Reddy et al. established a method for preparing analytically pure crocetin on a small scale using saffron as raw material. The raw material (C. sativus stigma) was sonicated, followed by alkalization and acidification of the supernatant. The resulting precipitate was dissolved in ethyl acetate, and analytically pure crocetin was obtained from ethyl acetate solution (Reddy et al., 2020). In addition, the authors prepared a gram scale for extracting crocetin from saffron raw material. The raw material was extracted with methanol: water, and the obtained extract was hydrolyzed, neutralized, and separated to obtain crocetin (Reddy et al., 2020). Lautenschläger et al. performed enzymatic deglycosylation to extract crocetin from saffron. Two different enzyme preparations were used: RöhmEnzym^® and Rohament CL^®. Further purification was performed using medium pressure liquid chromatography (Lautenschläger et al., 2014).

Using Amberlite D140 resin chromatography, gardenia yellow pigment was obtained from the 60% ethanol extract of gardenia fruit, which was then alkali-hydrolyzed and acidified. The resulting precipitate was mixed with methanol to remove impurities, and crocetin was crystallized from dimethylformamide (Qian et al., 2010). In another study, the SPE-007A enzyme was selected for enzymolysis of gardenia fruit. After enzymolysis, the obtained materials were alkali-hydrolyzed and then acidified. Crude crocetin was separated using a silica gel column. Finally, crocetin was purified by recrystallization (Zhang W. et al., 2017).

Tan et al. studied the effect of a specific aldehyde dehydrogenase (CsALDH3) on the oxidation of crocetin dialdehyde to crocetin. The authors predicted that four CsALDH genes encode enzymes responsible for catalyzing crocetin dialdehyde conversion to yield crocetin. To characterize the function of candidate CsALDH genes, nucleotide sequence analysis was performed to identify the full-length transcripts. Accordingly, three cDNAs (CsALDH1, CsALDH2, and CsALDH3) were predicted as candidate genes involved in crocetin biosynthesis. Codon-optimized CsALDHs were individually introduced into the zeaxanthin-producing yeast. Expression of the recombinant CsALDH3 protein in crocetin-producing yeast strains resulted ina39% increased yield (Tan et al., 2019). Song et al. optimized the overproduction of crocetin in yeast. By blocking genes related to citric acid synthase (CIT2) in the glyoxylate cycle, the crocetin titer could be elevated by 50% when compared with the starting strain. Accordingly, the crocetin yield was further elevated by 44% by introducing the forward fusion enzyme PsCrtZ-CsCCD2. Finally, the crocetin titer was 12.43 ± 0.62 mg/L in a 5 L bioreactor (Song et al., 2020). In addition, the resulting engineered strain was characterized by overexpression of CrtZ and CCD genes. The engineered strain displayed higher efficiency in crocetin production, and the concentration of crocetin reached 1.17 mg/L after fermentation for 108 h (Xiao et al., 2019). Lou et al. introduced a plant expression vector carrying crtRB and ZCD1 genes into C. vulgaris; crtRB and ZCD1 genes encode key enzymes that control crocetin biosynthesis. Crocetin can be produced in transgenic C. vulgaris but not in the wild-type species (Lou et al., 2016).

Obviously, G. jasminoides fruit is more cost-effective than saffron for crocetin production. In addition, alkali hydrolysis is simple and easy, and enzymolysis is considered more eco-friendly than other methods. In the 21st century, bioengineering can broaden prospective resources for crocetin extraction and production.

Fang et al. applied for a patent on crocetin synthesis via organic chemistry. The method used 3,7-dimethyloctatrienemthanal and methyl 2-bromopropionate as raw materials to synthesize crude crocetin as a dimethyl ester via a three-step reaction, the refined crocetin was obtained after hydrolysis, decoloration, and recrystallization (Fang and Wang 2007).

Microbial glycosyltransferases [GTs; bacterial GTs (Bs-GT)] extracted from Bacillus subtilis 168 by Ding et al. showed a high degree of carboxyl glycosylation activation for crocetin. The molecular conversion rate approached 81.9%, affording 476.8 mg/L crocin, thus indicating the efficient production of crocin. Rare crocin-5 and crocin-3 are specifically produced by Bs-GT (Ding et al., 2018).

Yang et al. applied for a patent for the preparation of crocetin diammonium salt. Crocetin diammonium salt was extracted from G. jasminoides with ammonia water and concentrated to a thick paste by adding organic solvents (methanol, isopropanol). The diammonium salt of crocetin was completely precipitated after chilling (owing to the low solubility of the diammonium salt of crocetin in organic solvent). The crude product of the diammonium crocetin salt was obtained by filtration. Then, HPD-100 resin was used to separate the diammonium salt of crocetin (Yang 2012).

Crocetin dialdehyde was synthesized by reacting 2,7-dimethylocta-2,4,6-trienedial with diethyl 3-(5,5-dimethyl-1,3- dioxane-2-yl) but 2-enylphosphonate via the Horner-Wadsworth-Emmons reaction. This method yielded a 41% crocetin dialdehyde (Zhang and Luo 2016).

