RESEARCH ARTICLE
Evaluation of 90 day repeated dose oral toxicity and reproductive/developmental toxicity of 3’-hydroxypterostilbene in experimental animals Muhammed Majeed1, Sarang Bani2, Sankaran Natarajan3, Anjali Pandey2, Naveed S4*
a1111111111 a1111111111 a1111111111 a1111111111 a1111111111
1 Sami Labs Limited, Bangalore, Karnataka, India, 2 Biological Research Department, Sami Labs Limited, Bangalore, Karnataka, India, 3 Research and Development Center, Sami Labs Limited, Bangalore, Karnataka, India, 4 ClinWorld, ClinWorld Private Limited, Bangalore, Karnataka, India * [email protected]
Abstract OPEN ACCESS Citation: Majeed M, Bani S, Natarajan S, Pandey A, S N (2017) Evaluation of 90 day repeated dose oral toxicity and reproductive/developmental toxicity of 3’-hydroxypterostilbene in experimental animals. PLoS ONE 12(3): e0172770. doi:10.1371/journal. pone.0172770 Editor: David L. McCormick, IIT Research Institute, UNITED STATES Received: October 21, 2016 Accepted: February 9, 2017 Published: March 3, 2017 Copyright: © 2017 Majeed et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
3’-Hydroxypterostilbene (3’-HPT) is one of the active constituents of Sphaerophysa salsula and Pterocarpus marsupium. Despite many proposed therapeutic applications, the safety profile of 3’-HPT has not been established. The present work investigated 90 day repeated oral dose and reproductive (developmental) toxicity of 3’-HPT as a test substance in rats as per OECD guidelines. 90 day toxicity was conducted in sixty Sprague Dawley rats of each sex (120 rats), grouped into six dosage groups of 0 (control), 0 (control recovery), 20 (low dose), 80 (mid dose), 200 (high dose) and 200 (high dose recovery) mg/kg bwt/day (body weight/day) respectively. For the reproductive toxicity study forty Wistar rats of each sex (80 rats) divided into four dosage groups received 0 (vehicle control), 20 (low dose), 100 (mid dose) and 200 (high dose) mg/kg bwt/day of 3’-HPT respectively for a period of two weeks while pre-mating, mating, on the day before sacrifice, in females during pregnancy and four days of lactation period. Results showed no significant differences in body weight, food intake, absolute organ weight, haematology, with no adverse effects (toxicity) on biochemical values nor any abnormal clinical signs or behavioural changes were observed in any of the control/treatment groups, including reproductive and developmental parameters, gross and histopathological changes. In conclusion, the results suggested a No-ObservedAdverse-Effect-Level (NOAEL) of 200 mg/kg bwt/day in rats after oral administration, implying 3’-HPT did not exhibit any toxicity under the study conditions employed.
Data Availability Statement: All relevant data are within the paper. Funding: This work was sponsored by Sami Labs Limited. There was no external funding involved. The funder provided support in the form of salaries for authors of both Sami Labs Limited and ClinWorld (P) Limited [MM, SB, AP, SN and NVS], but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of all authors affiliated to Sami Labs
Introduction 3’-Hydroxypterostilbene (3’-HPT) is a naturally occurring stilbenoid molecule present in the plants Sphaerophysa salsula [1], Pterocarpus marsupium and also in Honey Bee propolis (bee glue) [2]. Sphaerophysa salsula (Pall.) DC (Leguminosae) is a shrub widely distributed in the Central-Asia and in the northwest of China. It is used as a folk medicine for the treatment of hypertension in China [1]. 3’-HPT is an analogue of another natural stilbenoid pterostilbene
PLOS ONE | DOI:10.1371/journal.pone.0172770 March 3, 2017
1 / 14
3’-hydroxypterostilbene: 90 day oral, reproductive/developmental toxicity
or ClinWorld are articulated in the ’author contributions’ section. Competing interests: The authors have declared that no competing interests exist. This does not alter our adherence to PLOS ONE policies on sharing data and materials.
Fig 1. Stilbenoid analogues of resveratrol. doi:10.1371/journal.pone.0172770.g001
which belongs to a class of compounds known as phytoalexins. Since pterostilbene is widely recognized for its anti-cancer and anti-diabetic properties, it is important to evaluate the toxicological role of its metabolites, as 3’-Hydroxypterostilbene has been found be one of the urinary metabolites of pterostilbene in mice [3]. Stilbene compounds like (trans-) resveratrol and piceatannol are widely distributed in nature. In view of their extensive range of biological activities such as cardioprotection and cancer chemopreventive properties, they have become of special interest to biologists and chemists. The trans-resveratrol-like natural stilbenoids have shown remarkable anti-cancer activity higher than resveratrol (Fig 1). 3’-HPT for example, is reported to be 50 to 97 times more potent than trans-resveratrol in eliciting significant apoptotic action on tumour cell lines and is markedly more active than pterostilbene in inducing apoptosis in sensitive and multi drug-resistant (MDR) leukemia cells [4]. Based on structure activity relationships, the importance of 3,5-dimethoxy motif in conferring pro-apoptic activity on leukemia cells has been recognized [5]. Pterostilbene and 3’-HPT possess the necessary 3,5-dimethoxy motif. Among these, 3’-HPT is an interesting natural compound which may be useful in treating different types of haematological malignancies as it is reported to possess a ‘privileged’ structure [4] motif of 3,5-dimethoxy group in addition to incorporating a piceatannol skeleton. 3’-HPT is a trans-stilbene molecule containing two methoxy groups in one ring and two hydroxy groups in the other ring. Recent research also revealed that 3’-HPT enhances the SIRT1 polypeptide activity and inhibits adipogenesis more effectively than resveratrol or its natural methoxylated derivative pterostilbene [6]. The antioxidant activity of 3’HPT is approximately 30 times more potent than resveratrol, 70 times more than vitamin E, 80 times more than vitamin C and 90 times than BHT (butylated hydroxytoluene, the commonly used synthetic antioxidant) [7]. An important factor that hampers the utility of resveratrol as a dietary supplement is its questionable stability on storage [8]. In evaluating the structure-activity correlation, 3’-HPT possesses the skeleton structure of piceatannol, a known and active metabolite of resveratrol. The dimethoxy-substitution seems to confer relative stability on the molecule [9, 10]. 3’-HPT has been reported to be an excellent anti-proliferative and anti-apoptotic compound [5] and less toxic on normal haemopoietic stem cells than on leukemia and lymphoma cells [4]. In spite of the beneficial pharmacological and health promoting properties of 3’-HPT, there is a lacuna in information on its safety (toxicity) profile. Hence, the current study targeted at a
PLOS ONE | DOI:10.1371/journal.pone.0172770 March 3, 2017
2 / 14
3’-hydroxypterostilbene: 90 day oral, reproductive/developmental toxicity
90 day repeated dose oral toxicity as well as a reproductive/developmental toxicity of 3’- HPT was performed.
