INTRODUCTION:

In anemia erythrocyte count and hemoglobin level gets decreased. Body’s ability to carry oxygen diminished which impairs exercise capacity, endurance and stamina ¹. During endurance exercise consumption of oxygen is increased by 20 folds due to high muscle blood flow and increased skeletal muscle level respiration². Several studies showed that in exercised animals, level of oxidative stress markers get increased because of increased oxidative damage and decreased level of free radical scavenger enzymes in muscle tissue³. Human and animal studies have shown that intake of antioxidants improves endurance performance when used in treating exercise induced oxidative stress⁴.

Skeletal muscle contraction leads to change in intracellular redox level and results in increased generation of free oxygen radicals. Chronic generation of reactive oxygen species and its subsequent effect is associated with muscle hypertrophy and sarcopenia⁵. Antioxidant containing vegetables and fruits are more useful than the single antioxidant like vitamin E⁶. The components present in antioxidant-rich vegetables and fruits, may play a positive role in reducing oxidative stress⁷.

Red beetroot is famous for its medicinal values and commonly known as beet, chard, spinach beet⁸. Beta vulgaris L, also known as beetroot belongs to the Amaranthaceae family (earlierly classified in the family Chenopodiaceae)⁹. Beetroot consist of bioactive compounds like betalains, phenols, carotenoids, B-vitamins, fibres and minerals including potassium (K), copper (Cu), magnesium (Mg), zinc (Zn), calcium (Ca), phosphorous (P), and sodium (Na). Pigment betalain such as betacyanines and betaxanthins impart red color to beet root^10,11. Different parts of this plant are useful in reducing oxidative stress, as mood elevators, anti-bacterial, antifungal, anti-inflammatory, diuretic, expectorant and carminative, hepatoprotective, and cardioprotective. It is traditionally known to increases athletic performance^12,13,14. The objective of this study is to assess the effect of Beetroot aqueous extract (BRAE) on anemia and muscle endurance in experimental animals.

MATERIALS AND METHODS:

Chemicals:

2-2-diphenyl-1-picrylhydrazyl (DPPH) (Sisco research lab, Mumbai, India), methanol, (Research lab, Mumbai), ascorbic acid (Sisco research lab, Mumbai, India) Phenylhydrazine (Sigma Aldrich, Mumbai), Orofer XT (Emcure), Oxymetholone (Anadrol). Throughout the study analytical grade chemical and reagents were used to ensure accurate and reliable results.

Collection and authentication of plant:

Fresh beetroots (Beta vulgaris L) were obtained from local area Ichalkaranji, Kolhapur District, Maharashtra, India. A specimen was submitted to the herbarium for the process. The plant was authenticated by botanist, Dr. Vikas B. Awale, Bharati Vidyapeeth’s Dr. Patangrao Kadam Mahavidyalaya, Sangliwadi, Sangli, and Shivaji University, Kolhapur, Maharashtra. [VSK 03]

Preparation of Extract:

Beta vulgaris L was washed under running tap water to eliminate adhered mud. The cleaned roots were then sliced and shed dried at room temperature for the one month duration. The dehydrated Beta vulgaris L was powdered and subjected to maceration process. The filtrate was slowly concentrated and then stored at cool place (4-8°C), throughout the study period. The aqueous extract of beetroot was named as BRAE. The percentage yield of the extract was calculated.

Preliminary phytochemical investigations:

BRAE was screened for the secondary metabolites like alkaloids, glycosides, tannins, saponins, triterpenoids, flavonoids, phenolics etc. using conventional laboratory reagents¹⁵.

In-vitro measurement of an antioxidant property

Diphenyl-1-picrylhydrazyl (DPPH):

1, 1-Diphenyl-2, Picryl-Hydrazyl (DPPH) was used for evaluation of antioxidant efficacy of the BRAE. 100μL of BRAE (1mg/ml) was mixed with 100μL of 0.1% methanolic DPPH and incubated for half an hour in dark place. The mixture was then observed for color change from purple to yellow and purple to pale pink; considering strong and weak positive, respectively. Absorbance was recorded using spectrophotometer at wavelength 490nm^16,17.