Purified crocetin was added to a sodium hydroxide solution at a molar ratio of 1:2. After the reaction was complete, crocetin sodium salt was obtained by filtration, sterilization, and freeze-drying. The total crocetin yield was 1.15% (Zhang 2017).

The gardenia yellow pigment was added to anhydrous methanol and a sodium methoxide solution. After changing to an ester, crocetin dimethyl ester was obtained. The purity of crocetin dimethyl ester was 98.8% by recrystallization (Fang and Wang 2007). In the synthesis experiment designed by Sun et al., crocetin dimethyl ester was obtained using the Wittig reaction, combining 2,7-dimethyl-2,4,6-octatriene-1,8- dialdehyde and γ-chloro methyl tiglate to achieve a crocetin dimethyl ester yield of 78.6%. Of these reagents, 2,7-dimethyl-2,4,6-octatriene-1,8- dialdehyde was synthesized via the Wittig-Horner reaction using dimethoxyacetone and 1,4-dibromo-2-butene as raw materials; γ-chloro methyl tiglate was synthesized from chloroacetaldehyde and 2-bromo methyl propionate (Sun et al., 2012).

Crocetin was mixed and reacted with oxalyl chloride and triethylamine, followed by the addition of phenylethylamine. The reaction solution was extracted with an organic solvent, and crocetin amide derivatives were obtained by recrystallization (Zhu et al., 2012). In another method designed by Wang et al., crocetin was added to HOBt and EDCl, followed by Et3N and 4-fluorobenzylamine. Synthetic crocetin derivatives were acquired by vacuum evaporation, and purified crocetin derivatives were obtained by column chromatography. After structural modification, the formation of hydrogen bonds increased, along with the solubility of obtained crocetin derivatives (Wang MZ. et al., 2020).

Dimethylformamide and organic amine were added to crocetin as the reaction solution, followed by ethyl acetate and petroleum ether. Organic amine salt crystals were obtained by precipitation, filtration, and recrystallization (Yang et al., 2011).

GTs can specifically transfer sugar groups to receptor molecules (Modenutti et al., 2019). He et al. applied to patent the preparation of crocetin glucose ester using glucose as the donor. GT from B. subtilis was used as a glycosyl donor to synthesize crocetin glucose ester. Crocetin and UDP-Glc were added to a phosphate buffer solution or glycine NaOH buffer solution to perform the reactions (He et al., 2017).

Overall, salinization and esterification are the main derivative strategies. However, comparisons examining the pharmacokinetics, bioavailability, and pharmacological activities of these derivatizations were insufficient.

Studies have shown that crocetin plays a potential role in prevention and treatment of cardiovascular diseases such as hypertension, myocardial hypertrophy, myocardial ischemia, atherosclerosis (Table 2).

Several theories exist regarding cancer occurrence, and the theory of “oxidative stress” is worthy of further attention (Sosa et al., 2012). Higher ROS levels in cancer cells have been found and used to explain the mechanisms of tumor growth, proliferation, and metastasis. Numerous studies have demonstrated the anticancer effects of crocin, crocetin, and other anticancer agents via the regulation of antioxidant activity, reduced cyclooxygenase (COX)-2 production and inflammation, induction of cell apoptosis, and antiproliferative activity (Hashemi S. et al., 2018; Zou et al., 2017). Studies have shown that crocetin can inhibit the synthesis of DNA, RNA, and proteins in cancer cells (Colapietro et al., 2019). Azarhazin et al. confirmed that crocetin, as an anticancer drug, interacted with Dickerson DNA through van der Waals forces and hydrogen bonds, and the active site was found to be located in the small groove of DNA (Azarhazin et al., 2017). In addition, crocetin reportedly influences the growth of cancer cells by blocking the growth factor signaling pathway, arresting the cell cycle, and inducing apoptosis (Gutheil et al., 2012).

In vivo and in vitro experiments have revealed that crocetin has therapeutic effects against breast, skin, gastrointestinal, liver, cervical, and ovarian cancers (Colapietro et al., 2019; Hashemi and Hosseinzadeh 2019) (Table 3).

Although the pathogenesis of nervous system disease remains unclear, the potential role of crocetin has been discussed in subsequent studies (Table 4).

