Phytochemical Analysis, Cytotoxic and In vitro Antioxidant Activity of Erythrina variegate Bark

Aim: The objective of the present study was to investigate the phytochemical analysis, cytotoxic and antioxidant activities of the bark extracts of Erythrina variegate. Place and Duration of Study: The plant, E. variegate was collected from Botanical Garden, Mirpur, Dhaka and also from Curzon Hall, University of Dhaka, Bangladesh in June 2014 and then the sample was identified by the National Herbarium of Bangladesh, Mirpur, Dhaka (DACB; Accession Number36148). Methodology: Preliminary phytochemical screening was done for determining the nature of phytoconstituents. Brine shrimp lethality bioassay technique was done to study the cytotoxic Short Research Article Shahriar et al.; EJMP, 11(3):1-5, 2016; Article no.EJMP.18866 2 properties. Aluminum chloride colorimetric method was applied for the determination of flavonoids and the total antioxidant ability was assessed by the phospho-molybdenum method. Results: Primary phytochemical analysis confirmed the presence of alkaloid, carbohydrate, glycosides and flavonoids. The extracts showed some toxicity to A. salina with LC50 values ranging from 1.41 to 3.66 μg/ml which was compared with standard vincristine sulphate (VS, LC50 value 0.92 μg/ml). n-Hexane extract of E. variegate was found to contain highest amount of flavonoids (3.77±1.97 mg/gm; quercetin equivalent) and ethanol extract of E. variegate was found to have highest antioxidant acitivity (2.03±0.09 mg/gm ascorbic acid equivalent). Conclusion: It can be concluded that the plant extracts of E. variegate possess several antioxidant activities which justifies its use as folk medicine.


INTRODUCTION
Traditionally medicinal plants are widely used for the treatment of many diseases. The plants may contain chemical compounds which have the property of drugs or can act as precursor for the synthesis of useful drugs [1]. The ancient literatures include many plants for their medicinal values and they have been found to be effective for the treatment of various diseases which play significant role in providing primary healthcare services to rural and poor people who cannot afford costly and rare synthetic drugs. Moreover, these plants can act as a major source of income by exporting medicinal plants to other countries [2].
Erythrina variegate belongs to Fabaceae family. The genus, Erythrina includes about 110 species which are collectively known as "coral tree". The coral tree is cultivated particularly as an decorative tree and as a shade and also as a soil improvement tree (it fixes nitrogen) for other trees and crops such as coffee and cacao [3]. The plant parts from E. variegate are widely used for the treatment of different disease complications. The leaves are employed for their antispasmodic, anthelmintic, laxative, diuretic, galactagogue and emmenagogue properties. They are also applied externally for dispersing venereal buboes, relieving pain and inflammation of the joints [4]. Its juice is used to the ear to relieve ear ache and is used as an anodyne in toothache. The bark is used for its astringent, febrifuge anti-bilious and anthelmintic properties; It is also useful in dysentery and as a collyriumin ophthalmia. The roots are emmenagogue [5].
The objective of the present study was to investigate the phytochemical properties, in vitro cytotoxic activity and in vitro antioxidant activities of the bark extracts of E. variegate.

Collection, Identification and Processing of Plant Sample
The bark extracts of Erythrina variegate was collected from the Botanical garden, Dhaka and Curzon Hall, University of Dhaka in June 2014 and was taxnomically identified with the help of the National Herbarium of Bangladesh, Mirpur-1, Dhaka (DACB; Accession Number-36148). Bark was sun dried for seven days. The dried bark were then ground in to coarse powder using high capacity mixer grinding machine (Jaipan designer mixer grinder, jaipan, India), which was then stored in air-tight container with necessary markings for identification and kept in cool, dark and dry place for the investigation.

Extraction Procedure
The powdered plant parts (500 gm) were successively extracted in a soxhlet extractor at elevated temperature using 250 ml of distilled methanol (40-60)ºC which was followed by ethanol, chloroform, petroleum and n-hexane. After extraction all extracts were labeled and kept in refrigerator at 4ºC for future investigation.

