Assessment of Antioxidant and Antiangiogenic Activities of Ethanolic Root extract of Cassia occidentalis

 

G. V. N. Kiranmayi*, L. Anil Ricky, L. Sandeep Kumar, M. Lalitha Kala, M. Krishna Vamsi, M. Sai Sureshma, M. Vishnu, M. Kiran Sai

Aditya College of Pharmacy, ADB Road, Surampalem, 533437, East Godavari District India

 

ABSTRACT:

Context: The process by which new blood vessels grow is termed as Angiogenesis. In the adult, except for a few physiological processes such as menses, wound healing, and placental formation, all angiogenic processes are pathologic. Angiogenic imbalance in which proangiogenic factors predominate over antiangiogenic factors is tumor angiogenesis. Inhibition of blood vessel growth, is a way to prevent tumour growth and other angiogenesis-dependent diseases is antiangiogenesis. Aim: The present study aims to determine the antioxidant and antiangiogenic activities of ethanolic root extract of Cassia occidentalis. Materials and methods:  Antioxidant activity was evaluated against various free radicals like Nitric oxide scavenging, Hydroxyl radicals and Phosphomolybdenum assay using Ascorbic acid as standard. The most common study used to evaluate the antiangiogenic activity is chick embryo chorioallantoic membrane (CAM) , an in vivo assay. Results: Ethanolic root extract of Cassia occidentalis exhibited a significant antioxidant status which is evident from their IC50 values 21.42 ug/mL in Nitric oxide scavenging method, 22.3 ug/mL in hydroxyl radical. In phosphomolybdenum assay, there is concentration-dependent increase in absorbance. The results obtained are similar to that of standard drug ascorbic acid. In anti-oxidant activity, percentage inhibition was determined by calculating IC₅₀ values . By decreasing the capillary networks proliferation in a dose (50 to 150 μg/egg) dependent manner which is probably related to the inhibition of neovascularization. The present study results indicated significant antiangiogenic effect on chorioallantoic membrane. Conclusion: The plant extract has shown good free radical scavenging activity indicative from their percentage inhibition. It also exhibits significant antiangiogenic activity, and this is a possible rationale for its folkloric use as an anticancer agent.

 

 

 

 

 

INTRODUCTION:

Tumor angiogenesis is the consequence of an angiogenic imbalance in which proangiogenic factors predominate over antiangiogenic factors.^1,2 Angiogenesis is the process by which new blood vessels grow. In the adult, except for a few physiological processes such as menses, wound healing, and placental formation, all angiogenic processes are pathologic.^3-5 By blocking the development of new blood vessels, one hopes to cut off the tumor’s supply of oxygen and nutrients and, therefore, its growth and spread to other parts of the body.^6-8 The current ongoing researches are approving the plant can be useful in malignancies and at various levels and with different mechanisms.⁹

 

C. occidentalis is a small herb that grows all over India. The plant is generally found at an altitude of 1,000 m in regions of Himalaya. The plant has been found in Andaman & Nicobar Island, Assam, Bihar, Kerala, Maharashtra, Rajasthan, Tamil Nadu, Tropical America and it is Native of South America; naturalized in Asia. Cassia species includes Cassia javanica, Cassia grandis, Cassia roxburghii, Cassia abbreviata, and Cassia notabilis respectively.

 

Therefore, the present investigation carried out an in-depth study regard the C. occidentalis ethanolic root for its antioxidant and antiangiogenic activities.

 

MATERIALS AND METHODS:

Collection of plant:

Cassia occidentalis roots were collected from Peddapuram area of East Godavari dt., of Andhra Pradesh. The plant authentication was done by Dr. T. Raghuram Taxonomist, Maharani College, Peddapuram.

 

Preparation of extract:

The freshly collected Cassia occidentalis plant roots were washed, cleaned, then dried under shade for about 30 days and shade dried roots were chopped into small pieces and ground well to coarse powder. The dried powder material was then subjected to maceration with ethanol (95%) for 72hours and extracted by hot percolation. The obtained concentrated product was dried using desiccators.

 

Chemicals and instruments:

Ascorbic acid, Hydrogen peroxide, Methanol, Sulphanilamide, ortho phosphoric acid, naphthylethyl enediamine dihydrochloride, sulphuric acid, Sodium phosphate, Ammonium molybdate, Sodium salicylate, Ferrous sulphate, prednisone. Incubator, U.V Spectroscopy, Colorimeter, Hot air oven.

