GC-MS analysis of the methanolic extracts of Smilax china and Salix alba and their antioxidant activity

Smilax china L. (family Smilacaceae) and Salix alba L. (family Salicaceae) are plants that have been traditionally used to treat various ailments in Indian and Chinese medicine. A quantitative estimation of the methanolic extracts of these plants was performed by GC-MS analysis to obtain insight into its phytoconstituents responsible for therapeutic action. The antioxidant potential of the methanol extracts of Smilax china (MESC) and Salix alba (MESA) were assessed with DPPH by using a UV spectrophotometer at a wavelength of 517 nm. The prevailing compounds found in MESC were lactam sugars including 2,5-dimethyl-2,4-dihydroxy-3(2H)-furanon (1.40%), 1,5-anhydro-6-deoxyhexo-2,3-diulose (4.33%), and alpha-methyl-1-sorboside (1.80%); the two alkaloids found were 1,4-methane-4,4a,5,6,7,8,9,9a-octahydro-10,10-dimethyl cyclohepta[d] pyridazine (0.87%) and 1,3,7-trimethyl-2,6-dioxopurine(0.54%); terpenes included deltacadinene (0.39%), terpineol, (+)-cedrol (22.13%), 3-thujanol (0.77%), and 9,10-dehydro-cycloisolongifolene (0.34%); fatty acids included cis-vaccenic acid (4.98%) and telfairic acid (1.10%); esters included 1,2,3-propanetriol diacetate (7.56%), 7-hexadecenoic acid, methyl ester (1.77%), eicosanoic acid, and methyl ester (0.95%); and glycerol included 1,2,3-propanetriol (28.75%). The interesting compounds found in MESA were reducing sugars like D-allose (4.40%) and pyrogallol (10.48%), alkaloids like caffeine (63.49%), and esters like methyl octadecanoate (0.53%). Both fractions revealed considerable antioxidant activity. The reported existing phenolic compounds and terpenes are responsible for the antioxidant activity of the plant extracts.


Introduction
Herbal drugs have attracted mankind since ancient times. The WHO estimates that about 70% to 80% of the populations of some Asian and African countries rely on traditional medicine as the main source of primary health care [1]. The unique perceived benefits of these medicines include their effectiveness, easy availability, limited or no side effects, and success in the treatment of multiple chronic diseases. All these factors play a significant role in the widespread acceptance of natural or alternative therapies by the international community. To harness the benefits and employ intense applications in healthcare, they have been included * Correspondence: aqilmalik@yahoo.com This work is licensed under a Creative Commons Attribution 4.0 International License.
in the pharmacopoeias of different countries for quality control purposes. In fact, many countries are making separate standard books for these herbs [2]. There is a sharp rise in the acceptance of herbal-based medicines due to lower toxicity issues and fewer or no side effects, which is a major issue in synthetic-based medicines.
The amalgamation of modern science (delivery systems) with herbal drugs ensures standardization, consistent and safer use, and regulation of quality control [3]. Unlike synthetic medicines, where combinations are usually avoided, herbal medicines have found popularity as polyherbal formulations. Literature survey reports reveal that these polyherbal preparations have multiple active constituents, which act in a synergistic manner to give a combined effect within the human body [4]. Smilax china L. (family Smilacaceae) and Salix alba L. (family Salicaceae) are two popular herbal drugs used in various polyherbal preparations in the Indian and Chinese systems of medicine. They have been incorporated for their abundant therapeutic effects. S. china is a deciduous tree with rounded leaves and red berries [5]. Quercetin is reported to be the bioactive component, which is also a marker compound of this plant [6]. S. alba is ordinarily known as willow bark or white willow [7]. Salicin is the major component of this plant. It is a metabolic precursor of salicylic acid, which possesses activity similar to aspirin. It is reported to possess antipyretic and analgesic effects [8]. Gas chromatography coupled with mass spectrometry (GC-MS) makes it a high-throughput dual analytical tool. This combination leads to high chromatographic tenacity and peak intensity and provides compositional information for nearly all volatile and semivolatile materials, including organic acids, amino acids, and so on. In this context, the bioactive components present in Smilax china and Salix alba were identified by GC-MS analysis in the present study [9]. The vital components in these plants can be characterized and explored as valuable assets in the pharmaceutical and nutraceutical industries. In the present study, extraction of these plant materials was carried out followed by screening of the antioxidant potential of the extracts.

Extraction of plant material
Plant material (200 g) was defatted with 500 mL of n-hexane for 24 h with occasional shaking. Afterwards, the drug was filtered, dried, and loaded in a Soxhlet apparatus for the extraction process. The drug was extracted in segments; as one batch, 40 g of crude drug was loaded in the Soxhlet apparatus with 120 mL of methanol at 55-60°C and continuously extracted for 6 h. Similarly, another batch of crude drug was extracted. Following complete extraction, the solvent was evaporated under reduced pressure to obtain a solid mass (extract) and then lyophilized to obtain the solid powder form (18.7 g MESC and 9.9 g MESA). A reported method was used to analyze minor quantities (1 g) of both extracts via GC-MS [10].

Antioxidant activity
The radical scavenging potential of MESC and MESA against 1,1-diphenyl-2-picrylhydrazyl (DPPH) were determined using an analytical technique and UV spectrophotometer at 517 nm. Aliquots (40, 60, 80, 100, 120, and 140 µ g/mL for MESC and MESA) were dissolved in test tubes containing 3 mL of methanol and 0.5 mL of 1 mM DPPH. Vitamin E ( α -tocopherol) was used as the standard with the same concentrations as the test samples. A placebo solution with equal volume of methanol and DPPH was made and this solution mixture was incubated at room temperature for 30 min [13]. The antioxidant activity was quantified using the following equation: % Scavenging activity = ((Ab -As)/Ab) ×100, where Ab denotes the absorbance of the blank and As denotes the absorbance of the sample.
All experimentation was done in triplicate. The values of the calculated IC 50 are expressed as mean ±SD.
Pyrogallol is a naturally occurring potent antipsoriatic and antioxidant drug with beneficial effects in the treatment of various skin disorders [20].

Antioxidant activity
Antioxidant activities of MESC and MESA were assessed by the DPPH assay, in which the utilization of a stable free radical (DPPH) was measured. When a solution of DPPH (used as an indicator) is mixed with an antioxidant compound that donates a hydrogen atom, it contributes to the reduced form of diphenyl picrylhydrazine (nonradical). The color of the reaction mixture changed from purple to yellow and its absorbance was measured at a wavelength of 517 nm. The standard α -tocopherol compound was taken to compare the antioxidant activity of both extracts. The IC 50 of DPPH scavenging activity was calculated graphically ( Figure   5). Both MESC and MESA at a concentration of 100 µg/mL showed 63.5% and 78.0% activity, comparable to that of the positive control ( α -tocopherol: 84.8%). It was found that the antioxidant activity of both extracts was directly proportional to the concentration of extracts. Lower IC 50 values indicate higher antioxidant activity. Oxidative stress is the foremost aspect involved in neurogeneration. There is evidence that reactive    The phytoconstituents existing in the extracts verified by GC-MS are rich in antioxidant properties, which are responsible for neuroprotective activity [21].

Conclusions
The methanolic extracts of both Smilax china and Salix alba contain polar as well as nonpolar components, which were qualitatively identified by chemical tests and GC-MS analysis. The analysis exhibited varied and Salix alba (bark) extracts could be helpful in inhibiting or slowing the progress a variety of oxidative stress-related ailments.