Evaluation of the antioxidant and anti-inflammatory activities of solvent extracts of Tricholepis chaetolepis (Boiss) Rech. f. whole plant

Abstract The whole plant, Tricholepis chaetolepis, powder was investigated using proximate and fluorescence analysis along with determining the extractive values. Total phenolics, flavonoids and total protein contents of n-hexane, chloroform and methanolic extracts of the whole plant were also determined. The anti-diabetic activity of all the three extracts of the plant was determined by in vitro alpha-amylase inhibition assay. The anti-oxidant potential was evaluated using Phosophomolybdenum and DPPH methods. The anti-inflammatory potential of all extracts were determined by carrageenan-induced rat paw oedema model. The evaluation of the plant extracts exhibited the anti-diabetic, anti-oxidant, and anti-inflammatory activities in dose dependent fashion. The research concludes that Tricholepis chaetolepis extracts contain phenol, flavonoids, and tannins that show observable anti-oxidant and anti-diabetic potential. It is also concluded that the methanol extract of the plant showed the maximum effect against inflammation induced by carrageenan in rat paw oedema as compared with n-hexane and chloroform extracts. Graphical Abstract


Introduction
Interest in the search for new natural antioxidants has grown dramatically over the past years because reactive oxygen species (ROS) production and oxidative stress have been shown to be linked to a large number of human degenerative diseases, including cancer, cardiovascular diseases, inflammation and diabetes (Tundis et al. 2012;Sharif et al. 2016). It has been reported that flavonoids have role of antiinflammatory and radicals scavenging, in addition, they were a-glucosidase, aldose reductase and glycation inhibitor (Gou et al. 2016).
Tricholepis chaetolepis (Bioss). Rech. f. belongs to a family of Asteraceae and it is called as 'Barham Dandi' (Ahmad et al. 2010). The Asteraceae family is the largest plant family in Pakistan with over 650 species (Tanvir et al. 2014). Tricholepis chaetolepis is an annual herb with dense leaves. It's all or some parts are considered to be crucial from the pharmaceutical point of view. Particularly, the aerial parts are considered of having an important role conventionally. These parts of the plant are known for their different pharmaceutical and therapeutic potentials like skin infections, nerve tonic and antipyretic properties (Khan et al. 2011). The aim of the present study was to evaluate phenolic content, flavonoids, antioxidant activity, in vitro a-amylase inhibitory potential and in vivo anti-inflammatory potential of the Tricholepis chaetolepis for the first time as no study has been performed on this plant.

Qualitative analysis
The moisture content was found to be 5.083 occurring within the standard range exhibiting good defensing property against microorganism. The total ash value of the whole plant powder was 11.50%, which was high, indicating the presence of high inorganic content. The swelling index is 8.6, it may be because of the existence of gum. Foaming index is less than 100. The swelling is responsible for drug release. Fluorescence was seen in plant powder after treating with iodine solution and methanol.
n-Hexane, chloroform and methanol extracts confirmed the presence of alkaloids, tannins, flavonoids, triterpenoids, cardiac glycosides, carbohydrates and proteins. Phytochemicals exists in edible plants as secondary metabolites . All extracts showed the absence of saponins. The gum and mucilages were present in n-hexane and chloroform extracts and absent in methanol extract.
The nutritive value of Tricholepis chaetolepis (TC) was found to be high (198.054) which showed that this plant can be used as a food. The plant found to be a good reservoir for the crude fiber (37.116%) than crude fat (2.57%). The result are presented in Table 1.

Determination of total phenolic content
The total phenolic content for n-hexane, chloroform and methanol extracts were estimated by Folin Ciocalteu's method using gallic acid as standard. The gallic acid solution of concentration (10-100 ppm) conformed to Beer's Law at 760 nm with a regression co-efficient (R 2 ) ¼ 0.9963.The plot has a slope (m) ¼ 0.016 and intercept ¼ 0.0306. The equation of standard curve is y ¼ 0.016x þ 0.0306.

Determination of total flavonoid content
The total flavonoid content for n-hexane, chloroform and methanol extracts were measured with the aluminium chloride colorimetric assay using quercetin as standard.
The quercetin solution of concentration (100-1000 ppm) conformed to Beer's Law at 415 nm with a regression co-efficient (R 2 ) ¼ 0.9966. The plot has a slope (m) ¼ 0.0103 and intercept ¼ 0.0179. The equation of standard curve is y ¼ 0.0103x þ 0.0179. Several studies revealed flavonoids as antioxidants because they inhibit the formation of free radicals by inhibiting enzymes involved in the synthesis of free radicals and also scavenge free radicals hence enhances the antioxidant defence system (Agati et al. 2012)

Total antioxidant capacity Phosphomolybdenum method
The phosphomolybdenum method was used to know the total antioxidant action of n-hexane, chloroform, and methanol extracts. The results showed that the methanol extract has highest total antioxidant activity (0.163 ± 0.002). The antioxidant activity of various plant extracts was decreased in the following order: methanol extract > chloroform extract (0.117 ± 0.003) > n-hexane extract (0.07 ± 0.001). Ascorbic acid showed total antioxidant activity of 0.636 ± 0.02.

Evaluation of antioxidant activity by DPPH radical scavenging method
The IC 50 value of DPPH radical scavenging assay (fig. S1B) for n-hexane, chloroform and methanol extracts was found to be 550.70, 28.570, 15.250 mg/ml respectively. The IC 50 value of DPPH radical scavenging assay for ascorbic acid was found to be 3.118 mg/ml. It was found that methanol extract shows maximum antioxidant activity followed by chloroform and n-hexane extract.

In vitro inhibitory alpha amylase assay
The IC 50 value of a-amylase inhibition for n-hexane, chloroform and methanol extracts was found to be 413.20, 63.87, 123.10 mg/ml respectively (fig. S1A). The IC 50 value of a-amylase inhibition for acarbose was found to be 57.74 mg/ml. It was found that CHCl 3 extract shows maximum a-amylase inhibitory activity followed by methanol and n-hexane extracts. The present study is in accordance with the previous studies in which flavonoids were found to be the main cause for the hypoglycemic potential of the plant by inhibiting alpha amylase (Shu et al. 2009).

Anti-inflammatory activity by carageenan-induced rat paw oedema
The confirmation potential of extracts against inflammation was done by carrageenan induced rat paw oedema assay. A dose dependent effect is seen with extract ( Figure S2). The chloroform extract (1000 mg/kg) and n-hexane extract (1000 mg/kg) doses exhibits better anti-inflammatory effect as compared with chloroform extracts (250 and 500 mg/kg) and n-hexane extracts (250 and 500 mg/kg) doses. So, extracts showed dose-dependent potential on inflammation, i.e. 39.55%, 47.01% and 50.01% inhibition by chloroform extract and 32.84%, 33.57% and 35.46% inhibition by n-hexane extract at the 250, 500 and 1000 mg/kg doses at 5 th hour. It was previously reported that phenolic contents present in the extracts demonstrarte antiinflammatory potential (Kassim et al. 2010).

Conclusion
Qualitative test, quantitative analysis, antioxidant, alpha amylase inhibitory assay and anti-inflammatory potential of the plant is screened in this research. TC chloroform extract showed maximum antidiabetic activity whereas the results indicated that high anti-inflammatory potential was shown by methanol extract owing to the phenolic and flavonoid contents present in them.

Disclosure statement
No potential conflict of interest was reported by the authors.