw palmetto a conversion into fatty acid bromophenacyl esters using authenticity. 2,4’-dibromoacetophenone and dicyclohexano-18-crown-6

Table 3: Comparison among GC Methods to Determine Fatty Acids in Saw Palmetto Extracts. Method Sample Run time Column Comments preparation timea Booker58 150 min 24 min 5% Phenyl/ 95% Modified method of the German Society methylpolysiloxane for Fat Science. Mikaelian35 210 min 14 min Polyethylene glycol Modified USP sample preparation method. Ph. Eur.29 25 min 32 min Poly(dimethyl)siloxane Accepted standard method. Penugonda59 155 min 66 min Cyanopropyl Validation data is lacking. Priestap60 Unclear 44 min Polyethylene glycol Validation data is lacking. Priestap60 10 min 84 min Phenyl arylene Direct (without derivatization) analysis of fatty acids. Oleic, linoleic, and linolenic acids not resolved. Broad peak shape. Validation data is lacking. De Swaef63 85 min 36 min Cyanopropyl Good separation, validated for lauric acid and ethyl laurate. USP33 210 min 17 min Polyethylene glycol Accepted standard method. Wang65 50 min 41 min 5% Phenyl/ 95% The peaks of linolenic and oleic acids methylpolysiloxane overlap, and the peak shape is not optimal. Validation data available.

aThe sample preparation time is based on the reported duration of various sample preparation steps provided in the experimental section of the corresponding paper and the estimated duration of e.g., weighing, dilution, centrifugation, etc., listed in the ABC-AHP-NCNPR Botanical Adulterants Prevention Program’s Skullcap Adulteration Laboratory Guidance Document.66

8 Saw Palmetto Extract - Laboratory Guidance Document • 2019 • www.botanicaladulterants.org 11.2.4 Gas chromatography long and the peaks are broader and less well-resolved than Numerous methods described in the following literature those of the corresponding methyl esters. Conversion into were evaluated in this review: Bedner et al.,55 Booker et fatty acid methyl esters is done by methanolysis under acidic 33,35,58,59,64 al.,58 Mikaelian and Sojka,35 Ph. Eur. 9.1,17,29 Penugonda or alkaline conditions, or by using specific and Lindshield,59 Priestap et al.,60 Sorenson and Sullivan,61 methylation reagents such as trimethylsulfonium hydrox- 17,29,63 60 Srigley and Haile,62 de Swaef and Vlietinck,63 USP,18,33,64 ide, diazomethane, or m-trifluoromethylphenyl 65 and Wang et al.65 trimethylammonium hydroxide. Methanolysis takes more time since it involves heating the samples for up to two Comments: Gas chromatography has been the method hours to complete the reaction. Some of the methylating of choice to analyze fatty acids, fatty alcohols, and phytos- reagents represent a convenient alternative, but are consid- terols in saw palmetto extracts. The determination of the ered more hazardous to health. Particular caution should be qualitative and quantitative fatty acid content has been the used when using diazomethane due to its acute toxicity and major focus in the analysis of saw palmetto extracts. risk of explosion. Separation of the fatty acid methyl esters has been done Fatty acid analysis: Measuring fatty acids by GC is usually on a number of stationary phases, with methylpolysilox- done after converting the free and bound fatty acids into ane-, cyanopropyl-, or polyethylene-coated columns being fatty acid methyl esters. An exception is one of the meth- the most commonly used. Run times vary between 14 35 59 ods by Priestap et al., where the fatty acids are determined minutes and 66 minutes (see Table 3), not including the without derivatization using a nonpolar column (Table 3). time to re-establish initial temperature and column equili- While sample preparation is quick and easy, the run time is bration. Separation of the fatty acid methyl esters has been

Figure 5: Saw palmetto fatty acid analysis after conversion into methyl esters by GC-FID. Conditions as detailed in the United States Pharmacopeia. Image provided by Valensa International (Eustis, FL).

