Effect of solvent on yield, phytochemicals and in vitro antioxidant potential of Rhododendron arboreum
Shefali Sharma1, Rupali Chaudhary1, Rajan Rolta1, Nitin Sharma2, Anuradha Sourirajan1, Kamal Dev1, Vikas Kumar1*
1Faculty of Applied Sciences and Biotechnology, Shoolini University of Biotechnology and Management Sciences, Bajhol, PO Sultanpur, Distt. Solan-173229 (HP), India.
2Department of Biotechnology, Chandigarh College of Technology, CGC Landran, 140307,
Mohali, Punjab, India.
*Corresponding Author E-mail: vikaskmr59@gmail.com
ABSTRACT:
Rhododendron arboreum (family- Ericaceae) is one among the medicinally and economically important plant species from Himalayan region. Aim of present study was to study the effect of solvent on extraction yield, phytochemicals and antioxidant potential of various parts of R. arboretum. Extract of various solvents (n-hexane, methanol and water) were prepared using cold maceration method. Spectrophotometric method was used to quantify total phenolic and flavonoid content of different extracts. DPPH, FRAP and ABTS method was used to evaluate antioxidant activity of different extracts of various parts of R. arboreum. Among all solvent extract of different part of R. arboretum, highest extraction yield was observed in aqueous extract of leaf (10.78%) followed by methanolic extract of flowers (10.42%). Methanolic extract of flower showed higher amount of TPC (107.46±8.74 mg/g GAE) and TFC (254.26±29.33 mg/g RE). DPPH, FRAP and ABTS assay showed lowest IC50 value in flower extracts (methanolic and water) and hence validating the utilization of flower of this tree as source of natural antioxidants.
KEYWORDS: Rhododendron arboretum, Cold maceration, extraction yield, antioxidant activity.
INTRODUCTION:
People from hilly areas, use the flower of R. arboreum in the preparation of local brew, jellies, jams and squash. It is a very common and pleasing drink as a refreshing appetizer and tonic [2]. Flower extract is also effective against stomach diseases and snuffed to stop nasal bleeding [3]. Flower petals and leaves of R. arboreum also used for the treatment of a headache, fever, dysentery, rheumatism, wounds, and nose bleeding [4]. Consuming its root decoction cures early stage of cancer diseases [5]. Fresh leaves in combination with thuja/ pine/ juniper leaves are burnt for making smoke that is believed to be holy and help in purifying surrounding air [6]. Rhododendron arboreum flower, bark, and Young Shoots are used for the treatment of a digestive and respiratory disorder, tonic for the heart [7].
The various phytocompounds isolated from leaves of R. arboretum are quercitirine, epicatechin, synergic acid and quercitine-3-O-galactoside [8], while quercitine, coumaric acid and rutin [9]. Phytocompounds such as taraxerol, ursolic acid acetate, betulinic acid, leuco-pelargonidin have been identified in various extracts of bark [10]. Leaves of this tree are reported to contain glucoside, ericolin (arbutin), ursolic acid, α-amyrin, epifriedelinol, campanulin, quercetin and hyperoside [11]. Study from [12] showed the presence of hyperoside (3-D -galactoside of quercetin), ursolic acid and epifriedelinol. The leaves are also reported to contain the flavone glycoside and dimethyl ester of terephthalic acid and certain flavonoids 13. Flowers are reported to possess Quercetin-3-rhamnoside [14], quercetin, rutin and coumaric acid [15]. Keeping in view the importance of this medicinal tree, the present study was designed to compare the effect of solvent on extraction yield and in vitro antioxidant activity among different parts of R. arboreum.
MATERIAL AND METHODS:
Chemicals and solvents:
The chemicals such as 2,2-Diphenyl-1-(2,4,6-trinitrophenyl) hydrazyl (DPPH), 2,4,6-Tri(2-pyridyl)-s-triazine (TPTZ), 2,2′-Azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) were obtained from Sigma-Aldrich Co. LLC, Mumbai. The solvents such as n-hexane, methanol and dimethyl sulfoxide (DMSO) used were procured from Loba Chemie Pvt. Ltd., Mumbai.
