Safety Evaluation of Crocin (a constituent of saffron) Tablets in Healthy Volunteers.

1Saffron (Crocus sativus, L.) has been used traditionally as a coloring or flavoring agent, as well as a herbal remedy. It contains four major bioactive constituents which are crocin (color), crocetin (color), picrocrocin (taste) and safranal (aroma).

2Crocin is a carotenoid of constituent of saffron has also shown various pharmacological activities such as antioxidant, anticancer, memory improvement, antidepressant, cerebral, kidney, heart, skeletal muscle) anti-ischemia, hypotensive, aphrodisiac, genoprotective and antidote activities. Crocin also inhibit morphine withdrawal syndrome and morphine-induced reinstatement of place preference in mice.

3The acute and sub-acute toxicity of crocin was evaluated in mice and rats. At pharmacological doses, crocin did not exhibit marked damages to all major organs of the body. With high doses (3 g/kg, IP or orally) after 24 and 48 hr no mortality was seen by crocin in mice. According to Loomis and Hayes classification, chemical substances with LD50 values within the range of 1–5 g/kg are considered as practically low-toxic.

4Since, there is insufficient data on toxicological evaluation of crocin in human for giving safety assurance in developing this constituent of saffron as a medicine, in this study crocin was evaluated for short-term safety and tolerability in healthy adult volunteers.

5Study design

6This study was a double-blind, randomized, placebo-controlled study consisting of one month treatment. The study was approved by the Ethics Committee of MUMS (Mashhad University of Medical Sciences). All subjects were asked to submit a signed consent form before participating in the study. This study was carried out at Imam Reza Hospital in association with Pharmaceutical Research Center, Department of Pharmacology and Toxicology of Mashhad University of Medical Science from May 2009 to January 2010.

7Forty two volunteers who fulfilled the following inclusion and exclusion criteria were enrolled in the study. All participants provided written informed consent prior to the start of the study. This form consisted of demographic data, previous medical history, drug history and familial history. The volunteers were randomized into 2 groups of 22 each who received 20 mg crocin tablets or placebo for 1 month. Before and after treatment clinical examination and laboratory tests (hematology, blood biochemistry and hormone and urine analysis) were carried out. During clinical trial the volunteers were evaluated for adverse drug reactions in both groups.

8The dose of crocin (20 mg) was selected based on acute and sub-acute toxicity of crocin in mice and rats as well as a recent study on the oral administration of crocetin (15 mg, 8 days) that attenuated physical fatigue in men compared with placebo.

9Inclusion & exclusion criteria

10Healthy adult volunteers who were willing to give voluntary written informed consent were included in this study. The volunteers were proven to be healthy through clinical examination by the physician along with hematological and biochemical investigations.

11Volunteers were excluded from study if any of the following criteria occurred at the time of study: 1- History of allergy to saffron 2- History of blood disease, for example: Iron deficiently, anemia, hemophilia, 3- History of cardiovascular disease including vascular or congestive cardiac disease, hypertension, orthostatic hypotension, 4- History of renal disorder or electrolytes disorder. 5- History of endocrine disease such as thyroid disorder, diabetic mellitus, dyslipidemia, 6- History of gynecologic or any menstrual disturbance, 7- Pregnant or lactating women 8- Addiction to smoking or any other substance. 9- Volunteers taking any drugs.

12Blood sampling and laboratory investigations

13Five ml blood sample was taken from each volunteer. 1.5 ml was used for hematologic tests and blood sample of 3.5 ml were collected in sterile tubes, centrifuged at 2500 rpm for 10 min, and immediately cooled to −20°C for 30 min, and then stored at −70°C until the time of analysis.

14The following laboratory tests were determined in both groups before and after treatment:

15Hematologic tests consisted of complete blood count with differentiation (CBC diff), serum iron plasma lipids (total cholesterol (HDL-c, LDL-c), triglyceride), liver function tests (serum glutamic oxaloacetic transaminase (SGOT), serum glutamic pyruvic transaminase (SGPT), serum bilirubin, total and direct, albumin), kidney function tests (urea, creatinine, urine analysis) electrolytes (calcium, phosphorus, sodium, potassium), coagulation tests (PTT, PT, INR), endocrine evaluation tests (fasting blood glucose, T4, TSH, cortisol, testestron), enzymes (alkaline phosphatase, lactate dehydrogenase, creatinine phosphokinase, amylase, lipase), inflammation biomarkers (hs-CRP, ESR ) and uric acid.

16Crocin extract and purification

17Stigmas of C. sativus L. from Novin Saffron (collected from Ghaen, Khorasan province, Northeast of Iran) was obtained and analyzed in accordance with the ISO/TS 3632-2. Crocin was extracted and purified as defined by Hadizadeh and colleagues) ).

18Statistical analysis

19Data (the diference between after-before treatment) are expressed as mean±SD. Statistical analysis was done using two independent sample test with SPSS 11.5 software. The P-values less than 0.05 were considered to be statistically significant.

20Adverse effects

21There were two drop outs during study period in crocin group. No major adverse events were reported during the trial. However, three volunteers showed adverse effects in crocin group. One volunteer claimed that had a burning symptom on his kidney. He had a history of kidney stone. Another volunteer demonstrated somnolence and urinary frequency and had occasional a burning sensation in his body for twenty min 30 min after receiving crocin tablet. Also, one woman volunteer had heavy and prolonged menstrual bleeding during her menstrual period. She had an early menstrual period. These symptoms were omitted without any need for discontinuation of treatment.

22Characteristics of the study population

23Demographic data including age, sex, weight, and body mass index were reported in Table 1. All volunteers have normal range of laboratory tests before entering the study.