Radical scavenging activity was calculated by the following equation:

DPPH scavenged (%) = [(Absorbance of control - Absorbance of test sample)/(Absorbance of control)] X100

HPTLC fingerprinting:

HPTLC finger printing studies were carried out according to the method of Wagner, Baldt and Harbone using Camag HPTLC system. 7μL of BRAE were spotted in the form of bands having band width of 5mm on precoated silica gel 60 F254 HPTLC plate (10 × 10 cm, 250um thickness) (E. Merck, Mumbai, India). The mobile phase optimized and used was Ethyl Acetate: Water: Ethanol: Formic Acid (2: 7: 1: 3: 0.5 v/v/ v/v/v). Densitometric scanning was performed with a TLC scanner equipped with DESKTOP-5IHGUM1, version 3.1.21109.3 software (Camag) in reflectance absorbance. Plates were subjected to scanning at wavelength 254nm which was chosen experimentally on the basis of characteristic absorption spectra of the compounds between 200 and 400nm. Each plate was placed in photo-documentation chamber (CAMAG) and images were captured under visible light and under UV at wavelength 366nm and 254nm. The peaks were recognized with their respective peak numbers, with its height and area, peak display, and peak densitogram. DESKTOP-5IHGUM1, version 3.1.21109.3 software was used to record retention factor (Rf)¹⁸.

Pharmacological evaluation:

Approval of research protocol:

Experimental protocol was approved by the Institutional Animal Ethics Committee (IAEC) constituted as per guidelines of the CPCSEA (Committee for Purpose of Control and Supervision of Experimental Animals), India¹⁹. (IAEC/Sangli/2022-23/01).

Experimental Animals:

Female Wistar rats required for the study were obtained from CPCSEA certified vendor. Rats with weight ranging between 200gm±20% and age between 8-12 weeks were chosen for the study. Animals were retained at temperature 24℃±1℃, 45-55% relative humidity, and 12:12 hour’s light/dark cycle.

Acute toxicity study

Acute oral toxicity study of the BRAE was conducted as per OECD 420 guideline at maximum single dose 2000 mg/kg. After administration of the dose, the signs of toxicity and/or mortality were recorded during 14 days of observation period^20,21.

Phenylhydrazine-induced Anemia:

Animals were divided into five groups each containing 6 rats as shown in table 1. The blood samples were collected under mild pet ether anesthesia on the first, third, seventh and fourteenth day by the retro-orbital route into EDTA coated vials. Blood samples were analyzed for erythrocyte, hemoglobin and hematocrit (HCT)²².

Table 1: Phenyl Hydrazine Induced Anaemia (n=6)

Sr No.	Name of Group	Treatment	Oral Doses, frequency	Blood withdrawal
1	- ve control	Vehicle	0.5ml, daily	Retro orbital sinus, 0.5-1 ml on 1^st, 3^rd,7^th and 14^th day using Pet. Ether.
2	+ ve control	Vehicle + PHZ	60mg/Kg, 20 mg for 3 days
3	Standard	Orofer-XT+ PHZ	10mg/kg, daily
4	BRAE 200 mg/kg	BRAE + PHZ	200mg/Kg, once daily
5	BRAE 400 mg/kg	BRAE + PHZ	400mg/Kg, once daily

Note: Except –ve control, all groups were administered with PHZ 20 mg/Kg intraperitoneally as inducer for 3 days.

Rota rod test:

On day 1, animals were placed on the rotating rod with the adjusted speed of 20 rotations/min for 300 seconds. The two trials were conducted and the animals staying on the rotating rod for 300 seconds during these successive two trials were selected for this study. Animals were grouped into Control group: received Normal saline solution, Standard group: received standard drug Oxymetholone (Anadrol) 50mg/kg and two test groups: received BRAE at dose of 200mg/kg and 400mg/kg respectively for seven days. On seventh day, 45min after the treatment, rats were placed on a rod rotating at speed of 20rotations/min. The duration of staying on the rod of the individual animal were recorded with cut off time of 300sec²³.