Crocetin displayed protective effects against aflatoxin B1-induced hepatotoxicity in rats by elevating the cytosolic GSH, as well as GST and GSH-Px activities (Wang et al., 1991). Sreekanth et al. examined the protective effect of crocetin on dengue virus (DENV)-infected liver damage in mouse models. Crocetin (50 mg/kg) was found to balance antioxidant enzymes (SOD and CAT), reduce the expression of pro-inflammatory cytokines, and inhibit nuclear translocation of NF-κB. The results showed that crocetin treatment could not reduce DENV replication in the liver of DENV-infected mice; however, crocetin could improve liver injury by reducing hepatocyte apoptosis (Sreekanth et al., 2020). In a study by Gao et al., the hepatoprotective effect of crocetin on paraquat (PQ) poisoned rats was investigated. The authors revealed that 50 mg/kg crocetin exerted hepatoprotective effects in PQ-poisoned rats, which may be achieved by reducing the levels of inflammatory factors in the blood and inhibiting the activities of caspase-8, -9, and -12, as well as the expression of iNOS and NF-κB in liver tissues (Gao et al., 2016). Liu et al. evaluated the protective effect of crocetin on arsenic trioxide (ATO)-induced hepatic injury and showed that 50 mg/kg crocetin could alleviate weight loss and hepatic pathological injury in rats with hepatic injury. Crocetin reversed the increase in alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase. In addition, crocetin enhanced antioxidant and anti-inflammatory effects in the body by activating the Nrf2 signaling pathway (Liu P. et al., 2020). Guo et al. found that crocetin promoted autophagy in injured hepatocytes and reduced further hepatocyte damage (Guo et al., 2018). In addition, crocetin impacted non-alcoholic fatty liver cells and showed that the TG content in fatty liver cells was decreased, and lipid deposition was effectively alleviated; the underlying mechanism might be related to the reduction in cellular oxidative stress (Liao et al., 2011). Gao et al. found that crocetin can be used as a preventive drug for fulminant hepatic failure (FHF). The authors revealed that crocetin pretreatment improved the liver tissue morphology, decreased total bilirubin production, and reduced the activities of ALT and AST in FHF rats. Moreover, crocetin reduced hepatocyte apoptosis, p53 mRNA expression, and caspase family protein expression. In addition, crocetin decreased the secretion of inflammatory cytokines by inhibiting NF-κB activation and suppressing liver oxidative stress (Gao et al., 2019). Crocetin effectively alleviated the degree of liver injury and fibrosis in liver fibrosis mice, which might be related to the downregulation of p38MAPK protein expression (Wang X. et al., 2017).

Michael et al. reported that I/R-induced renal damage was reduced following treatment with 50 mg/kg crocetin. The results showed that crocetin could suppress inflammatory components and the degree of epithelial injury, as well as induce the expression of miR21, miR127, and miR132 (Michael et al., 2020). Wang et al. administered 50 mg/kg crocetin through the duodenum to rats with hemorrhagic shock and resuscitation. Crocetin improved renal dysfunction caused by hemorrhagic shock and resuscitation by restoring T-SOD activity and quenching the superoxide anion/free radical, inhibiting NF-κB activation, and preventing TNF-α and IL-6 production (Wang et al., 2012). Liu et al. found that crocetin could prevent ATO-induced renal injury by inhibiting oxidative stress, inflammation, and apoptosis, which may be associated with activation of the PI3K/Akt signaling pathway (Liu Y. et al., 2020).

Accumulated evidence has revealed that saffron and its extracts are beneficial for treating diabetes and its complications (Hashemi and Hosseinzadeh 2019; Kumar and Gupta 2019). The underlying mechanisms may involve stimulating glucose uptake by peripheral tissues, inhibiting endogenous glucose production, reducing insulin resistance, and stimulating islet β cells to release more insulin (Farkhondeh and Samarghandian 2014).

Elgazar et al. found that aqueous saffron extract significantly increased body weight and serum insulin levels, decreased blood glucose levels, improved lipid levels, as well as liver and kidney functions in alloxan-induced diabetic rats (Elgazar et al., 2013). Xi et al. reported that crocetin has a regulatory effect on high-fructose diet-induced insulin resistance and free fatty acid-induced insulin insensitivity. Crocetin restored the levels of adiponectin (an insulin-sensitizing adipocytokine), TNF-α, and leptin in the experimental group (Xi et al., 2007). In addition, Sheng et al. showed that crocetin accelerated the uptake and oxidation of TGs and non-esterified fatty acids in the liver, thereby increasing insulin sensitivity (Sheng et al., 2008). In addition, crocetin suppressed the palmitate-induced activation of c-Jun NH (2)-terminal kinase (JNK) and inhibitor kappaB kinase beta (IKKbeta) by inhibiting protein kinase Ctheta (PKCtheta) phosphorylation and improving insulin sensitivity in 3T3-L1 adipocytes (Yang et al., 2010).

Endothelial progenitor cell (EPC) dysfunction is an important risk factor for diabetic vascular complications; thus, Cao et al. investigated the role of crocetin in diabetic EPC dysfunction. EPCs were isolated from the bone marrow of diabetic mice. Crocetin (5 μM) treatment alleviated diabetic EPC proliferative damage. Furthermore, crocetin augmented LDH release, cell apoptosis, and caspase-3 activity. The mechanism of crocetin against the impairment in diabetic EPCs could involve enhanced NO bioavailability by regulating the PI3K/AKT-eNOS and ROS pathways (Cao et al., 2017). Similarly, crocetin (0.1, 1.0 μM) prevented high glucose-induced apoptosis of HUVECs, possibly associated with p-Akt activation, following upregulated eNOS and NO production (Meng and Cui 2008).

