CLICK HERE FOR PDF - 03/14/2022 Updated PDF file with correction to author name
By Aboli Girme, PhD,a Amit Mirgal, PhD,a Bhaumik Darji,b Stefan Gafner, PhD,c and Lal Hingorani, PhDa
aPharmanza Herbal Pvt. Ltd., Borsad-Tarapur Road, Near Vadadla Patiya, Kaniya-388435, Gujarat, India
bVerdure Sciences, 17150 Metro Park Court, Noblesville, Indiana, 46060
cAmerican Botanical Council, PO Box 144345, Austin, TX 78714
Citation (JAMA style): Girme A, Mirgal A, Darji B, Gafner S, Hingorani L. Adulteration of saffron and saffron extracts. Botanical Adulterants Prevention Bulletin. Austin, TX: ABC-AHP-NCNPR Botanical Adulterants Prevention Program; 2022.
Keywords: adulterant, adulteration, calendula, Calendula officinalis, Carthamus tinctorius, corn, Crocus sativus, Curcuma longa, food dyes, safflower, saffron, turmeric, Zea mays
Goal: The goal of this bulletin is to provide timely information on issues of adulteration regarding saffron (Crocus sativus L., Iridaceae) and its extracts to the international herbal industry and extended natural products community in general by presenting data on the occurrence of adulteration, the market situation, and potential consequences for the consumer and the industry.
1 General Information
Crocus sativus L., known as saffron, is a plant whose dried stigmas* are a popular spice and food condiment renowned for its color, aroma, and flavor. Saffron is generally a cultivated crop known for labor-intensive harvesting of the stigmas from the flowers, prominently cultivated in India (Kashmiri saffron), Spain (Spanish saffron), Iran (Persian saffron), Afghanistan, and Greece, with suppliers offering various grades of saffron. Saffron has a rich traditional heritage for its medicinal use in addition to worldwide usage as a coloring and flavoring agent. The main bioactive constituents of saffron are carotenoids such as crocetin esters (or crocins), crocetin, and terpenoids like picrocrocin and safranal (Figure 1).
The phylogeny of saffron has been the subject of several investigations. Based on morphological and cytological criteria, Mathew1 noted that C. cartwrightianus is a species similar to C. sativus. Further work reported that C. sativus likely emerged from its ancestor C. cartwrightianus by fusion of two genomes from two different C. cartwrightianus genotypes (autotriploidy).1 Recent studies indicated that the DNA composition of C. sativus is similar to C. cartwrightianus.2,3
1.1 Common name: Saffron
1.2 Common name in other languages: The name of saffron (C. sativus) is similar in many languages globally, derived from the Arabic word z'aferan, meaning yellow. Saffron was named A-Zupiru, which means queen of herbal drugs in Anatolia during the Hittite period. The plant is called koricos in ancient Greek, crocum in the Roman period, and kurkum in some parts of the Middle East. Ancient Greeks named saffron "blood of Hercules" and they used it as incense and incorporated C. sativus in amulets for protection from disease and evil.4 Common names in other languages are indicated below.5
Assamese: kungkum (কুংকুম)
Chinese: fan honghua (番紅花), xi honghua (西红花), or zanghonghua (藏紅花)
Hindi: zaffran, kesar (केसर)
Japanese: safuran (さふらん)
Kashmiri: kong or koung (کونگ)
Persian: zaefaran (زعفران)
Russian: shafran (шафран)
Sanskrit: kunkuma (कुंकुम)
Urdu: kisar (کیسر)6
1.3 Accepted Latin binomial: Crocus sativus L.7
1.4 Synonyms: Crocus autumnalis Sm., Crocus officinalis (L.) Honck, Crocus pendulus Stokes, Crocus setifolius Stokes7
1.5 Botanical family: Iridaceae6,7
1.6 Distribution: Saffron (C. sativus L.) is one of the oldest spices with a documented history of usage, mainly as a culinary and food ingredient throughout antiquity. The origin and domestication of saffron are not certain. Some authors indicate that saffron originated in the Middle East, while other authors mention Central Asia or the islands of southwestern Greece.8 From this region, its use spread to India, China, and the Middle Eastern countries. Among the latter countries, the Arabic region is credited with the saffron distribution throughout the Mediterranean basin, including Morocco. It was established as a high-value secondary crop in the Old World from India to Britain.8-10
Countries with traditional cultivation are Azerbaijan, France, Greece, India, Iran, Italy, Spain, China, Israel, Japan, and Mexico. At present, the cultivation range has expanded and includes many regions around the world (section 2.2)
1.7 Plant part and form: The flower is formed by a perianth of six tepals (often erroneously called petals in the scientific literature) which contain anthocyanin pigments.11 It has three stamens with yellow anthers, 15-20 mm long. The style is colorless to yellow, connected to a three branched stigma of intense red color, 25-50 mm long (Figure 2).12,13 Now, saffron tepals are mainly used to dye wool, in accordance with historical documentation of its use as a dye.14 Saffron's stigma has the highest market value, while tepals are by-products and sometimes discarded after harvesting.
