Improvement of Blood Parameters of Male Rats Exposed to Different Injection Doses of Liquid Chlorophyll

Introduction Chlorophylls are natural pigments in our everyday diet, especially with customers' rising preference for more natural and healthful habits. The antioxidant capabilities of both classes of lipophilic substances have been researched since disrupting antioxidant equilibrium appears to be linked to the development of several diseases. Methods This research aimed to evaluate the effect of injection with chlorophyll (30 and 60 mg/ml) on enhancing the blood parameters of rats. Twenty-one white male rats were included in this study and divided into three groups: control, 30 mg/ml, and 60 mg/ml. Results Treatment with liquid chlorophyll significantly increased white blood cells (WBCs), red blood cells (RBCs), granulocytes, lymphocytes, hemoglobin (Hgb), hematocrit (Hct), mean corpuscular Hgb concentration (MCHC), and platelets. However, it nonsignificantly increased mean corpuscular volume (MCV). These results confirm a great increase in important hematological parameters in response to exogenous injectable chlorophyll with concentrations of 30 and 60 mg/ml and at two different time points, 14 and 28 days after injection. The platelet count was significantly (p<0.001) increased after 30 mg/ml and 60 mg/ml. Conclusion These results show a significant increase in important hematological parameters in response to exogenous injectable chlorophyll. The liquid chlorophyll is recommended to increase blood parameters and improve blood characteristics avoiding anemia.


Introduction
Chlorophylls are natural pigments in our daily diet, especially with consumers' increasing tendency toward more natural and healthy behaviors [1]. Dietary chlorophyll can be found as chlorophyll a and chlorophyll b in fresh fruits and vegetables, and as metal-free pheophytins and pyropheophytins in thermally processed fruits and vegetables [2]. Chlorophyll in the form of underutilized greens in fresh vegetables, supplements, liquid solutions, extracts, or tablets can be used effectively as a healthy and beneficial nutrient supplement [3].
Chlorophyll is the most prevalent plant photopigment in nature, with chlorophyll-a (Chl-a) accounting for nearly 75% of the green pigments found in plants [4]. Chl-a is a totally unsaturated asymmetric macrocyclic molecule with a hydrophobic nature, which contributes to its poor solubility in hydrophilic fluids [4]. Therefore, Chl can be effectively used as a nutrient in the form of underutilized greens in fresh vegetables, supplements, liquid solutions, extracts, or pills; addition to micellar copolymers, such as P123, which has been shown to be biocompatible, is essential for in vivo and in vitro analyses, as they guarantee the monomerization of the hydrophobic PS and the maintenance of its photophysical properties [5].
Chl-a and its metabolites have been shown to build up in several tissues, including the liver and gut [6,7], which suggests that these organs might be affected by these compounds. Chlorophylls have several positive benefits, and one of these is antioxidant activity, which helps to prevent oxidative DNA damage and lipid peroxidation by decreasing reactive oxygen species (ROS) and chelating metal ions [4,[8][9][10]. The chemical nature of porphyrin allows chlorophylls to function as hydrogen donors, stopping the chain process [11]. Chlorophyll and other pigments, mostly isolated from sea algae, were investigated for their biological functions and potential health advantages [9]. Natural pigments, especially chlorophylls, provide several health benefits. They have been shown to have anti-inflammatory, anti-obesity, anti-angiogenic, and neuroprotective properties [9].
This research aims to evaluate the effect of two concentrations of liquid chlorophyll in enhancing the hematological parameters of experimental rats, including blood features.

Liquid chlorophyll
The chlorophyll used is a liquid chlorophyll ES (Extra-strength), Nature's sunshine product, Inc., Spanish Fork, UT84660 (Nature sunshine, Inc., USA) [12]. Liquid chlorophyll ES is a water-soluble extract obtained from alfalfa by extraction of chlorophyll (sodium-copper salt of chlorophyll). Liquid chlorophyll is a concentrated source of both Chl-a and Chl-b in addition to several nutrients of natural origin, including beta-carotene, vitamins C, E, and K. It is also rich in minerals and trace elements. Twenty-one white male rats were included in this study and divided into three groups: control, 30 mg/ml, and 60 mg/ml (A0, A1, and A2). Blood parameters were measured after 14 and 28 days of injection.

Sample size calculations
This research was performed to evaluate the effect of different concentrations of liquid chlorophyll on various blood parameters at two different time points, 14 and 28 days; a repeated-measures analysis of variance (ANOVA) has been proposed. A minimum total sample size of 21 white rats was sufficient to detect the effect size of 0.386, according to Cohen (1988), at a power (1-β=0.80) of 80% at a significance probability level of p <0.05, and a partial eta squared of 0.12. According to sample size calculations, each treatment group (A0, A1, and A2) including a control group, 30 mg/ml, and 60 mg/ml and time of investigations (T0, T1) would be represented by a minimum of seven rats, as shown in Table 1 and Figure 1. The sample size was calculated according to G*Power software version 3.1.9.6 [13][14][15].

