Evaluation of In-vitro Antioxidant Potential of Bryophillum pinnatum Leaf Juice in Phenyl Hydrazine Treated Goat Erythrocytes

 

O. Sreenivasa Krishna1, S. Naveen Kumar2, K. Vijay Kumar3,

C. Rajeswari4, CH. Akhil5

1Assistant Professor, Department of Pharmacology,

Jntua Oil Technological and Pharmaceutical Research Institute, Anantapur.

2,4M. Pharm Students, Department of Pharmacology JNTUA-OTPRI Anantapur, India.

3,5M. Pharm Students, Department of Pharmaceutical Chemistry JNTUA-OTPRI Anantapur India.

*Corresponding Author E-mail: krishna.vlcp@gmail.com

 

ABSTRACT:

Phytochemicals are known to contain a high concentration of therapeutically active principles, which, when consumed in sufficient quantities, can have drug-like effects. Bryophillum pinnatum (Lam.) is a succulent-ethno medicinal xerophyte with a long history of use in traditional Indian medicine. Because reactive oxygen species are the most common etiological factor in many degenerative diseases, controlling them with antioxidants may both treat and prevent disease progression. The current study will look at the ability of fresh leaf juice of Bryophillum pinnatum (Lam.) (FLJBP) to prevent oxidative injury and subsequent hemolysis in goat erythrocytes as a model test system. The catalytic activity of membrane bound enzymes like sodium potassium-ATPase, Ach-Ease, and metabolic enzymes like hexokinase, as well as the NO concentration in goat RBCs, were measured. Phenyl hydrazine was used first, followed by FLJBP at concentrations of 20, 40, 60, 80, and 100g/ml. A dose-dependent (PHZ) increase in Pi liberation by hexokinase and Na+/K+ ATPase, as well as increased formation of thiocholine iodide and enhanced NO scavenging potential, has indicated that flip has good anti-oxidant and anti-haemolytic activity. To record the functional food status of Bryophillum pinnatum (Lam.) species, a more rigorous and stringent battery of pharmacological, phytochemical, and bio analytical studies, followed by observational studies in humans, will be conducted.

 

KEYWORDS: Oxidative injury, Bryophillum pinnatum (Lam.), Phenyl hydrazine (PHZ), Hexokinase, Na+/K+ ATPase, Acetyl cholinesterase.

 

 

INTRODUCTION:

In the drug discovery process, intensive in-vitro pharmacological profiling of new chemical entities is required to identify undesirable off-target activity that could impede or halt the development of new candidate drugs, anit has recently become an essential tool for predicting clinical adverse effects.1

 

The challenge for pharmacologists will always be to correlate in vitro data with in vivo findings, remembering the old adage "In vitro simplicit as, in vivo veritas."2. Plants are the only source of therapeutics with myriad activities like immunomodulatory, anti-inflammatory, analgesis and antiinfective potential3-4

 

Crassulacean acid metabolism in this genus was discovered to be responsible for the production of medicinally useful principles in xerophytic geographical areas such as Sub-Saharan Africa5. Rosmarinic acid, resveratrol and sulquinovosyl diacyl glycerools are known to scavenge ROS effectively in various in -vitro studies6-7. Aerial root or stem modifications of various plants like Ficus and some commonly consumed vegetables if consumed in regular have protective effects aginst certain chronic  degenerative disorders8

 

Fig no: 1 Bryophillum pinnetum

 

MATERIALS AND METHODS:

Chemicals used:

The chemicals including solvents used in the preparation of various reagents Ammonium Molybdate, ammonium sulfate, ATP (sodium salt), Bovine serum albumin, Coomassie brilliant blue, imidazole, in the studywere of analytical grade andobtained from CDH, SRL, Sigma- Aldrich, SD-fine Ltd, Mumbai, Qalinge’s.

 

Chemical constituents:

Bufadienolide compounds isolated from Bryophyllum pinnatum include Bryophillin A, bersaldegenin-3-acetate, and Bryophillin C. Bryophillin C also showed insecticidal properties and prominent antioxidant activites as determined by various antioxidant assays9-10 Phytochemical constituents of broyophyllum pinnata have identified the presence of triterpenes, steroid, phenanthrene, flavonoid, flavones, chalcones, taraxasterol, aurones, phenolic acid, caffeic acid, syringic acid, malic, oxalic and ferulic acid. Bufadienolides and phenanthrene are toxic compounds. The plant Bryophyllum calycinum contains high quantity of flavonoids of proven anti-inflammatory potential11.

 

Preparation of Bryophillum pinnatum (Lam.) leaf juice:

Freshly collected Bryophillum pinnatum (Lam.) leaves are thoroughly washed to remove adherent impurities, ground to a paste with a mortar and pestle, the mass is squeezed through a muslin cloth to obtain juice, and the volume is measured after centrifugation at 5000rpm for 15 minutes at room temperature.

