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Open Chemistry

formerly Central European Journal of Chemistry

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Volume 15, Issue 1


Volume 13 (2015)

Phenolic profiling and therapeutic potential of local flora of Azad Kashmir; In vitro enzyme inhibition and antioxidant

Muhammad Asam Raza
  • Corresponding author
  • Center of Natural Product Department of Chemistry, Hafiz Hayat Campus, University of Gujrat, Gujrat, Pakistan
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/ Muhammad Danish
  • Center of Natural Product Department of Chemistry, Hafiz Hayat Campus, University of Gujrat, Gujrat, Pakistan
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/ Mahvish Mushtaq
  • Center of Natural Product Department of Chemistry, Hafiz Hayat Campus, University of Gujrat, Gujrat, Pakistan
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/ Sajjad Hussain Sumrra
  • Center of Natural Product Department of Chemistry, Hafiz Hayat Campus, University of Gujrat, Gujrat, Pakistan
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/ Zenab Saqib
  • Center of Natural Product Department of Chemistry, Hafiz Hayat Campus, University of Gujrat, Gujrat, Pakistan
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/ Shafiq Ur Rehman
  • Center of Natural Product Department of Chemistry, Hafiz Hayat Campus, University of Gujrat, Gujrat, Pakistan
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Published Online: 2017-12-29 | DOI: https://doi.org/10.1515/chem-2017-0041


The current study supports the phytochemical screening, evaluation of antioxidant and enzyme inhibition potential and correlations between antioxidant activities and phenolics of Rumex dentatus (Family: Polygonaceae), Mentha spicata (Family: Lamiaceae), Withania somnifera (Family: Solanaceae), Nerium indicum (Family: Apocynaceae) and Artemisia scoparia (Family: Asteraceae). The herbal materials were extracted in ethanol (90%) and partitioned between several solvents based on polarities. Total phenols were determined with FC method and ranged 21.33 ± 1.53 - 355.67 ± 6.03 mg GAE/ mg of the extract. Antioxidant activities (DPPH, total iron reducing capacity, phosphomolybdate assay & FRAP) and enzyme inhibition potential (Protease, AChE & BChE) were performed by the standard protocols. The results showed that all extracts exhibited significant DPPH activity ranging from 12.67 ± 2.08 - 92.67 ± 1.53%. The extracts that were active in DPPH activity also potrayed marvelous FRAP, total iron reducing and phosphomolybdate values. Correlation studies of antioxidant activities and the content of phenolic compounds in plant materials exhibited positive correlation between them. The outcome of enzyme inhibition activity exhibited that about 80% of the fractions under surveillance plants intimated more than 50% inhibition. Isolation of bioactive compounds from these plants is in progress.

Keywords: Azad Kashmir; Antioxidants; Medicinal Plants; Enzyme Inhibition

1 Introduction

The word ‘medicinal’ is a term that is used for plants having therapeutic effects on humans and animals. Plant materials are important sources which combat against serious diseases. Several compounds and their therapeutic derivatives obtained from herbs have demonstrated activities against various disorders and ailments [1]. Medicinal plants (herbs) are considered as important sources for sthe pharmaceutical, agriculture and food industries. Even though the pharmaceutical industries create a lot of synthetic drugs that facilitates chronic disease attenuation, with the inception of the synthetic epoch, the usage of these synthetic drugs comes up with the number of side effects and microbial resistance towards these drugs. Secondly, these drugs are very costly and hence inaccessible for the majority of population. Research studies on medicinal plants have become more renowned these days. The natural therapeutics have gained enormous significance as they are risk-free, profitable, effective and possess remarkable advantages due to the of fusion of medicinal ingredients with vitamins and minerals [2].

In recent times, there is a rising trend in research studies on biological activities of herbal plants. Many researchers have delineated about the effectual and chemotherapeutic role of medicinal plants as remedies for multiple diseases [3,4]. The role of plants makes them indispensable for human life. With the passage of time, medicinal importance of plants has been explored. Natural products obtained from plants offer novel basis of biological molecules that may have an enormous impact on infectious diseases and overall human health [2,3]. The historical background of drug discovery showed that the plants are rich sources of new active compounds, and a lot of synthetic drugs owed their origin to plant-based medicine [4]. According to WHO (1993), 80% of the world’s population is dependent on the traditional medicines and a major part of traditional therapies involve the use of plant extracts [5,6].