Collection of plant materials and Extract preparation:
The flower, leaf and bark samples of R. arboreum were collected in the month of March (2019) from Kullu, Himachal Pradesh, India. All the plant materials (flower, leaf, and bark) were washed twice and allowed to dry at 40 °C. The dried plant material was converted into powder using electric grinder. Cold macerated method was used to prepare extracts of different solvents (n-hexane, methanol and water) [16 -18]. The dried extracts were stored at 4 °C in airtight bottles till further use.
Determination of total phenolic and flavonoid content
Total phenolic content (TPC) in various extracts of different parts of R. arboreum was determined by using Folin-ciocalteau reagent method [19] whereas total flavonoid content (TFC) of different solvent extracts of different parts of R. arboreum were quantified by using aluminum chloride (AlCl3) method [20].
In vitro antioxidant activity in various solvents extracts of different parts of R. arboreum
Antioxidant activity of various solvent extracts of different parts of R. arboreum was determined using methods such as DPPH, FRAP and ABTS method. To analyze the antioxidant potential of various extracts of different parts of R. arboreum, all the extracts were dissolved at a concentration of 1 mg/ml in ethanol and then diluted in order to prepare different concentrations (10-80 µg/ml) for antioxidant assays. Ascorbic acid was used as standard antioxidant compound for comparative analysis in all assays.
2, 2-Diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity
DPPH radical scavenging activity of various extracts of different parts of R. arboreum was measured by the method described by Barros et al. (2007) [21] and Rolta et al [22, 23]. The capability of scavenging DPPH radical was calculated using the following equation:
% DPPH radical scavenging activity =
{A(control)-A(sample)] / A(control)}× 100
where A (control)- Absorbance of control and A (sample) - absorbance of the test/standard.
Ferric Reducing Antioxidant Power (FRAP) assay
FRAP activity was calculated according to the method described by Benzie and Strain [24]. The antioxidant capacity of extract/standard was calculated from the linear calibration curve of FeSO4 (10 to 80 μM) and expressed as μM FeSO4 equivalents per gram of the extract.
ABTS scavenging assay:
ABTS scavenging activity was calculated using method described by Re et al. [25] Percentage ABTS scavenging activity was calculated as-
ABTS radical scavenging activity (%) =
[(Acontrol − Asample)]/(Acontrol)]× 100
where Acontrol is the absorbance of ABTS radical + methanol; Asample is the absorbance of ABTS radical + sample extract /standard.
Statistical analysis:
All analysis was done at least in duplicate/triplicates, and these values were then presented as average values along with their standard derivations. Subsequently, the IC50 values of the antioxidant assay were calculated from the linear regression method.
RESULTS AND DISCUSSION:
Extraction Yield
The extraction with solvents of increasing polarity resulted in extraction of phytocompounds of a plant according to their degree of solubility. The yield percent of hexane, methanol and water extracts obtained from various parts of R. arboreum was shown in Fig. 1. Methanolic extract of flower (11.225±1.14 %) showed highest % yield followed by water extract of leaf 10.5±0.4%, and water extract of flower (5.335±0.63%). Among all parts, bark showed least extract yield with all solvents. This shows that the extraction yield increases with increasing polarity of the solvent used in extraction.
The result of present study was in line with study from[26] on Orthosiphon stamineus and [27] on Limnophila aromatica.
Figure 1: Percentage extraction yield in solvent extracts of different parts of R. arboreum.
