Journal of Scientific & Industrial Research, Vol. 72, May 2013, pp. 307-311
Bioactivity guided extraction of Seabuckthorn (Hippophae rhamnoides L. ssp. turkestanica) leaves 1
Singh Amrit Kumar, 1Attrey Dharam Paul* and 2Naved Tanveer
1
Amity Institute of Seabuckthorn Research, Amity University Uttar Pradesh, Sector 125, Noida–201303, India 2 Amity Institute of Pharmacy, Amity Univsersity Uttar Pradesh, Sector 125, Noida–201303, India Received 02 May 2012; revised 16 November 2012; accepted 05 March 2013
This study represents antioxidant (DPPH scavenging) activity guided extraction of Seabuckthorn (SBT) leaves and its correlation with total phenol content. 75% ethanolic and acetone extracts showed significantly higher antioxidant activity and total phenol content in comparison with other solvent extracts. Phenol contents correlated well (R2 = 0.952) with the antioxidant activities. It suggests that these phenolic compounds are responsible, at least in part, for the antioxidant activities observed. HPLC analysis also appears to support the correlation of total phenol content and antioxidant activity. Keywords: Seabuckthorn, Bioactivity guided extraction, DPPH Scavenging Activity, Total Phenol Content
Introduction Oxidative stress is an imbalance between oxidants and antioxidants, towards oxidants. Reactive oxygen species are the major cause of oxidative stress, which play an important role in various degenerative or pathological processes like ageing, cardiovascular diseases, cancer, neurodegenerative disorders etc.1-3 Plant derived antioxidants have the potential to reduce the incidence of oxidative stress by shifting the balance towards antioxidants.4 Also, there are reports of increased interest on plant based antioxidants.5 Seabuckthorn (SBT) (Hippophae rhamnoides L. ssp. Turkestanica; Family: Elaeagnaceae), found growing in the wild in North-West Himalayas at high altitudes (7000–15,000 feet), is a dwarf to tall (3–15 feet), branched, and thorny nitrogen fixing deciduous shrub, native to Europe and Asia. All parts of the plant are considered to be good source of a large number of bioactive substances.6 SBT is a good source of antioxidant compounds.7-8 Also, the medicinal effects of SBT have been suggested to be due to the presence of high antioxidant contents.6 SBT has been reported to be an excellent source of phenolic compounds. Phenolic compounds are known to show various properties including antioxidant property.9-10 Although a number of researchers have investigated SBT leaf extracts,6,11-12 but no report has been found —————— *Author for correspondence: E-mail:
[email protected],
[email protected]
on the bioactivity guided extraction of SBT leaves. The present work was undertaken to study the bioactivity (Antioxidant activity) guided extraction of SBT leaves, its correlation with total phenol content and HPLC analysis of suitable extract. Materials and Methods Plant material
Plant materials i.e. SBT leaves were collected during the month of September from Leh, Ladakh, India and were authenticated by National Institute of Science Communication and Information Resources (NISCAIR). Leaves were cleaned, dried, powdered and stored at ambient temperature till extraction. Extract preparation
Powdered SBT leaves were extracted with solvents of different polarities like pet ether, chloroform, ethyl acetate, acetone, methanol, ethanol (100, 75, 50 and 25%) and 100% aqueous solvents by cold percolation method.6 The powdered dry leaves were soaked in above solvents (1: 8 w/v) at room temperature. After 24 h, the supernatant was decanted and the residue was re-soaked in fresh solvent. This process was repeated 5 times for complete extraction. After completion of the extraction process, the supernatants were pooled and filtered through 250 mesh nylon cloth. This filtrate was dried under reduced pressure till a solid mass was obtained. Same procedure was repeated for preparation of all the above extracts.