Several reports reveal that natural compounds are capable of protecting against reactive oxygen species (ROS) [7,8,9]. Therefore, these may have potential application in prevention and curing of diseases [10]. The antioxidant constituents of plants are gaining the interest of food scientists and food manufactures, as the future demand will be on functional foods with specific health effects. The antioxidant property of plant extracts has been attributed to their polyphenol contents. They improve the defensive system of plants against infection and injury. They are usually found as glycosylated derivatives, inorganic salts with sulfates counter ion or organic acids. They play a key role as antioxidants due to the presence of hydroxyl constituents and their aromatic nature which enable them to act as reducing agents, hydrogen donors, and singlet oxygen quenchers. In addition, they have a metal chelation potentials as well [11].

Mentha spicata also known as mint, are given to the patient as an indoor remedy to eradicate stomach ache and also provide sleeping assistance. Mint tea acts as diuretic, essential oils of this plant is being vigorously used as antiseptic, insecticide to kill wasps, hornets, ants, and cockroaches. Many phytochemical compounds have been isolated form M. spicata like menthol, piperitone, α-pinene, 1,8-cineole, pulegone, menthofuran, menthone, cadinine, caryophylline, iso-caryophylline, carnone, limonene, piperitenone epoxide, 5, 6, 4’-trihydroxy-7,8,3-trimethoxyflavone etc. [12]. Rumex dentatus has allopathic activity, such as antibacterial, astringent, antagonistic to dermatitis, anti-inflammatory, antitumor, diuretic and antifungal activities [13]. Nerium indicum is used as diuretic, anticonvulsant, antioxidant, sedative, cardiotonic, emetic, anti-HIV, anticancer, anthelmintic, expectorant and diaphoretic [14,15]. It is also useful for joint pain, stomache, leprosy, diabetes, ulcer and reduces swelling as well as inflammation [16,17]. Artemisia scoparia is used to cure Jaundice, hepatitis, fever, high blood pressure, itching and gallbladder diseases. It is also utilized as cholagogue, antipyretic, antiseptic, antibacterial, antiviral, diuretic, vasodilator and antibacterial [18]. Withania somnifera is a plant used in medicine from the time of Ayurveda, have many pharmacological activities like antioxidant, anxiolytic, antiparkinsonian, antivenom, antiinflammatory, antitumor etc [19].

The deficiencies or hyper functions of enzymes cause a number of diseases. Enzymes activities can be blocked or modulated by specific inhibitors. Enzyme inhibition is an important area of pharmaceutical research since the studies in this field have already led to the discovery of wide range of drugs useful for a number of diseases. Specific inhibitors interact with enzymes and block their activity towards their corresponding natural substrates. The importance of enzyme inhibitors as drugs is enormous since these molecules have been used for treating a number of physiological conditions. Natural products can provide good pharmacophore templates for new drugs associated with various diseases. Development of new drugs from plant origin seeks much attention in order to overcome the undesirable side effects caused by synthetic medicines and for the emergence of multi-drugs. Based on literature reviews of the medicinal plants, the present work was designed to check their potentials in antioxidant and inhibitory activities against key enzymes.

2 Materials and methods

2.1 Chemical used

Methanol, n-hexane, ethanol, ethyl acetate and n-butanol were used for extraction/fractionation of plants. All solvents were commercial grade and distilled before use. Gallic acid, TPTZ, DPPH, FeSO4.7H2O, FeCl3 (Merck chemicals, Germany) were used for the studies. Rotary evaporator was used for the recovery of solvents.

2.2 Extraction of material

Rumex dentatus, Mentha spicata, Withania somnifera, Nerium indicum and Artemisia scoparia were collected from different areas of Azad Kashmir. These plants were identified by Dr. Khalid Hussain (Taxonomist) at the department of botany, the university of Gujrat (Gujrat). Fresh plant material was dried at room temperature for 20 days, powdered and stored in polythene bags for further process. Extraction of pulverized plant material was carried out by soaking in aqueous ethanol (ethanol/water, 90:10) for 7 days and stirring at regular interval. The extracts were filtered through filter paper and concentrated under vacuum on rotary evaporator to get residue (crude extracts). Crude extracts were dissolved in distilled water, stirred and filtered. The filtrate was partitioned with a variety of solvents using solvent extraction technique according to the scheme 1 mentioned below.

2.3 Total Phenols

Total phenol contents (TPC) of all fractions were calculated using Folin-Ciocalteu (FC) reagent [20]. A portion of 200 μl of each of the extracts (2 mg/ml) were mixed with 100 μl of FC reagent and 200 μl of sodium bicarbonate solution (10%). The mixture was left for 30 min at room temperature and absorbance was measured at 765 nm using UV/Vis Spectrophotometer. The amount of total phenolic content of each extract/fractions was articulated as mg/g equivalent to Gallic acid.