Materials and methods Chemicals and analysis of 3’-HPT The chemicals and solvents used throughout the study were of analytical grade. The test item 3’- HPT was provided by Sami Labs Ltd., (Bengaluru, Karnataka, India). The 3’- HPT as provided was obtained through the steps disclosed in the US patent no. US 9458075 B1 [11]. The 3’-HPT as obtained had the following specifications and structural analysis was performed using IR spectrum, proton NMR, carbon NMR and mass spectrum. Analysis using HPLC indicated that the compound obtained was 98.31% w/w pure and was off white in color. Microbiological profile revealed negative for total yeast, E. coli, Salmonella spp., Staphylococcus aureus, Pseudomonas aeruginosa and Enterobacteriaceae when tested. Obtained compound was stable at 25 ± 2˚C and 60 ± 5% RH conditions when tested for a period of three years. Compound was also found stable when tested at accelerated conditions 40 ± 2˚C and 75 ± 5% RH for a period of 6 months. Its molecular formula, the molecular weight being C16H16O4 and 272.30 respectively, having the CA index name: 1,2-Benzenediol, 4-[(1E)-2-(3,5-dimethoxyphenyl)ethenyl]- (9CI).
Animals For reproductive/developmental toxicity study—young healthy adult Wistar rats aged 12–13 weeks at the time of mating as sourced from Dabur Research Foundation (DRF) animal facility and for 90 day repeated dose oral toxicity study- Sprague Dawley (SD) rats aged 6–8 weeks sourced from Bioneeds animal facility were used. These were housed in a controlled environment of temperature (22 ± 3˚C) and relative humidity (RH) (50–60%), with photo periods of 12/12 h light-dark cycles. Provided with pelleted diet (Golden Feeds, New Delhi, India), filtered water ad libitum. Housing in cages: For reproductive/developmental toxicity study, five animals of same sex per cage before mating, two animals per cage of each sex during mating and pregnant females were individually housed with soft nesting material. For 90 day repeated dose oral toxicity study, two animals of same sex per cage were housed during acclimatization and study period.
Ethics Both 90 day repeated dose oral toxicity and reproductive/developmental toxicity studies were performed in strict accordance with the recommendation of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) guidelines for laboratory animal facility, and as per OECD guideline 408, 421 respectively [12, 13]. The individual protocol was approved by the Institutional Animal Ethics Committee (IAEC) of Bioneeds (Registration No. 969/bc/06/CPCSEA) and Dabur Research Foundation (DRF) (Registration No.64/PO/br/ s/99/CPCSEA) respectively. The animals were euthanized using carbon dioxide asphyxiation, and all efforts were made to minimize suffering.
Dosing The test item 3’- HPT was freshly prepared and daily administered orally after suspending it in corn oil (mg/kg/bwt).
PLOS ONE | DOI:10.1371/journal.pone.0172770 March 3, 2017
3 / 14
3’-hydroxypterostilbene: 90 day oral, reproductive/developmental toxicity
Table 1. Dosage groups for repeated dose 90 day oral toxicity study in SD rats. Groups
Dose (mg/kg/d)
Concentration (mg/ml)
No. of Rats/group, Sex (n) Male
Female
NG1
Vehicle Control (VC) (Corn oil)
0
NA
10
10
NG2
Control Recovery (CR) (Corn oil)
0
NA
10
10
NG3
Low dose (LD)
20
2
10
10
NG4
Mid dose (MD)
80
8
10
10
NG5
High dose (HD)
200
20
10
10
NG6
High dose Recovery (HDR)
200
20
10
10
NA- Not Applicable; n = number. doi:10.1371/journal.pone.0172770.t001
90 day repeated dose oral toxicity study Experimental design. The dose formulations, vehicle were administered to the treatment and recovery group animals (Table 1). Dose formulations, vehicle were administered by oral gavage once daily, to the respective groups, at the same time each day (varying by ± 2 h) for a period of 90 days with the actual volume calculated based on the recent body weight recorded at weekly intervals. There was no administration of test item formulations / vehicle during the 28-day recovery period in order to permit evaluation of the persistence, reversibility or delayed occurrence of toxic effects. Clinical observations. All treated, control rats were observed twice (morning and afternoon) daily for clinical signs and mortality for a total period of 90 days. The body weight of each rat was recorded prior to treatment on day 1, weekly thereafter and at end of the 90 day study. Group mean body weights were calculated. The food consumption of all rats in each cage was recorded from day of commencement of treatment and average weekly consumption was calculated. During post-treatment period, food consumption was recorded weekly for recovery group rats. Neurobehavioural assessment. The neurological examination was carried out on all the animals in twelfth week of the study. The neurobehavioural assessment (functional examination) of sensory reactivity to different types of auditory, visual and proprioceptive stimuli (landing foot splay, righting reflex, and gait), assessment of grip strength and motor activity assessment were recorded (Table 2). At the end of treatment all surviving animal were