Preliminary Phytochemical Screening
One gram of the methanol extract of Erythrina variegate was dissolved in 100 ml of methanol and was subjected to preliminary phytochemical screenings for determining nature of phytoconstituents [5,6].

Brine Shrimp Lethality Bioassay
Brine shrimp lethality bioassay [7,8] technique was applied for the determination of general toxic properties of the plant extracts. Dimethylsulfoxide (DMSO) solutions of the samples were applied against Artemia salina in a 1-day in vivo assay.
For the experiment, 1 mg of each extracts were added with 5 ml of sea water. Concentration was found to be 200 µg/ml. Then 50 µl DMSO was added to these and sample was prepared. Then the solution was serial diluted to 100, 50, 25, 12.5, 6.25, 3.125, 1.563 µg/ml with sea water. Then 2.5 ml of plant extract solution was added to 2.5 ml seawater containing 10 nauplii. Vincristine Sulphate (VS) was used as positive control.

Determination of Total Flavonoids Content
Aluminum chloride colorimetric method was used for flavonoids determination [9].

Determination of Total Antioxidant Capacity
The total antioxidant capacity was evaluated by the phosphomolybdenum method [10,11]. 0.3 ml of extract and sub-fraction in ethanol, ascorbic acid used as standard (5-200 µg/ml) and blank (ethanol) were combined with 3 ml of reagent mixture separately and incubated at 95ºC for 90 minutes. After cooling to room temperature, the absorbance of each sample was measured at 695 nm against the blank. The antioxidant activity is expressed as the number of equivalents of ascorbic acid and was calculated by the following equation: where, A = total content of Antioxidant compounds, mg/gm plant extract, in Ascorbic acid Equivalent, c = the concentration of ascorbic acid established from the calibration curve, mg/ml, V = the volume of extract in ml, m = the weight of crude plant extract, gm.

Preliminary Phytochemical Screening
In primary phytochemical screening, methanol, ethanol, pet-ether and chloroform extracts of E. variegate was found to contain alkaloid, carbohydrate, glycosides and flavonoids although the saponin was absent in chloroform, pet-ether and n-hexane extracts. Phenols, steroids and tannins were absent in all the extracts (Table 1).

Brine Shrimp Lethality Bioassay
In the brine shrimp lethality bioassay the LC 50 value of the test samples after 24 hours was obtained by a plot of percentage of the shrimps died against the logarithm of the sample concentration. The best-fit line was obtained from the curve data by means of regression analysis.
Vincristine Sulphate (VS) was used as positive control, LC 50 was found to be 0.92 µg/ml.

Total Flavonoid Content
Aluminium chloride colorimetric method was used to determine total flavonoid content of different extracts of E. variegate. Total flavonoids content was determined using the standard curve of quercetin (y = 0.690x -0.689; R² = 0.761) and was expressed as Quercetin Equivalents (QE) per gram of the plant extract. N-Hexane extract of E. variegate was found to contain the highest amount of flavonoids (Table 3).

Total Antioxidant Capacity
Total antioxidant capacity of the different extracts of E. variegate was evaluated by the phosphomolybdenum method and was expressed as ascorbic acid equivalents per gram of plant extract. Total antioxidant capacity of the test samples was calculated using the standard curve of ascorbic acid (y = 1.076x -0.808; R 2 = 0.953). The ethanol extract of was found to possess highest amount of antioxidant (Table 4).

CONCLUSION
After the screening of the phytoconstituients showed the presence of glycosides, carbohydrates, alkaloids, saponins, flavonoids and steroids. The n-hexane extract of the plant was found to possess the maximum (6.65 mg/gm) antioxidant capacity. The extracts revealed some total antioxidant capacity. On the other hand the methanol extract was found to contain highest (11.21 mg/gm) amount of flavonoids. From the results of the present study, it can be concluded that the plant extracts of E. variegate possess some antioxidant activities and this finding justifies its use as folk medicine.

CONSENT
It is not applicable.

ETHICAL APPROVAL
It is not applicable.