Zheng et al. investigated the therapeutic effect of crocetin on STZ-induced gestational diabetes mellitus (GDM) in rats. Crocetin reduced blood glucose levels and increased body weight in GDM rats. In addition, crocetin treatment increased the levels of antioxidant enzymes, including SOD, GSH-Px, GSH, and CAT, decreased expression levels of IL-6, TNF-α, and IL-1β, and suppressed the levels of intercellular adhesion molecule-1 (ICAM-1), COX-2, and PGE₂. In addition, crocetin treatment enhanced levels of Bcl-2 and reduced levels of Bax and caspase-3 in rats. In summary, crocetin showed significant therapeutic effects against GDM by improving the status of endogenous antioxidant enzymes, inhibiting the inflammatory reaction, and suppressing mitochondrial pathway apoptosis (Zheng et al., 2021).

Mahdavifard et al. found that MB-92 (a combination of some amino acids and crocetin) has potential therapeutic effects for inhibiting glycation and oxidation products, atheromatous plaque formation, and inflammation in diabetic atherosclerotic rats (Mahdavifard et al., 2016).

Previous studies have shown that advanced glycation end-products (AGEs) are key pathogenic factors in diabetic angiopathy. Crocetin can inhibit the migration of AGE-induced VSMCs by suppressing receptor advanced glycation end (RAGE) expression, resulting in the reduction of protein levels of TNF-α and IL-6, as well as the suppression of MMP-2/9 activity (Xiang et al., 2017). Xiang et al. investigated the effect of crocetin on AGE formation and the expression of RAGE protein in diabetic rats. STZ-induced diabetic rats were intragastrically administered crocetin (50 mg/kg) for 21 days. Crocetin markedly reduced the content of fructosamine (FMN) and glycosylated hemoglobin (GHb), intermediate AGE products. In addition, the deposition of AGEs in the aortic and mesenteric vascular beds decreased, while the expression of RAGE was significantly decreased. Therefore, crocetin could afford a protective effect on blood vessels of diabetic rats (Xiang et al., 2006).

Mesenchymal stem cells (MSCs) play an important role in bone repair. Studies have reported that crocetin can effectively promote osteogenic differentiation of MSCs (Kalalinia et al., 2018). For example, Li et al. induced arthritis by administering intraperitoneal Complete Freund’s adjuvant in rats. The authors showed that crocetin could adjust paw edema and body weight in rat models in a dose-dependent manner. Crocetin protected rat models of arthritis by reducing HO-1/Nrf-2 expression and inhibiting inflammatory mediators (Li et al., 2018). Regulatory T cells (Tregs) are key regulatory factors in asthma. Ding et al. used crocetin to treat ovalbumin (OVA)-induced asthma in mice. Crocetin alleviated the asthma severity in mice. A possible mechanism underlying this effect is that crocetin activates Foxp3 through TIPE2 in Treg cells (Ding et al., 2015). In addition, crocetin has a potential therapeutic effect on scleroderma; crocetin (0.1, 1, or 10 μmol) inhibited the proliferation and differentiation of skin fibroblasts isolated from patients with systemic scleroderma in a concentration-dependent manner. Intraperitoneal injection of 50 mg/kg crocetin reduced skin and lung fibrosis in bleomycin-induced scleroderma mice, mainly owing to the reduction of endothelin-1 (ET-1) (Song et al., 2013). Crocetin has a protective effect against 2,4,6-trinitrobenzene sulfonic acid-induced colitis in mice. Studies have shown that 50 mg/kg crocetin (i.g.) significantly improved diarrhea and destruction of colon structure, as well as reduced the degree of neutrophil infiltration and lipid peroxidation in the inflammatory colon, thus suggesting that crocetin plays beneficial roles in experimental colitis (Kazi and Qian 2009). Previous findings have shown that saffron (C. sativus L.) extract has antinociceptive effects. Erfanparast et al. showed that crocetin injection into the cerebral fourth ventricle improved formalin-induced orofacial pain in rats, and the antinociceptive effect was related to central H₂ histaminergic and α₂ adrenergic receptors (Erfanparast et al., 2020).

1) Animal/cell research

In a report by Zhang et al., following intragastric administration of 25 mg/kg crocetin, Sprague Dawley (SD) rats reached the highest blood concentration (3.56 μg/ml) after 1.7 h; however, its oral bioavailability was only 11.25%. The area under the concentration-time curve from time zero to the last measurable concentration (AUC_0-t) was 92.242 μg/L·h, AUC_0-∞ was 92.244 μg/L·h (Zhang 2017).

Liu et al. administered crocetin (50 mg/kg) to 10 rats via intragastric administration. The content of crocetin in the plasma was determined using HPLC. The pharmacokinetic parameters were obtained by calculation; the half-life was approximately 30 min, the peak time was approximately 65 min, the maximum plasma concentration was 50 μg/ml, AUC_0-t was 845 ± 109 μg·min·ml⁻¹, and volume of distribution (V_D) was 50 ± 08 L kg⁻¹ (Liu and Qian 2003).

