In vitro inhibitory potential of methanolic extract of Celosia argentea var. cristata on tyrosinase, acetylcholinesterase and butyrylcholinesterase enzymes

This study was conducted to evaluate the pharmacological potential of plant in the inhibition of three to Abstract In the current study, methanol extract of Celosia argentea var. cristata was tested for its inhibitory potential against tyrosinase, acetylcholinesterase and butyrylcholinesterase enzymes at the concentration of 0.5 mM by ELISA microtiter plate assays. A significant tyrosinase inhibitory activity (63.6%), acetylcholinesterase inhibitory activity (80.3%) and butyrylcholinesterse inhibitory activity (68.24%) was shown by crude methanolic extract of C. argentea var . cristata with respective IC 50 values of 268.5 ± 0.2 µg/mL, 73.6 ± 0.1 µg/mL and 132.8 ± 0.9 µg/mL. The result of this study reveals the use of C. argentea var. cristata in skin hyperpigmentation, Parkinson’s disease and neurodegenerative disorders like Alzheimer’s disease and dementia. Keywords:

Collection and authentication of plant material: Whole plant of C. argentea var. cristata was collected from botanical garden of Bahauddin Zakariya University Multan and identified with the kind cooperation of an expert taxonomist (Prof. Altaf Dasti) from the Institute of Pure and Applied Biology at Bahauddin Zakariya University Multan, Punjab, Pakistan. A sample voucher (STW-231) was submitted to the herbarium of Institute of Pure and Applied Biology at Bahauddin Zakariya University Multan, Punjab, Pakistan.
Extraction of crude extract: The plant material was shadedried and rendered free from soil and adulterated material and coarsely ground by electrical device. The powdered material was soaked into aqueous-ethanol (80%) for 72 hours with occasional shaking. The soaked material was rendered free from plant debris by passing through a muslin cloth and fluid portion was filtered through a fine filter paper (Williamson et al., 1998). The above mentioned extraction procedure was repeated twice on the plant debris and filtrate was subsequently combined before subjecting to evaporation under reduced pressure on a rotary evaporator to thick paste like mass of dark brown color, i.e., crude extract of C. argentea var. cristata, yielding approximately 11.1% (w/ w).
Preliminary phytochemical analysis: Qualitative phytochemical analysis of crude extract was done for the presence of alkaloids, anthraquinones, coumarins, saponins, flavonoids and tannins as reported elsewhere (Janbaz and Fatima., 2015) Tyrosinase inhibition activity: Total volume of reaction mixture 100 μL containing 60 μL phosphate buffer (100 mM), pH 6.8, 10 μL mushroom tyrosinase enzyme (5 units) and 10 μL 0.5 mM test compound mixed in 96well plate (Lee et al., 2009). Contents were pre incubated for 5 minute at 37 o C. After incubation, 20 μL of 10 mM L-dopamine was added as a substrate. Contents were mixed and incubated for further 30 min. Absorbance was taken at 490 nm using Synergy HT Biotek 96well plate reader.
The enzyme inhibition (%) was calculated by the bellow formula: %Inhibition = 100 -(abs of test sample / abs of control × 100) Acetylcholinesterase inhibition activity: The AChE inhibition activity was performed according to the method (Ellman et al., 1961) with slight modifications. Total volume of the reaction mixture was 100 µL. It contained 60 µL Na2HPO4 buffer with concentration of 50 mM and pH 7.7. Ten microliter test compound (0.5 mM/well) was added, followed by the addition of 10 µL (0.005 unit per well) enzyme. The contents were mixed and pre-read at 405 nm. Then contents were pre-incubated for 10 min at 37ºC. The reaction was initiated by the addition of 10 µL of 0.5 mM/well substrate (acetylthiocholine iodide), followed by the addition of 10 µL DTNB, 0.5 mM/well. After 30 min of incubation at 37ºC absorbance was measured at 405 nm using 96-well plate reader Synergy HT, biotek, USA. All experiments were carried out with their respective controls in triplicate. Eserine (0.5 mM/well) was used as a positive control. The percent inhibition was calculated by the help of following equation.

Inhibition (%) = Control -Test x 100 Control
Butyrylcholinesterase inhibition activity: The BChE inhibition activity was performed according to the method (Ellman et al., 1961) with slight modifications. Total volume of the reaction mixture was 100 µL containing 60 µL, Na2H PO4 buffer, 50 mM and pH 7.7. The 10 µL test compound 0.5 mM per well was added followed by the addition of 10 µL (0.5 unit per well) BChE (Sigma Inc.). The contents were mixed and pre-read at 405 nm and then pre-incubated for 10 min at 37ºC. The reaction was initiated by the addition of 10 µL of 0.5 mM/well substrate (butyrylthiocholine chloride). Followed by the addition of 10 µL DTNB (5,5'-dithiobis-(2-nitrobenzoic acid)), 0.5 mM well -1 . After 30 min of incubation at 37ºC, absorbance was measured at 405 nm using 96-well plate reader Synergy HT, Biotek, USA. All experiments were carried out with their respective controls in triplicate. Eserine (0.5 mM/well) was used as positive control. The percent inhibition was calculated by the help of following equation.