2.4 Ferric Reducing Antioxidant Power (FRAP)

The FRAP assay was conducted according to Shahwar (2012). The stock solutions constituted 300 mM acetate buffer (pH 3.6), 10 mM TPTZ (2, 4, 6-tripyridyl-s-triazine) solution in 40 mM HCl, and 20 mM FeCl3.6H2O solution. The fresh working solution was prepared by mixing 25 ml of acetate buffer, 2.5 ml of TPTZ solution, and 2.5 ml of FeCl3.6H2O solution. The extracts (150 μl) was then allowed to react with 2.5ml of the FRAP solution for 5 min. Absorbance readings of the colored solutions [ferrous tripyridyltriazine complex] was recorded at 593 nm [21].

2.5 Antiradical activity

For the estimation of the antiradical potential, DPPH radical scavenging activity of all the extracts/fractions was conducted using DPPH method [22]. A portion of 100 μl of extract (2 mg/ml) were mixed with 2 ml of DPPH solution (25 mg/l) and left for 30 min at room temperature. Methanol was used as the blank and the absorbance of the fractions was recorded at 517 nm using UV/Vis Spectrophotometer. Gallic acid was used as standard and the antioxidant activity of all extracts was expressed as percentage inhibition using the following formula; %Inhibition=Absorbance(blank)Absorbance(test)Absorbance(blank)×100

2.6 Total Antioxidant Capacity

The total antioxidant capacity of the plant extracts was evaluated by the method of Shahwar et al., 2011. An aliquot of 0.2 mL (500 ug/mL) of the sample solution was mixed with 2 ml of the reagent solution (600 mM sulfuric acid, 28 mM sodium phosphate and 4 mM ammonium molybdate). The reaction mixture was heated at 95 °C for 1h and absorbance was measured at 695 nm against a blank contained 2 mL of reagent solution [22].

2.7 Total Iron Reducing Power

Total iron reducing power of fractions/extracts was carried out by taking 100 μl of extract (2 mg/ml) in individual test tubes and mixing it with 2.5 ml of buffer (basic), 1.5 ml of potassium ferrocyanide (1%) and 100 pl of FeCl3 The reaction mixture was left for 30 min and absorbance was recorded at 700 nm by using UV/VIS Spectrophotometer [23].

2.8 Correlation Studies

A quantitative relationship between TPC vs antioxidant activities was developed using MS Excel software. In this graph the % inhibition acted as a function of total phenolic content (mg/g GAE). The percentage inhibition and FRAP values were taken on x-axis and TPC values of the extract were taken on y-axis. The relative dependence of antioxidant activity on the amount of phenolics was determined by explaining their correlation coefficient (R2)[24].

2.9 Enzyme Inhibition Activity

2.9.1 Acetyl/butyrylcholine Esterase

Spectrophotometric method was used to determine the inhibitory potential of the extracts/fractions against acetyl/butyryl choline esterase enzyme. Acetyl/ butyrylthiocholine iodide was used as a substrate. A portion of 2 ml of tris buffer of pH 7.8 was taken in a test tube and 0.2 ml of compound (2 mg/mL) and 30 μl of enzyme was added to it. The reaction mixture was then allowed to stand for 15 min. The coloring agent (50 μl) was added and the substrate ( 30 μl) was incubated at 37 °C for 20 min [25]. The absorbance was measured at 412 nm and % inhibition was calculated using the following formula: %Inhibition=Absorbance(blank)Absorbance(test)Absorbance(blank)×100

2.9.2 Protease Inhibition Activity

The protease inhibitory potential of extracts was evaluated using the colorimetric method of Shahwar et. al,. 2012. A mixture of tris buffer (100 mM) of pH 7.5 (1.0 mL), trypsin (0.3 ml), and extracts (0.1 ml) was incubated at room temperature for 10 min. Nα-benzoyl-DL-arginine-paranitroanilide hydrochloride BApNA (50 μl) was added to the reaction mixture and incubated at 37 °C for 30 min. Absorbance was recorded at 410 nm [26].

The % inhibition was calculated by the following formula; %Inhibition=Absorbance(blank)Absorbance(test)Absorbance(blank)×100

2.9.3 Statistical Analysis

All experiments were conducted in triplicates. Standard deviation and correlation studies (R2) was calculated using MS. Excel (2011) while ANOVA (one way) was applied using MiniTab with p < 0.05.

Ethical approval

The conducted research is not related to either human or animals use.