Saw Palmetto Extract - Laboratory Guidance Document • 2019 • www.botanicaladulterants.org 9 done on a number of stationary phases, with methylpoly- acids methyl esters from fatty acids bound to glycerin. siloxane-, cyanopropyl-, or polyethylene-coated columns Conversely, when methanolic sulfuric acid (or other strong being the most commonly used. Detection is achieved by acid) is used for the reaction, methyl esters of both free and FID17,18,29,33,59,60,63,64 and/or MS.60,65 Chromatograms bound fatty acids are obtained. By calculating the differ- were presented in only two publications: de Swaef and Vlie- ence between total and glycerin-bound concentrations for tinck have a good separation of all the fatty acid methyl each individual fatty acid, the concentration of free fatty and ethyl esters.63 In the case of Wang et al., the peaks acids can be determined. of linolenic and oleic acids overlap, and show an apparent Designer blends that are made with mixed vegetable fronting.65 oils or fatty acids derived from animal fats may be present Based on thorough validation of GC methodology and when phytosterol or fatty alcohol concentrations are outside easy sample preparation, the Ph. Eur. method is a good the specifications. In other cases, the use of stable isotope choice for the analysis of saw palmetto fatty acids. Sample measurements has proven helpful to detect such fraud.1,12 preparation time in the USP method (Figure 5) is longer, but the shorter GC run time is advantageous. In addition, Phytosterol analysis: Due to the need for a hydrolysis step USP has detailed a specific range for the ratio of nine fatty (some of the phytosterols occur as fatty acid esters in the acids relative to lauric acid, which can be used to detect extract) with subsequent derivatization with a silylating adulteration with vegetable oils, unless these are mixed in agent, the sample preparation for sterols is lengthy, involv- a way to mimic the saw palmetto fatty acid composition. ing many manipulations. Hydrolysis is achieved by heating A simple additional sample preparation method for the the sample in ~2M potassium hydroxide solution. Sterols are determination of free fatty acids in saw palmetto extract either recovered by partitioning the aqueous solution with has been developed and submitted in 2017 to USP as a toluene55,61 or diethyl ether,62 or by adsorbing the solution Saw Palmetto Extract monograph revision.35 This method onto diatomaceous earth, followed by elution with methy- uses methanolic sodium hydroxide to hydrolyze the mono-, lene chloride.29,33 Both Ph. Eur. and USP use the same di, and triglycerides in order to selectively provide fatty GC-FID method on a dimethylpolysiloxane column. The

Figure 6: 1H NMR spectrum of saw palmetto (ethanol extract) in deuterated chloroform. The insert shows the characteristic α/α› and β-protons of triglycerides at 4.20 and 5.24 ppm, respectively, in coconut oil. The α/α’ protons at 4.15 ppm in the saw palmetto extract overlap with the β-protons of 1,3-diacylglycerides. Saw palmetto NMR spectrum provided by Indena SpA (Milan, Italy).

