Variation in Antioxidant Activity of a Rattan Species, Plectocomia himalayana Griff. by DPPH assay based on two different methods of Methanol Extraction.

 

Raksha Mukhia1, Bharat Basistha2, Dhani Raj Chhetri1*

1Department of Botany, Sikkim University, Tadong, Gangtok, Sikkim, India 737102

2Department of Science and Technology, Govt. of Sikkim, Gangtok, Sikkim, India 737102

*Corresponding Author E-mail: drchhetri@cus.ac.in

 

ABSTRACT:

Antioxidant plays an important role in inhibiting and scavenging free radicals, which provides protection to human against infection and degenerative diseases. At present research is directed towards “natural antioxidants” from the plants due to safe therapeutic. Therefore, in the present paper we have investigated antioxidant activity by DPPH method from the shoot extracts of Plectocomia himalayana. The solvent extracts of its shoots were found to have free radical scavenging activity by DPPH (2, 2-diphenyl-1-picrylhydrazyl) assay. Hot methanol and cold methanol extracts of the dried powder of the shoots of Plectocomia himalayana were used as sample for antioxidant profiling which was compared with two standards namely BHT (Butylated Hydroxyl Toluene) and Ascorbic acid. Studies suggested that the percentage inhibition at 100 μg/μl which was the lowest concentration was higher in cold extract and that of 800 μg/μl was slightly higher in case of hot extract. In general, shoot extracts of Plectocomia himalayana showed a relatively good free radical scavenging activity even when compared to BHT and ascorbic acid. Furthermore, the IC50 value, the half maximal inhibitory concentration, for cold methanol extract and hot methanol extract of the shoots of Plectocomia himalayana was found to be 117.73 (μg/μl) and 163.16 (μg/μl) respectively.

 

KEYWORDS: Plectocomia himalayana, antioxidant, DPPH, free radical, hot and cold methanol extract.

 

 


INTRODUCTION:

‘Rattans’ are referred to the spiny, climbing species of palms[1]. They belong to the family Arecaceae and sub family calamoideae [2, 3]. In India four genera of rattans are found, viz. Calamus, Daemonorops, Korthalisa and Plectocomia with 60 species of which 32 species are endemic to the country.

 

The rattan genus Plectocomia is known to have a total of 16 species, and is found in Himalayas and South China to Western Malaysia[4]. Among its species himalayana is known from Bhutan, China, India, Lao, Nepal and Thailand[5].

 

Plectocomia himalayana Griff. regionally known as ‘bet’ (Nepali), is commonly cultivated from the wild for its edible shoot. It has a climbing habit and is found in the Himalayas at an elevation ranging from 1500–2500 m. In India it has been reported to occur in Sikkim, West Bengal and Arunachal Pradesh (Kurung Kumey District) [6]. This species is harvested both for its edible shoot and also for its cane, which is used in making strong ropes, split into strips for tying fences and for making rough baskets. Thicker cane is used for making furniture like walking sticks, chair, baskets, small bag, container, decorative, etc.[7]. Inner pith of the young shoots are eaten which are locally known as bet tussah (Nepali).

 

Worldwide rattan has many beneficial uses like food, craftsmanship, housing material etc., similarly it has potent medicinal properties too, generally, roots and fruits of rattan are mostly used for medicinal purposes. Johnson, 2010 reported that roots of Calamus casteneus, C. longispathus, Daemonorops didymophylla are traditionally used as medicine[8]. The resin of rattan is used for the coagulation of blood during cuts and wounds. Similarly, young shoots of C. erectus, C. floribundus and C. latifolius is a cure for stomach ulcer and muscular sprain[9]. Equally leaves are used for treating blood impurities, hypertension, cancer, anti-aging, congestive heart failure, antioxidants and cardiac arrhythmia[3]. Raw seed are used for curing common diseases like cough, edema, herpes, diabetes, rabies etc. [10]. Even the pulp of ripe fruit of Calamus floribundus is eaten during dyspepsia disease[9]. Apart from its traditional use in medicines rattan are also used in bone replacement material because of properties like porosity, pore size and mechanical stability which can be used in development of synthetic porous hydroxyapatite[11].

