Lycopene improves on basic hematological and immunological parameters in diabetes

doi:10.21203/rs.2.16409/v1

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Lycopene improves on basic hematological and immunological parameters in diabetes

https://doi.org/10.21203/rs.2.16409/v1

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published 12 Dec, 2019

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Objective Diabetes is associated with an upset of hematological and immunological parameters in humans, however information on the effects of Lycopene is scarce. The aim of the study was to gain information on basic changes in hematological parameters as markers for safety since anemia as a complication in diabetic chemotherapy has been reported.

Results Lycopene had anti-anemic effects and improved on the immune status of diabetic rats and these observations were dose independent. There was a decrease in neutrophil, low neutrophil-lymphocyte ratio and platelet counts and stable albumin, globulin levels.

Gastroenterology & Hepatology

Antioxidants

diabetes

anemia

Carotenoids

blood.

Anemia has been identified as a major complication of diabetes mellitus (DM) in humans [1] and vitamin supplementation using folic acid and vitamin B12 has been associated with improved patient outcomes, although a lot remains to be established in this field [2]. In blood, a significant reduction in vitamin B12 (cobolamin) due to its malabsorption in Type 1 Diabetes Mellitus (T1DM) would be associated with anemia since this is a maturation factor needed during erythropoiesis [3]. Metformin use has also been associated with low vitamin B12 levels [4] demonstrating a need for novel therapies to manage complications in DM therapy. Lycopene is a phytochemical carotenoid, red in color, lipophilic and naturally occurring in many natural products especially in tomatoes [5]. Carotenoids have been associated with strong antioxidant activity and this is responsible for their therapeutical benefits in humans following a heavy vegetable intake [6]. Lycopene has established hypoglycemic and pancreatic protective effects [7], however information on its effects on hematological parameters in anemia is scarce. Hematological indices i.e. mean corpuscular hemoglobin concentration (MCVH) has been shown to be higher in diabetic patients and the mean corpuscular hemoglobin concentration negatively correlated with blood pressure demonstrating the importance of hematological indices in diabetes [8]. In severe diabetic human patients, anemia has been associated with low hemoglobcin (Hb) levels [9] demonstrating its importance as a clinical marker in this regard. Since glycated hemoglobin (A1C) is not recommended as a test in clinical situations due to its ability to interfere with Hb metabolism, the glycated albumin reflects short-term glycemia and is not influenced by situations that falsely alter A1C levels [10], however, information on Lycopene induced albumin changes is scarce. Serum albumin can be estimated using the bromocresol green (BCG) and bromocresol purple (BCP) methods and these methods may lead to serious confusions especially at low albumin levels and an overestimation has been associated with serum acute phase proteins [11]. High globulin levels have also been reported as a risk factor to diabetes [12] and these would be associated with beta globulins while gamma globulins are decreased in diabetes, however the role of Lycopene created a rationale for the study.

Study design

Male Wistar rats of both sexes (N = 30) weighing 150–200 g were kept as previously described with 24hr of day light [13]. Diabetes mellitus was induced by using 60 mg/kg of Streptozotocin (Sigma^®) in olive oil and after confirmation of hypoglycemia, rats were divided into experimental groups as previously described [14] i.e. 5 rats per experimental group. Thirty rats were divided into a negative control which was administered with olive oil, and diabetic rats were used in positive control with olive oil at 0.5 ml/kg, the comparative control administered with glibenclamide at 2 mg/kg and three experimental groups’ i.e. low, medium and high dose of lycopene at 10mg/kg, 20 mg/kg and 40 mg/kg of Lycopene.

Blood Sample Collection and Serum Preparation

After the last day of treatment (28 days) all animals were euthanized using sodium pentobarbitone injected intraperitoneally [13] and 5 ml of blood was collected through cardiac puncture into heparinized sample bottles for hematological analysis,erythrocyte osmotic fragility test and erythrocyte malondialdehyde concentration. The other portion of blood sample collected into specimen bottles and allowed to clot, and separated by centrifugation at 2000 g for 10 minutes using Centrifuge Hettich (Universal 32, Made in Germany). The supernatant obtained was used for the determination of the physiological parameters.

Determination of Serum Total Protein, Albumin, Globulin and Albumin-Globulin Ratio

The serum total protein was determined using a Eurochem® total protein test kit according to the manufacturer’s recommendations [15]. Serum albumin which quantitatively binds bromocresol green (BCG) to form an albumin-BCG complex was measured as an endpoint reaction at 596 nm [11]. Globulin was obtained by subtracting the albumin from the total protein and the albumin-globulin ratio was obtained subsequently

Determination of Hematological Parameters

These were determined using an automated analyzer using the manufacturer’s manual [16]. Parameters of interest were red blood cells (RBC) counts, white blood cells (WBC) counts, haemoglobin (Hb) concentration, packed cell volume (PCV), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH) and mean corpuscular haemoglobin concentration (MCHC).

Data analysis

This was done using Graph Pad Prism Version 6 and information was presented as figures and a Table. ANOVA test was done and Tukey’s test was used to determine sources of variation and significant differences (P < 0.05) were indicated with different superscripts i.e. letters a, b, c.