Grip-strength measurement:

Kondziela's inverted screen method was used to measure grip strength. Animals were grouped into Control group received normal saline solution; Standard group received standard drug oxymetholone (Anadrol) 50 mg/kg and two test groups received BRAE at dose of 200mg/kg and 400mg/kg respectively for seven days. On day 7 after BRAE treatment, animals were placed on wire mesh screen and screen was inverted. The time when animal releases their hind limb and falls off is being recorded²⁴.

Data Analysis:

The findings were presented as mean±SEM. A one-way ANOVA was used to evaluate the data, and Dunnett's t test was then used to compare the groups with the positive control groups. Results were expressed at different precision levels in terms of p values (p<0.05, p<0.01, or p<0.001). The confidence level of greater than 95% (p<0.05) was considered sufficient to propose the precision of the hypothesis in each case.

RESULTS:

Qualitative analysis:

The percentage yield of the extract was 3.7%. Phytochemical study of the BRAE showed presence of phytoconstituents such as flavonoids, glycosides, phenolics, tannins and triterpenoids.

In-vitro antioxidant activities:

Diphenyl-1-picrylhydrazyl (DPPH):

Standard ascorbic acid showed 49.795±0.69% inhibition, while aqueous extract showed 46.24±3.06% of DPPH radical scavenging. According to results, the DPPH scavenging effect of BRAE increased significantly with rising concentration. This clearly indicates its antioxidant activity.

HPTLC Fingerprinting:

Flavonoids and phenolic compounds

Chromatogram was developed for BRAE aqueous extract under mobile phase saturated condition where Ethyl Acetate: Water: Ethanol: Formic Acid (2: 7: 1: 3: 0.5v/v/ v/v/v) used as mobile phase.

Figure 1: HPTLC fingerprinting of BRAE for phenolic & flavonoids content.

The HPTLC fingerprinting of BRAE aqueous extract showed presence of 6 peaks with RF values for phenolic compounds in the ascending order of 0.010, to 0.958 end values. The phytoconstituents with highest area percentage was found to be 64.72% and its consequent RF value was 0.196. Also HPTLC fingerprinting of BRAE aqueous extract showed presence of 6 peaks with RF values for flavonoid compounds in the ascending order of 0.011, to 0.533 end values [Figure 1]. The highest area percentage of the phytoconstituents was found to be 41.04% and its resultant RF value was 0.533. When HPTLC plate spotted, developed and then exposed to UV light at 254nm, distinct light and dark bands were seen. Exposure to UV light at absorbance 366nm, showed multi-colored bands of different intensities. This revealed presence of hydrophilic and hydrophobic constituents in the BRAE. In the developed HPTLC fingerprinting, blue colored bands indicated presence of phenolic compounds and yellow colored zones indicated presence of flavonoids in BRAE aqueous extract.

Anti-anemic activity:

Experimental protocol was approved by IAEC of Biocyte Institute of Research and Development, Sangli. (IAEC/Sangli/2022-23/01).

Acute toxicity study:

During the two weeks study the toxicity signs were not observed and there were no mortality recorded. As the doses used in the later study were 5-10 times smaller than the fixed dose used in the acute toxicity study, we can consider that later study was conducted using safe doses.

Induction of anemia in participated group was done using PHZ method. RBC count, hemoglobin content and hematocrit percentage was determined in the rat blood sample collected in EDTA containing vials using MISPA VIVA (KT21092350) semi-autoanalyser. BRAE, 400 mg/kg group and standard group (Orofer XT 10mg) showed significant activity (P<0.01) against positive control group on 14th day. [Table 2].

Table 2: Effect of BRAE aqueous extract on Red blood cell count, Hemoglobin count and Hematocrit % in phenylhydrazine induced anemia.