fasted overnight with water given ad libitum. Ophthalmological examination. Ophthalmological examination was carried out at the beginning and at termination of the study for all the groups. Haemotology and clinical chemistry. Blood samples were collected in EDTA tubes for determination of haematological parameters (Table 3) and heparin tubes for clinical chemistry (Table 4). Necropsy/Gross pathology and organ weights. On completion of 90 days and 118 days in case of treatment and recovery periods respectively, all surviving rats were fasted overnight, subjected to complete necropsy, weighed before exsanguination, and gross pathological examination was performed. The organs thus collected (Table 5) from all animals were trimmed of adherent tissue, fat as appropriate and weighed wet as soon as possible to avoid drying. Histopathology. Histopathological examination was performed on tissues of all animals from the control and the high dosage group, sacrificed at scheduled termination. The tissues / organs (Mesenteric lymph node, thymus, trachea, lungs, heart, thyroid, stomach, small intestine, large intestine (colon), liver, spleen, kidneys, adrenals, epididymides / uterus, testes / ovaries and
PLOS ONE | DOI:10.1371/journal.pone.0172770 March 3, 2017
4 / 14
3’-hydroxypterostilbene: 90 day oral, reproductive/developmental toxicity
Table 2. Effect of 90 day oral administration of 3’-HPT on functional observation battery (neurobehavioural assessment) tests in male and female SD rats. Groups
Functional observation battery Dose (mg/kg/d) Visual Response
Auditory Response
Neurobehavioural response
Response to Proprioceptive Stimuli Gait
Righting Reflex
Landing Foot splay (cm) Male
Female a b c d
Locomotor Activity
Grasping strength
NG1 (VC)
0
0
0
0
3.4 ± 0.9 3.8 ± 0.9 0 0 0 0
0
0
NG2 (CR)
0
0
0
0
3.9 ± 0.7 4.3 ± 1.2 0 0 0 0
0
0
NG3 (LD)
20
0
0
0
3.6 ± 1.0 3.6 ± 0.9 0 0 0 0
0
0
NG4 (MD)
80
0
0
0
3.5 ± 0.8 4.0 ± 0.8 0 0 0 0
0
0
NG5 (HD)
200
0
0
0
4.1 ± 0.9 4.1 ± 0.9 0 0 0 0
0
0
NG6 (HDR)
200
0
0
0
3.5 ± 0.6 3.9 ± 1.0 0 0 0 0
0
0
n = 10; 0: Normal; a: on back; b: body tilted; c: head tilted; d: dropped. Values are in Mean ± SEM. doi:10.1371/journal.pone.0172770.t002
Table 3. Effect of 90 day oral administration of 3’-HPT on haematology data of male and female SD rats. Parameters
Groups / Dose (mg/ kg/d)
NG1 (VC) / (0)
NG2 (CR) / (0)
NG3 (LD) / (20)
NG4 (MD) / (80)
NG5 (HD) / (200)
NG6 (HDR) / (200)
M
14.9 ± 2.8
15.6 ± 3.2
14.4 ± 3.1
14.9 ± 2.3
15.5 ± 2.7
17.7 ± 1.5
F
10.0 ± 2.9
9.5 ± 2.0
8.5 ± 1.3
8.7 ± 2.5
11.2 ± 2.9
9.1 ± 2.8
Total Erythrocyte count (106 cells/μl)
M
7.5 ± 0.3
7.5 ± 0.4
7.5 ± 0.4
7.3 ± 0.8
7.7 ± 0.5
8.0 ± 0.6
F
6.6 ± 0.4
7.0 ± 0.8
7.0 ± 0.6
6.6 ± 0.6
6.8 ± 0.7
6.7 ± 0.6
Haemoglobin (g/dl)
M
13.2 ± 0.6
13.1 ± 0.6
13.2 ± 0.8
13.0 ± 0.5
12.9 ± 0.7
13.7 ± 1.0
F
12.4 ± 0.8
13.0 ± 1.2
13.1 ± 1.2
12.5 ± 1.4
12.8 ± 1.2
12.6 ± 0.8
Haematocrit (%)
M
42.9 ± 1.9
42.5 ± 2.2
42.9 ± 2.3
41.1 ± 4.9
42.7 ± 2.7
44.5 ± 3.2
F
38.1 ± 2.2
39.8 ± 3.8
39.9 ± 3.2
38.0 ± 4.1
38.8 ± 3.6
38.4 ± 2.2
M
57.4 ± 2.1
56.4 ± 1.9
57.0 ± 1.6
55.9 ± 0.9
55.6 ± 1.9
55.8 ± 2.4
F
57.6 ± 1.3
56.6 ± 1.6
57.0 ± 2.0
57.1 ± 2.3
57 ± 2.1
57.2 ± 3.4
Mean corpuscular haemoglobin (pg)
M
17.8 ± 0.6
17.4 ± 0.5
17.6 ± 0.8
17.9 ± 2.9
16.9 ± 0.6
17.1 ± 0.8
F
18.8 ± 0.8
18.6 ± 0.9
18.8 ± 1.1
18.7 ± 0.9
18.8 ± 0.8
18.8 ± 1.2
Mean corpuscular haemoglobin concentration (g/dl)
M
30.9 ± 0.4
30.9 ± 0.4
30.8 ± 1.1
32.1 ± 5.6
30.3 ± 0.4
30.7 ± 0.9
F
32.6 ± 0.7
32.8 ± 0.2
33.0 ± 1.2
32.8 ± 0.4
32.9 ± 0.6
Platelet Count (103 cells/μl)
M
688.7 ± 123.7
624.0 ± 85.5
633.5 ± 100.1 618.3 ± 169.6 714.4 ± 83.8
F
641.2 ± 81.3
659.6 ± 109.4
621.8 ± 81.3
Blood clotting time (Sec)
M
72.9 ± 16.9
70.3 ± 14.3
66.7 ± 8.3
67.1 ± 18.2
60.7 ± 13.6
67.9 ± 15.7
F
55.8 ± 22.5
62.9 ± 18.8
51.0 ± 11.5
53.5 ± 14.5
57.2 ± 18.2
59.4 ± 11.2
Neutrophils (%)
M
15.6 ± 5.5
20.6 ± 7.0
19.2 ± 5.4
16.1 ± 4.7
17.2 ± 4.8
17.3 ± 5.9
F
20.4 ± 4.9
21.4 ± 5.1
17.6 ± 6.3
20.7 ± 5.8
20.9 ± 6.6
23.3 ± 6.1
Lymphocytes (%)
M
84.2 ± 5.8
81.0 ± 6.3
80.7 ± 5.4
83.7 ± 5.0
82.4 ± 5.2
82.6 ± 5.9
F
79.4 ± 4.7
78.2 ± 5.5
82.0 ± 6.6
79.0 ± 6.2
78.8 ± 6.8
76.6 ± 6.1
Eosinophils (%)
M
0.1 ± 0.3
0.0 ± 0.0
0.0 ± 0.0
0.0 ± 0.0
0.1 ± 0.3
0.1 ± 0.3
F
0.0 ± 0.0
0.1 ± 0.3
0.1 ± 0.3
0.3 ± 0.5
0.0 ± 0.0
0.0 ± 0.0
M
0.1 ± 0.3
0.1 ± 0.3
0.1 ± 0.3
0.2 ± 0.6
0.3 ± 0.7
0.0 ± 0.0
F
0.2 ± 0.6
0.3 ± 0.7
0.3 ± 0.7
0.2 ± 0.6
0.3 ± 0.7
0.1 ± 0.3
Sex Total Leucocyte Count (103 cells/μl)
Mean corpuscular volume (fL)
Monocytes (%)
32.9 ± 0.7 703.8 ± 61.9
563.6 ± 144.6 639.5 ± 96.0 593.8 ± 120.4
n = 10; M, male; F, female. Values are in Mean ± SD. doi:10.1371/journal.pone.0172770.t003
PLOS ONE | DOI:10.1371/journal.pone.0172770 March 3, 2017
5 / 14
3’-hydroxypterostilbene: 90 day oral, reproductive/developmental toxicity
Table 4. Effect of 90 day oral administration of 3’-HPT on biochemical data of male and female SD rats. Parameter
Groups / Dose (mg/ kg/d)
NG1 (VC) / (0)
NG2 (CR) / (0)
NG3 (LD) / (20)
NG4 (MD) / (80)
NG5 (HD) / (200)
NG6 (HDR) / (200)
Sex Glucose (mg/dl)
M
112.70 ± 18.18 109.40 ± 18.90 105.70 ± 15.09 100.39 ± 16.39 101.80 ± 12.84
98.70 ± 12.28