Lycopene improves on hematological indices during diabetes

Lycopene administration was associated with a significant increase in the packed cell volume, hemoglobin levels, and red blood cell counts (Fig 1. A-C) while no significant differences were seen in the mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH) and mean corpuscular hemoglobin concentration (MCHC). There were no significant effects in lycopene activity at higher concentrations demonstrating that effects observed are dose independent as shown in Table 1.

Figure 1 Here

Figure 1. Changes in hematological indices following Lycopene administration

Lycopene improves on the immune status in diabetic rats

Lycopene administration was associated with an increase in white blood cells and lymphocyte counts (Fig 2. A, B), however, a decrease in neutrophils was observed although no significant differences were observed (Fig. 2C). Lycopene was also associated with significant decrease in neutrophil-lymphocyte ratio and platelet countss (Fig2. D, E). Furthermore, Lycopene was associated with significant increase in blood total protein and globulin levels (Fig 2. G, I) however, no significant differences were found in the blood albumin levels and fragility tests (Fig 2. H, F).

Figure 2 Here

Figure 2. Changes in immunological parameters following Lycopene administration

Table 1 Inferential analysis on hematological parameters following Lycopene administration in diabetes.

Tukey's multiple comparisons test	PCV	Hb	RBC counts	MCV	MCH	MCHC	WBC	LYMP	NEUT	N:L	Platelets	Total protein	Albumin	Globulin
Tukey's multiple comparisons test	Adjusted P values
Normal + Olive oil vs. DM + Olive oil	0.0006	< 0.0001	< 0.0001	0.9469	0.6628	0.8080	0.4535	0.3125	0.8834	0.0001	0.0032	0.0005	0.9357	0.0202
Normal + Olive oil vs. DM + 10mg Lycopene	0.9737	0.9984	0.9491	> 0.9999	> 0.9999	0.9973	0.8742	> 0.9999	0.0190*	0.1455	0.2215	0.8463	0.9099	> 0.9999
Normal + Olive oil vs. DM + 20mg Lycopene	0.9849	0.9999	0.9998	0.9999	> 0.9999	0.7016	> 0.9999	> 0.9999	0.5622	0.6296	0.9999	0.8907	0.9998	0.9825
Normal + Olive oil vs. DM + 40 mg Lycopene	> 0.9999	0.9836	0.9931	0.7344	0.3103	0.9968	> 0.9999	> 0.9999	0.5846	0.1947	0.8684	0.8463	0.8423	0.9997
Normal + Olive oil vs. Comparative control	> 0.9999	0.4128	0.0370	0.3898	0.9827	0.0588	0.5872	0.4467	0.6953	0.9995	0.9693	0.1483	0.0328*	0.6926
DM + Olive oil vs. DM + 10mg Lycopene	0.0037	< 0.0001	0.0004	0.9733	0.6364	0.5423	0.0664	0.2702	0.1818	0.0603	< 0.0001	< 0.0001	0.4054	0.0252
DM + Olive oil vs. DM + 20mg Lycopene	0.0030	< 0.0001	< 0.0001	0.8667	0.6098	0.1136	0.3617	0.3958	0.9909	0.0061	0.0057	< 0.0001	0.8423	0.0040
DM + Olive oil vs. DM + 40 mg Lycopene	0.0004	< 0.0001	0.0002	0.2446	0.0149	0.9684	0.4115	0.4281	0.9932	0.0427	0.0002	< 0.0001	0.3179	0.0389
DM + Olive oil vs. Comparative control	0.0008	0.0014	0.1049*	0.0832	0.2804	0.5049	0.0209	0.0065	0.9990	0.0003	0.0005	< 0.0001	0.0037*	0.3498*
DM + 10mg Lycopene vs. DM + 20mg Lycopene	> 0.9999	> 0.9999	0.9886	0.9989	> 0.9999	0.9170	0.9319	0.9998	0.4529	0.9129	0.1441	> 0.9999	0.9720	0.9687
DM + 10mg Lycopene vs. DM + 40 mg Lycopene	0.9522	0.8851	0.9995	0.6552	0.3314	0.9363	0.9024	0.9995	0.4320	> 0.9999	0.8281	> 0.9999	> 0.9999	> 0.9999
DM + 10mg Lycopene vs. Comparative control	0.9887	0.6581	0.2117	0.3208	0.9871	0.0216*	0.9946	0.5014	0.3346	0.2556	0.6344	0.7363	0.2431	0.7489
DM + 20mg Lycopene vs. DM + 40 mg Lycopene	0.9698	0.9418	0.9996	0.8571	0.3532	0.4187	> 0.9999	> 0.9999	> 0.9999	0.9565	0.7497	> 0.9999	0.9357	0.9224
DM + 20mg Lycopene vs. Comparative control	0.9945	0.5506	0.0643	0.5294	0.9906	0.0020*	0.6884	0.3575	> 0.9999	0.8122	0.9086	0.6738	0.0569	0.3021
DM + 40 mg Lycopene vs. Comparative control	0.9999	0.1374	0.1176	0.9915	0.7016	0.1505	0.6325	0.3283	> 0.9999	0.3288	0.9993	0.7363	0.3179	0.8480