 

There is a dearth of information regarding the phytochemical constituents of rattans in general and Plectocomia himalayana in particular. However, phytochemical analysis of the related genera, Bambusa arundinacea revealed the presence of tannins, phlobatannins, flavonoids, cardiac glycosides, reducing sugar, phenols in its seed extract[12]. The shoots were rich in proteins, saccharides, amino acids, minerals, low in fat, inorganic salts and the water content found as high as 90%. Studies revealed that shoots were rich in dietary fiber and low in calories[13]. In addition, bamboo leaf is a good source of natural antioxidants and the ethanolic extract from the leaves of Bambusa arundinacea showed relatively high DPPH scavenging activity[14].

 

Many mechanical, morphological and anatomical studies have been carried out in Plectocomia himalayana but available information on the biochemical and phytochemical properties on this particular species is very less and research is needed to determine such properties to better understand and utilize it. Therefore shoots of Plectocomia Himalayana is used in this experiment for screening the anti-oxidant potential through DPPH method.

 

MATERIALS AND METHODS:

Collection of Plant Material:

Edible rattan shoots were brought from Dzongu, North Sikkim. The barks were removed; the core region was then washed properly and cut into small pieces. The initial weight was taken (W1), and then they were left for shade drying. After 15 days of drying, the final weight was taken (W2). The dried material was ground to fine powder which was used for further extraction [15].

 

Extraction:

Hot methanol Extraction:

14.209gms of the powdered material was taken in a conical flask and 100ml of methanol was added to it. Then the extraction was carried out in soxhlet apparatus which was run continuosly for 8-hours at 80˚C. Then it was left for a couple of minutes at room temperature for cooling and then was filtered in Whatman no. 1 filter paper and the extract obtained was collected. The extract was further left for evaporation after which the semi-solid extract obtained was stored until further studies.

 

Cold Methanol Extraction:

14.209gms of the powdered material was transferred in a round bottom flask and 100ml methanol was added and it was kept at room temperature for 24hrs. Then the extract was taken out, cooled at room temperature and filtered in Whatman no.1 filter paper.  The filtrate was evaporated in the rotatory evaporator (Buchi) at 30˚C till all the solvent was evaporated to give a thick semi solid extract. It was then dried in open air at room temperature till all the solvent was evaporated and the solid extract was weighed and stored for further studies [15].

 

Antioxidant Assay by DPPH method:

Both hot and cold solid extracts of Plectocomia himalayana was taken 50mg each, separately and dissolved in 50ml of methanol to make a concentration of 1mg/ml and used for free radical scavenging activity by DPPH assay. Likewise, standard solutions of BHT and Ascorbic acid were prepared by dissolving them separately in methanol to give 1mg/ml concentration respectively.

 

Free radical scavenging activity was carried out following the method of Cheel et al; 2007 [16], with slight modifications. 0.006 mM of DPPH solution was freshly prepared in methanol and stored in dark reagent bottle. Different concentration of hot and cold extract sample solution was prepared by adding 100μl, 200μl, 300μl, 400μl, 500μl, 600μl, 700μl, and 800 μl in test tubes respectively. Thereafter methanol was added to make the volume up to 1 ml. After that 2ml of freshly prepared DPPH solution was added to all the test tubes, shaken well, incubated in dark for 30 minutes and the absorbance measured at 517nm using Thermo Fisher Scientific, UV-Visible Spectrophotometer (Evolution 201). Methanol was used as blank and the solution of BHT and Ascorbic acid was taken as the standard and same procedure was carried out .The percentage scavenging activity was calculated using the formula given below:-

 

DPPH radical scavenging capacity (%) =

[(Abs control – Abs sample)] / (Abs control)] × 100,

 

Where,

Abs control is the absorbance of DPPH radical + methanol;

Abs sample is the absorbance of DPPH radical with sample extract or standard.