10 Saw Palmetto Extract - Laboratory Guidance Document • 2019 • www.botanicaladulterants.org AOAC method (Sorenson and Sullivan; Bedner et al.)55,61 geographical origin of the local water. The 13C/12C ratio in and the method by Srigley and Haile62 use a phenyl- plants depends on the type of photosynthesis that a plant methylpolysiloxane stationary phase, although AOAC also utilizes. While most plants exclusively use the Calvin cycle, permits a dimethylpolysiloxane column. Run times are some plants (e.g., corn [Zea mays, Poaceae] or sugar cane 33-66 minutes. The conditions established by Srigley and [Saccharum officinarum, Poaceae]) have additional photo- Haile, with a run time of 66 minutes, allow quantification synthetic pathways, leading to a slightly higher 13C/12C of up to 18 common phytosterols. All methods provide a ratio in the latter. The 13C/12C isotopic ratio of animal good separation of the saw palmetto phytosterols and have fats is known to be correlated with their diet, e.g., animals been extensively validated. As mentioned in section 11.2.3 that feed exclusively on corn will have a higher 13C/12C above, the analysis of phytosterols as a stand-alone method ratio than those that ingest a wider variety of plants. The is not sufficient to rule out adulteration, but it is an excel- 18O/16O ratio depends on the temperature, freshwater lent choice as a complementary method since deviations input, and other climatic factors. Results are expressed as from pharmacopeial specifications (not less than 0.2% total the ratio difference (δ2H, δ13C, δ18O) of the material to be sterols, not less than 0.1% β-sitosterol) are a good indication analyzed and a standard with a known isotopic ratio, e.g., of ingredient adulteration. the Pee Dee Belemnite (based on a Cretaceous marine fossil from the Pee Dee Formation in South Carolina), which is Fatty alcohol analysis: USP is the only compendial stan- one of the standards used for the 13C/12C ratio, and the dard to measure fatty alcohols in saw palmetto extracts.33 Vienna Standard Mean Ocean Water (VSMOW), which Sample preparation and analysis conditions are the same defines the 2H/1H and 18O/16O composition of fresh water. as for the sterols, which is convenient as both classes of Isotope ratios can be measured using gas chromatogra- compounds can be measured in a single run. As with the phy with an isotope mass spectrometer. In the approach by sterol analysis, the determination of fatty alcohols by itself Perini et al., the addition of a single-quadrupole mass spec- is insufficient to detect adulteration, but it is considered a trometer allowed identification of individual compounds at valuable complementary means of verifying the authenticity the same time as the isotope ratios were measured. While of saw palmetto extracts. reported stable isotope ratios of some of the vegetable oils overlap with those of saw palmetto, measuring the δ18O 11.2.5 Nuclear magnetic resonance may provide valuable information about the possible risk Two methods described in the literature were evaluated in of adulteration since the δ18O of most vegetable oils is this review: Booker et al.,58 and de Combarieu et al.67 The lower than the range observed in saw palmetto. Fatty acids NMR parameters outlined by de Combarieu et al. were also derived from animal sources have a δ18O and a δ2H below used by Perini et al.1 those reported for saw palmetto, and therefore can be readily detected as adulterants, as evidenced in the publications Comments: Even though the 1H NMR spectrum of saw by Perini et al.1,12 palmetto extract is relatively simple compared to extracts of Measuring the stable isotope ratios of bulk fatty oils is other botanicals, a lot of useful information can be obtained a helpful means to detect adulteration, especially when a by visual evaluation of the spectrum. Adulteration with number of isotopes are measured and analyzed using appro- vegetable oils can be readily distinguished by the presence priate statistical tools. Further research needs to be done to of the signals of the α/α’ and β-protons of triglycerides verify the ability of SIR analysis to detect other potential (Figure 6), which are much smaller in saw palmetto extracts adulterants, and to determine the limit of detection of this than in vegetable oils.68 Using data from the PCA load- technique. Due to the availability and ease-of-use of more ings plot, Booker et al.58 and Perini et al.1 noticed that the established methods, the application of stable isotope anal- regions between 4.1-4.2 ppm, and between 5.3-5.5 ppm ysis may be best suited as an orthogonal assay to confirm were important for clustering of the samples (commercially adulteration, and to determine the origin of the adulterant. available finished products). The assessment of three principal components allowed for authentic saw palmetto to be 12. Conclusion distinguished, even from animal fat-based ‘designer blends’ Identification of saw palmetto extract adulteration has matching the saw palmetto fatty acid profile.1 Based on all been achieved using a number of analytical techniques. the data, 1H NMR represents a valuable tool to detect saw Macroscopic and organoleptic assessment may provide the palmetto adulteration, but is often not part of the instru- first indication of adulteration by observing the color and ments found in a botanical ingredient or dietary supplement strongly aromatic and foul odor. Absence of the character- manufacturing quality control laboratory. istic odor is a good indication that the oil is adulterated. In practice, several assays are needed to confirm the authen- 11.2.6 Stable isotope ratio ticity of saw palmetto extract. Gas chromatography for Stable isotope analysis for the authentication of saw measuring fatty acid, fatty alcohol, and phytosterol profiles, palmetto extracts has been described in two separate publi- combined with a visual and organoleptic inspection of the cations by Perini et al.1,12 liquid and determination of the acid value, provides a robust affirmation of saw palmetto extract authenticity. 1H NMR Comment: Variations in the stable isotopic ratios (SIRs) spectroscopy (with or without chemometric data analysis) in plants and animals may occur for a number of reasons. provides a suitable option for those companies with access For example, the 2H/1H ratio in plants is influenced by the to an NMR instrument.