F
107.40 ± 18.65 114.50 ± 14.18 106.90 ± 10.80 105.10 ± 19.93
99.00 ± 13.78
103.10 ± 12.27
M
42.10 ± 6.47
40.20 ± 7.94
37.80 ± 8.99
43.00 ± 9.76
38.30 ± 9.51
41.10 ± 7.71
F
57.50 ± 12.84
58.10 ± 8.52
50.70 ± 6.57
48.00 ± 8.94
47.40 ± 9.49
48.50 ± 7.81
Creatinine (mg/dl)
M
0.86 ± 0.15
0.78 ± 0.04
0.77 ± 0.07
0.82 ± 0.09
0.77 ± 0.12
0.78 ± 0.14
F
0.98 ± 0.06
0.99 ± 0.03
0.98 ± 0.13
0.97 ± 0.18
0.93 ± 0.13
0.96 ± 0.08
Total Protein (mg/dl)
M
5.62 ± 0.88
5.62 ± 0.38
5.55 ± 0.25
5.28 ± 0.44
5.64 ± 0.52
5.69 ± 0.37
F
6.85 ± 0.35
6.99 ± 0.43
6.78 ± 0.43
7.01 ± 0.52
7.15 ± 0.39
7.00 ± 0.49
M
3.51 ± 0.22
3.31 ± 0.17
3.28 ± 0.25
3.28 ± 0.19
3.38 ± 0.17
3.47 ± 0.28
F
3.90 ± 0.27
3.93 ± 0.47
3.89 ± 0.23
4.12 ± 0.27
4.03 ± 0.18
3.96 ± 0.36
Triglycerides (mg/dl)
M
60.05 ± 13.71
68.70 ± 14.00
65.00 ± 14.41
69.40 ± 20.66
50.40 ± 12.37
52.00 ± 12.34
F
69.30 ± 14.85
71.70 ± 21.15
55.90 ± 20.37
59.40 ± 13.33
55.90 ± 21.12
52.50 ± 16.22
Urea Nitrogen (mg/dl)
M
13.42 ± 2.42
14.64 ± 1.65
14.62 ± 2.76
15.35 ± 3.24
16.03 ± 1.90
16.01 ± 1.89
F
18.33 ± 4.23
19.91 ± 5.21
15.75 ± 2.51
15.60 ± 2.29
17.54 ± 2.50
19.27 ± 3.79
Total bilirubin (mg/dl)
M
0.33 ± 0.08
0.36 ± 0.07
0.33 ± 0.07
0.30 ± 0.08
0.32 ± 0.09
0.29 ± 0.07
F
0.37 ± 0.13
0.39 ± 0.12
0.32 ± 0.04
0.35 ± 0.07
0.33 ± 0.11
0.30 ± 0.05
Alanine aminotransferase (U/L)
M
77.50 ± 20.46
67.60 ± 19.08
60.30 ± 14.65
61.20 ± 12.78
54.20*± 18.08
53.30*± 10.63
F
73.00 ± 16.15
78.00 ± 14.73
74.40 ± 18.07
65.30 ± 10.06
56.10*± 10.65
57.40*± 7.83
Aspartate aminotransferase (U/L)
M
166.60 ± 17.52 168.40 ± 15.09 163.90 ± 25.05 149.00 ± 23.49
139.30*± 9.17
140.00*± 12.82
F
172.70 ± 20.34 169.40 ± 16.71 171.60 ± 17.76 167.30 ± 19.45
147.50*± 20.64
143.20*± 21.47 27.43 ± 5.01
Total Cholesterol (mg/dl)
Albumin (mg/dl)
High density lipoprotein (mg/ dl)
M
22.67 ± 1.95
22.56 ± 3.64
21.00 ± 5.49
24.07 ± 3.50
21.37 ± 4.80
F
27.85 ± 6.20
26.63 ± 4.87
26.60 ± 4.58
27.31 ± 4.80
30.82 ± 6.10
Alkaline phosphatase (U/L)
M
143.10 ± 18.79 152.70 ± 22.07 151.80 ± 25.68 170.66 ± 23.42 144.70 ± 33.46
160.15 ± 21.45
F
144.44 ± 19.94 139.84 ± 27.01 145.68 ± 29.32 154.78 ± 31.17 163.13 ± 19.77
159.60 ± 22.95
Low density lipoprotein (mg/ dl)
M
17.26 ± 2.37
13.86 ± 2.29
14.95 ± 3.80
16.47 ± 2.39
19.94 ± 4.67
17.69 ± 3.54
F
16.36 ± 3.03
14.95 ± 7.12
15.60 ± 3.30
15.00 ± 3.40
12.14 ± 2.10
14.55 ± 3.16
Calcium (mg/dl)
M
9.26 ± 0.77
8.77 ± 1.12
9.09 ± 0.90
9.18 ± 0.86
9.09 ± 0.90
8.87 ± 0.96
F
9.14 ± 0.71
8.91 ± 1.09
17.51 ± 26.89
8.92 ± 0.97
8.79 ± 0.84
9.31 ± 1.12
M
3.97 ± 0.62
3.75 ± 0.56
3.67 ± 0.57
4.94 ± 0.90
4.66 ± 1.40
4.55 ± 1.64
F
4.19 ± 0.59
4.14 ± 0.32
4.31 ± 0.51
4.20 ± 0.69
4.76 ± 0.58
4.72 ± 0.93
Sodium (mmol/L)
M
141.40 ± 4.38
140.60 ± 4.67
140.20 ± 3.19
139.20 ± 7.11
140.80 ± 1.93
140.60 ± 4.72
F
141.30 ± 2.67
139.80 ± 4.87
140.90 ± 3.73
140.80 ± 3.74
141.50 ± 3.63
141.60 ± 2.59
Potassium (mg/dl)
M
4.28 ± 0.57
4.18 ± 0.70
4.14 ± 0.67
4.11 ± 0.28
3.94 ± 0.39
4.10 ± 0.34
F
4.19 ± 0.34
4.11 ± 0.52
4.18 ± 0.47
4.16 ± 0.68
4.11 ± 0.28
4.06 ± 0.43
Phosphorous (mg/dl)
31.59 ± 4.69
n = 10; M, male; F, female. Values are in Mean ± SD; * Statistically significant (P
Evaluation of 90 day repeated dose oral toxicity and reproductive/developmental toxicity of 3’-hydroxypterostilbene in experimental animals Muhammed Majeed1, Sarang Bani2, Sankaran Natarajan3, Anjali Pandey2, Naveed S4*
a1111111111 a1111111111 a1111111111 a1111111111 a1111111111
1 Sami Labs Limited, Bangalore, Karnataka, India, 2 Biological Research Department, Sami Labs Limited, Bangalore, Karnataka, India, 3 Research and Development Center, Sami Labs Limited, Bangalore, Karnataka, India, 4 ClinWorld, ClinWorld Private Limited, Bangalore, Karnataka, India * [email protected]
Abstract OPEN ACCESS Citation: Majeed M, Bani S, Natarajan S, Pandey A, S N (2017) Evaluation of 90 day repeated dose oral toxicity and reproductive/developmental toxicity of 3’-hydroxypterostilbene in experimental animals. PLoS ONE 12(3): e0172770. doi:10.1371/journal. pone.0172770 Editor: David L. McCormick, IIT Research Institute, UNITED STATES Received: October 21, 2016 Accepted: February 9, 2017 Published: March 3, 2017 Copyright: © 2017 Majeed et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
3’-Hydroxypterostilbene (3’-HPT) is one of the active constituents of Sphaerophysa salsula and Pterocarpus marsupium. Despite many proposed therapeutic applications, the safety profile of 3’-HPT has not been established. The present work investigated 90 day repeated oral dose and reproductive (developmental) toxicity of 3’-HPT as a test substance in rats as per OECD guidelines. 90 day toxicity was conducted in sixty Sprague Dawley rats of each sex (120 rats), grouped into six dosage groups of 0 (control), 0 (control recovery), 20 (low dose), 80 (mid dose), 200 (high dose) and 200 (high dose recovery) mg/kg bwt/day (body weight/day) respectively. For the reproductive toxicity study forty Wistar rats of each sex (80 rats) divided into four dosage groups received 0 (vehicle control), 20 (low dose), 100 (mid dose) and 200 (high dose) mg/kg bwt/day of 3’-HPT respectively for a period of two weeks while pre-mating, mating, on the day before sacrifice, in females during pregnancy and four days of lactation period. Results showed no significant differences in body weight, food intake, absolute organ weight, haematology, with no adverse effects (toxicity) on biochemical values nor any abnormal clinical signs or behavioural changes were observed in any of the control/treatment groups, including reproductive and developmental parameters, gross and histopathological changes. In conclusion, the results suggested a No-ObservedAdverse-Effect-Level (NOAEL) of 200 mg/kg bwt/day in rats after oral administration, implying 3’-HPT did not exhibit any toxicity under the study conditions employed.