 

Preliminary phytochemical screening:

Preliminary phytochemical screening of Cassia occidentalis extract was done to test the presence of the active chemical constituents such as alkaloids, flavonoids, tannins, phenolic compounds, saponins, fixed oils and fats¹⁰.

 

Quantitative Phytochemical testing:

Aliquots of extract was prepared by dissolving 10mg of individual extracts in 10 mL of methanol to get 1000μg/ml.

 

Estimation of Phenolic Contents:

The phenolic content of ethanolic root extract of Cassia occidentalis (1 mg/ml, aliquots) was determined by using the method Folin-Ciocalteu¹¹. 0.5ml aliquots of extract was mixed with 3ml Folin-Ciocalteu reagent (1:10 v/v) and the mixture allowed to stand for 5min. 4ml of 20% w/v of sodium carbonate solution was added into the mixture tube. The tubes were kept aside for 15 min at 30⁰C for colour development. The absorbance was measured at 765nm by spectrophotometer. Phenolic content was estimated from the calibration curve using standard gallic acid in methanol and the results were expressed as gallic acid equivalent mg/100mg dry weight of extract.

 

Estimation of total Flavanoids:

Total flavonoid content of ethanolic root extract of Cassia occidentalis (1 mg/ml, aliquots) was determined by aluminium chloride method¹². To 0.6ml of aliquots of extract,1.8ml of methanol, 0.1mL of 10% aluminium chloride, 0.1ml of 1M sodium acetate and 3ml of distilled water were added and left at 30⁰C. The absorbance was measured individually after 30 min at 415 nm. Total flavonoid was estimated from the calibration curve using standard quercetin in methanol and the results were expressed as quercetin equivalent mg/100 mg dry weight of extract.

 

Estimation of alkaloids:

Alkaloid content in the extract was determined by using the method of Fazel et al., The ethanolic root extract of Cassia occidentalis (1 mg/mL, aliquots) was dissolved in 2N Hydrochloric acid and filtered. To the filtrate was added 0.1 N NaOH, from this solution 1ml was transferred to a separating funnel and then 5 ml of bromo cresol green solution was added along with 5 ml of phosphate buffer. The mixture was shaken extracted with chloroform. The absorbance was noted at 470 nm. Alkaloid content was estimated from the calibration curve using standard atropine calibration curve, measuring the concentration of alkaloid content in atropine equivalents using the units mg/100mg dry weight of extract¹³.

 

Evaluation of In-vitro anti-oxidant activity:

Hydroxyl Radical Scavenging Assay:

The scavenging ability of the five sample extracts on hydroxyl radicals was determined according to the method described by Smirnoff and Cumbes¹⁴ with some modifications. Briefly, individual sample extracts (1 mL) at different concentrations (50, 100, 300, and 500 μg/ml) were added to the reagent containing 1 mL 1.5 mM FeSO4, 0.3 mL 20 mM sodium salicylate and 0.7 mL 6 mM H2O2. Later on the sample was incubated at a temperature of 37°C for 1 h and absorbance of the reaction mixture was read at 562 nm.

 

Scavenging ability on hydroxyl radicals (%) =

[(Ao–A1)/Ao] ×100

 

 

Where, Ao indicates the control reaction (containing all reagents except the sample extract) absorbance, and A1 is the sample extract absorbance. Ascorbic acid was used as positive controls.

 

Phosphomolybdenum Antioxidant Assay:

The antioxidant activity of the extract was evaluated by the phosphomolybdenum method according to the procedure¹⁵. In this method, occurs the reduction of Mo (VI)–Mo (V) by the extract and subsequent formation of a green phosphate/Mo (V) complex at acid pH. 0.3 ml of extracts (50, 100, 300, and 500 μg/ml) was combined with 3 ml of reagent solution (0.6 M sulfuric acid, 28 mM sodium phosphate, and 4 mM ammonium molybdate). The reaction solution was incubated at a temperature of 95°C for 90 min. The solution absorbance using spectrophotometer against blank after cooling to room temperature at 695 nm was measured.

 

NO scavenging activity:

The scavenging effect of the extract on NO was measured according to the method of Marcocci and colleagues¹⁶. Briefly, sodium nitroprusside (5 mM) in phosphate-buffered saline(PBS) (pH 7.4) was mixed with different concentrations of the test sample (100–1000 μg/ml) and incubated at 25°C for 150 minutes. After incubation, nitrite produced from sodium nitroprusside was measured by Griess reagent (1% sulfanilamide in 5% phosphoric acid and 0.1% 1-naphthylethylenediamine dihydrochloride in water). The absorbance was immediately read at 570 nm. Catechin was used as a positive control.