The most prized part of the flower is its pistil, composed of three parts: ovary, style, and stigma with fibrous aspects and deep red coloring (Figure 3). Powdered saffron is produced by grinding dried stigmas.15 Bloom collection must be performed carefully to facilitate the separation of stigma and stamens from tepals. When separated from the flower, the stigma must be devoid of other parts of the flower, dirt, and insects.16
1.8 General uses: Saffron tepals are used as a natural dye, including dyeing of wool fibers. People in several geographical regions have utilized saffron for various functions such as perfume, spice, and a dye.10,17
Saffron tinctures and extracts, but also saffron-containing teas, ointments, poultices, and baths, have documented traditional use over centuries to treat several diseases and symptoms, such as the healing of wounds, fever, lower back pain, as a digestive aid, and as a teething aid for infants.15 The modern phytotherapeutic and functional effects of saffron extracts have been focused on the relief of mild- to moderate depression, anxiety and stress.18,19 Furthermore there is growing evidence from clinical trials of the beneficial effects on sleep.20-23 Saffron has also been used to treat eye diseases, skin diseases,24 heal fractures, reduce joint pain, and heal wounds.15 Additionally, saffron extracts and their chemical constituents have been tested to improve neurodegenerative diseases, metabolic syndrome, sexual dysfunction, premenstrual syndrome, appetite, and glucose and lipid metabolism.25,26
Reports from ancient times describe the use of saffron for cosmetic purposes. In traditional Iranian medicine, saffron is utilized to treat erysipelas and improve complexion. In traditional Greek medicine, it is used to refresh facial skin, treat wounds, acne, and skin diseases.24 Saffron-containing forumlations are also used in Ayurvedic medicine for the topical treatment of acne.27,28 Saffron extract and some of its chemical constituents are authorized in the European Union as cosmetic ingredients29 and used in many countries as skin conditioners, antioxidants, colorants and perfume.10.
Much of the cultivated saffron is used for in food and beverages (see section 2.3). A number of well-known dishes use saffron as a spice. For example, saffron is used as a spice in bouillabaisse, a classic spicy French fish soup, and in a Spanish seafood stew called zarzuela. It is frequently used to add color and flavor to rice dishes: Examples include the paella in Spain, risotto Milanese, a common dish in Italy, the Iranian national dish, chelow kabab, or biryanis, traditional dishes made with rice in Bangladesh, India and Pakistan.8,30 Safranlı zerde is a saffron-containing Turkish rice pudding served as a dessert, while saffron ice cream is popular in Iran, and in India, desserts/sweets such as gulabjaman and kulfi, along with the popular tea known as kahwa are also made with saffron.30 In Morocco, saffron is used in tea instead of mint.8,30
2.1 Importance in the trade: As per data from 2015, around 300 metric tons of dried whole threads and powder are gleaned yearly, of which 50 metric tons are top grade saffron.31,32 Saffron global production was estimated at 418 metric tons per year in 2018. In 2019, global production was up to approximately 472 tons according to Future Market Insights, a market research organization out of Pune, India.33 Saffron is the most expensive spice in the world.24,31 Saffron prices at wholesale and retail rates range from US $500 to US $5,000 per pound, or US $1,100–11,000 per kg, equivalent to £2,500/€3,500 per pound or £5,500/€7,500 per kg. The price in Canada recently rose to CAD $18,000 per kg. A pound contains between 70,000 and 200,000 threads.