Data Availability Statement: All relevant data are within the paper. Funding: This work was sponsored by Sami Labs Limited. There was no external funding involved. The funder provided support in the form of salaries for authors of both Sami Labs Limited and ClinWorld (P) Limited [MM, SB, AP, SN and NVS], but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of all authors affiliated to Sami Labs
Introduction 3’-Hydroxypterostilbene (3’-HPT) is a naturally occurring stilbenoid molecule present in the plants Sphaerophysa salsula [1], Pterocarpus marsupium and also in Honey Bee propolis (bee glue) [2]. Sphaerophysa salsula (Pall.) DC (Leguminosae) is a shrub widely distributed in the Central-Asia and in the northwest of China. It is used as a folk medicine for the treatment of hypertension in China [1]. 3’-HPT is an analogue of another natural stilbenoid pterostilbene
PLOS ONE | DOI:10.1371/journal.pone.0172770 March 3, 2017
1 / 14
3’-hydroxypterostilbene: 90 day oral, reproductive/developmental toxicity
or ClinWorld are articulated in the ’author contributions’ section. Competing interests: The authors have declared that no competing interests exist. This does not alter our adherence to PLOS ONE policies on sharing data and materials.
Fig 1. Stilbenoid analogues of resveratrol. doi:10.1371/journal.pone.0172770.g001
which belongs to a class of compounds known as phytoalexins. Since pterostilbene is widely recognized for its anti-cancer and anti-diabetic properties, it is important to evaluate the toxicological role of its metabolites, as 3’-Hydroxypterostilbene has been found be one of the urinary metabolites of pterostilbene in mice [3]. Stilbene compounds like (trans-) resveratrol and piceatannol are widely distributed in nature. In view of their extensive range of biological activities such as cardioprotection and cancer chemopreventive properties, they have become of special interest to biologists and chemists. The trans-resveratrol-like natural stilbenoids have shown remarkable anti-cancer activity higher than resveratrol (Fig 1). 3’-HPT for example, is reported to be 50 to 97 times more potent than trans-resveratrol in eliciting significant apoptotic action on tumour cell lines and is markedly more active than pterostilbene in inducing apoptosis in sensitive and multi drug-resistant (MDR) leukemia cells [4]. Based on structure activity relationships, the importance of 3,5-dimethoxy motif in conferring pro-apoptic activity on leukemia cells has been recognized [5]. Pterostilbene and 3’-HPT possess the necessary 3,5-dimethoxy motif. Among these, 3’-HPT is an interesting natural compound which may be useful in treating different types of haematological malignancies as it is reported to possess a ‘privileged’ structure [4] motif of 3,5-dimethoxy group in addition to incorporating a piceatannol skeleton. 3’-HPT is a trans-stilbene molecule containing two methoxy groups in one ring and two hydroxy groups in the other ring. Recent research also revealed that 3’-HPT enhances the SIRT1 polypeptide activity and inhibits adipogenesis more effectively than resveratrol or its natural methoxylated derivative pterostilbene [6]. The antioxidant activity of 3’HPT is approximately 30 times more potent than resveratrol, 70 times more than vitamin E, 80 times more than vitamin C and 90 times than BHT (butylated hydroxytoluene, the commonly used synthetic antioxidant) [7]. An important factor that hampers the utility of resveratrol as a dietary supplement is its questionable stability on storage [8]. In evaluating the structure-activity correlation, 3’-HPT possesses the skeleton structure of piceatannol, a known and active metabolite of resveratrol. The dimethoxy-substitution seems to confer relative stability on the molecule [9, 10]. 3’-HPT has been reported to be an excellent anti-proliferative and anti-apoptotic compound [5] and less toxic on normal haemopoietic stem cells than on leukemia and lymphoma cells [4]. In spite of the beneficial pharmacological and health promoting properties of 3’-HPT, there is a lacuna in information on its safety (toxicity) profile. Hence, the current study targeted at a
PLOS ONE | DOI:10.1371/journal.pone.0172770 March 3, 2017
2 / 14
3’-hydroxypterostilbene: 90 day oral, reproductive/developmental toxicity
90 day repeated dose oral toxicity as well as a reproductive/developmental toxicity of 3’- HPT was performed.