 

NO scavenging activity (%) = [(Ao–A1)/Ao] ×100

 

Where,

Ao indicates the control reaction (containing all reagents except the sample extract) absorbance, and

A1 is the sample extract absorbance. Ascorbic acid was used as positive controls.

 

Chicken egg Chorioallantoic Membrane (CAM) Assay ¹⁷

This is a novel in ovo angiogenesis assay used for the estimation of angiogenic and antiangiogenic agents. Fertilized leghorn chicken eggs were collected from a local hatchery at day’0’ and checked for the damage. They were randomly grouped, each containing 6 eggs. The eggs were cleaned with 70 % ethanol and incubated under condition of constant humidity at 37⁰C.

 

 

 

On the 3rd day a small hole was drilled and 2-3 ml of albumin was withdrawn. Later on the particular window was sealed with transparent tape and again kept for incubation. Again one 7th day, a small square window was opened in the shell and sterile gel foam was placed in the membrane.

 

The vehicle control group was impregnated with sterile normal saline; the standard and test groups were impregnated with their respective doses. The eggs were returned to the incubator and they were incubated undisturbed till day 14. On 14th day of incubation the eggs were removed from the incubator and the CAM tissues directly beneath each sponge was removed from control and treated CAM samples. Later on Tissues were placed in formalin followed by staining with hematoxylin and eosin, and then observed under Trinocular microscope.

 

The vessel branch points in the square region equal to the area of each sponge were counted and findings from CAM preparations were analyzed for each treatment group.

 

Eggs were divided into the following groups-

·      Group 1: Normal control (0.9% NaCl )

·      Group 2: Positive control (Prednisone 5mg/ml)

·      Group 3: EECO 25μg/egg (EECO – Ethanolic extract of Cassia occidentalis )

·      Group 4: EECO 50 μg/egg

·      Group 5: EECO 75 μg/egg

·      Group 6: EECO 150 μg/egg

 

RESULTS:

Preliminary and Quantitative Phytochemical screening:

The results implies that extract contains alkaloids, flavonoids, saponins, carbohydrates, proteins and Amino acids which are the main phytochemical groups with biological activities. The results of Quantitative Phytochemical screening was tabulated (Table 1).

 

 

Table 1: Quantitative phytochemical determination of Ethanolic root extract of Cassia occidentalis

S. No	Total Phenolics mg/g	Total flavanoids mg/g	Total alkaloids mg/g
Ethanolic root extract of Cassia occidentalis	11.04±0.25	42.06±0.43	35.12±0.38

All the values are expresses ad mean ± SEM, n=3

 

 

 

In-vitro anti-oxidant activity:

Table 2: Effect of Ethanolic Cassia occidentalis root Extract against NO and hydroxyl radical

Tested Material	Concentration ( µg/ml)	No method		Hydroxyl method
		%Inhibition	IC50 (ug/ml)	%Inhibition	IC50 (ug/ml)
Cassia occidentalis Ethanolic root extract	50	50.71±0.001	44.77	44.8±0.06	57.27
	100	62.22±0.005		62.76±0.16
	300	75.41± 0.004		80.52± 0.04
	500	83.74± 0.007		85.36±0.15
Ascorbic acid	50	55.36 ±0.22	33.11	69.47±0.32	1.69
	100	60.32±0.27		82.55±0.26
	300	70.85±0.13		83.46±0.12
	500	80.32±0.12		87.65±0.16

Values are indicated in terms of Mean ± SEM; n=3 in each concentration;

 

Table 3: Effect of Ethanolic Cassia occidentalis root Extract using Phosphomolybdenum method

Tested Material	Concentration (µg/ml)	Phosphomolybdenum method
Cassia occidentalis Ethanolic root extract	50	0.012±0.0015
	100	0.027±0.0013
	300	0.102±0.0009
	500	0.198±0.0012
Ascorbic acid	50	0.181±0.032
	100	0.381±0.018
	300	0.621±0.023
	500	0.973±0.036

Values are indicated in terms of Mean ± SEM; n=3 in each concentration;

 

Table 4: Antiangiogenic activity of ethanolic extract of Cassia occidentalis root

Tested Material	Egg 1	Egg 2	Egg 3	Average No. of Vessels	Egg 1	Egg 2	Egg 3	Average No. of Vessels
	No. of Vessel in untreated CAM				No. of Vessel in treated CAM
Normal control (0.9% NaCl )	8	10	7	8.33	9	10	7	8.67
Positive control (Prednisone 5mg/ml)	12	9	10	10.33	3	4	3	3.33
EECO 25μg/egg	11	9	8	9.33	9	7	6	7.33
EECO 50μg/egg	10	10	12	10.67	7	5	5	5.67
EECO 75 μg/egg	8	7	9	8.00	5	4	4	4.33
EECO 150 μg/egg	8	9	9	8.67	4	3	4	3.67