2.2 Supply sources: Today, the main supply sources of saffron are in Iran, India, Afghanistan, and Spain (Figure 3)34-37 However, Greece is the largest saffron producer in Europe. According to a report from 2011, 38 up to 90% of saffron sold as Spanish Saffron is not coming from Spain but rather from Iran, Greece, or Morocco, where it is less expensive. Spanish companies can sell saffron bought from other countries and sell it as Spanish saffron.38
In Iran, most of the cultivation is done in the Khorasan province. The largest cultivation areas in Afghanistan are in Herat province, accounting for 90% of the country’s production in 2020.39 In India, saffron is widely cultivated in the Jammu and Kashmir regions (Figures 4 and 5). In Spain, it is cultivated in the Castilla-La Mancha region. Small-scale cultivation is documented in Greece (Kozani, western Macedonia), Azerbaijan (Aspheron peninsula), Italy (Sardinia, Abruzzo), Morocco (Taliouine area), France, Turkey, Israel, Pakistan, China, Egypt, United Arab Emirates, Japan, United States, New Zealand, and Australia.9,17,40
Iran reportedly accounts for around 90–93 % of global production and exports.41,42 In 2005, Iran produced 230 MT of saffron; second-ranked Greece produced 5.7 MT, while Morocco and India tied for the third rank, each producing 2.3 MT.43 According to a report by the Iranian Ministery of Industry, Mine, and Trade, Iran produced 376 MT of saffron in the 2017 season.44 A 2021 report estimates Iran’s annual harvest at ca. 350 MT.45 A few of Iran's drier eastern and southeastern provinces, including Fars, Kerman, and those in the Khorasan region, make up the bulk of modern global production.46 The collection of approximately 1 lb (454 g) of dry saffron stigmas requires the harvest of 50,000–75,000 flowers. An experienced picker can collect approximately 150,000 flowers in 40 hours of labor,47 although lower amounts have been reported from trials in Central Otago, New Zealand.46
2.3 Market dynamics:
According to data from the International Trade Center, the global value of the saffron market has increased between 2013–2016. At the same time, the exported quantity has decreased since 2014. The increase in saffron prices is believed to be largely due to the exchange rate fluctuations in the United States dollar faced by Iran. While Iran produces more than 90% of the world's saffron, it accounts for only 40% of direct global exports. European countries (e.g., Spain, France, Italy), Hong Kong, and the United Arab Emirates buy large quantities of Iranian saffron and resell the saffron at a higher price. The saffron price can vary substantially during a year, depending on weather, economic, and political conditions. For example, saffron prices nearly tripled between 2006–2009, to drop back to the historical average in the following year. Reasons for the price spike were allegedly the low saffron production in the Iranian saffron fields due to heavy frost during these years, especially in April 2007,48 and possibly because of Iranian traders purposefully limiting the availability of saffron on the international market.42 Other factors impacting saffron yield are fungal diseases (Fusarium oxysporum f. sp. gladioli, Penicillium spp., Helicobasidium purpureum) and the adverse effects of climate change leading to droughts in some of the production areas.49 According to Maximize Market Research Pvt Ltd., the global saffron market was valued at about US $390 million in 2017 and about US $430 million in 2018.50
According to Julia Diaz (Pharmactive Biotech Products SL, Madrid, Spain), food and beverages processing (color, taste, and fragrance) represent 28.3% of the saffron market, followed by its use as a food additive with 16.4%, food service and hotel industries with 15.1%, dietary supplements with 14.1%, cosmetic and personal care products with 7.7%, and the textile industry with 6.5% where saffron is used as a fabric dye (Email to S. Gafner, February 3, 2021). As mentioned above, the vast majority of saffron stigmas (Figure 5) is destined for food use.16 The beverage industry, which uses saffron as a flavoring agent, is experiencing a steep increase in demand, e.g., in dairy products in India and other Asian markets. However, the market is increasingly constrained due to saffron's high price. In addition to the competition in demand, the short harvesting season and storage requirements influence the supply chain of saffron. This, and the high prices may have encouraged the chemical synthesis of saffron constituents rather than extracting them from saffron. For example, the monoterpenoid safranal, which substantially contributes to saffron's aroma, has become available as a synthetically manufactured ingredient at competitive prices and could substitute natural safranal from saffron.16