3 Results and Discussion

Drug’ dependence on plants is well known since early civilization. The continuing research in this field showing that the natural extracts hold chemical compounds that exhibit pharmacological and biological activities.

Medicinal plants and their extracts are being exercised for therapeutic purposes as they comprise of natural products which are being used to heal various diseases. The globe possesses about 2, 50,000 higher plants and 80,000 plants have medicinal importance among them. The extracts of medicinal plants are of great need for pharmaceutical and clinical use. The plant species Rumex dentatus (Polygonaceae), Mentha spicata (Lamiaceae), Withania somnifera (Solanaceae), Nerium indicum (Apocynaceae) and Artemisia scoparia (Asteraceae) were collected from different areas of the Azad Kashmir in 2012 and identified by Dr. Khalid Hussain (UOG). They were extracted, concentrated and partitioned with different solvents successively using the scheme 1.

3.1 Total Phenolic Contents

Total phenolic contents (TPC) of R. dentatus, M. spicata, W. somnifera, N. indicum and A. scoparia were enumerated by using Gallic acid as standard. TPC calculated as mg GAE/g of dry weight in M. spicata ranged from 74.33 ± 3.06 to 322.33 ± 7.09 mg GAE/g of dry weight in R. dentatus ranged 97.67 ± 2.08 to 355.67 ± 6.03 mgGAE/g of dry weight in N. indicum ranged from 46.33 ± 1.53 to 236.67 ± 2.52 mg GAE/g of dry weight in W. somnifera ranged from 34.00 ± 2.00 to 251.67 ± 5.03 mg GAE/g of dry weight in A. scoparia ranged from 21.33 ± 1.53 to 168.00 ± 4.00 (Table 1). The order of TPC in R. dentatus, M. spicata, W. somnifera, N. indicum, A. scoparia are given below: MM6>MM2>MM12>MM3>MM8>MM5>MM11>MM14>MM9>MM15>MM4>MM1>MM7>MM10>MM13

Table 1

Total Phenolic Contents of Selected Plants.

Previous studies have shown that phenolic compounds are good antioxidants and higher the phenolic contents better the antioxidant properties [27]. In the above order, it is clear that n-butanol and ethyl acetate fractions have shown good results as compared to n-hexane as phenolics are often extracted in higher amounts in more polar solvents [28,29]. Our results also showed close agreement with previously reported data which show higher the polarity of the solvent better the scavenging of radicals as phenolic compounds have the affinity towards polar solvents.

3.2 Total Iron Reducing Power

Total Iron Reducing Power of various extracts (leave and branch) of R. dentatus, M. spicata, W. somnifera, N. indicum and A. scoparia were accomplished and it was observed that all extracts exhibited significant activity ranging from 5.00 ± 0.00(minimum; MM13) to 92.33 ± 2.52 maximum; MM3) (see table 2). There seems a direct relationship between total iron reducing power and antioxidant activities [23].

Table 2

Antioxidant Activity of Selected Plants.

3.3 Free Radical Scavenging Potential

Radical scavenging potential of plant extracts was tested spectrophotometrically taking DPPH as free radical and Gallic acid as standard. The results indicated that all the extracts/fractions illustrated very good scavenging potential. The maximum activity i.e. 92.67 ± 1.53 % was shown by MM8 fraction and the lowest activity (12.67 ± 2.08 %) was shown by MM10 as given in table 2.

3.4 Evaluation of Total Antioxidant Capacity

Phosphomolybdate method was also carried out to test antioxidant potential of plant extracts. Most of the extracts exhibited excellent activity while a few showed poor activity. The antioxidant activity index (AAI) of plant extracts is given in table 2.

3.5 Ferric Reducing Antioxidant Power (FRAP)

The FRAP assay of all extracts of R. dentatus, M. spicata, W. somnifera, N. indicum and A. scoparia was accomplished using standard protocol [21].The extracts showed excellent result against ferric reducing antioxidant power except (MM13; 7.67 ± 0.58%). The maximum activity was exhibited by (MM3; 66.00 ± 1.00%) and the order is given as below:


3.6 Correlation of Antioxidant Activities

The quantitative analysis was used for investigating the correlation between antioxidant activities and phenolic contents in all the extracts. Correlation graphs were plotted against different antioxidant activities (including of DPPH, phosphomolybdate, total iron reducing, FRAP) and phenolic contents as shown in table 3.

Table 3

Correlation Coefficient.

As shown in Figures 1 and Table 3, significant positive correlations were observed between total phenolic content and different antioxidant assays, indicating the significant contribution of phenolics to these antioxidant assays. Among the above correlations, the total phenols vs FRAP exhibits lower R2 value compare to others. This result indicates that it exerts less inhibitory effect.