Quantification of TPC in various solvents extracts of different parts of R. arboretum:
TPC was calculated from standard curve of gallic acid (y = 0.023x - 0.0054) and expressed as mg/g GAE. Order of TPC was methanolic extract of Flower (107.46±8.74 mg/g GAE)>methanolic extract of bark (70.29±13.45 mg/g GAE)>water extract of flower (64.93±8.36 mg/g GAE)>hexane extract of flower (48.08±1.06 mg/g GAE)>water extract of bark (37.42±14.42 mg/g GAE)>hexane extract of bark (21.02±1.08 mg/g GAE)>water extract of leaf (17.98±2.88 mg/g GAE)>hexane extract of leaf (13.64±0.77 mg/g GAE)>methanol extract of leaf (9.70±0.58 mg/g GAE) (Fig. 2). In contrast to our results,[28] showed higher amount of TFC in methanolic extract of leaves (150.0±0.00 mg/g RE per gram extract), flower (103.8±2.16 mg/g RE per gram extract) and bark (51.3±6.49 mg/g RE per gram extract). The amount of TPC in Ethanolic extract of flowers was 65.50 ± 1.12 mg GAE/ g). [29]
Figure 2: Total phenolic content in various solvents extracts of different parts of R. arboreum. Gallic acid was used as standard. Phenolic content was expressed as Gallic acid equivalents.
Quantification of TFC in various solvents extracts of different parts of R. arboretum:
Amount of flavonoid was calculated from standard curve of rutin (y = 0.0044x + 0.007) and expressed as mg/g RE. Order of TFC was methanolic extract of Flower (254.26±29.33 mg/g RE)>water extract of leaf (244.26±15.15 mg/g RE)>methanolic extract of leaf (155.114±2.41 mg/g RE)> )>hexane extract of flower (135.12±2.42 mg/g RE) hexane extract of flower (105.68±15.27 mg/g RE)>methanolic extract of bark (95.68±1.12 mg/g RE)>water extract of bark (60.52±12.45 mg/g RE)>hexane extract of bark (35.33±1.08 mg/g RE)>water extract of flower (29.83±6.03 mg/g RE (Fig. 2). The amount of TPC in various parts of R. arboreum was also quantified by [28]. In contrast to our results, they showed higher amount of TPC in methanolic extract of leaves (495.0±8.66 mg/g GAE per gram extract), flower (440.0±0.00 mg/g GAE per gram extract) and bark 240.0±0.00 mg/g GAE per gram extract). Ethanolic extract of flowers showed 33.25 ± 0.89 mg RE/g flavonoid content. [29]
Figure 3: Total flavonoid content in various solvents extracts of different parts of R. arboreum. Rutin was used as standard. Flavonoid content was expressed as Rutin equivalents.
DPPH scavenging assay:
All the solvent extracts of different parts of R. arboreum showed concentration-dependent % DPPH radical scavenging activity (Fig. 4 A-C). Ascorbic acid was used as standard antioxidant compound. DPPH radical scavenging activity was expressed in terms of IC50 value. Among all extracts, methanolic (121.55±30.98 µg/ml) and water extract (141.98±3.7 µg/ml) of flower of R. arboreum showed least IC50, indicating higher % DPPH radical scavenging activity of methanolic and water extract of flower. Least antioxidant activity in terms of %DPPH scavenging was identified in hexane extract of leaf (232.07±12.53 µg/ml) and water extract of bark (229.06±15.44 µg/ml). (Ascorbic acid showed IC50 value of 55.58±1.07 µg/ml with DPPH radical scavenging assay (Table-1).
Figure 4: % DPPH radical scavenging activity of various extracts of different parts of R. arboreum. A: Flower; B: Leaf; C: Bark.
Figure 5: FRAP activity of various extracts of different parts of R. arboreum. A: Flower; B: Leaf; C: Bark.
Figure 6: ABTS radical scavenging of various extracts of different parts of R. arboreum. A: Flower; B: Leaf; C: Bark.
FRAP assay
FRAP activity also increased with increase in concentration of solvent extracts of different parts of R. arboreum as shown in Fig. 5 A-C. Among all extracts, methanolic (22.76±0.39µM) and water extract (33.78±1.39 µM) of flower of R. arboreum showed highest FRAP activity as indicated by least value of IC50. Least FRAP activity was shown by methanolic extract of leaf (333.52±30.14µM) and water extract of leaf (168.39±35.73µM). Ascorbic acid showed IC50 value of 56.29±1.97 µg/ml with FRAP assay (Table-1).