The use of saffron as a dietary supplement in the United States is limited based on sales data from market research company SPINS. It has not ranked within the top 100 dietary supplement ingredients over the past ten years. The majority of sales are in the natural channel, with annual sales volumes between US $110,000 and $350,000 in the years 2012–2017 (T. Smith [American Botanical Council] e-mail to S. Gafner, September 2, 2015, September 3, 2015, and February 6, 2018. K. Kawa [SPINS] e-mail to S. Gafner, July 11, 2016.). Nevertheless, there is a continued interest in saffron for use in the nutraceutical industry in eye health markets, women's health, sports energy, men's health, anti-aging, well-being support, skincare, cognitive health, mood, sleep, and gut health. Products have been launched in the United States, Europe, and the Australian dietary supplement and nutraceutical market. The interest and demand within this market may have increased due to the positive results from several clinical investigations carried out with different saffron extracts.51-54
3.1 Known adulterants: Concerns regarding adulterated saffron have existed for centuries, driven by the high price and production limits. In his book Naturalis Historia,55 Roman naturalist Pliny the Elder (23/24–79 CE) wrote that nothing is as much adulterated as saffron. Saffron has been subjected to various types of economically motivated adulteration. The National Dispensatory, published in 1884, mentioned the undeclared admixture of considerable portions of yellow style to the stigmas.56 The stigmas are attached to a slender white style that turns pale yellow when dried. The style curls and is hardly noticeable after drying, but if it is left attached to the red stigmas, it adds 30% to 50% weight to the saffron material. The style of the saffron plant has no culinary value, meaning no flavor or color. The 1884 publication also lists exhausted saffron being sold instead of genuine saffron. Also, partially exhausted saffron has been reported to be mixed or immersed in oils, glycerin, or honey as a means of increasing weight.56 Other adulterants mentioned in the older literature are chalk, gypsum, or heavy spar (barium sulfate), which were added to the saffron powder.57 Another common type of fraud in saffron production is the addition or admixture of extraneous filaments, either as a whole, or processed into small pieces, which substantially hinders morphological identification. The filaments, or coarse powders made thereof used for such adulteration may be corn (Zea mays, Poaceae) stigmas colored with beet (Beta vulgaris, Amaranthaceae) root, pomegranate (Punica granatum, Lythraceae) fruit peel, or pomegranate fruit fibers, red-dyed silk fibers, and the stigmas from safflower (Carthamus tinctorius, Asteraceae) and calendula (Calendula officinalis, Asteraceae).57-59
Confusingly, the dried flowers of safflower, also known as kasubha, are often labeled as “saffron flowers,” “bastard saffron,” or “Philippine saffron,” possibly misleading consumers into thinking that these are products made with actual saffron.60
Adulteration of saffron powder is mainly a form of intentional economic adulteration. In Britain, adulterated saffron was sold at £277/pound compared to £3750/pound for top-grade saffron in 2013.61 According to an article from 2018, authentic saffron sold for €3.50/gram on Moroccon markets while adulterated saffron was available at €1/gram.62 Increasing the bulk quantity and adding plant parts of similar physical and chemical characteristics to saffron are common forms of adulteration. Adulterants in powdered saffron reportedly are turmeric powder (Curcuma longa, Zingiberaceae) and paprika (Capsicum annuum, Solanaceae) as bulking materials due to similar color and UV/Vis absorbance chatracteristics.59,63 Since the fruits of cape jasmine (Gardenia jasminoides, syn. G. augusta, Rubiaceae) produce the same chemical compounds as saffron, crocetin esters (Figure 1) derived from it have been investigated as a potential source of adulteration in saffron extracts. Prices for gardenia extract (32 €/kg) are around 62–66 fold lower than for saffron extract (Spanish origin) (J. Diaz email to S. Gafner, February 3, 2021). Further, the dye extracted from Buddleja officinalis (Scrophulariaceae) flowers also contains constituents that are often added as chemical adulterants.63 Saffron powder is also intentionally adulterated with artificial dyes, including water-soluble colorants such as erythrosine, ponceau 4R, tartrazine, and non-polar compounds like Sudan dyes.64
Safranal (Figure 1) is an apocarotenoid responsible for the aroma of saffron. The first chemical synthesis was described in 193665 and it is currently a low cost source of an aroma similar to saffron,66 in particular in China. Some known adulterants of saffron are listed in Table 1 and Figures 7 and 8.