 

Process for obtaining whole RBCs from goat blood12:

To obtain the packed RBCs, goat blood was collected from a local Anantapur Municipal Corporation-approved slaughterhouse in heparin-coated vacutainers and cold centrifuged at 3000rpm for 10 minutes. Aspiration was used to remove the buffy coat and plasma, and the RBCs were washed three times with isotonic saline.

 

Incubation of whole RBCs with PHZ13:

500l of whole RBCs were mixed with 1mM phenyl hydrazine (PHZ) in 50mM sodium phosphate buffer (pH 7.4) to make a final volume of 1.0ml and incubated at 370C in a shaking water bath for 1hour. The incubation was stopped by adding 100l of 16mM EDTA, and the PHZ-treated red blood cells were washed three times with 0.9% NaCl solution before lysis and membrane preparation.

 

The tests with whole RBCs were then divided into seven groups, which are listed below:

Group I: Normal Control group contains the untreated RBC preserved in Sodium phosphate buffer to measure the activity of Na+/K+ATPase and the liberation of inorganic phosphate.

Group II: Phenyl hydrazine treated can serve as the negative control. The purpose of this group is to know the level of damage induced by Phenyl hydrazine to the RBC membrane and the membrane-bound Na+/K+ ATPase.

Groups III: PHZ treated + concentration of FLJBP at 20µg/ml.

Group IV: PHZ treated + concentration of FLJBP at 40µg/ml.

Group V: PHZ treated + concentration of FLJBP at 60µg/ml.

Group VI: PHZ treated + concentration of FLJBP at 80µg/ml.

Group VII: PHZ treated + concentration of FLJBPat 100µg/ml.

Group III to Group VII was employed to determine the effect of the protectiveness of leaf extract on Phenyl hydrazine-induced haemolysis.

 

Another group with washed RBC and FLJBP at 100µg/ml was also included to observe any effect of leaf extract on the activity of membrane-bound Na+/K+ ATPase to assess whether any inhibitors of sodium pump were present in the juice which may reveal the discovery of digoxin like molecules serendipitously.

 

Preparation of erythrocyte membrane:14

Haemoglobin-free erythrocyte membrane (either normal or treated) was prepared according to the method of Arduini et al. The erythrocytes (100µl) are washed and subjected to hypotonic lysis in 40 volumes of 5Mm Na3PO4 buffer (pH 8.0) and centrifuged at 14,000rpm for 20min at 4°C. The supernatant was discarded and obtained the pellet was washed at least five times in the same buffer till a colorless pellet was obtained. The erythrocyte ghosts were suspended in the same buffer and stored at –20°C.

 

Determination of erythrocyte membrane-bound enzyme Na+ K+ ATPase activity15:

The Na+/K+ ATPase was measured using Svoboda and Mosinger's (1981) method with minor modifications. 250 l of 10mM tris Hydrochloride buffer, 50 l of 600 l mM sodium chloride, 50 l of 50 mM potassium chloride, 50 l of 1mM Sodium EDTA, and 50 l of 80mM Adenosine triphosphate The reaction mixture was preincubated for 10 minutes at 37°C. Following that, 25 l of 10% homogenate was added to the test alone and incubated at 37°C for 1 hour. By adding 10% TCA, the reaction was immediately stop the control reaction rate was determined by adding 25 l of 10% homogenate only after the reaction had been stopped. This was centrifuged for 10 minutes at 3500rpm to remove the precipitate. The intensity of the blue colour was measured using a spectrophotometer at 640nm with a blank that contained all of the reagents except the supernatant. The results are displayed in grammes of inorganic phosphate (Pi).

 

Estimation of inorganic phosphate16:

The protein-free filtrate reacts with acid molybdatesolution, resulting in the formation of phosphomolybdic acid from any phosphate present by adding 1,2,4-amino-napthosulphonic acid reagent to the phosphomolybdic acid, a blue colour is produced whose intensity corresponds to the amount of phosphate present. The following solutions were prepared for the estimation of inorganic phosphate using the Fiske and Subbarao method (1925) Amount of inorganic phosphate released per mg protein per minute = Control pi minus Test

 

In-vitro Acetylcholinesterase activity:17,18,19

The basic idea behind the method is to "measure the rate of thiocholine production when acetylthiocholine is hydrolyzed." The yellow anion of 5-thio-2-nitrobenzoic acid is produced by continuously reacting the thiol with 5-dithiobis-2-nitrobenzoate ion(I) (II). At 412nm, photometry measures the rate of colour production.