Treatment group (A)
Total sample size A 0 A 1 A 2 Time of investigation (T) Total sample size 7 7 7 21

Animals and experiment design
According to sample size calculations, experiments were carried out on 21 non-purebred white male rats weighing 220-230 g. The experimental rats were divided into three groups: Group-I (A0) untreated control group received 10 ml of isotonic saline solution. Group-II (A1) experimental rats received 10 ml of 30 mg/ml liquid chlorophyll through tail-vein injection. Group-III (A2) Group-II (A1) experimental rats received 10 ml of 60 mg/ml liquid chlorophyll through tail-vein injection. Animals were fed and followed up regularly, including changes in body weight and other parameters following standard care. The peripheral blood sample was obtained 14 and 28 days following treatment.

Statistical analyses
Statistical analyses were applied to compare different treatment groups (A0, A1, and A2) at different times of investigations (T1 and T2). The data were collected, checked, revised, and organized in tables and figures using Microsoft Excel 2016. Data were subjected to outliers' detections and statistical normality tests to detect whether the data were parametric or nonparametric. Data were analyzed for descriptive statistics, both graphical and numerical descriptions. Inferential statistics for evaluating and comparing three different treatments (A0, A1, and A2) and time of investigations (T1 and T2) was performed by repeatedmeasures ANOVA or corresponding nonparametric analyses at significance levels of 0.05. ANOVA was followed by Duncan's multiple-range tests (DMRTs) to compare treatment groups or corresponding post hoc test for nonparametric data. Data analyses were carried out using the computer software Statistical Package for Social Sciences (SPSS ver. 28.0 for Mac OS; IBM Corp, Armonk, NY) [17].

Results
Various measured blood parameters are presented in tables and figures as mean±standard deviation (±SD).
The WBC showed a highly significant increase after injection with liquid chlorophyll and also after the time of injection, as revealed by repeated-measures ANOVA (p<0.001) ( Table 2; Figure 2). Moreover, the difference between groups at 14-day time points was highly significant (p<0.001) as revealed by one-way ANOVA. Also, the difference between groups at 28-day time points was highly significant (p<0.001) as revealed by one-way ANOVA.

Parameter Time
Mean±SD   Granulocytes count also showed a significant increase with a 30 mg/ml dose of chlorophyll, as revealed by Duncan's multiple range test and one-way ANOVA.  Figure 2).

FIGURE 3: Various blood parameters in experimental male rats after treatment with 30 and 60 mg/ml.
Bars followed by different letters are significantly different according to DMRTs.
The HCT (%), MCV, MCH, MCHC, RDW, stable neutrophils, PCT (%), and basophils showed a nonsignificant response to exogenous injectable liquid chlorophyll, as revealed by a two-way ANOVA ( Table 3).  The segmented neutrophils showed a highly significant difference with chlorophyll injection with concentrations of 30 and 60 mg/ml, as revealed by two-way ANOVA.

Variables
Chlorophyll

Discussion
The liquid chlorophyll in this study showed a significant appositive correlation with WBCs, lymphocytes, monocytes, lymphocytes (%), granulocytes, RBCs, Hgb, Hct, MCV, MCH, MCHC, and blood platelets. These significant positive effects of chlorophyll concentrations tested indicate a high health benefit of using chlorophyll from biological sources. These results agree with those of Pangestuti and Kim [9] who listed various significant effects of natural pigments, including chlorophyll. However, it disagrees with the study by Cugliari et al. [18], in order to verify the effects of the protracted intake of chlorophyll on blood count parameters and iron levels in endurance athletes, investigating supposed anti-anemic properties. They reported no significant difference in blood parameters, including hemoglobin; however, they reported an increase in blood platelets [18].
The increase in platelet-related measures could positively influence endurance performance by reducing pain and fatigue. However, the supposed ergogenic effects and anti-anemic properties are recommended for further study [18]. Platelet-rich plasma has anti-inflammatory and anabolic effects [19] and several studies show its effectiveness in the healing process of muscle injury [20], tendon injury [21], and in the treatment of osteoarthritis [22]. A recent study shows a significant correlation between MPV and the running time in a half marathon [23], while in short-term performance at the maximum intensity, it appears to have no significant relationship between PLTS, MPV, and PDW with VO 2 Max, resistance, and running speed [24].
These results suggest that platelets may play a role in the medium-to a long-term performance by promoting the gradual release of growth factors and thereby relieving muscular pain and/or fatigue, or that MPV increase could be attributed to a more significant turn-over of platelets that could reflect other chronic physical adaptation without necessarily having a direct ergogenic effect. In this study, however, only the experimental group obtained a significant increase, indicating chlorophyll's role in modifying the above factor.
The effect of chlorophyll on improving blood parameters includes antioxidant activities. Antioxidants may have a positive effect on human health as they can protect the human body against damage by ROS, which attack macromolecules such as membrane lipids, proteins, and DNA, leading to many health disorders such as cancer, diabetes mellitus, aging, and neurodegenerative diseases [25].