Another type of economically motivated adulteration is the mislabeling of the Designation of Origin by selling saffron obtained from countries not listed on the label. As stated in section 2.2, much of the saffron labeled to originate in Spain is actually from Greece, Iran, or Morocco.38 One example of such practice was documented in 2021, when a criminal organization dedicated to the fraudulent sale of Iranian saffron labeled to be of Spanish origin was dismantled. About 500 kg of Iranian material was mixed with floral remains, saffron styles, and even colorants not authorized for consumption by the European Union or the United States.67
3.2 Sources of information supporting the confirmation of adulteration: The main official document with quality control methods for saffron filaments and its powder currently is ISO 3632-2,which uses a UV-spectrophotometric method. This is a well established analytical approach for the identification and grading of saffron, but has low sensitivity and specificity.73 This method has been used in the study of Khilare et al.74 to evaluate 36 whole commercial saffron samples from India, along with microscopic examination and DNA barcoding using Sanger sequence analysis. The study revealed the presence of adulterants like Pistacia vera (Anacardiaceae), Morus alba (Moraceae), Triticum aestivum (Poaceae), and Eriochloa spp. (Poaceae) in 10 adulterated samples, while 24 samples were found consistent with characteristics of saffron species and two samples found inconclusive.74,75
Another study by the Forensic Science Laboratory of Jammu and Kashmir (India) found adulterated saffron in samples from the local market. The researchers discovered two adulterated saffron samples in the four samples tested using solubility, the sulphuric acid spot test, and the ISO method in combination with thin-layer chromatography (TLC) fingerprinting.76 TLC and electrospray ionization mass spectrometry (ESI-MS) were used together in another published study to test 104 commercial whole saffron samples collected from 16 countries, out of which 20 samples were found to contain the synthetic dyes magenta III and rhodamine B.77
Investigations and reports provided and supported by government organizations have also reported incidences of saffron fraud. The Indian Council of Agricultural Research (ICAR) initiated an analysis of 113 market samples of whole saffron from retail markets in the Kashmir region in India, which classified 71 samples as grade III, indicating low-quality saffron according to ISO standards. Poor post-harvest processing practices and adulteration were the main reasons for poor quality saffron.78 The European authorities reported two cases of regulatory enforcements against individuals producing adulterated saffron.79,80 In one case, the Spanish Guardia Civil seized 87 kg of adulterated saffron with an estimated market value of €783,000 (US $897,475 per the January 2019 conversion rate). The adulterated material was a mixture of saffron fibers with stamens from other sources.79,81,82 In the other case, three people were arrested for selling substandard saffron. No additional details on the saffron composition were provided.79
A study using DNA barcoding with Sanger sequencing utilized three genomic regions, trnH-psbA, rbcL-a, and ITS2, to distinguish between saffron and its adulterants. Four out of 12 whole saffron (C. sativus L.) samples obtained from 12 different China provinces were contaminated with Carthamus tinctorius or Chrysanthemum × morifolium.83
In Germany, 15 saffron samples (collected mostly from internet trade) were analyzed using 1H nuclear magnetic resonance (NMR) spectroscopy to detect adulteration. Thirteen samples consisted of natural saffron material, although one of these samples was colored with tartrazine. The remaining two market samples, procured from a bazaar in Egypt, were colored paper.84
3.3 Frequency of occurrence: While the extent of saffron adulteration remains unknown, available data suggest it to be prevalent. Hensel and Rösing85 cite two studies by Czygan documenting adulteration in over 90% of 198 commercial samples of powdered and whole saffron analyzed in 1980 and 1986, respectively. According to the report, adulteration is more prominent in powdered samples than in whole saffron stigmas. Currently, saffron is among the most frequently adulterated food items. In an effort to catalog a scholarly work on food fraud, Moore et al. performed a comprehensive literature search regarding the adulteration of food ingredients. The number of articles on saffron adulteration retrieved ranked fourth behind olive oil, cow's milk, and honey.86 Similarly, Everstine reported that saffron ranked third behind chili powder and turmeric in the herb/spice category regarding the number of fraud records retrieved between 2010-2019.87