 

Method for estimating RBC-bound acetylcholinesterase activity:

The number of moles of substrate hydrolysed per minute per RBC = (4.41) (l0-14)A/ RBC In which case, 4.41 (l0-1: The acetylcholinesterase activity on the RBC membrane is measured using a fairly stable suspension made from whole blood or washed human erythrocytes. The Aches test was performed as follows: The blood cells were suspended in phosphate buffer (pH 8.0, 0.1 M). A cuvette was filled with 3.0 ml of the suspension. A total of 25 ul of DTNB reagent was added. The photometer was loaded with the cuvette. The photometer's slit was adjusted so that the absorbance of the suspension in the cuvette at 412nm was zero. This cuvette received 20ul of substrate. Changes in absorbance at 412nm were measured every 2 minutes for 20 minutes intervals. 4) = dilution factor, extinction coefficient, and unit changes A = absorbance change per minute, RBC = number of red blood cells (in millions per mm3).

 

Statistical analysis:

Using the trial version of Graph Pad Prism, San Diego version (Prism graph pad version 8.0.2), the experimental data were statistically analysed using ordinary one-way ANOVA followed by Tukey's multiple comparisons test (263, GraphPad Software, Inc. La Jolla, CA USA).

 

RESULTS:

Table: The Percentage yieldand weight/ml of fresh leaf juice of Bryophillum pinnatum (Lam.)

S. No.

Material

% Yield

(ml/100 gm)

wt/ml

1.

Leaf juice of Bryophillum pinnatum (Lam.).

51.6 ± 2.13

200 mg/ml of juice
The value expressed were the Mean ± SEM of 3 observations, (n=3).

 

Table No. Effect of FLJBP treatment on Na+/K+ ATPase, acetylcholinesterase and hexokinase activity in liberating inorganic Phosphate (Pi)

S. No.

Group

Na+/K+ ATPase (Amount of Pi (µg of Pi liberated/min/mg of protein)

Acetylcholinesterase activity (Units/min/mg of protein)

Hexokinase activity (Units/min/mg of protein)

1

Normal Control

60.247±0.061

66.767±1.271

5.480±0.315

2

PHZ Treated

10.745±0.495

23.610±0.370

1.033±0.109

3

FLJBP Treated (20 µg/ml)

23.117±0.536****

26.27±0.615ns

1.337±0.066 ns

4

FLJBP Treated (40 µg/ml)

31.370±0.494 ****

34.153±0.483 ns

1.440±0.053 ns

5

FLJBP Treated (60 µg/ml)

38.467±0.371****

41.473±0.596****

1.900±0.072 ns

6

FLJBP Treated (80 µg/ml)

48.600±0.775****

53.40811±0.918****

1.840±0.062 ns

7

FLJBPTreated(100µg/ml)

58.153±0.654****

60.513±0.376****

2.503±0.093****
The values expressed were the Mean ± SEM of 3 observations (n=3)

Indicates significant when compared with PHZ     treated., (p<0.0001)

 

No activity: Effect of leaf juice of Bryophillum pinnatum (Lam.). on nitric oxide concentration in Goat RBCs20

Table No. Effect of FLJBP treatment on NO concentration in Goat RBCs

S. No.

Group

No concentration (µmoles of nitrate/mg of protein)

1

Normal Control

6.210±0.184

2

PHZ Treated

18.470±0.447

3

FLJBP Treated (20µg/ml)

17.943±0.509 ns

4

FLJBP Treated (40µg/ml)

15.853±0.302 ns

5

FLJBP Treated (60µg/ml)

14.353±0.262 ns

6

FLJBP Treated (80µg/ml)

12.597±0.697****

7

FLJBP Treated (100µg/ml)

10.267±0.378****

 

 

 

 

A. Effect of FLJBP treatment on Na+/K+ATPase activity in liberating inorganic phosphate

 

B. Acetylcholinesterase activity in FLJBP treated goat RBC membranes

 

C. Nitric oxide concentration in FLJBP treated in Goat RBC

D. effect of FLJBP treatment on hexokinase activity treated goat RBCs

Fig: Effect of Bryophillum pinnatum leaf juice on activity of various enzyme activities and NO scavenging activity.

 

DISCUSSION:

The plant Bryophillum pinnatum (Lam.) belonging to Crassulaceae family with numerous reported pharmacological activities and fresh leaf juice of this plant was investigated for in -vitro anti haemolytic study as crude consumption of certain leaf juices is the continued trend since ancient times. The leaves of the plant were fleshy hence the good yield of juice from the leaves (51.6ml/100gm isobvious and the quantity of the juice is not an indication of availability of proteins and other phytochemicals. The wt./ml value obtained i.e., 200mg/ml enabled to prepare intended dilutions of FLJB over a specific concentration range like 20, 40.60, 80 and 100μg/ml for in-vitro studies. The total protein concentration of FLJBP was approximately 10.65mg/ml confirms the presence of proteins and hence serves as source of energy. RBCs are among the most vulnerable cells to oxidative stress because of their high concentration of polyunsaturated fatty acids (PUFA) in the membrane and oxygen transport, both of which promote ROS generation. The purpose of this study was to see if FLJBP.is capable of protecting the cytoskeletal architecture of RBC membranes from phenyl hydrazine-induced oxidative stress in goat RBCs.