Saw Palmetto Extract - Laboratory Guidance Document • 2019 • www.botanicaladulterants.org 11 Table 4. Comparison among the Different Chemical Methods to Authenticate Saw Palmetto Extract

Method Analyte Pro Contra HPTLC Free fatty acids Quick Available methods unlikely to detect sophisticated types of adulteration Sterols Basic systems affordable

Triglycerides HPLC-MS Free fatty acids Quick sample preparation for Equipment expensive sterol analysis Sterols Complicated sample preparation for fatty acid analysis

MS identification of fatty acids not essential since reference standards are available

Reliance on solely fatty acid or sterol determination not sufficient for the detection of mixtures mimicking the saw palmetto fatty acid profile GC-FID Fatty alcohols Standard equipment in many Mainly quantitative method laboratories Total fatty acids Need for derivatization of target analytes Basic systems affordable Free fatty acids Total fatty acid analysis is not sufficient for Ability to measure three classes the detection of mixtures mimicking the saw Sterols of compounds provides robust palmetto fatty acid profile approach for the detection of saw palmetto adulteration GC-MS Free fatty acids Qualitative and quantitative Equipment expensive

Need for derivatization of target analytes

MS identification of fatty acids not essential since reference standards are available

Not sufficient for the detection of mixtures mimicking the saw palmetto fatty acid profile Infrared Fingerprint No derivatization needed for Mostly qualitative sample analysis Little data available data on adulterant Affordable detection

State-of-the-art statistical Need to build-up reference library evaluation possible

NMR Fingerprint No derivatization needed for Equipment expensive sample analysis Triglycerides Need to build-up reference library Ability to detect most/all adulterants

State-of-the-art statistical evaluation possible Stable Fingerprint Ability to detect most Equipment expensive isotope ratio adulterants Free fatty acids Analysis offered by limited number of contract State-of-the-art statistical laboratories evaluation possible