3.4 Possible safety/therapeutic issues: Common adulterants found in saffron are synthetic dyes, and some of these colorants such as Sudan dye and Rhodamine B may be harmful for health. These chemicals reportedly may cause asthma, allergic reactions, and DNA damage.88,89 In their assessment, the Scientific Panel on Food Additives, Flavourings, Processing Aids and Materials in Contact with Food, commissioned by the European Food Safety Authority (EFSA), both Sudan I-IV and Rhodamine B are considered potentially genotoxic and carcinogenic.88 Therefore, saffron adulteration may cause a safety issue due to the presence of synthetic colorants.15,59
3.5 Analytical methods to detect adulteration: Many methods for detecting saffron adulteration have been reported, including visual inspection and solubility.73,90,91 High-performance liquid chromatography with photodiode array detection (HPLC-PDA), HPLC with electrospray mass spectrometric detection (HPLC-ESI-MS),70 thin layer chromatography (TLC),77,92 electronic nose system coupled to multivariate statistical analysis,68 1H nuclear magnetic resonance (1H NMR) metabolomics,93,94 and economic, fast, non-destructive techniques such as mid-range infrared (IR) spectroscopy95 and ultraviolet/visible (UV/Vis) spectrophotometry96 have been proposed as analytical methods to distinguish saffron and its adulterants.
3.5.1 Spot tests: Based on the water-solubility of the crocetin esters, a simple test sprinkling some saffron powder on water can provide information on its authenticity. In case of true saffron, the whole or powdered saffron is quickly surrounded by a clear yellow color. In case of complete adulteration, either no color, or an opaque reddish color slowly diffusing into the water is observed. Exposure of saffron to methanol or acetonitrile has also been used to detect synthetic colorants. A dark red or pink color after exposing the material to these solvents indicates the presence of artificial dyes.76,77,90 Another simple screening technique proposed is to rub the material between the fingers and visually inspect the fingers for color residues. The above methods are quick and inexpensive, but the approach is limited to detecting undeclared dyes.
3.5.2 Botanical microscopy: A microscope can aid in detecting admixture with whole or powdered materials from other plants. Characteristic for saffron is the red contents of the parenchyma cells (Figure 6A), the papillae on the epidermis cells (Figure 6B), and the pollen's size and shape (Figure 6C), which can be used to distinguish saffron from calendula flowers and safflower.74,91,97 In addition, the European Pharmacopoeia monograph Crocus sativus for homeopathic preparations, which contains a detailed description of the positive microscopic characteristics of Crocus stigma,r equires that “no parts with rough walls, no crystals and no pollen grains containing 3 germinal pores are present.”98
3.5.3 Genetic methods: Some publications have investigated the capability of DNA barcoding to distinguish saffron from its adulterants, including closely related Crocus species, but also safflower and other known adulterants.72,83,99,100 Barcodes for the matK, rbcL, ITS2, and trnH-psbA regions readily distinguished saffron and turmeric, calendula, gardenia, safflower, and buddleia (Buddleja spp., Scrophulariaceae).63 Gismondi et al. showed that the matK and rbcL regions are highly conserved within the Crocus genus and therefore do not allow differentiation among species from that genus. However, ITS2 was useful to identify the various Crocus species.99 The use of DNA markers has allowed the detection of low amounts (as little as 1 %) of several bulking materials, including safflower and turmeric.83 Combining DNA barcoding with high-resolution melting (HRM) analysis permitted distinguishing among Crocus species and saffron and turmeric, calendula, corn, safflower, and hemerocallis (Hemerocallis spp., Asphodelaceae).99
PCR-based DNA work was developed to detect and quantify using DNA-based methods to detect and quantify safflower as an adulterant in saffron. The assays resulted in absolute and relative sensitivities of 2 pg of safflower DNA (~1.4 DNA copies) and 0.1% of safflower in saffron, respectively.71 However, these methods have not been assessed in their ability to detect undeclared plant materials in saffron extracts. While DNA mini-barcodes have shown success in finding smaller-sized DNA fragments101 that can be present in herbal extracts, evidence for a successful application of this approach has not been put forward in the case of saffron. Additionally, DNA-based methods cannot detect the undeclared addition of food dyes or synthetic saffron constituents such as safranal.