Materials and methods Chemicals and analysis of 3’-HPT The chemicals and solvents used throughout the study were of analytical grade. The test item 3’- HPT was provided by Sami Labs Ltd., (Bengaluru, Karnataka, India). The 3’- HPT as provided was obtained through the steps disclosed in the US patent no. US 9458075 B1 [11]. The 3’-HPT as obtained had the following specifications and structural analysis was performed using IR spectrum, proton NMR, carbon NMR and mass spectrum. Analysis using HPLC indicated that the compound obtained was 98.31% w/w pure and was off white in color. Microbiological profile revealed negative for total yeast, E. coli, Salmonella spp., Staphylococcus aureus, Pseudomonas aeruginosa and Enterobacteriaceae when tested. Obtained compound was stable at 25 ± 2˚C and 60 ± 5% RH conditions when tested for a period of three years. Compound was also found stable when tested at accelerated conditions 40 ± 2˚C and 75 ± 5% RH for a period of 6 months. Its molecular formula, the molecular weight being C16H16O4 and 272.30 respectively, having the CA index name: 1,2-Benzenediol, 4-[(1E)-2-(3,5-dimethoxyphenyl)ethenyl]- (9CI).
Animals For reproductive/developmental toxicity study—young healthy adult Wistar rats aged 12–13 weeks at the time of mating as sourced from Dabur Research Foundation (DRF) animal facility and for 90 day repeated dose oral toxicity study- Sprague Dawley (SD) rats aged 6–8 weeks sourced from Bioneeds animal facility were used. These were housed in a controlled environment of temperature (22 ± 3˚C) and relative humidity (RH) (50–60%), with photo periods of 12/12 h light-dark cycles. Provided with pelleted diet (Golden Feeds, New Delhi, India), filtered water ad libitum. Housing in cages: For reproductive/developmental toxicity study, five animals of same sex per cage before mating, two animals per cage of each sex during mating and pregnant females were individually housed with soft nesting material. For 90 day repeated dose oral toxicity study, two animals of same sex per cage were housed during acclimatization and study period.
Ethics Both 90 day repeated dose oral toxicity and reproductive/developmental toxicity studies were performed in strict accordance with the recommendation of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) guidelines for laboratory animal facility, and as per OECD guideline 408, 421 respectively [12, 13]. The individual protocol was approved by the Institutional Animal Ethics Committee (IAEC) of Bioneeds (Registration No. 969/bc/06/CPCSEA) and Dabur Research Foundation (DRF) (Registration No.64/PO/br/ s/99/CPCSEA) respectively. The animals were euthanized using carbon dioxide asphyxiation, and all efforts were made to minimize suffering.
Dosing The test item 3’- HPT was freshly prepared and daily administered orally after suspending it in corn oil (mg/kg/bwt).
PLOS ONE | DOI:10.1371/journal.pone.0172770 March 3, 2017
3 / 14
3’-hydroxypterostilbene: 90 day oral, reproductive/developmental toxicity
Table 1. Dosage groups for repeated dose 90 day oral toxicity study in SD rats. Groups
Dose (mg/kg/d)
Concentration (mg/ml)
No. of Rats/group, Sex (n) Male
Female
NG1
Vehicle Control (VC) (Corn oil)
0
NA
10
10
NG2
Control Recovery (CR) (Corn oil)
0
NA
10
10
NG3
Low dose (LD)
20
2
10
10
NG4
Mid dose (MD)
80
8
10
10
NG5
High dose (HD)
200
20
10
10
NG6
High dose Recovery (HDR)
200
20
10
10
NA- Not Applicable; n = number. doi:10.1371/journal.pone.0172770.t001
90 day repeated dose oral toxicity study Experimental design. The dose formulations, vehicle were administered to the treatment and recovery group animals (Table 1). Dose formulations, vehicle were administered by oral gavage once daily, to the respective groups, at the same time each day (varying by ± 2 h) for a period of 90 days with the actual volume calculated based on the recent body weight recorded at weekly intervals. There was no administration of test item formulations / vehicle during the 28-day recovery period in order to permit evaluation of the persistence, reversibility or delayed occurrence of toxic effects. Clinical observations. All treated, control rats were observed twice (morning and afternoon) daily for clinical signs and mortality for a total period of 90 days. The body weight of each rat was recorded prior to treatment on day 1, weekly thereafter and at end of the 90 day study. Group mean body weights were calculated. The food consumption