 

The half maximal inhibitory concentration (IC50) for scavengers i.e., the concentration of antioxidant sample required for 50% reduction in DPPH activity was calculated using Microsoft excel 2007.

 

 

Figure-1: Percentage scavenging activity of BHT and Ascorbic acid as standard and cold and hot methanol extract of shoots of Plectocomia himalayana.

 

Table 1:IC50 value of cold methanol and hot methanol extract of Plectocomia himalayana.

Sl. No.

Sample

IC50 (μg/μl)

1

Cold extract

117.73

2

Hot Extract

163.16

 

RESULTS:

Both cold methanol and hot methanol extract of the shoots of Plectocomia himalayana showed more or less similar antioxidant activity. Percentage inhibition of 100 μg/μl conc. of the cold extract which was the lowest concentration is 78.09 ± 0.026 and that of 800 μg/μl which was the highest concentration is 96.17 ± 0.001. Similarly in case of the hot methanol extract of the shoot of Plectocomia himalayana, the percentage inhibition of the lowest concentration 100 μg/μl was 77.02 ± 0.005 and that of highest concentration 800 μg/μl is 96.93 ± 0.014. Shoot extract of Plectocomia himalayana has less potential in free radical scavenging activity compared to the standards namely BHT and Ascorbic acid. Further the IC50 value for cold methanol extract and hot methanol extract of Plectocomia himalayana was found to be 117.73 (μg/μl) and 163.16 (μg/μl) respectively.

 

DISCUSSION:

Evaluation of antioxidant activity in plants is vital for discovery of natural antioxidants. A variety of free radical scavenging antioxidants is found in dietary sources like fruits, vegetables and tea, etc. [17] and regular consumption of antioxidants reduces the risk of chronic diseases and is vital for maintaining optimum health and well being[18].

 

DPPH method of screening the antioxidant is the most common and widely accepted method.  It is based on the ability of free radical 2, 2–diphenyl-1-picryl hydrazyl to react with hydrogen donors [19]. In this study percentage inhibition at 100 μg/μl which was the lowest concentration was higher in cold extract and that of 800 μg/μl was slightly higher in case of hot extract. Shoot extract of Plectocomia himalayana has shown relatively good potential in free radical scavenging activity. Further the IC50 value for cold methanol extract of Plectocomia himalayana was found to be 117.73 (μg/μl) which is indicative of higher antioxidative capacity as compared to the hot methanol extract which was 163.16 (μg/μl). In an experiment conducted by Karanja et al, 2015 [20] Bambusa vulgaris shoots had the highest IC50 value of 2.0mg/ml followed by Dendrocalamus giganteus at 3.0 mg/ml and Yushania alpina at 3.8 mg/ml. Similarly, IC50 value of the leaves of Artemisia dracunculus and Salvia sclarea was found to be 98.20 (μg/μl) and 96.70 (μg/μl) respectively and that of Dactylorhiza hatagirea flower was found to be 97.40 (μg/μl) which is comparatively higher than what we found in our sample[21].

 

Limited work has been done on the phytochemical aspect of Plectocomia himalayana and these observations could help in developing new drugs for various therapeutic uses in human beings. Results of this study suggest that the shoot extracts show relatively high antioxidant activities, which might be helpful in preventing or slowing the progress of various oxidative stress-related diseases. Further studies regarding the isolation and characterisation of the active principles responsible for these activities is currently in progress.

 

ACKNOWLEDGEMENT:

We thankfully acknowledge Department of Science and Technology (DST), Sikkim and Department of Botany, Sikkim University for the necessary assistance.

 

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Received on 13.02.2018       Modified on 23.02.2018

Accepted on 16.04.2018       ©A&V Publications All right reserved

Res.  J. Pharmacognosy and Phytochem. 2018; 10(2):175-178.

DOI: 10.5958/0975-4385.2018.00027.4