12 Saw Palmetto Extract - Laboratory Guidance Document • 2019 • www.botanicaladulterants.org 13. References can Herbal Pharmacopoeia: Botanical Pharmacognosy—Micro- scopic Characterization of Botanical Medicines. Boca Raton, FL: 1. Perini M, Paolini M, Camin F, et al. Combined use of isoto- CRC Press; 2011. pic fingerprint and metabolomics analysis for the authentica- 23. Barrett CF, Baker WJ, Comer JR, et al. Plastid genomes reveal tion of saw palmetto (Serenoa repens) extracts. Fitoterapia. support for deep phylogenetic relationships and extensive rate 2018;127:15-19. variation among palms and other commelinid monocots. New 2. Gafner S, Baggett S. Adulteration of saw palmetto (Serenoa Phytol. 2016;209(2):855-870. repens), version 3. Botanical Adulterants Prevention Bulletin. 24. Barrett CF, Bacon CD, Antonelli A, Cano Á, Hofmann T. An Austin, TX: ABC-AHP-NCNPR Botanical Adulterants introduction to plant phylogenomics with a focus on palms. Prevention Program; 2018:1-7. Bot J Linnean Soc. 2016;182(2):234-255. 3. McGuffin M, Kartesz JT, Leung AY, Tucker AO. Herbs of 25. Couvreur TL, Forest F, Baker WJ. Origin and global diver- Commerce. 2nd ed. Silver Spring, MD: American Herbal sification patterns of tropical rain forests: inferences from Products Association; 2000. a complete genus-level phylogeny of palms. BMC Biology. 4. Melzig MF, Hiller K, Loew D. Sabalis serrulatae fructus. In: 2011;9(1):44. Blaschek W, ed. Wichtl — Teedrogen und Phytopharmaka. 26. Baker WJ, Savolainen V, Asmussen-Lange CB, et al. Complete Stuttgart, Germany: Wissenschaftliche Verlagsgesellschaft generic-level phylogenetic analyses of palms (Arecaceae) with mbH; 2016:572-574. comparisons of supertree and supermatrix approaches. Syst 5. The Plant List. Version 1.1. http://www.theplantlist.org/ Biol. 2009;58(2):240-256. tpl1.1/search?q=serenoa+repens. Accessed June 12, 2017. 27. Joint WHO/FAO Codex Alimentarius Commission. Codex 6. Anderson MK, Oakes T. Plant guide for saw palmetto Alimentarius: Standard for named vegetable oils. Vol CODEX (Serenoa repens). Davis, CA: USDA-Natural Resources STAN 210-1999. Rome, Italy: World Health Organiza- Conservation Service, National Plants Data Team; 2012. tion and Food and Agriculture Organization of the United 7. Nelson G. The Shrubs and Woody Vines of Florida: A Reference Nations; 2015:1-13. and Field Guide. Sarasota, FL: Pineapple Press, Inc; 1996. 28. Harbaugh Reynaud DT. The DNA toolkit: a practical user’s 8. Medicinal Plant Names Services (MPNS), Version 7.0 guide to genetic methods of botanical authentication. In: Royal Botanic Gardens, Kew; 2017. http://mpns.kew.org/ Reynertson K, Mahmood K, eds. Botanicals. Boca Raton, FL: mpns-portal/?_ga=1.239114563.1577664092.1475222805. CRC Press; 2015:43-68. Accessed June 6, 2017. 29. Sabalis serrulatae extractum. European Pharmacopoeia (Ph. 9. The Plant List. Version 1.1 http://www.theplantlist.org. Eur. 9.1). Strasbourg, France: European Directorate for the Accessed May 19, 2017. Quality of Medicines and Health Care; 2014:1509-1511. 10. National Plant Germplasm System. Germplasm Resources 30. Mikaelian G, Hill WS, Nguyen U, Holzer SJ. Preliminary Information Network [Internet]. United States Department quality examination of saw palmetto extract. Nutra Bus Tech- of Agriculture, Agricultural Research Service. https://www.ars- nol. 2006;2:64-65. grin.gov/npgs/index.html. Accessed November 29, 2017. 