ABTS radical scavenging activity:
Fig. 6 A-C showed concentration dependent ABTS scavenging activity of different parts of R. arboreum. Ascorbic acid was used as standard antioxidant compound. Among all solvents extracts, methanolic (11.49±1.25 µg/ml) and water extract (15.64±1.26 µg/ml) of flower of R. arboreum showed highest ABTS radical activity as indicated by least value of IC50. Methanolic extract of leaf (30.39±0.544 µg/ml) and water extract of bark (27.24±2.17 µg/ml). Ascorbic acid showed IC50 value of 54.05±2.40 µg/ml (Table-1).
Different part of R. arboreum contains high value flavonoids and phenolics which can be used in nutraceuticals and preventive medicines [28]. Ethanolic extract of flower of R. arboreum possesses maximum antioxidant and nitric oxide synthase (NOS) activation properties which might be utilized as promising source of therapeutics [30]. However, ethanolic extract of leaves of R. arboreum also have in vitro antioxidant activity as evidence by the low IC50 values in the scavenging of DPPH (19.91 ± 1.73 μg ml−1) and ABTS (9.50 ± 0.50) hydroxyl radical methods [31]. Methanolic extract of leaves (50%) also show antioxidant activity as it contains high phenol [32]. Antioxidant nature of leaves of R. arboreum was also in agreement with study from [33, 34] Antioxidant activity of ethanolic extract of flower was also determined by using DPPH (134.1±2.34 mM TE/g) and FRAP assay (140.6±2.76 mM TE/g) by [35]. In the present study, flower extracts (methanolic and water) showed high antioxidant activity among all solvent extracts of various parts of R. arboreum, which may be due to higher amount of TPC and TFC in methanolic extracts of flower.
Table-1: Half maximal inhibitory concentration (IC50) of different extracts of R. arboreum in different antioxidant assays.
|
Plant part |
Type of extract |
IC50 |
||
|
DPPH* |
FRAP** |
ABTS* |
||
|
Ascorbic acid |
55.58±1.07 |
56.29±1.97 |
54.05±2.40 |
|
|
Flower |
Hexane extract |
184.01±4.9 |
57.04±6.58 |
25.82±7.09 |
|
Methanolic extract |
121.55±30.98 |
22.76±0.39 |
11.49±1.25 |
|
|
Water extract |
141.98±3.7 |
33.78±1.39 |
15.64±1.26 |
|
|
Leaf |
Hexane extract |
232.07±12.53 |
87.34±6.7 |
21.26±2.71 |
|
Methanolic extract |
192.51±38.18 |
168.39±35.73 |
30.39±0.544 |
|
|
Water extract |
174.02±4.98 |
333.52±30.14 |
23.83±0.30 |
|
|
Bark |
Hexane extract |
147.18±25.39 |
51.07±1.01 |
24.32±3.42 |
|
Methanolic extract |
178.61±21.53 |
39.27±4.08 |
25.79±1.11 |
|
|
Water extract |
229.06±15.44 |
98.58±16.44 |
27.24±2.17 |
|
Values are expressed as mean ± S.D. of three independent experiments.
*-µg/ml; **-µM Fe (II) equivalents
CONCLUSIONS:
It can be concluded from the present study that extraction yield was highest in methanol and water solvent. The results of present study showed highest antioxidant activity in methanolic and water extract of flower, thereby validating the traditional use of R. arboreum as source of natural antioxidants.
ACKNOWLEDGEMENT:
The authors acknowledge Shoolini University, Solan, for providing infrastructure support to conduct the research work. Authors also acknowledge the support provided by Yeast Biology Laboratory, School of Biotechnology, Shoolini University, Solan, India.
CONFLICT OF INTEREST:
The authors declare no conflict of interest.
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Received on 11.12.2019 Modified on 19.02.2020
Accepted on 30.03.2020 © RJPT All right reserved
Research J. Pharm. and Tech. 2021; 14(1):311-316.
DOI: 10.5958/0974-360X.2021.00057.3