 

Table 5: Anti-angiogenic effect of of ethanolic extract of Cassia occidentalis root

Tested Material	Egg 1	Egg 2	Egg 3	Average Percentage of vessels inhibition (%)
	Percentage of vessels inhibition (%)
Normal control (0.9% NaCl)	-12.5	0	0	4.17±4.17
Positive control (Prednisone 5mg/ml)	75	55.55	70	66.85±5.83
EECO 25μg/egg	18.18	22.22	25	21.8±1.98
EECO 50μg/egg	30	50	58.33	46.11±8.41
EECO 75 μg/egg	42.8	28.5	55.55	42.28333±7.82
EECO 150 μg/egg	50	66.66	55.55	57.40333±4.90

Values are expressed as mean ± SD, (n=3)

 

DISCUSSION:

Phytochemical tests are very helpful in identifying therapeutically valuable new source of compounds having importance and to make the natural wealth available for their best use. For this, different phytochemicals were extracted and highlighted by different methods; The results implies that extract contains alkaloids, flavonoids, saponins, carbohydrates, proteins and Amino acids which are the main phytochemical groups with biological activities. The results of Quantitative Phytochemical analysis was tabulated in table 1.

 

The results of antioxidant activity were expressed in terms of IC₅₀ values using different antioxidant methods. The calculated IC50 values using Nitric oxide and Hydroxyl radicals method for Cassia occidentalis are 44.77 μg/ml and 57.27 μg/ml and for ascorbic acid it is 33.11 and 1.69 μg/ml . The results are indicated in Table 3. There is an concentration dependent increase in absorbance in Phophomolybdenum antioxidant assay. From the results obtained it may be postulated that Cassia occidentalis root extract reduces the radicals to the corresponding hydrazine when it react with the hydrogen donor in the antioxidant principles. Free radical scavenging activity of the Cassia occidentalis is concentration dependent. Lower IC 50 value reflects better protective action.

The angiogenic properties of Cassia occidentalis root extract may be attributed due to the phytochemical present in these plants.

 

Angiogenesis is defined as new blood vessels Formation from the existing vascular bed, which plays an hallmarks of cancer. Because of various factors like genetically instability and tumor blood vessels to normal vessels difference, in all types of cancer they are potential targets in therapy. In many other pathological conditions, including diabetic retinopathy, inflammation, hemangiomas, arthritis, psoriasis and atherosclerosis, cancer seems to be driven by persistent upregulated angiogenesis.^18,19 Angiogenesis is a strictly controlled process in normal human body and regulated by a variety of endogenous angiogenic and angiostatic factors.²⁰

 

The investigation is based on the need for newer antiangiogenic agents from natural source with potent activity to substitute chemical therapeutics. Realizing the fact this study was carried out to evaluate the antiangiogenic activity of Cassia occidentalis root extract using CAM model in this direction.

 

Using chick CAM model, the new pharmacological effects of Cassia occidentalis root have been confirmed by the proven inhibition of angiogenesis. Cassia occidentalis root extract had significant antiangiogenic activity at all doses of treatment studied (Table 4); where these extracts of Cassia occidentalis root reduce neovascularization of the CAM as well as distortion of existing vasculature.

 

Folkman et al have reported, by blocking the development of new blood vessels, one hopes to cut off the tumor’s supply of oxygen and nutrients and, therefore, its growth and spread to other parts of the body.^21-23. This may be due to the induction of apoptosis by phytochemical present in these plants.²⁴

 

The results of the present study suggest that Cassia occidentalis root extracts exhibits strong antiangiogenic action in concentration dependent manner and also may have the potential to be a useful inhibitor of tumors and other angiogenesis-dependent diseases. It is due to the presence of active principles such as flavonoids and tritrepenoids may responsible for this activity. Hence, Cassia occidentalis can be used as a potent antiangiogenic agent. Nevertheless, further studies needed to substantiate these findings.

 

CONCLUSION:

The plant extract has shown good free radical scavenging activity indicative from their percentage inhibition. The plant used in this study could be potential sources of new anticancer agents.

 

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Received on 05.01.2019         Modified on 27.01.2019

Accepted on 09.02.2019      © RJPT All right reserved