To evaluate the effect of crocetin on cerebral I/R injury, six rats in each group were intravenously administered crocetin (0.33 mg/kg), and the total urine and feces samples were collected every 8 h after administration. Crocetin was not excreted in the urine or feces following intravenous administration. Moreover, it did not exhibit any anticipated pharmacological effects. Therefore, oral administration of crocetin is superior to intravenous administration (Zhang et al., 2019a).

Oliveira et al. determined the gastrointestinal absorption of major carotenoids (crocetin, crocin-1, and crocin-2) in G. jasminoides by assaying the transport using MKN-28 and Caco-2 cells lines. In general, crocetin showed the greatest efficiency in terms of gastrointestinal transport (Oliveira et al., 2017).

Lautenschläger et al. examined the intestinal permeation of trans-crocetin using a Caco-2 monolayer cell culture. The results showed that trans-crocetin permeated the intestinal barrier by transcellular passage, with approximately 32% of the substrate transported within 2 h. In addition, porcine brain capillary endothelial cells (BCECs) and the blood-cerebrospinal fluid barrier (BCSFB) were used to study the permeation characteristics of trans-crocetin across the blood-brain barrier (BBB). Trans-crocetin permeates the BBB to enter the central nervous system (CNS) at a slow but constant velocity over a 29-h period (Lautenschläger et al., 2015).

2) Clinical research

In a study by Almodóvar et al., 13 healthy human volunteers were administered different concentrations of saffron extract (56 and 84 mg), and blood samples were collected every 30 min after the first 3 h madministration. Crocin, safranal, and picrocrocin levels were undetectable in mplasma. Only sufficient concentrations of crocetin could be detected in blood samples to be identified and quantified. HPLC- photodiode-array detection and electrospray (PAD)/mass spectroscopy (MS) was used for identification and quantification. Approximately 60–90 min after oral administration, the maximum concentration (C_max) of crocetin in blood could be detected, and the kinetics of the reaction was dose-dependent. According to the two doses, the mean C_max and the estimates of the pharmacokinetic parameters (AUC_0-3h) of crocetin approximately ranged between 0.26 and 0.39 μg/ml and 21.07–26.15 μg·h/ml, respectively (Almodóvar et al., 2020).

The C_max of crocetin was 0.28 μg/ml with a single oral dose of 22.5 mg (Umigai et al., 2011). These data closely correlated with the C_max detected by Almodóvar et al. using saffron extract containing only 23 mg crocin, thus confirming that crocetin derived from saffron-extracted crocin was more bioavailable than the pure crocetin following oral administration. This finding could be explained by the greater bioavailability of crocin into enterocytes for later absorption than that of crocetin (Almodóvar et al., 2020).

The value of T_max after crocetin administration was smaller than that of other carotenoids, indicating that the absorption and detection of crocetin in plasma were more rapid than that of other carotenoids (Umigai et al., 2011).

Miller et al. used absorption and fluorescence techniques to study the binding of crocetin to human and bovine plasma albumin. The results showed that crocetin binds to plasma albumin by occupying the binding site of free fatty acid binding, indicating that plasma albumin may be a transporter of crocetin (Miller et al., 1982). Hydrophobic interactions are one mechanism of crocetin and plasma albumin interaction (Kanakis et al., 2007).

Once in circulation, given the weak interaction between crocetin and plasma albumin, crocetin can reach different tissues and cross the BBB in a concentration-independent manner by passive transcellular diffusion mechanism, as demonstrated in an in vitro study. In addition, it should be noted that the typical transporter saturation effect could not be determined owing to the poor solubility of crocetin (Lautenschläger et al., 2015). However, similar studies have shown that crocetin is easily absorbed by intestinal epithelial cells, and its uptake is positively correlated with increased drug concentration, demonstrating that crocetin enters cells through passive diffusion (Wang HF. et al., 2018).

To determine whether absorption and transport of crocetin in the Caco-2 cell model is related to P-glycoprotein (P-gp), Lautenschläger et al. suggested that crocetin serves as the substrate of the PGP efflux pump and enters the BBB via passive transcellular diffusion (Lautenschläger et al., 2015). However, in another experiment, the apparent permeability coefficient of crocetin in the Caco-2 cell model was 5.06 × 10⁻⁶ cm·sec⁻¹ and permeability damage rate (PDR) was 1.52, which indicated that crocetin was moderately absorbed in the Caco-2 cell model. After the addition of verapamil (P-gp inhibitor), the values of PappAP-BL and PDR did not significantly differ from those previously reported, which indicated that crocetin uptake and transport were not mediated via P-gp (Wang S. L. et al., 2018). This result contradicts the previous research by Lautenschläger. Accordingly, whether P-gp transporters or other intestinal transporters, such as MRPs and PEPT1, mediate crocetin absorption in the intestine warrants further study.

Christodoulou et al. examined the oral and intravenous administration of saffron (C. sativus L.) aqueous extract in C57/BL6J mice by assessing the kinetics of crocetin and its metabolites, exhibiting a one-compartment pharmacokinetic model with first-order absorption after oral administration. After intravenous administration of the aqueous saffron extract, the one-compartment pharmacokinetic model described the kinetics of crocetin, while the first-order kinetic parameters described the rate of crocetin to that of its metabolite (Christodoulou et al., 2019).