Group

RBC count (lacs/mm3)

Hemoglobin count g/dl

HCT count %

Day 1

Day 3

Day 7

Day 14

Day 1

Day 3

Day 7

Day 14

Day 1

Day 3

Day 7

Day 14

-ve control

64.0 ±5.0

63.4±

6.7

62.3±

2.8

64.6±

4.2

13.31±

0.34

12.68±

0.42

12.89±

0.12

13.27±

0.18

38.12±

1.21

41.42±

1.67

39.33±

1.78

40.79±

1.34

+ve control

65.8±

4.3

31.8±

8.7

39.5±

4.5

41.9±

8.3

13.08±

0.46

6.35±

0.28

6.92±

0.34

7.23±

0.35

39.89±

2.63

24.31±

2.04

27.24±

1.56

30.29±

1.45

Standard

68.0±

6.7

39.1±

5.6

58.9±

6.7

69±

3.5**

12.93±

0.35

8.14±

0.31

11.46±

0.41

14.23±

0.18**

40.18±

1.67

23.48±

1.45

33.67±

2.03

40.23±

1.56**

BRAE 200 mg/kg

62.3±

0.54

37.8±

0.67

49.1±

0.40

68.9±

0.81**

12.85±

0.31

8.67±

0.46

10.92±

0.37

12.76±

0.36*

40.73±

2.03

24.05±

1.85

30.52±

2.43

37.85±

2.03*

BRAE 400 mg/kg

64.6±

0.38

38.1±

0.23

52.9±

0.42

76.4±

0.34

***

13.08±

0.46

8.13±

0.45

11.56±

0.45*

13.78±

0.28**

41.12±

1.98

22.76±

1.56

34.86±

1.72*

39.62±

2.43**

Values are expressed as mean ± SEM followed by one-way ANOVA *P<0.05, ** P<0.01, BRAE: Beetroot aqueous extract, RBC: Red blood cell, HCT: Hematocrit.

Figure 2: Grip strength test: Time taken for rats to release hind limb and drop down. All the values are expressed as mean ± SEM (n=6)

Rota rod test:

On seventh day, both the standard drug and BRAE at doses 200mg/kg and 400mg/kg demonstrated significant increase in the time spent by the animals on the rotating rod compared to the control group. BRAE demonstrated dose dependant increase in muscle strength at doses 200mg/kg and 400mg/kg, showing values of 68.5±2.75 and 78.16±3.02 respectively when compared to control group. Results of rotarod test showed that administration of BRAE extract significantly improved the muscle strength among the tested animals.

Grip strength measurement:

Grip strength of the animals was measured after the administration of the test drug (200mg/kg and 400 mg/kg) at the end of day 7.

In grip strength measurement the release limb time and drop down time from wire mesh was significantly increased in the test group when compared to control and standard group [Figure 2].

DISCUSSION:

Natural substances are useful as protective and therapeutic agent because of presence of enriched phytoconstituent in them. Recent studies suggested that the active constituents present in red beet root has antioxidative, anti-inflammatory, anticancer, antihypertensive, lipid lowering, antidiabetic and anti-obesity effects²⁵. BRAE revealed the presence of flavonoids, glycosides, phenolics, tannins and triterpenoids, which are valuable for different therapeutic activities. Phenolic compounds are known for its antioxidative effect and having free radical scavenging activity. Also research study showed that antioxidant effect of flavonoids is responsible for their pharmacological effects²⁶. Phenolic compounds function as antioxidants by neutralizing free radicals. These compounds provide protective effect when incorporated in diet²⁷. In HPTLC fingerprinting, after derivatization, the presence of flavonoids confirmed by development of blue-violet colored zones, while polyphenols confirmed by development of blue and light brown color zone²⁸. Raktavardhak Kadha exhibited the presence of phytoconstituents with significant antioxidant activity. Also, its antianemic activity was demonstrated using a phenylhydrazine-induced model. The authors concluded that the antianemic activity could be linked to the presence of flavonoids²⁹. Research proved that secondary metabolites present in plants react with free radicals intermediate and stop the further progressing reaction. Thus they prevent oxidative damage induced by generation of unstable free radicals³⁰. DPPH stable free radical method provides convenient, quick and sensitive approach to study the antioxidant activity of a specific compound or plant extracts³¹. BRAE scavenges the free radical which is associated with oxidative stress generated by DPPH method. Plant materials containing phenolic compounds have the ability to slow down oxidative breakdown of lipids. Thus they enhance the nutritional value and benefits of food³². Phenolic compounds, flavonoids are naturally found in plants shown positive effect on human health³³. Phenyl hydrazine through oxidative stress induces hemolytic anemia by destroying matured red blood cells. It is characterized by erythropenia, lowered hemoglobin level and reduced hematocrit level in the blood^34,35. Hikmawanti NPE et al. conducted hematopoietic activity of an ethanolic, alkaloid-free, and dichloromethane extract of beet root using phenylhydrazine-induced anemia model at a dose of 200mg/kg. All three extracts [EEBR>AF-EEBR>DE] improved all hematological parameters [except platelets] when compared with the normal control group³⁶. In this study the hematological parameters were reversed by oral treatment with BRAE at the doses 200mg/kg and 400mg/kg. It has been reported that Phenyl hydrazine enhances the production of free radicals and leads to structural and functional impairment in red blood cells. While flavonoids exhibit powerful antioxidant potential and have ability to prevent such damage to RBCs³⁷. Phytochemical analysis and HPTLC fingerprinting of BRAE confirmed the presence of phenolic compounds and flavonoids. Additionally its DPPH antioxidant activity suggests a potential connection to the hematinic activity seen in BRAE. Endurance exercise decreases erythrocytes, ferritin, hematocrit, and hemoglobin count. These changes are often observed in athletes³⁸. Past research had shown that natural products can enhance athletic performance and reduce or prevent fatigue without inducing any adverse effects³⁹. Muscles never function independently but are controlled by the signals from brain, local or higher spinal reflexes, through the neuromuscular junctions and work within the restrictions set by other muscles and the skeletal system ³². To assess effect of BRAE on muscle coordination we had used two models namely rota rod apparatus and grip strength measurement. Oxymetholone commonly known as Anadrol is an anabolic androgenic steroid has ability to improve muscle mass and muscle strength. It improves muscle development and is being used to enhance athletic performance⁴⁰. Anemia causes weakness, fatigue, poor work productivity, or difficulty concentrating⁴¹ and reduced exercise capacity due to diminished oxygen-carrying capacity⁴² and lowers physical performance⁴³. The administration of M. oleifera leaf extract resulted in an enhancements of motor coordination when compared to the effects seen with the standard drug. This effect is influenced by the markers responsible for protein balance in the skeletal muscles⁴⁴. In our research study it was observed that administration of BRAE 200mg/kg and 400mg/kg showed similar dose dependant augmentation in the duration of time spent on the rotating rod, and a positive impact on grip strength. Oral administration of BRAE extract demonstrated anti-anemic activity and exhibited a positive impact on muscle coordination.

CONCLUSION:

Research findings highlights on multifaceted advantages of BRAE extract. It not only showed antioxidant and hematinic effect but also have positive influence on muscle coordination. These dual functionalities suggest the potential of BRAE in anemia related concerns and improving muscular performance. Further research into its mechanism and applications could establish the approach for novel therapeutic advancements.

ACKNOWLEDGEMENTS:

Authors are thankful to Sanjay Ghodawat University, School of Pharmaceutical Sciences for providing facilities to carry out this research work. Authors are also thankful to Biocyte Instutute of Research & Development, Sangli and Poona College of Pharmacy, FDA Approved Public Testing Laboratory, Pune for analytical help. We acknowledges Dr. Shirishkumar D. Ambavade, Associate professor, Department of Pharmaceutical Sciences and Technology of ICT Mumbai for support.

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Received on 09.10.2023 Modified on 12.02.2024

Research J. Pharm. and Tech 2024; 17(8):3593-3598.

DOI: 10.52711/0974-360X.2024.00561