of all rats in each cage was recorded from day of commencement of treatment and average weekly consumption was calculated. During post-treatment period, food consumption was recorded weekly for recovery group rats. Neurobehavioural assessment. The neurological examination was carried out on all the animals in twelfth week of the study. The neurobehavioural assessment (functional examination) of sensory reactivity to different types of auditory, visual and proprioceptive stimuli (landing foot splay, righting reflex, and gait), assessment of grip strength and motor activity assessment were recorded (Table 2). At the end of treatment all surviving animal were fasted overnight with water given ad libitum. Ophthalmological examination. Ophthalmological examination was carried out at the beginning and at termination of the study for all the groups. Haemotology and clinical chemistry. Blood samples were collected in EDTA tubes for determination of haematological parameters (Table 3) and heparin tubes for clinical chemistry (Table 4). Necropsy/Gross pathology and organ weights. On completion of 90 days and 118 days in case of treatment and recovery periods respectively, all surviving rats were fasted overnight, subjected to complete necropsy, weighed before exsanguination, and gross pathological examination was performed. The organs thus collected (Table 5) from all animals were trimmed of adherent tissue, fat as appropriate and weighed wet as soon as possible to avoid drying. Histopathology. Histopathological examination was performed on tissues of all animals from the control and the high dosage group, sacrificed at scheduled termination. The tissues / organs (Mesenteric lymph node, thymus, trachea, lungs, heart, thyroid, stomach, small intestine, large intestine (colon), liver, spleen, kidneys, adrenals, epididymides / uterus, testes / ovaries and
PLOS ONE | DOI:10.1371/journal.pone.0172770 March 3, 2017
4 / 14
3’-hydroxypterostilbene: 90 day oral, reproductive/developmental toxicity
Table 2. Effect of 90 day oral administration of 3’-HPT on functional observation battery (neurobehavioural assessment) tests in male and female SD rats. Groups
Functional observation battery Dose (mg/kg/d) Visual Response
Auditory Response
Neurobehavioural response
Response to Proprioceptive Stimuli Gait
Righting Reflex
Landing Foot splay (cm) Male
Female a b c d
Locomotor Activity
Grasping strength
NG1 (VC)
0
0
0
0
3.4 ± 0.9 3.8 ± 0.9 0 0 0 0
0
0
NG2 (CR)
0
0
0
0
3.9 ± 0.7 4.3 ± 1.2 0 0 0 0
0
0
NG3 (LD)
20
0
0
0
3.6 ± 1.0 3.6 ± 0.9 0 0 0 0
0
0
NG4 (MD)
80
0
0
0
3.5 ± 0.8 4.0 ± 0.8 0 0 0 0
0
0
NG5 (HD)
200
0
0
0
4.1 ± 0.9 4.1 ± 0.9 0 0 0 0
0
0
NG6 (HDR)
200
0
0
0
3.5 ± 0.6 3.9 ± 1.0 0 0 0 0
0
0
n = 10; 0: Normal; a: on back; b: body tilted; c: head tilted; d: dropped. Values are in Mean ± SEM. doi:10.1371/journal.pone.0172770.t002
Table 3. Effect of 90 day oral administration of 3’-HPT on haematology data of male and female SD rats. Parameters
Groups / Dose (mg/ kg/d)
NG1 (VC) / (0)
NG2 (CR) / (0)
NG3 (LD) / (20)
NG4 (MD) / (80)
NG5 (HD) / (200)
NG6 (HDR) / (200)
M
14.9 ± 2.8
15.6 ± 3.2
14.4 ± 3.1
14.9 ± 2.3
15.5 ± 2.7
17.7 ± 1.5
F
10.0 ± 2.9
9.5 ± 2.0
8.5 ± 1.3
8.7 ± 2.5
11.2 ± 2.9
9.1 ± 2.8
Total Erythrocyte count (106 cells/μl)
M
7.5 ± 0.3
7.5 ± 0.4
7.5 ± 0.4
7.3 ± 0.8
7.7 ± 0.5
8.0 ± 0.6
F
6.6 ± 0.4
7.0 ± 0.8
7.0 ± 0.6
6.6 ± 0.6
6.8 ± 0.7
6.7 ± 0.6
Haemoglobin (g/dl)
M
13.2 ± 0.6
13.1 ± 0.6
13.2 ± 0.8
13.0 ± 0.5
12.9 ± 0.7
13.7 ± 1.0
F
12.4 ± 0.8
13.0 ± 1.2
13.1 ± 1.2
12.5 ± 1.4
12.8 ± 1.2
12.6 ± 0.8
Haematocrit (%)
M
42.9 ± 1.9
42.5 ± 2.2
42.9 ± 2.3
41.1 ± 4.9
42.7 ± 2.7
44.5 ± 3.2
F
38.1 ± 2.2
39.8 ± 3.8
39.9 ± 3.2
38.0 ± 4.1
38.8 ± 3.6
38.4 ± 2.2
M
57.4 ± 2.1
56.4 ± 1.9
57.0 ± 1.6
55.9 ± 0.9
55.6 ± 1.9
55.8 ± 2.4
F
57.6 ± 1.3
56.6 ± 1.6
57.0 ± 2.0
57.1 ± 2.3
57 ± 2.1
57.2 ± 3.4
Mean corpuscular haemoglobin (pg)
M
17.8 ± 0.6
17.4 ± 0.5
17.6 ± 0.8
17.9 ± 2.9
16.9 ± 0.6
17.1 ± 0.8
F
18.8 ± 0.8
18.6 ± 0.9
18.8 ± 1.1
18.7 ± 0.9
18.8 ± 0.8
18.8 ± 1.2
Mean corpuscular haemoglobin concentration (g/dl)
M
30.9 ± 0.4
30.9 ± 0.4
30.8 ± 1.1
32.1 ± 5.6
30.3 ± 0.4
30.7 ± 0.9
F
32.6 ± 0.7
32.8 ± 0.2
33.0 ± 1.2
32.8 ± 0.4
32.9 ± 0.6
Platelet Count (103 cells/μl)
M
688.7 ± 123.7
624.0 ± 85.5
633.5 ± 100.1 618.3 ± 169.6 714.4 ± 83.8
F
641.2 ± 81.3
659.6 ± 109.4
621.8 ± 81.3
Blood clotting time (Sec)
M
72.9 ± 16.9
70.3 ± 14.3
66.7 ± 8.3
67.1 ± 18.2
60.7 ± 13.6
67.9 ± 15.7
F
55.8 ± 22.5
62.9 ± 18.8
51.0 ± 11.5