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DPPH scavenging activity
Statistical analysis
The DPPH (1, 1-diphenyl-2-picrylhydrazyl) scavenging activity was determined using the method described by Blois.13A 0.1mM solution of DPPH was prepared by using methanol. 2 ml of this solution was added to 2 ml solution of extract with a range of different concentrations (1–100 µg/ml) and the mixture was kept in dark for 20 min. After 20 min. absorbance of the color developed was recorded at 517nm with the help of a UV-Visible spectrophotometer against blank. Control was prepared by adding 2 ml of the DPPH solution to 2 ml methanol. Same procedure was repeated for estimation of antioxidant activity in all the above extracts and IC50 values were calculated using the formula:
All measurements were performed at least in triplicate, and values were averaged and reported along with the standard deviation. To determine whether there was any difference between antioxidant activities or phenolic contents of various extracts, a one-way analysis of variance (ANOVA) was applied. Values of p < 0.05 were considered as significantly different. The Statistical Package SPSS 12.0 for Windows was used to analyze the data.
Percentage Scavenging = {Absorbance (Control-Test)/ Absorbance of Control} × 100 Total phenol content
The total phenol content was estimated in above prepared SBT extracts by Folin-Ciocalteu Reagent (FCR) based assay.14 To the aliquot (50µl) taken from stock solution (1mg/ml) of the extract, 3.5 ml distilled water and 250µl of FCR was added, the mixture was kept at room temperature for 1–8 min and 750µl of 20% sodium carbonate solution was added. Mixture was kept at room temperature for 2 hrs and absorbance of the color developed was recorded at 765nm with the help of a UV-Visible spectrophotometer against blank. Total phenols (mg/g) in the SBT leaf extracts were expressed as Gallic Acid Equivalent (GAE), using standard curve (R2 = 0.986) prepared from gallic acid (0.1 mg/ml) solution. Same procedure was repeated for estimation of total phenolic content in all the extracts. HPLC analysis
HPLC analysis of the 75% ethanolic extract of SBT leaves was carried out as per Yuangang Zu et al15 using HIQ SIL C18V reversed-phase column. The mobile phase used, was methanol–acetonitrile–water (40:15:45, v/v/v) containing 1.0% acetic acid. This mobile phase was filtered through a 0.45µm membrane filter (Millipore), then deaerated ultrasonically prior to use. Flow rate and injection volume were 1.0 ml/min and l0µl, respectively. Quercetin and Isorhamnetin were quantified by diode array detector following reverse phase HPLC separation at 368 nm. All chromatographic operations were carried out at ambient temperature.
Results and Discussion Antioxidant activity and Total phenol content
There are various in-vitro methods which are being used to measure the antioxidant activity of natural compounds. The DPPH method is one of them. It is one of the most widely used, simple, rapid and reproducible method independent of sample polarity for screening of many samples for radical scavenging activity.16-17 On analysis of antioxidant activity of various SBT leaf extracts, the maximum activity was found in case of 75% ethanolic extract, followed by acetone, 25% ethanolic and 100% aqueous extracts. However, DPPH being a surrogate model for radical activity, assessment of total antioxidant activity should be further validated in cell/tissue based models. The total phenol contents of various SBT leaf extracts studied in this work were found to vary from 35.62 ± 4.75 to 402.19 ± 2.20 mg/gm extract in terms of gallic acid equivalent. Maximum phenol content was observed in 75% ethanolic leaf extract followed by acetone, 25% ethanolic and 100% aqueous leaf extracts (Table 1). Variation in the total phenol content observed in extracts of different solvents appears to be due to the difference in solubility of the phenolic compounds. It may be due to varying Table 1—DPPH Scavenging activities (IC50 values) and total phenol contents of SBT leaf extracts Solvents Pet. Ether Chloroform Ethyl acetate Acetone Methanol 100% aqueous 25% ethanolic 50% ethanolic 75% ethanolic 100% ethanolic
IC50 (µg/ml)
Total phenol content (GAE mg/gm)
48.12 ± 1.77 56.07 ± 2.02 14.71± 1.67 6.01 ± 0.02 11.31 ± 0.26 7.88 ± 0.16 7.56 ± 0.25 8.74 ± 0.23 5.99 ± 0.25 29.88 ± 0.30
44.29 ± 2.65 35.62 ± 4.75 265.14 ± 3.22 398.86 ± 2.63 304.77 ± 1.05 373.62 ± 2.72 377.52 ± 1.35 348.76 ± 1.74 402.19 ± 2.20 143.33 ± 1.84
