ANTIOXIDANT ACTIVITY OF COMPOUNDS ISOLATED FROM PYCNANTHUS ANGOLENSIS ( WELW . ) WARB AND BYROPHYLLUM PINNATUM ( LAM . ) OKEN

Pycnanthus angolensis (Welw.) Warb and Byrophyllum pinnatum (Lam.) Oken are two herbal recipes employed in native medicine for the treatment /management of gastrointestinal disorders, skin infections and more especially cancers, wounds and many other inflammatory conditions. Before now, seven compounds (NG-1b, NG-2, NG-3b, NG-4c, NG-5a, KF-1a and KF-2) have been obtained from the extracts of these plants by column and /or preparative thin-layer chromatographies. The search for new and more active antioxidant agents necessitated the screening of these compounds for potential activity. Generally the isolates gave (IC50) of between 0.50 and 0.60 μg mL-1. However, NG1b demonstrated a significant activity at 0.48 μg mL-1 which compare favourably with that shown by Vitamin A (a standard antioxidant drug) at 0.49 μg mL-1. These compounds, most especially NG-1b could be further worked on with the aim of improving on the observed antioxidant activity through in-vitro structural activity relationship studies (SARS) and probably in-vivo clinical trials. .


Introduction
Free radicals occur in the human body in the forms of reactive oxygenated species (ROS), super-oxides, peroxides, hydroxyl and nitrogenous species.These chemical species are natural by-products of cellular metabolism and also form an integral part normal physiology. 1 Furthermore, they can also be produced by bacterial leucocytes, atmospheric pollutants, drugs, xenobiotics and in mitochondrial respiration.In addition, chemical conversion of fats during lactation, exercise, fever, infections and even fasting can result in increased radical activity leading to damage of the immune and nervous systems.The stress hormones (adrenalin and noradrenalin) secreted by the adrenal glands under conditions of continuing and excessive emotional stress are metabolized into simpler, albeit, free radical molecules which can attack biological moieties such as proteins, lipids, enzymes, DNA and RNA resulting in cellular or tissue or organ injury associated with degenerative diseases. 2The consumption of fruits and vegetables containing antioxidants has been found to offer protection against these diseases. 3Besides playing an important role in the physiological systems, antioxidants have been used in the food industry to prolong the shelf life of foods especially those rich in polyunsaturated fats.Phytochemicals such as flavonoids, terpenes and polyphenols have been reported to inhibit free radical reactions, protect the human body from disease [9][10] and retard lipid oxidative rancidity. 11Therefore, researches are presently focused on obtaining antioxidants from plant sources.Two of such plants namely, Pycnanthus angolensis (Welw.)Warb and Byrophyllum pinnatum (Lam.)Oken are employed in folkloric medicine for the treatment of gastrointestinal disorders, skin diseases and more especially cancers, ulcers, wounds and inflammations.3][14][15] This present study was done by subjecting the crude extracts and the seven isolated compounds to antioxidant tests with a view of confirming or disproving the reported uses in ethno-medicine for the treatment/management of inflammatory conditions which arise from the debilitating incidence of free-radicals.

Experimentals
Fresh leaves of P. angolensis and B. pinnatum were collected in the month of July, 2016 within the University of Uyo Campus, Nigeria.The plants had previously been identified in studies [16][17] for which voucher specimens No. H 045 and No. H 047 were deposited in the Herbarium Unit of the Faculty of Pharmacy.Immediately after collection, the plant materials were separately dried in a laboratory oven (Gallenkamp, England) at 40 0 C for 48 h and the resultant materials powdered on an electric mill (Uniscope, England).

Extraction and processing of plant material
The powders were extracted with cold 96 % ethanol at room temperature (27 ± 2 0 C) for 72 h.The obtained filtrates were then evaporated to dryness in-vacuo on a rotary evaporator (R205D, Shensung BS & T, China).The extracts, NG-1b, NG-2, NG-3b, NG-4c, NG-5a, KF-1a and KF-2 previously isolated from chromatographic separations of organic fractions of the two plants [12][13][14][15] were kept in appropriately labelled amber bottles and then stored in a refrigerator at -4 0 C prior to the antioxidant tests.