31. Olennikov DN, Zilfikarov IN, Khodakova SE. Phenolic 11. The Biology of Brassica napus L. (canola/rapeseed). Canadian compounds from Serenoa repens fruit. Chem Nat Compd. Food Inspection Agency; 2017. http://www.inspection.gc.ca/ 2013;49(3):526-529. plants/plants-with-novel-traits/applicants/directive-94-08/ 32. Marti G, Joulia P, Amiel A, et al. Comparison of the biology-documents/brassica-napus-l-/eng/1330729090093/13 phytochemical composition of Serenoa repens extracts 30729278970. Accessed February 28, 2019. by a multiplexed metabolomic approach. Molecules. 12. Perini M, Paolini M, Pace R, Camin F. The use of stable 2019;24(12):2208. isotope ratio analysis to characterise saw palmetto (Serenoa 33. Saw palmetto extract. USP 41-NF 36. Rockville, MD: United repens) extract. Food Chem. 2019;274:26-34. States Pharmacopeial Convention; 2018:4860-4861. 13. Hiermann A, Hübner WD, Schulz V. Serenoa. In: Hänsel R, 34. Mikaelian G, Sojka M, Minatelli J. The ultimate way to win Keller K, Rimpler H, Schneider G, eds. Hager’s Handbuch the fight against saw palmetto extract adulteration. Nutra Bus der Pharmazeutischen Praxis. Drogen P-Z. Vol 2. Heidelberg, Technol. 2009;1:46-50. Germany: Springer Verlag; 1994:680-687. 35. Mikaelian G, Sojka M. Authenticating saw palmetto extract : 14. Peng TS, Popin WF, Huffman M. Systematic investigation a new approach. Nutra Bus Technol. 2009;5:24-27. on quality management of saw palmetto products. In: Ho 36. Schantz MM, Bedner M, Long SE, et al. Development of saw CT, Zheng QY, eds. Quality Management of Nutraceuticals. palmetto (Serenoa repens) fruit and extract standard reference Vol 803. Washington, DC: American Chemical Society; materials. Anal Bioanal Chem. 2008;392(3):427-438. 2002:117-133. 37. Giammarioli S, Boniglia C, Di Stasio L, Gargiulo R, Mosca 15. Little DP, Jeanson ML. DNA barcode authentication of saw M, Carratù B. Phytosterols in supplements containing palmetto herbal dietary supplements. Sci Rep. 2013;3:3518. Serenoa repens: an example of variability of active prin- 16. Identifying commonly cultivated palms. Florida Department ciples in commercial plant based products. Nat Prod Res. of Agriculture and Consumer Service; 2011. http://idtools. 2019;33(15):2257-2261. org/id/palms/palmid/. Accessed February 28, 2019. 38. Ham B, Jolly S, Triche G, Williams PR, Wallace F. A study 17. Sabalis serrulatae fructus. European Pharmacopoeia (Ph. Eur. of the physical and chemical properties of saw palmetto berry 9.1). Strasbourg, France: European Directorate for the Quality extract. Chemistry Preprint Archive. 2002(2):106-121. of Medicines and Health Care; 2014:1512-1513. 39. Suzuki M, Ito Y, Fujino T, et al. Pharmacological effects 18. Saw palmetto. USP 41-NF 36. Rockville, MD: United States of saw palmetto extract in the lower urinary tract. Acta Pharmacopeial Convention; 2018:4856-4858. Pharmacol Sin. 2009;30(3):227-281. 19. Fructus Serenoae repentis. WHO Monographs on Selected 40. O’Brien RD. Fats and Oils: Formulating and Processing for Plants. Vol 2. Geneva, Switzerland: World Health Organiza- Applications. 3rd ed. Boca Raton, FL: CRC Press; 2009. tion; 2002:285-299. 41. Gunstone FD, Harwood JL, Dijkstra AJ. The Handbook of 20. Henderson A, Galeano G, Bernal R. Field Guide to the Palms Lipids. Boca Raton, FL: CRC Press; 2007. of the Americas. Princeton, NJ: Princeton University Press; 42. Przbylski R, Mag T, Eskin NAM, McDonald BE. Canola oil. 1995. In: Shahidi F, ed. Bailey’s Industrial Oil and Fat Products. Vol 21. Zona S. The genera of Palmae (Arecaceae) in the southeastern 2. 6 ed. Hoboken, NJ: John Wiley & Son, Inc.; 2005. United States. Harvard Papers in Botany. 1997;2:71-107. 43. Orsavova J, Misurcova L, Ambrozova JV, Vicha R, Mlcek J. 22. Upton R, Graff A, Jolliffe G, Länger R, Williamson E. Ameri- Fatty acids composition of vegetable oils and Its contribu-

