Microwave-Assisted Efficient Extraction of Different Parts of Hippophae rhamnoides for the Comparative Evaluation of Antioxidant Activity and Quantification of Its Phenolic Constituents by Reverse-Phase High-Performance Liquid Chromatography (RP-HPLC) † UPENDRA K. SHARMA, ‡ KAPIL SHARMA, § NANDINI SHARMA, ‡ ABHISHEK SHARMA, ‡ HARSH P. SINGH, § AND ARUN K. SINHA* ,‡ Natural Plant Products, and Hill Area Tea Science Divisions, Institute of Himalayan Bioresource Technology (IHBT), Post Box 6, Palampur-176061, Himachal Pradesh, India The outcome of different extraction procedures (microwave, ultrasound, Soxhlet, and maceration) on the antioxidant activity of seeds, leaves, pulp, and fruits of Hippophae rhamnoides (sea buckthorn or SBT) was investigated by two different bioassays: 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) and 2,2′-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assays. The SBT extracts were found to possess strong antioxidant activity measured in terms of TEAC (2.03–182.13 and 6.97–282.75 mg/g) with ABTS and DPPH assays, respectively. In general, the antioxidant capacity of microwave-assisted extracts was found to be significantly higher than those obtained by ultrasound-assisted extraction (UAE) and maceration while being slightly higher than Soxhlet extracts. Further, microwave extracts of seeds were found to possess maximum antioxidant capacity followed by leaves, fruits, and pulp. Also, the chemical composition of extracts, studied in terms of the total phenolic content, was found to be in the range of 1.9–23.5 mg/g Gallic acid equivalent (GAE), which indicates a strong correlation between antioxidant activity and phenolic content present in the SBT. In addition, some of its bioactive phenolic constituents, such as rutin (1), quercetin-3- O-galactoside (2), quercetin (3), myricetin (4), kaempferol (5), and isorhamnetin (6), were also quantified in different extracts by reverse-phase high-performance liquid chromatography (RP-HPLC). KEYWORDS: Hippophae rhamnoides; sea buckthorn; antioxidant activity; DPPH; ABTS; HPLC INTRODUCTION In recent years, intensive research on natural antioxidants has received considerable importance because synthetic antioxidants, such as butylated hydroxyanisole (BHA) and butylated hy- droxytoluene (BHT), have been shown to have one or the other side effects (1, 2). Several studies have revealed that plants have potent antioxidants in the form of vitamins, flavonoids, and other phenolic compounds that act as scavengers of free radicals and inhibitors of lipid peroxidation (3–5). Among the various plants reported for antioxidant activity, sea buckthorn (Hippophae rhamnoides L., Elaeagnaceae) stands out. This plant is a native of Eurasia and has been domesticated in several countries (India, China, Nepal, Pakistan, Myanmar, Russia, Britain, Germany, Finland, Romania, France, etc.) at an altitude of 2500–4300 m (6). It has been recognized as a versatile nutraceutical crop with diverse uses, from controlling soil erosion to being a source of horse fodder, nutritious foods, drugs, and skin-care products (7). Different parts of this plant are used in traditional medicine for the treatment of diseases, such as flu, cardiovascular diseases, mucosal injuries, and skin disorders (8, 9). Various studies of alcoholic and hydroalcoholic extracts of fruits, seeds, and leaves of sea buckthorn have confirmed its medicinal and nutritional value (6, 10–14). All parts of this wonder plant are considered to be a good source of a large number of bioactive compounds, including carotenoids, tocopherols, sterols, flavonoids, lipids, vitamins, tannins, minerals, etc. (15–20), which contribute to its wide usage as a natural antioxidant. Nowadays, there has been a huge upsurge for developing rapid, reliable, and reproducible methods for the efficient extraction of bioactive compounds from plants to increase their therapeutic functionality. In the literature, different extraction techniques, such as maceration, Soxhlet, ultrasound-assisted † IHBT Communication number 0747. * To whom correspondence should be addressed. Telephone: +91-1894-230426. Fax: +91-1894-230433. E-mail: aksinha08@ rediffmail.com. ‡ Natural Plant Products Division. § Hill Area Tea Science Division. 374 J. Agric. Food Chem. 2008, 56, 374–379 10.1021/jf072510j CCC: $40.75  2008 American Chemical Society Published on Web 12/29/2007