In a study by Zhang et al., cerebral I/R injury rats were administered crocin orally or intravenously, and neither crocin nor crocetin was detected in the cerebral tissue, indicating that crocin does not affect the cerebral tissue through its prototype or metabolite. More importantly, the effects of crocetin in the circulatory system might mediate the cerebral-protective effects (Zhang X. et al., 2019). However, Lautenschläger et al. demonstrated that trans-crocetin could cross the BBB to reach the CNS. The authors employed porcine BCECs and BCSFB as suitable models for monitoring the permeation characteristics of trans-crocetin across the BBB. The results showed that trans-crocetin bypassed the BBB at a slow but constant speed within 29 h (Lautenschläger et al., 2015). Other studies have revealed that after oral administration of crocetin (100 mg/kg) for 90 min, the brain concentration was approximately 2.43 nmol/g (Wong et al., 2020). Therefore, future investigations need to examine whether crocetin can cross the BBB and determine the effect of the crocetin configuration on BBB permeation.

It is well-established that crocetin can pass through the intestinal barrier. Several studies have shown that crocetin can be rapidly absorbed into the blood via the gastrointestinal tract, reaching peak plasma concentration for a short period (Colapietro et al., 2019). In a study by Christodoulou et al., following oral and intravenous administration of an aqueous saffron extract (60 mg/kg) to C57/Bl6J mice, crocetin (derived from in vivo crocin hydrolysis) tissue levels were measured using the HPLC-PDA method, and non-compartmental pharmacokinetic analysis was performed. Crocetin was extensively distributed in the liver and kidney, the main organs for crocetin biotransformation and elimination. The levels of crocetin could not be detected in the lungs and heart, possibly because of rapid glucuronidation to form its conjugated forms (Christodoulou et al., 2019). However, Du et al. showed that orally administered crocetin is widely distributed in the body. The crocetin concentration in tissues, ranging from high to low, was found to occur in the following order: liver, lung, ovary, kidney, fat, and testis (Du et al., 2004). Additional research is needed to determine the distribution of crocetin in the body.

Crocetin-γ-cyclodextrin was administered to SD rats via intravenous and intraperitoneal injections. Crocetin-γ-cyclodextrin can enter the brain by crossing the BBB and is distributed in the stomach, large intestine, small intestine, liver, spleen, kidney, lung, and heart. The bioavailability of intravenous injection was greater than that of intraperitoneal injection, and the drug concentration in each tissue reached a maximum after 5 min of intravenous injection. After 4 h, nearly no residual drug could be detected, and the metabolic rate was relatively elevated (Wong et al., 2020). In a study examining the inhibitory effect of crocetin on proliferative vitreoretinopathy in rabbits, 0.4 μmol crocetin was administered as an intravitreal injected into PVR rabbit eyes. The half-life of crocetin was determined as 4.231 h by HPLC. One hour after intravitreal injection, the C_max of crocetin was 36.77 ± 3.39 μg/ml (Wang S. L. et al., 2018).

The whole in vitro digestion process (salivary, stomach, and duodenal steps) designed by Almodóvar et al. showed no increase in the crocetin concentration when compared with the original composition of saffron (Almodóvar et al., 2020). The results indicated that saffron extract was not metabolized into crocetin in these parts of the body.

Almodóvar et al. speculated that crocetin formation in the blood could be attributed to the presence of enzymes in the epithelial cells of the gastrointestinal tract, which can hydrolyze the crocin isomers (Almodóvar et al., 2020). Enzymes of gastrointestinal tract epithelial cells include esterase or β-glycosidase (Németh et al., 2003).

Lautenschläger et al. also believed that the intestinal deglycosylation of different types of crocin was primarily attributed to enzymatic processes in the epithelial cells and only to a very small extent to deglycosylation by the fecal microbiome (Lautenschläger et al., 2015). In vivo and in vitro experiments by Zhang et al. have shown that crocin can be deglycosylated to crocetin in the intestinal content of normal rats; however, this transformation did not occur in pseudo-germ-free (pGF) rats, suggesting that the intestinal microbiota plays a key role transforming crocin into crocetin. A possible explanation might be that crocin was not absorbed by enterocytes following oral administration, and a considerable portion of crocin was retained in the intestinal tract, where it could be directly metabolized into crocetin by intestinal flora (Zhang Y. et al., 2019). Further studies are needed to confirm the precise intestinal section involved. Moreover, crocetin is metabolized to ester-type glucuronides in the liver or gut mucosa after oral administration. This form of crocetin showed stronger stability in plasma and could be considered a bioactive molecule or a carrier to deliver crocetin to target tissues (Mh and Hhb 2019).