3.5.4 Spectroscopic methods: The UV-Vis spectrophotometric method proposed by ISO 3632-273 is popular because of its simplicity, low costs, and short analysis time. It helps to detect saffron adulteration with common adulterants like safflower, turmeric, or calendula, but it is not specific enough for all adulterants and requires considerably high (about 200 mg/g) levels of adulterant to be present.64,70
Fourier transform infrared (FT-IR) spectroscopy with pattern recognition has been used to identify saffron adulteration with undeclared food colorants, including Tartrazine, Sunset Yellow, Azorubine, Quinoline Yellow, Allura Red, and Sudan-II. The spectral regions of 1800–1830, 2600–2900, and 3700–3850 cm-1 have been reported to be helpful for the differentiation of authentic saffron samples from adulterated materials. Genetic algorithm linear discriminant analysis (GA-LDA) based on clustering of variable concept has been applied to FT-IR spectra to distinguish authentic saffron from its adulterants.102
3.5.5 NMR-based methods: Several publications have provided evidence for the usefulness of detecting adulteration by nuclear magnetic resonance (NMR) spectroscopy, especially when coupled with multivariate statistical analysis like principal component analysis (PCA) or partial least square-discriminant analysis (PLS-DA) models. These methods successfully detected saffron adulteration with saffron stamens, safflower, turmeric, and gardenia, and synthetic colorants like Sudan I-IV, Tartrazine or Naphthol Yellow.93,103,104
3.5.6 TLC and HPTLC assays: Several publications used thin-layer chromatography to detect synthetic dyes such as magenta III or rhodamine B in saffron.76,77,105 Dyes were easily detected by the presence of pink or orange spots without using a derivatization reagent. In 2020, high-performance thin-layer chromatography (HPTLC) coupled with multivariate image analysis (MIA) was evaluated as a tool for authentic saffron and adulteration detection with safflower, saffron style, calendula, and madder (Rubia tinctorium, Rubiaceae). MIA analysis of the HPTLC images using PLS-DA at 5–35% (w/w) levels showed proper classification of saffron and adulterants with an error rate of 1.96%.92 A HPTLC method published in 2021 allows distinguishing the stigmas from the stamen and the tepals based on the apocarotenoid and flavonoid fingerprints.106 An official HPTLC method is available in the European Pharmacopoeia monograph Crocus for homeopathic preparations.98
3.5.7 HPLC-UV/Vis, HPLC-MS/MS, and GC-MS methods: Crocins are the main group of pigments in C. sativus, but Gardenia species also contain the same constituents. Many analytical methods have been developed to find species-specific chemical markers to determine the difference between the two plants' constituent profiles. The iridoid glycoside geniposide was identified as a marker compound for Gardenia species.107,108 An HPLC-MS method enabled the detection of up to 0.004% of adulteration with Gardenia based on geniposide concentrations.108 Additionally, a UHPLC-UV/Vis-ESIMS method showed the presence of crocetin esters, crocetins, kaempferol derivatives, safranal, and picrocrocin in C. sativus and Gardenia specific iridoids like gardenoside, genipin-1-O-gentibioside, geniposide, and 6"-O-trans-coumaroylgenipin-1-O-gentibioside, along with crocetin esters in Gardenia.109
Sabatino and co-workers separately characterized the HPLC-PDA-ESI-MS fingerprints of saffron extracts with extracts from other sources like calendula, safflower, and turmeric. Isorhamnetin 3-O-neohesperidoside was found to be characteristic of calendula; in the case of safflower, the specific marker molecules were anhydro safflor yellow B and carthamin. Turmeric could be easily detected by the presence of curcuminoids, i.e., demethoxycurcumin, bisdemethoxycurcumin, and curcumin (Figures 7 and 8).70
Aiello and associates, in 2018, described a rapid, simple, and reliable method for the quantitative analysis and constituent fingerprinting of saffron by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. The chemical profile of authentic saffron and samples adulterated with vegetable matter (C. longa and C. tinctoria) were identified by MS and tandem mass spectrometry (MS/MS).110 The same group also identified geniposide as a specific biomarker to detect saffron sample adulteration, with G. jasminoides. Nevertheless, there is still an ongoing demand for the development of faster, simple, and robust screening methods suited for identifying saffron adulteration, especially at levels that make economic sense.111