Saw Palmetto Extract - Laboratory Guidance Document • 2019 • www.botanicaladulterants.org 13 tion to dietary energy intake and dependence of cardiovas- 56. Fibigr J, Šatínský D, Solich P. A UHPLC method for the cular mortality on dietary intake of fatty acids. Int J Mol Sci rapid separation and quantification of phytosterols using 2015;16(6):12871-12890. tandem UV/Charged aerosol detection – A compari- 44. Siew WL, Ng W-L. Diglyceride content and composition as son of both detection techniques. J Pharm Biomed Anal. indicators of palm oil quality. J Sci Food Agric. 1995;69(1):73- 2017;140:274-280. 79. 57. Al-Achi A, Locklear AF, Fetterman L. Commercially available 45. Warner K, Vick BA, Kleingartner L, Isaac I, Doroff K. saw palmetto products: Quality control testing. Int J Drug Composition of sunflower NuSun (mid-oleic sunflower), Discovery Herbal Res. 2012;2(1):267-271. and high-oleic sunflower oils. Paper presented at: Sunflower 58. Booker A, Suter A, Krnjic A, et al. A phytochemical compari- Research Workshop.2003; Fargo, ND. son of saw palmetto products using gas chromatography and 46. International Olive Council. Trade standard applying to olive (1)H nuclear magnetic resonance spectroscopy metabolomic oils and olive pomace oils. Vol COI/T.15/NC No 3/Rev. 12. profiling. J Pharm Pharmacol. 2014;66(6):811-822. Madrid, Spain: International Olive Council; 2018:17. 59. Penugonda K, Lindshield BL. Fatty acid and phytosterol 47. Yorulmaz A, Erinc H, Tekin A. Changes in olive and olive content of commercial saw palmetto supplements. Nutrients. oil characteristics during maturation. J Am Oil Chem Soc. 2013;5(9):3617-3633. 2013;90(5):647-658. 60. Priestap H, Houle P, Bennett B. Fatty acid composition of 48. Boskou D, Blekas G, Tsimidou M. Chemistry, properties, fruits of two forms of Serenoa repens. Chem Nat Compd. health effects. In: Boskou D, ed. Olive Oil: Chemistry and 2011;47:511-514. Technology. 2 ed. Champaign, IL: AOCS Press; 2006:41-72. 61. Sorenson WR, Sullivan D. Determination of campesterol, 49. Vulcano I, Halabalaki M, Skaltsounis L, Ganzera M. Quan- stigmasterol, and beta-sitosterol in saw palmetto raw materi- titative analysis of pungent and anti-inflammatory phenolic als and dietary supplements by gas chromatography: single- compounds in olive oil by capillary electrophoresis. Food laboratory validation. J AOAC Int. 2006;89(1):22-34. Chem. 2015;169:381-386. 62. Srigley CT, Haile EA. Quantification of plant sterols/stanols 50. Cicerale S, Conlan XA, Sinclair AJ, Keast RSJ. Chemistry in foods and dietary supplements containing added phytoster- and health of olive oil phenolics. Crit Rev Food Sci Nutr. ols. J Food Comp Anal. 2015;40:163-176. 2008;49(3):218-236. 63. De Swaef SI, Vlietinck AJ. Simultaneous quantitation of 51. Serenoa repens, fruit. HPTLC Association; 2019. Accessed lauric acid and ethyl laurate in Sabal serrulata by capillary gas August 8, 2019. chromatography and derivatisation with trimethyl sulphoni- 52. Halkina T, Sherma J. Determination of sterols and fatty umhydroxide. J Chromatogr A. 1996;719:479-482. acids in prostate health dietary supplements by silica 64. Powdered saw palmetto. USP 41-NF 36. Rockville, MD: gel high performance thin layer chromatography with United States Pharmacopeial Convention; 2018:4858-4860. visible mode densitometry. J Liq Chromatogr Relat Technol. 65. Wang M, Avula B, Wang Y-H, Zhao J, Parcher JF, Khan 2007;30(15):2329-2335. IA. Fatty acid analysis of saw palmetto (Serenoa repens) and 53. Hanson BA, Ye T, Raftery DM. Assessing Serenoa repens pygeum (Prunus africana) in dietary supplements by gas chro- (Arecaceae) quality at the retail level using spectroscopic and matography/mass spectrometry in the selected ion monitoring chemometric methods. The 49th Annual Meeting of the Soci- mode. J AOAC Int. 2013;96(3):560-566. ety for Economic Botany; 2008; Durham, NC. 66. Gafner S. Skullcap adulteration laboratory guidance docu- 54. Villar A, Mulà A. Full traceability, high quality production ment. Austin, TX: ABC-AHP-NCNPR Botanical Adulterants and exhaustive analytical control – industry’s key tools to Prevention Program; 2015:1-12. avoid and prevent adulteration and fraud of botanical ingredi- 67. De Combarieu E, Martinelli EM, Pace R, Sardone N. Metab- ents. Adulteration and Fraud of Botanical and Natural Health olomics study of saw palmetto extracts based on 1H NMR Ingredients: Issues, Challenges and Prevention Tools for the spectroscopy. Fitoterapia. 2015;102:56-60. Industry; 2018; Frankfurt, Germany. 68. Gafner S, Blumenthal M, Foster S, Cardellina II JH, Khan 55. Bedner M, Schantz MM, Sander LC, Sharpless KE. Develop- IA, Upton R. Botanical ingredient adulteration – how some ment of liquid chromatographic methods for the determina- suppliers attempt to fool commonly used analytical tech- tion of phytosterols in Standard Reference Materials contain- niques. Acta Hort. 2019:in press. ing saw palmetto. J Chromatogr A. 2008;1192(1):74-80.

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