24Effect of crocin on blood cells and inflammatory biomarkers in healthy adult volunteers

25The difference in RBC count, WBC, and platelet and their indices as well as inflammatory biomarkers after 1 month treatment were compared between 2 groups. There was a significant reduction of MXD count in crocin group in comparison with control group but there were no significant differences between 2 groups with respect to other blood cells (Table 2).

26The difference of PT and aPTT after 1 month treatment were compared between 2 groups. There was a significant reduction of aPTT in crocin group in comparison with control group but there were no significant differences between 2 groups with respect to PT (Table 2).

27Effect of crocin on some of important enzymes in healthy adult volunteers

28The difference of enzymes activity (alkaline phosphatase, lactate dehydrogenase, Creatinine phosphokinase, amylase, lipase) after 1 month treatment were compared between 2 groups. There was a significant reduction of amylase activity in crocin group in comparison with control group but there were no significant differences between 2 groups with respect to other enzymes activity (Table 3).

29Effect of crocin on kidney function tests and serum electrolytes in healthy adult volunteers

30The difference of kidney function tests (BUN, serum Creatinine, urine analysis) and some important serum electrolytes (calcium and phosphorus) after 1 month treatment were compared between 2 groups. There were no significant differences between 2 groups with respect to these parameters (Table 4).

31Effect of crocin on liver function tests in healthy adult volunteers

32The difference of liver function tests (SGOT, SGPT, serum bilirubin-total and direct, Albumin) after 1 month treatment was compared between 2 groups. There were no significant differences between 2 groups with respect to these parameters (Table 5).

33Effect of crocin on some of endocrine laboratory tests in healthy adult volunteers

34The difference of some of endocrine laboratory tests (FBS, T4, TSH, cortisol, and testestron) after 1 mo treatment was compared between 2 groups. There were no significant differences between 2 groups with respect to these parameters (Table 6).

35Effect of crocin on serum lipid profile in healthy adult volunteers

36The difference of serum lipid profile (total cholesterol (HDL-c, LDL-c), triglyceride) after 1 month treatment was compared between 2 groups. There were no significant differences between 2 groups with respect to these parameters (Table 7).

37The result of present study showed that crocin tablet (20 mg/day, one month) could decrease the amylase, PTT and MXD in the healthy adult volunteers.

38Few studies have evaluated safety of saffron or its constituents in human. In one study, saffron (Crocus sativus) stigma tablets were evaluated for short-term safety and tolerability in healthy adult volunteers. In this study, volunteers had 200 mg and 400 mg saffron tablets for seven days. Saffron tablets changed some hematological and biochemical parameters. There are also some clinical trials about saffron for assessment of its activity such as antidepressant, improving male erectile dysfunction and anti-Alzheimer. However, it seems there is no safety evaluation about the constituents of saffron such as crocin, crocetin or safranal. In one double-blind, placebo-controlled, 3-way crossover study, the oral administration of crocetin (15 mg, 8 days) attenuated physical fatigue in men compared with placebo.

39In this study, crocin decreased MXD% and level (monocyte, eosinophil and basophil) without any effects on total white blood cells count as well as neutrophil and lymphocyte. Kianbakht and Ghazavi showed that saffron decreased the percentage of basophils but increased the percentage of monocytes compared with placebo after 3 weeks. However, these parameters returned to the baseline levels after 6 weeks. In animal study, the aqueous extract of saffron stigma did not change WBC after 14 days treatment in rats. In a recent study, the hematological parameters such as WBC, RBC, Hg and Ht did not alter by crocin (15-180 mg/kg) in rats.

40The administration of 20 mg/d crocin ay for one month decreased activated partial thromboplastine time (aPTT). In one placebo‐controlled clinical study was found the administration of 200 and 400 mg saffron tablets to healthy adult volunteers (n = 10 in each of the three groups) for 7 days significantly reduced the platelet count (only a dose of 200 mg/day). Saffron tablet at a dose of 200 mg also decreased platelets, INR and bleeding time. However, crocin prolonged blood coagulation time of mice and markedly inhibited, dose-dependently, thrombin- and ADP-induced blood platelet aggregation of rabbits in vivo. Crocetin inhibited platelet aggregation induced by ADP and collagen, without effect on platelet aggregation induced by arachidonic acid. Thus, further study in a larger sample size and longer time period is needed to clarify this discrepancy.

41A reduction in amylase level was considered in crocin group. Gardenia jasminoides whose main components contain geniposide and crocin reduced pancreatic edema, neutrophil infiltration, serum amylase and lipase levels, serum cytokine levels, and mRNA expression by cerulein-induced acute pancreatitis.

42Japanese researchers showed that crocins (orally) were hydrolyzed to crocetin before or during intestinal absorption. In another study, Xi et al, showed that the orally administrated crocin (single or repeated doses 6 days) is not absorbed via the intestinal tract of rats and is mainly excreated through the colon. The low concentrations of crocetin (by hydrolysis of crocin) were also detected in plasma. In mice, the oral administration of crocin up to 3 g/kg did not induce any mortality after 48 hr. Thus it seems that most dosage of crocin excretes via colon and a small amout converts to crocetin in animals. However, pharmacokinetic effect of crocin including its oral absorbtion in human needs to be studied.

43Crcoin selectively inhibited the activity of pancreatic lipase in the intestine and reduced serum triglyceride, total cholesterol, low density lipoprotein (LDL) cholesterol and very low density lipoprotein (VLDL) cholesterol level in the daily oral dose range of 25 to 100 mg/kg in rats. Thus, the oral administration of crocin will be suitable as a hypolipidemic medicine.

44This clinical safety evaluation showed a relatively safe and normal profile for crocin in healthy volunteers at the given doses within the trial period (20 mg/day, one month). However, further investigations need to be done with higher doses, longer period of study and larger number of participants.