Lozano and co-workers published an HPLC-UV/Vis method in 1999 for secondary metabolites from saffron and from three adulterants, calendula, red dodder (Cuscuta planiflora, Convolvulaceae), and safflower.112 Surprisingly, the authors were unable to extract any colorants from the three plant species and, therefore, detection of adulteration was not possible . Subsequently, the method was expanded by including extracts from madder, red beet, and saffron tepals in addition to the previously studied safflower. While madder (> 9.1%), red beet (>14.3%), and safflower (> 14.3%) extracts were readily identified, the authors were unable to detect the addition of saffron tepal extract.113
A thermal desorption-gas chromatographic mass spectroscopy (GC-MS) technique was developed for the volatile constituents in saffron; it was applied to 252 saffron samples from Spanish producers. Based on the fingerprint and content of safranal, adulteration with safflower could be detected. Also, the presence of β-cyclocitral was identified as a chemical marker for the presence of synthetic safranal.114 Chemometric analysis using multivariate curve resolution–alternating least squares (MCR–ALS) and multivariate pattern recognition methods such as PCA and k-means applied to GC–MS fingerprints of saffron has been reported by Aliakbarzadeh and co-workers.115 Iranian saffron was extracted by ultrasound-assisted solvent extraction (UASE) and dispersive liquid-liquid microextraction (DLLME), and 77 constituents determined with GC–MS. The elution profiles were used to distinguish five classes of saffron based on 11 compounds, including safranal, linoleic acid, and the long-chain fatty alcohol nonacosanol.115
In a recent report, a GC-MS technique was developed with solid-phase microextraction (SPME) pretreatment to classify genuine and adulterated saffron samples from three regions of Italy based on its aroma profile. The predictive performance of a PLS-DA model calibrated with 42 samples was tested using nine authentic and nine artificially adulterated samples. In this study, the nine samples of saffron adulterated with Calendula officinalis L. petals, Carthamus tinctorius L. petals, or Curcuma longa L. powdered rhizomes were correctly profiled.116
3.5.8 Other methods: An analytical technique named the electronic nose has been shown to detect and characterize complex odors using arrays of sensors. The electronic nose system successfully classified authentic and adulterated saffron (e.g., safflower, corn stigma, or beetroot) by pattern recognition.68
Saffron constituents like safranal have also been reportedly substituted with nature-identical molecules of synthetic origin. A 13C isotopic analysis of safranal was carried out with saffron samples from five different countries. Safranal was extracted using supercritical fluid extraction (SFE) in this study. While the samples' geographical origins could not be distinguished, there was a clear difference in 13C levels in safranal of natural origin compared to the same compound made by chemical synthesis.66 Similarly, Wakefield et al. were able to distinguish between samples of Iranian and Spanish origin using stable isotope ratios and measuring of trace elements. Differentiation of samples from various places in the Iranian province of Khorasan proved more difficult.117
As an expensive raw material, saffron is clearly at risk of adulteration. Cultivation and collection activities from different geographical areas lead to saffron with similar identity and quality characteristics from the major C. sativus-supplying countries. Economic adulteration includes materials of inferior quality, other plants, synthetic colorants, and the undeclared addition of oils, glycerin, or honey to increase weight; synthetic dyes can pose safety and health risks and their absence needs to be verified by appropriate testing. A complete set of orthogonal testing methods (which may include, among others, microscopy, HPTLC, HPLC, FT-IR, or NMR) needs to be used to authenticate saffron. Ideally such a combination of methods should be published by official compendia to enable appropriate monitoring of the authenticity of saffron ingredients throughout the supply chain.
*There are two acceptable plural forms of stigma: stigmas or stigmata. In the scientific literature on saffron, the term stigmas is more widespread and therefore used throughout this manuscript.
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