Spectrophotometric determination of antioxidant activity using DPPH reagent
Substances which are capable of donating electrons or hydrogen atoms (free-radical scavengers) can convert the purple-coloured DPPH radical (2, 2-diphenyl-1picrylhydrazyl hydrate) to its yellow-coloured non-radical form (1,1-diphenyl-2-picryl hydrazine). 2,22 his reaction can be monitored by spectrophotometry.[25][26] DPPH (4 mg) was weighed and dissolved in methanol (100 mL) to produce the stock solution (0.004 % w/v).Serial dilutions of the stock solution were carried out.The absorbance of each of the sample was taken at λm 512 nm using the UV-VIS spectrophotometer (Jenway 6405, USA).A solution of methanol without DPPH served as the blank.

Determination of the antioxidant activity of crude extract / isolate
In every case, 2 mg of the extract/isolate was dissolved in 50 mL of methanol.Serial dilutions were done to obtain the following concentrations; 0.0008 mg mL -1 , 0.0016 mg mL -1 and 0.0024 mg mL -1 using methanol.5 mL of each concentration was incubated with 5 mL of 0.004 % w/v methanolic DPPH solution for optimal analytical accuracy.After an incubation period of 30 minutes in the dark at room temperature (25 ± 2 0 C), observation was made for a change in the colour of the mixture from purple to yellow.The absorbance of each of the test samples was then taken at λm 512 nm.The Radical Scavenging Activity (RSA %) or Percentage Inhibition (PI %) of free radical DPPH was thus calculated: where Ablank is the absorbance of the control reaction (DPPH solution without the test sample and Asample is the absorbance of DPPH incubated with the extract/isolate /anti-oxidant drug.

DPPH assay of standard antioxidant drugs
Standard antioxidants namely, vitamin A (Emzor Drugs, Nigeria), vitamin C (Greenfield Drugs, Nigeria) and vitamin E (Gemini, Nigeria) were used.While vitamin C was in a tablet dosage form, vitamins A and E were formulated as gelatine capsules.The estimated weight of the formulations containing 2 mg of the standard antioxidant drugs were determined by proportionality and then diluted.Methanol was used to dissolve vitamin C, while n-hexane was used to dissolve vitamins A and E because solubility problems encountered with these two vitamins.Thus, methanolic and hexane solutions of 0.004 % w/v DPPH were used for incubation of vitamin C, vitamin A and E respectively for 30 minutes.
The absorbance value of each of the drugs was taken at wavelength at λm 512 nm and the IC50 determined.

Results and discussion
The plants were identified and collected observing basic rules of plant collection.0] Phytochemical investigations on the plant extracts indicated the presence of alkaloids, saponins, tannins, terpenes, flavonoids and cardiac glycosides while anthraquinones and cyanogenic glycosides were absent. 31The crude extracts and resultant sub-fractions were separately put through silica chromatographic separations and the exercises led to the isolation of compounds coded as NG-1b, NG-2, NG-3b, NG-4c, NG-5a, KF-1a and KF-2 respectively.[14][15]

Rapid thin-layer chromatographic analysis for antioxidant activity
The extracts, isolates and ascorbic acid showed white spots on purple background when the chromatogram was sprayed DPPH reagent.The observation of white spots (irrespective of initial spotted colour) was the evidence of reduction of DPPH reagent (discoloration) by the by free-radical scavenger in the samples.The Beer-Lambert Law is the basis of all absorption spectrophotometry.Therefore, a plot of absorbance against concentration for a cell of unit thickness (1 cm) should give a straight line passing through the origin.The reduction of the DPPH radical was determined by taking its absorption at a wavelength of λm 512 nm.It was observed that the absorbance of DPPH decreased as the concentration of added free radical scavenger extract /isolate /standard antioxidant drug) increased which suggested that the DPPH reagent was being reduced.The results of the reduction are as presented in Table 1.The Radical Scavenging Activity (RSA %) or percentage inhibition (PI %) and the IC50 values of extract and standard antioxidant drugs were computed as Table 2 shows.
The RSA % is an indicator of the antioxidant activity of extract/ isolate /standard antioxidant drug. 32All the isolated compounds gave largely moderate antioxidant activity (IC50) of between 0.50 μg mL -1 and 0. 60 μg mL -1 .However, NG-1b elicited a remarkable activity at 0.48 μg mL -1 .It should be stated that this activity compare favourably with that demonstrated by Vitamin A (a standard antioxidant drug) at 0.49 μg mL -1 .

Conclusion
The results of this present study indicate that compounds isolated from the plants have demonstrated antioxidant activities which can compare with those of standard antioxidant drugs in clinical practice.Also, these compounds are to be further investigated (in-vitro SARS) in our laboratories with a view to improving on the observed activities.

2
Determination of the antioxidant activity of extract/ isolate/ antioxidant drug