Lautenschläger et al. used mouse intestinal tissue and fecal homogenate for metabolism experiments. Fecal bacteria degraded the apocarotenoid backbone into smaller alkyl units, which did not demonstrate any typical ultraviolet (UV) absorption peaks of crocetin. Additional liquid chromatography (LC)-MS studies indicated the absence of specific degradation products (data not shown) (Lautenschläger et al., 2015).

Crocetin was not excreted in the urine or feces after intravenous administration. Orally administered crocetin is mainly excreted in feces (Zhang X. et al., 2019). Umigai et al. showed that crocetin was eliminated from human plasma with a half-life (T_1/2) ranging between 6.1 and 7.5 h (7.5, 15, and 22.5 mg in one-weekly interval) (Umigai et al., 2011). Zhang et al. reported that the plasma T_1/2 was calculated from 1.640 to 1.671 h, as detected by HPLC after oral administration of crocetin (25 and 100 mg/kg) in rats. After intravenous administration, the T_1/2 of crocetin (5 mg/kg) was 1.914 h (Zhang 2017).

Few studies have examined the pharmacokinetics of crocetin. Additional data are crucial to better understand the effectiveness and dose-response relationship of crocetin, and it is important to determine the doses, directions for use, and further development of crocetin.

In a clinical trial assessing the effect of crocetin on sleep quality, subjects were given crocetin capsules (7.5 mg) once daily for 14 days. No crocetin-induced adverse reactions were observed (Kuratsune et al., 2010).

In a study by Mori et al., the effect of crocetin was assessed in children with myopia. The subjects were given soft capsules containing 7.5 mg of crocetin, taken orally once a day for 24 weeks. No adverse reactions associated with crocetin were reported during the clinical study. All adverse events reported were unrelated to crocetin administration (Mori et al., 2019). In a clinical study evaluating the pharmacokinetics of crocetin, 10 healthy volunteers had no significant adverse events after oral administration of 22.5 mg crocetin (Umigai et al., 2011). In another clinical study evaluating crocetin for physical fatigue relief, healthy volunteers were orally administered crocetin (15 mg) for 8 days (Wang S. L. et al., 2018). Similar to previous studies, no significant discomfort was observed in healthy volunteers at the end of the study period. A phase II clinical trial assessing intravenous crocetin (0.25 mg/kg) for acute stroke was approved by the Food and Drug Administration (Abedimanesh et al., 2019).

Related animal experiments showed that the LD₅₀ of crocetin was 20.7 g/kg body weight following oral administration (Abdullaev 2002).

Crocetin exhibited selective toxicity against cancer cells and may be effective in cancer prevention. However, the cytotoxicity of crocetin on normal cells is negligible, and oral administration is non-toxic (Milajerdi et al., 2016). Jagadeeswaran et al. demonstrated that crocetin (5–20 μg/ml) had selective cytotoxic effects against human rhabdomyosarcoma cells, with poor cytotoxic effects on normal cells when compared with cisplatin-mediated cytotoxicity (Jagadeeswaran et al., 2000). Some studies have shown that 50–100 mg/kg crocetin could protect gastric cancer tissues in rats and had no cytotoxic effect in normal rats (Bathaie et al., 2013a). In addition, studies have shown that the percentage of cytotoxic effect of crocetin beta-D-glucosyl ester (31.25–1,000 mg/ml) ranged between 18.5 and 61.57% in MCF-7 cells when examined at different concentrations (Mam et al., 2020). Previous studies have shown that continuous doses exceeding 10 g can be toxic and cause uterine stimulation and miscarriage during pregnancy (Wüthrich et al., 2010). Martin et al. examined the teratogenic effect of crocetin in frog embryos, and the results showed that a high concentration of crocetin (200 μmol) exerted a teratogenic effect; however, the teratogenic effect was considerably less than that of all-trans-methyl acid (Martin et al., 2002).

Zhang et al. patented the production of crocetin injections. The raw material crocetin and auxiliary materials such as propylene glycol were mixed to dissolve the raw crocetin material. Subsequently, injection water and activated carbon were added. A qualified crocetin injection was then prepared. After filtration, filling, lamp inspection, and packaging, a qualified crocetin injection was prepared. The crocetin injection had good stability, simple preparation, and low cost. The bioavailability of crocetin can be improved by formulating a crocetin injection (Zhang et al., 2011). Another modification involves the preparation of crocetin salt injections. The injection was obtained by dissolving crocetin salt and sodium chloride in water, which can improve the bioavailability and treatment effects of crocetin (Wang and Li 2015).

NP-based drug delivery systems are promising new drug delivery systems that can improve drug delivery efficiency by improving the pharmacokinetics and overcoming the shortcomings of native drugs (Feng 2006).