Corelation studies between different activities.
Figure 1

Corelation studies between different activities.

It is well established that phenolic compounds are good antioxidants and there is a positive cointerrelation ship between the two [27].

3.7 Acetylcholine Esterase Studies

In the field of pharmaceutical research, enzyme inhibition is an important area which has resulted in the discovery of a number of useful drugs. Specific inhibitors interact with enzymes and block their activity towards their corresponding physiological substrates. The importance of enzyme inhibitors as drugs is enormous, since these molecules have been used for treating a large number of physiological conditions [30]. Acetyl cholinesterase plays a vital role in the central and peripheral nervous systems. It catalyzes the hydrolysis of the neurotransmitter acetylcholine (ACh) in cholinergic synapses, and subsequently it can affect a number of pathogenic processes [31]. AChE inhibitors are used to cure of various neuromuscular disorders and have provided the first generation of drugs for treatment of Alzheimer’s disease (AD) [32], which is a progressive physical disorder and causes increasingly severe impairment in the cognitive [33].

The AChE inhibition activity of plant extracts was evaluated according to the method of Shahwar et al. 2012 with slight modifications [21]. It was concluded that the greatest inhibitory potential against the enzyme AChE was shown by ethyl acetate fractions of all the plants except N. indicum, while moderate activity was exhibited by other fractions as shown in table 4.

Table 4

Enzyme Inhibition Activity of Selected Plants.

3.8 Butyrylcholine Esterase

The evaluation of BChE inhibition activity of plant extracts concludes that the greatest inhibitory potential against the enzyme BChE was found in alignment with the observation of AChE. The ethyl acetate fractions show the maximum activity except for R. dentus which shows the minimum activity instead of N. indicum in the case of AChE .( see table 4 and figure 2).

Enzyme inhibition potential of various extracts.
Figure 2

Enzyme inhibition potential of various extracts.

3.9 Protease Inhibition

Proteases refer to a group of enzymes, whose catalytic function is to hydrolyze peptide bond of protein. They play a key role in a variety of biological processes, both at the physiological level as well as in infection. Proteases are classified into 5 groups namely, serine, threonine, cysteine, aspartate, and glutamic acid. Serine proteases are most widely studied group of proteases. Many pathological disorders are caused by deficiencies in the normally exquisite regulation of the activity of proteolytic enzymes, resulting in abnormal tissue destruction and the aberrant processing of other proteins. Elastase, chymotrypsin, and trypsin are the subclass of serine proteases based on the type of substrate. Trypsin cleaves polypeptide chains on the C-terminal side of a positively charged side-chine containing arginine or lysine. Protease inhibition assay was carried out using UV/Vis spectrophotometer. Absorbance was measured at 410 nm and percentage inhibition was also determined. All extracts exhibited excellent activities and the maximum inhibitory potential was shown by MM4. The percentage inhibition is given in the following order:

MM4> MM15> MM8> MM5> MM1> MM14> MM2> MM3> MM9> MM11> MM12> MM6> MM13> MM10> MM7 (table 4 and figure2).

4 Conclusions

in recent years, tThere is an exponential increase in the research studies investigating the antioxidant properties of plantsin recent years. It is observed that the higher intakes of natural antioxidants containing phenolic compounds are associated with long-term health benefits. Free radicals play a significant role in pathogenesis of tissue damage, consequently having implications in many clinical conditions. Both endogenous and exogenous antioxidants play a protective role in repairing the damage caused by the free radicals. Exogenous antioxidant supplementation is increasingly used to fight against oxidative stress. All extracts of Rumex dentatus, Mentha spicata, Withania somnifera, Nerium indicum, and Artemesia scoparia exhibited good antioxidant as well as enzyme inhibition activities. Antioxidant potential of some plants have been reported while enzyme inhibition studies of all plants are not carried out previously. Further research studies are needed to determine the structure, composition, isolation methods and purification techniques of these phytochemicals and investigation of their relevant antioxidants, enzyme inhibition potentials and toxicological estimation with the idea of formulating novel chemotherapeutics.


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About the article

Received: 2017-08-08

Accepted: 2017-10-02

Published Online: 2017-12-29

Conflict of interest: Authors state no conflict of interest.

Citation Information: Open Chemistry, Volume 15, Issue 1, Pages 371–379, ISSN (Online) 2391-5420, DOI: https://doi.org/10.1515/chem-2017-0041.

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© 2017 Muhammad Asam Raza et al.. This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 License. BY-NC-ND 4.0

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