KEY: Superscripts (*) mean. No differences in RBC counts of DM + olive oil and comparative control (P > 0.05). 40 mg of Lycopene had significant differences against by increasing MCH values (P = 0.0149), although findings were not so strong. 10 mg and 20 mg Lycopene had significant differences above the comparative control (P < 0.05). Differences in WBC count (P = 0.0209), lymphocytes (P = 0.0065) between olive oil and comparative control. Neutrophils different in DM and normal rats (P = 0.0190). DM + olive oil and DM + 10 mg lycopene not different (P = 0.0603).

Fragility test non-liner regression

The effective concentration which had effects on 50% RBC activity was found to be 0.38, 0.44, 0.41, 0.39, 0.37 units for normal + olive oil, DM + olive oil, 10mg, 20mg, 40mg lycopene and comparative control demonstrating the safety of concentrations used in the study. Significant differences in normal and DM with olive oil against the comparative control for albumin. No significant differences in DM + olive oil and comparative control on globulin levels (P = 0.3498).

The study demonstrated that Lycopene had anti-anemic effects (Fig 1.)) demonstrating its importance in the management of anemia associated with diabetes. This is important since little is known about the role of carotenoids on hematological indices [1,2] and this study, Lycopene could upregulates erythropoiesis which is associated with the maturation factor [3]. In addition, the strong hematoprotective effects of Lycopene strengthen its therapeutical effects observed in diabetes and in pancreatic protection [7]. This is important since the conventional drug metformin interfears with vitamin B12 absorption [4], demonstrating its relevance in integrative and alternative medicine. No changes in the mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration (Table 1),, demonstrating the safety of Lycopene at varying concentrations on blood. In diabetes, these hematological indices are often high [8], thus the use of Lycopene in integrative medicine would help lessen side effects and toxicities associated with conventional therapies in diabetes.

In the body, white blood cells play a crucial role in immunity, thus an increase in WBC and lymphocyte counts (Fig 2. A, B) improves on the immune status of diabetic rats and makes them more resilient against opportunistic infections. Since these observations were dose independent in Lycopene, the strong antioxidant activity would be responsible for a favorable immune response as is common with many vegetables and fruits due to their high carotenoid content [6]. A decrease in neutrophil levels showed that no major inflammatory processes were ongoing in diabetic rats thus revalidating the therapeutical potential of Lycopene in diabetes. A significantly low neutrophil-lymphocyte ratio and platelet counts showed that the risk of liver and pancreas damage associated with diabetes was minimal [7]. In diabetes, differences in beta and gamma globulins and low albumin levels have been reported [11,12] and the ability of Lycopene to maintain these within normal ranges demonstrated its

Limitations

The study worked on basic markers hematological parameters thus a follow study on secondary messengers, antioxidant status, inflammatory cytokines would offer a clearer insight on the molecular mechanism of Lycopene activity in diabetes. In this study we offer basic information on the use of natural products rich in Lycopene in alternative medicine, however, effects of Lycopene in several body organs remains to be established.

BCG Bromocresol green

BCPBromocresol purple

DMDiabetes mellitus

HbHemoglobin

LYMPLymphocytes

MCH Mean corpuscular haemoglobin

MCHCMean corpuscular haemoglobin concentration

MCVMean corpuscular volume

mg/kgmilligrams per kilogram of body weight.

nmnanometers

PCV Packed cell volume

RBCRed blood cells

WBCWhite blood cells

Acknowledgements

Not Applicable.

Author contributions

EDE designed the study; EDE, AMA, JK, KIK collected the data; EDE, KIK conducted data analysis and interpretation. EDE, AMA, JK, KIK interpreted results, wrote, revised the initial and final manuscript. All authors read and approved the final manuscript.

Funding

Not applicable.

Data availability statement

Information used in the study can be accessed on https://figshare.com/s/472d34dc4f4de9ac93cc

Ethical approval and consent to participate

This was acquired from the Kampala International University Western Campus Ethics and Scientific review board. Consent to participate was not applicable to this study.

Consent to publish

Not applicable.

Competing interests

The authors declare that they have no competing interests.

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[4].Aroda VR, Edelstein SL, Goldberg RB, Knowler WC, Marcovina SM, Orchard TJ, et al. Long-term Metformin Use and Vitamin B12 Deficiency in the Diabetes Prevention Program Outcomes Study. J Clin Endocrinol Metab. 2016 Apr;101(4):1754–61.

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Journal Publication

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Editorial decision: Major revision
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Lycopene improves on basic hematological and immunological parameters in diabetes

Status:

Journal Publication

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Abstract

Figures

Introduction

Methods

Results

Discussion

Abbreviations

Declarations

Acknowledgements

Author contributions

Funding

Data availability statement

Ethical approval and consent to participate

Consent to publish

Competing interests

References

Status:

Journal Publication

Version 1