Neyshaburinezhad et al. encapsulated crocetin into poly (lactic-co-glycolic acid) nanoparticles using a single emulsion-solvent evaporation method. The particle size was determined as 239.8 ± 9 nm. The entrapment efficiency and loading capacity of crocetin NPs were approximately 79 ± 3% and 4.9 ± 0.2%, respectively (Neyshaburinezhad et al., 2019). Yang et al. prepared a nano-formulation of crocetin (CT-PLGA-NPs) using a double emulsion evaporation technique, which employed Span 60 and Tween 80 as the internal and external aqueous phases, respectively, and polyvinyl alcohol (1%) was used to stabilize the external aqueous phase (Yang 2019). In addition, Langroodi et al. used the solvent evaporation/double emulsion method to load crocetin and doxorubicin into PLGA NPs. The prepared NPs exhibited a particle size of 200–300 nm, and the drug loading efficiencies of crocetin and doxorubicin were 65 and 54%, respectively. In addition, the prepared NPs inhibited the growth of MCF-7 tumor cells more effectively (Langroodi et al., 2017).

Pradhan et al. prepared crocetin-loaded lipid NPs using glycerol monooleate (GMO), a synthetic, non-toxic, biocompatible, biodegradable material as the carrier material. The physical characteristics of crocetin-loaded NPs were obtained on measurement: particle size, 119 ± 4 nm; zeta potential, 18.3 ± 4.21 mV; polydispersity index, 0.426. The crocetin-loaded NPs exhibited an encapsulation efficiency of 80%. The low PDI indicated that the particles were uniformly distributed, and the negative zeta potential was conducive to the mutual exclusion of the formulations, which ensured particle stability and prevented particle aggregation (Jyotsnarani et al., 2018). Photodynamic therapy (PDT) is a new method for treating tumor diseases using a photosensitizer, commonly known as indocyanine green (ICG). NPs were used with ICG to overcome the high cytotoxicity at higher concentrations and instability in aqueous media. Sazgarnia et al. loaded crocetin into PLGA NPs to improve the efficacy of PDT with ICG against MCF-7 cells. Accordingly, PLGA-CRT NPs combined with ICG could improve PDT results more effectively. This method afforded low cytotoxicity for treating breast cancer (Sazgarnia et al., 2021).

Wong et al. encapsulated crocetin into γ-cyclodextrin using an ultrasonic method. Crocetin-γ-cyclodextrin inclusion complexes demonstrated good water solubility and were found to be suitable for intravenous injection. Based on the pharmacokinetics and biodistribution, the crocetin-γ-cyclodextrin inclusion complex can improve the crocetin bioavailability and promote BBB crossing, which is beneficial for treating neurosystemic diseases, such as AD (Wong et al., 2020).

Puglia et al. used softisan 100 (hydrogenated coco-glycerides) as solid lipid matrixes and solvent diffusion technology to prepare crocetin solid lipid NPs, exhibiting an average diameter of 280 nm and zeta potential value of −17.8 mV, implying that the NPs have good long-term stability. The nanodispersion displayed good stability, with an encapsulation efficiency of 80% (Carmelo et al., 2018). Current treatment methods have improved the impaired oxygen transportation in acute respiratory distress syndrome, which is beneficial for treating patients with severe respiratory complications of coronavirus disease (COVID-19). A phase I/II clinical trial of the liposomal nanocarrier encapsulating trans-crocetin enhanced the oxygenation of vascular tissue, indicating the potential to treat respiratory distress syndrome due to COVID-19. In addition, the liposomal formulations could increase the reoxygenation properties of free trans-crocetin in endothelial cells from 30 min to 48 h. The clinical experimental results showed that the proportion of partial arterial pressure of oxygen (O₂) to the inspired fraction of O₂ (PaO₂/FiO₂ proportion) improved by ≥25% in patients with acute respiratory distress syndrome under artificial respiratory support. Accordingly, liposomal encapsulation of trans-crocetin enhanced oxygenation in patients with COVID-19-related acute respiratory distress syndrome on mechanical ventilation (Mertes et al., 2021).

To increase the stability of crocetin, Zhou et al. used gum acacia as wall material and spray-drying technology to microencapsulate crocetin; the microencapsulation rate reached 85.03%. The deterioration rate was consistent with the first kinetic model (Hui et al., 2013).

Song et al. prepared crocetin solid dispersion using the solvent method and PVPK30 as the carrier material; the optimal dosage ratio of crocetin and PVPK30 was 1:4. Drug release was significantly higher from the prepared solid dispersion than from the bulk drug. Based on previous experiments, HPMC-K4M and HPMC-K15M (6:4) were used as sustained-release skeleton materials, and MCC was used as the filler to prepare crocetin solid dispersion sustained-release tablets. The drug content of the prepared sustained-release tablets was 98.02%. The release rates at 2, 6, 12, and 24 h were 8, 22, 27, and 34%, respectively, indicating good sustained-release effects in vitro. Finally, the pharmacokinetics of crocetin solid dispersion sustained-release tablets were assessed in beagle dogs. For the bulk drug, solid dispersion tablet, and sustained-release tablets, the AUC_0-24 values were 33.74, 39.64, and 86.06 μg·h/ml, the C_max values were 3.79, 10.95, and 11.05 μg·ml⁻¹, T_max values were 2, 0.5, and 4 h, respectively. The average bioavailability of sustained-release tablets was 255.07 and 217.10% when compared with bulk drugs and solid dispersions, respectively (Zhong 2014).