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polarity of the compounds resulting in differential solubility in different solvent systems. It further indicates the presence of varying nature of phenolic compounds with different polarities in SBT leaves. Since no significant difference was observed between acetone & 75% ethanolic extracts (Table 1), 75% ethanol and acetone appear to be the solvents of choice for the extraction of SBT leaves in order to get maximum antioxidant activity as well as total phenol content depending upon their relative commercial viability and residual effects. Correlation between total phenol content and antioxidant activity
Since phenolic compounds have been reported to show various activities including antioxidant activity, at least in in-vitro systems.9-10,18 it was hypothesized that total phenol content of the various SBT extracts may be responsible for the antioxidant activities observed. Further, on analysis of antioxidant activity and total phenol content, SBT leaf extracts with high levels of phenol content also showed higher antioxidant activity (Table 1). In fact, a strong correlation (R2 = 0.952) was found between the total phenolic content and the antioxidant activity (Figure 1), suggesting that these compounds are responsible, at least in part, for the antioxidant activity observed. Further, it has been reported that the dietary intake of antioxidant supplements could be a useful strategy to reduce the incidence of diseases associated with oxidative stress, such as neurodegenerative diseases.19
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Thus, 75% ethanolic and acetone extracts of SBT leaves appear to be useful as dietary supplement against oxidative stress related health disorders. HPLC analysis of 75% ethanolic extract
Although there was no significant difference between 75% ethanolic and acetone extracts, quantitatively higher amounts of both antioxidant activity as well as total phenols were found to be present in 75% ethanolic extract in comparison with all other extracts, including acetone extract (Table 1). Accordingly this extract was used further for estimation of quercetin & isorhamnetin through HPLC analysis. SBT leaves have been reported to be rich in flavonoides6 (Quercetin and Isorhamnetin being the major flavonoides).15 Results of HPLC analysis of 75% ethanolic extract (Figure 2A & Table 2) have, however, shown almost negligible quantities of these two flavonoides. Further, on acid hydrolysis, a quantum increase in content of these two flavonoides was observed in comparison with unhydrolyzed extract (Figure 2B & Table 2). Still the total flavonoid content was < 0.3%. These results (Table 2) indicate that 75% ethanolic extract contains very little amount of free flavonoides. They appear to be present, mainly, in the bound form. Other investigators20 have also reported similar findings in the case of SBT fruits. Since quantities of these two major flavonoides were observed to be too low, it appears that the antioxidant activity of this extract could be due to presence of phenolic
60 R2 = 0.9527
IC - 50 values
50 40 30 20 10 0 0
100
200
300
400
Total phenol content (mg/gm extract) in terms of gallic acid equivalent Fig. 1—Correlation between total phenol content and antioxidant activity
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A. Before acid hydrolysis
B. After acid hydrolysis
Fig. 2—HPLC profiles of 75% ethanolic SBT leaf extract
Table 2—HPLC analysis of 75% ethanolic SBT leaf extract Analytes
Quercetin Isorhamnetin
Retention Time
5.87 9.14
Amount Present in Leaf extract (%w/w) Without Hydrolysis
With Hydrolysis
0.0004 0.002
0.0389 0.282
funds for the project. The authors are also grateful to Dr. Ashok K. Chauhan, Hon’ble Founder President, Amity University Uttar Pradesh, Noida for providing facilities for conducting the research. References 1 2
compounds rather than quercetin and isorhamnetin. However, it needs further investigations on chemical characterization of this extract. Conclusion Among all the solvents used in the present study, 75% ethanol and acetone were found to be the solvents of choice for extraction of SBT leaves in order to get maximum antioxidant activity as well as total phenol content. A strong correlation was also observed between total phenol content and antioxidant activities. Further, HPLC analysis also appeared to support the above finding. Acknowledgement The authors are grateful to the Chief Controller R & D (Life Sciences) & Director, Life Sciences, DRDO HQ, DRDO Bhavan, New Delhi for providing
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