53.5 ± 14.5
57.2 ± 18.2
59.4 ± 11.2
Neutrophils (%)
M
15.6 ± 5.5
20.6 ± 7.0
19.2 ± 5.4
16.1 ± 4.7
17.2 ± 4.8
17.3 ± 5.9
F
20.4 ± 4.9
21.4 ± 5.1
17.6 ± 6.3
20.7 ± 5.8
20.9 ± 6.6
23.3 ± 6.1
Lymphocytes (%)
M
84.2 ± 5.8
81.0 ± 6.3
80.7 ± 5.4
83.7 ± 5.0
82.4 ± 5.2
82.6 ± 5.9
F
79.4 ± 4.7
78.2 ± 5.5
82.0 ± 6.6
79.0 ± 6.2
78.8 ± 6.8
76.6 ± 6.1
Eosinophils (%)
M
0.1 ± 0.3
0.0 ± 0.0
0.0 ± 0.0
0.0 ± 0.0
0.1 ± 0.3
0.1 ± 0.3
F
0.0 ± 0.0
0.1 ± 0.3
0.1 ± 0.3
0.3 ± 0.5
0.0 ± 0.0
0.0 ± 0.0
M
0.1 ± 0.3
0.1 ± 0.3
0.1 ± 0.3
0.2 ± 0.6
0.3 ± 0.7
0.0 ± 0.0
F
0.2 ± 0.6
0.3 ± 0.7
0.3 ± 0.7
0.2 ± 0.6
0.3 ± 0.7
0.1 ± 0.3
Sex Total Leucocyte Count (103 cells/μl)
Mean corpuscular volume (fL)
Monocytes (%)
32.9 ± 0.7 703.8 ± 61.9
563.6 ± 144.6 639.5 ± 96.0 593.8 ± 120.4
n = 10; M, male; F, female. Values are in Mean ± SD. doi:10.1371/journal.pone.0172770.t003
PLOS ONE | DOI:10.1371/journal.pone.0172770 March 3, 2017
5 / 14
3’-hydroxypterostilbene: 90 day oral, reproductive/developmental toxicity
Table 4. Effect of 90 day oral administration of 3’-HPT on biochemical data of male and female SD rats. Parameter
Groups / Dose (mg/ kg/d)
NG1 (VC) / (0)
NG2 (CR) / (0)
NG3 (LD) / (20)
NG4 (MD) / (80)
NG5 (HD) / (200)
NG6 (HDR) / (200)
Sex Glucose (mg/dl)
M
112.70 ± 18.18 109.40 ± 18.90 105.70 ± 15.09 100.39 ± 16.39 101.80 ± 12.84
98.70 ± 12.28
F
107.40 ± 18.65 114.50 ± 14.18 106.90 ± 10.80 105.10 ± 19.93
99.00 ± 13.78
103.10 ± 12.27
M
42.10 ± 6.47
40.20 ± 7.94
37.80 ± 8.99
43.00 ± 9.76
38.30 ± 9.51
41.10 ± 7.71
F
57.50 ± 12.84
58.10 ± 8.52
50.70 ± 6.57
48.00 ± 8.94
47.40 ± 9.49
48.50 ± 7.81
Creatinine (mg/dl)
M
0.86 ± 0.15
0.78 ± 0.04
0.77 ± 0.07
0.82 ± 0.09
0.77 ± 0.12
0.78 ± 0.14
F
0.98 ± 0.06
0.99 ± 0.03
0.98 ± 0.13
0.97 ± 0.18
0.93 ± 0.13
0.96 ± 0.08
Total Protein (mg/dl)
M
5.62 ± 0.88
5.62 ± 0.38
5.55 ± 0.25
5.28 ± 0.44
5.64 ± 0.52
5.69 ± 0.37
F
6.85 ± 0.35
6.99 ± 0.43
6.78 ± 0.43
7.01 ± 0.52
7.15 ± 0.39
7.00 ± 0.49
M
3.51 ± 0.22
3.31 ± 0.17
3.28 ± 0.25
3.28 ± 0.19
3.38 ± 0.17
3.47 ± 0.28
F
3.90 ± 0.27
3.93 ± 0.47
3.89 ± 0.23
4.12 ± 0.27
4.03 ± 0.18
3.96 ± 0.36
Triglycerides (mg/dl)
M
60.05 ± 13.71
68.70 ± 14.00
65.00 ± 14.41
69.40 ± 20.66
50.40 ± 12.37
52.00 ± 12.34
F
69.30 ± 14.85
71.70 ± 21.15
55.90 ± 20.37
59.40 ± 13.33
55.90 ± 21.12
52.50 ± 16.22
Urea Nitrogen (mg/dl)
M
13.42 ± 2.42
14.64 ± 1.65
14.62 ± 2.76
15.35 ± 3.24
16.03 ± 1.90
16.01 ± 1.89
F
18.33 ± 4.23
19.91 ± 5.21
15.75 ± 2.51
15.60 ± 2.29
17.54 ± 2.50
19.27 ± 3.79
Total bilirubin (mg/dl)
M
0.33 ± 0.08
0.36 ± 0.07
0.33 ± 0.07
0.30 ± 0.08
0.32 ± 0.09
0.29 ± 0.07
F
0.37 ± 0.13
0.39 ± 0.12
0.32 ± 0.04
0.35 ± 0.07
0.33 ± 0.11
0.30 ± 0.05
Alanine aminotransferase (U/L)
M
77.50 ± 20.46
67.60 ± 19.08
60.30 ± 14.65
61.20 ± 12.78
54.20*± 18.08
53.30*± 10.63
F
73.00 ± 16.15
78.00 ± 14.73
74.40 ± 18.07
65.30 ± 10.06
56.10*± 10.65
57.40*± 7.83
Aspartate aminotransferase (U/L)
M
166.60 ± 17.52 168.40 ± 15.09 163.90 ± 25.05 149.00 ± 23.49
139.30*± 9.17
140.00*± 12.82
F
172.70 ± 20.34 169.40 ± 16.71 171.60 ± 17.76 167.30 ± 19.45
147.50*± 20.64
143.20*± 21.47 27.43 ± 5.01
Total Cholesterol (mg/dl)
Albumin (mg/dl)
High density lipoprotein (mg/ dl)
M
22.67 ± 1.95
22.56 ± 3.64
21.00 ± 5.49
24.07 ± 3.50
21.37 ± 4.80
F
27.85 ± 6.20
26.63 ± 4.87
26.60 ± 4.58
27.31 ± 4.80
30.82 ± 6.10
Alkaline phosphatase (U/L)
M
143.10 ± 18.79 152.70 ± 22.07 151.80 ± 25.68 170.66 ± 23.42 144.70 ± 33.46
160.15 ± 21.45
F
144.44 ± 19.94 139.84 ± 27.01 145.68 ± 29.32 154.78 ± 31.17 163.13 ± 19.77
159.60 ± 22.95
Low density lipoprotein (mg/ dl)
M
17.26 ± 2.37
13.86 ± 2.29
14.95 ± 3.80
16.47 ± 2.39
19.94 ± 4.67
17.69 ± 3.54
F
16.36 ± 3.03
14.95 ± 7.12
15.60 ± 3.30
15.00 ± 3.40
12.14 ± 2.10
14.55 ± 3.16
Calcium (mg/dl)
M
9.26 ± 0.77
8.77 ± 1.12
9.09 ± 0.90
9.18 ± 0.86
9.09 ± 0.90
8.87 ± 0.96
F
9.14 ± 0.71
8.91 ± 1.09
17.51 ± 26.89
8.92 ± 0.97
8.79 ± 0.84
9.31 ± 1.12
M
3.97 ± 0.62
3.75 ± 0.56
3.67 ± 0.57
4.94 ± 0.90
4.66 ± 1.40
4.55 ± 1.64
F
4.19 ± 0.59
4.14 ± 0.32
4.31 ± 0.51
4.20 ± 0.69
4.76 ± 0.58
4.72 ± 0.93
Sodium (mmol/L)
M
141.40 ± 4.38
140.60 ± 4.67
140.20 ± 3.19
139.20 ± 7.11
140.80 ± 1.93
140.60 ± 4.72
F
141.30 ± 2.67
139.80 ± 4.87
140.90 ± 3.73
140.80 ± 3.74
141.50 ± 3.63
141.60 ± 2.59
Potassium (mg/dl)
M
4.28 ± 0.57
4.18 ± 0.70
4.14 ± 0.67
4.11 ± 0.28
3.94 ± 0.39
4.10 ± 0.34
F
4.19 ± 0.34
4.11 ± 0.52
4.18 ± 0.47
4.16 ± 0.68
4.11 ± 0.28
4.06 ± 0.43
Phosphorous (mg/dl)
31.59 ± 4.69
n = 10; M, male; F, female. Values are in Mean ± SD; * Statistically significant (P