 

Phenyl hydrazine (PHZ) known to enhance the levels of ROS in erythrocytes hence selected as model for our study. Administration of PHZ can induce peroxidative lipid damage of erythrocyte membrane, glutathione and ATP depletion, cation imbalances, reduced membrane deformability etc. resulting in the accumulation of MDA and alteration of cytoskeletal protein, subsequent haemolysis and hence an excellent in-vitro model model to ascertain the mechanism of haemolytic anaemia.

 

The Na+/K+ -ATPase is the intrinsic membrane protein responsible for pumping Na+ and K+ against their membrane gradient. Since the Na+/K+ -ATPase is an essential enzyme of the plasma membrane of animal cells and ROS are known as the to inhibit Na+/K+ -ATPase activity. The present study was to determine the structural integrity ofred blood cells by assessing the activity of Na+/K+ -ATPase inerythrocytes.

 

The amount of inorganic phosphate liberated is 62.21µg of Piliberated/min/mg of protein as measured by ANSA reagent in Normal group indicated that the membrane architecture was preserved. The lowest amount of inorganic phosphate liberated was 10.754µg of Pi liberated/min/mg of protein is reflective of extensive free radical induced damage of PHZ.

 

The dose dependent increase in inorganic phosphate liberation, i.e., 23.11, 3137, 38. 471, 48.66, and 58.153g of Pi liberated/min/mg of protein by various FLJBP treated groups at concentrations of 20, 40, 60, 80, and 100g/ml, clearly demonstrated the substance under investigation's (FLJBP) protective effect against PHZ induced erythrocyte membrane damage. The acetylcholinesterase activity of normal group goat RBC is 66.767Units/min/mg of protein, indicating that the membrane is intact and thus the enzyme is catalytically efficient. The AchEase activity of the PHZ-treated RBC is 23.610units. The dose-dependent increase in acetylcholinesterase activity, i.e., 26.27, 34.15. 41.47, 53.40, and 60.51 units at concentrations of 20.40, 60, 80, and 100g/ml, indicated that FLJBP protects against RBC membrane damage. The FLJBP's antihaemolytic activity is indicated by the decrease in NO quantification under its effect and the dose-dependent rise in inorganic phosphate quantification with respect to hexokinase activity.

 

In literature, Bryophillum pinnatum (Lam.) contains numerous pharmacologically active phytochemical constituents that, acting singly or in combination, may interfere with different molecular pathways to prevent haemolysis caused by phenyl hydrazine-induced oxidative stress

 

The combination of compounds in FLJBP that impart efficacies through their antioxidant phytochemicals may be the scientific basis for this anti-haemolytic activity. Therefore, FLJBP might havethe potential to protect the cellular membranes and other macro molecules by its potent anti-oxidant principles like flavonoids, tannins etc thereby reduce the toxic effects of PHZ, affords protection to RBCs maintains cellular homeostasis. Bryophillum pinnatum (Lam.), also known as Kalanchoe pinnatum, stone crop, or orpine, is a Crassulaceae family member with three genera and nearly 54 species claimed to possess myriad pharmacological activities purposed to have potent health benefits21-22. Hence the present study substantiates the folklore claim of Bryophillum pinnatum  (Lam.) as plant of potential health benefits specifically by containing oxidative stress. Future studies were essential to substantiate the in-vitro findings to isolate the component actually responsible for the evaluated activities by a battery of pharmacological, phytochemical and bio analytical studies.

 

CONCLUSION:

The current in-vitro pharmacological screening using fresh leaf juice of Bryophillum pinnatum (Lam..) has revealed that a dose dependent increase in the catalytic activity of certain membrane bound and cytosolic metabolic enzymes such as Na+/K+ATPase, Acetylcholinesterase, hexokinase, and reduced NO concentration h indicates that fresh leaf juice of Bryophillum pinnatum (Lam.According to Hippocrates 400 BC (The Father of Medicine), "Let thy food be thy medicine, and medicine be thy food," this in vitro study requires further validation and supportive claims through a battery of rigorous pharmacological, photochemical, and bio analytical studies to fortify the intended claim about fresh leaf juice of Bryophillum pinnatum (Lam.).

 

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Received on 13.12.2022         Modified on 18.02.2023

Accepted on 21.04.2023     ©A&V Publications All right reserved

Res.  J. Pharmacology and Pharmacodynamics.2023;15(3):103-108.

DOI: 10.52711/2321-5836.2023.00019