%Inhibition = Control -Test x 100 Control
Statistical analysis: In all of the enzyme inhibition activities, the experiments were performed three times and the results were expressed as mean ± standard error of mean (S.E.M) of three parallel measurements and the respective.IC50 values were calculated using EZ -Fit Enzyme kinetics software (Perrella Scientific Inc. Amherst, USA). Amherst USA software. The data was analyzed by an analysis of variance p<0.05. Results were processed by computer programs: Excel and Ezi Fit software.

Results
The result of preliminary phytochemical analysis showed that extract of C. argentea var. cristata contains alkaloids, saponins, flavonoids, tannins and phenolic compounds.
Very moderate antityrosinase activity was seen with the methanolic extract of Celosia argentea var. cristata at the dose of 0.5 mg/mL which showed 63.6% inhibition of tyrosinase with 50% inhibition (IC50) at a concentration of 268.9 ± 0.2 µg/mL (p<0.05) ( Table I). The standard drug kojic acid, showed significant tyrosinase inhibition activity with the 0.5 mg/mL concentration showing a 93.5% inhibition of tyrosinase(IC50 6.0 ± 0.1 µg/mL). The %tyrosinase inhibition activity of crude extract and kojic acid are shown in a Figure 1.
The IC50 values of the extract was found to be greater than the standard, eserine which showed an IC50 of acetylcholinesterase at concentration of 0.04 ± 0.0 µg/ mL and IC50 of butyrylcholinesterase at 0.9 ± 0.0 µg/mL. The %acetylcholinesterase and butyrylcholines-terase inhibition activity of crude extract and standard, eserine are shown in a Figure 2.

Discussion
The skin is an important barrier that protects our body from damage due to its direct contact with the outside environment. Melanin is the important pigment in the skin. It protects our skin from UV damage by absorbing sunlight and removing reactive oxygen species. About 10% of skin cells in the innermost layer of epidermis produce melanin. Upon exposure to UV radiation, melanogenesis is initiated by the enzyme tyrosinase resulting in skin darkening (Vamos, 1981). As discussed earlier, tyrosinase is involved in the transformation of  tyrosine to o-dopaquinone. The hyperpigmented skin is observed in various dermatological disorders namely melasoma, solar lentigines and ephelides (Maeda and Fukuda, 1991). The reactive oxygen species produced in the human body enhances the DNA damage, melanin biosynthesis and induces the proliferation of melanocytes (Yasui and Sakurai, 2003) which results in hyper pigmentation disorders. The production of melanin in the body can be reduced by several mechanisms including direct inhibition of tyrosinase enzyme, inhibiting the transport of melanosome to the stratum corneum, the supplementation of antioxidants (Pawelek and Kormer, 1982) as well.
The most common skin lightening and depigmentation agents available commercially are kojic acid, arbutin, catechins, hydroquinone (HQ) and azelaic acid (Maeda and Fukuda, 1996). Some adverse effects of these synthetic compounds are irreversible cutaneous damage, ochronosis etc. These adverse effects have led to the search for safer plant-based skin lightening ingredients. The aqueous methanolic extract of C. argentea var. cristata showed a moderate anti-tyrosinase activity with 63.64% inhibition. The preliminary chemical examination of the crude extract of C. argentea var. cristata has demonstrated the presence of phenolic and flavonoid compounds which may contribute to its tyrosinase inhibitory effect as reported earlier (Kubo et al., 2003;Nagendra et al., 2009). Anti-oxidant activity also affect tyrosinase activity (Kim et al, 2008). The potent skin whitening and antioxidant ability of C. argentea var. cristata make it a suitable candidate for the remedy of hyper pigmentation disorders of skin.
AChE (EC 3.1.1.7) comprise a family of enzymes which include serine hydrolases. They share about 55% of amino acid sequence identity and have similar catalytic properties. The different specificities for substrates and inhibitors are due to the difference in amino acid residues of the active sites of AChE and BChE (Cyglar et al., 1993). Acetylcholinesterase plays a prominent role as it participate in termination of acetylcholine based signal transmission through neurosynaptic cleft or neuromuscular junction (Ballard et al., 2005;Khaled et al., 2010).
Butyrylcholinesterase (BChE) is a non-specific ester hydrolyzing enzyme with no knownendogenous physiological substrate (Quinn, 1987). It can hydrolyze various esters of choline and other compounds (Sun et al., 2001), e.g., cocaine, some organophosphorus compounds. BChE is produced in liver and enriched in blood circulation. In addition, it is also present in adipose tissues, intestine, smooth muscle cells, and white matter of the brain (Schwarz et al., 1995). H1 and H2 receptor antagonists have been shown to possess AChE inhibitory activities.
A deficit of ACh levels in CNS leads to conditions such as Alzheimer's disease (AD) which is the most common form of dementia, a progressive neurologic disease of the brain that leads to loss of mental ability severe enough to interfere with normal activities of daily living and decline in cognitive functions such as remembering, reasoning and planning. It affects parts of the brain that control thought, memory, and language. It is characterized by nerve-cell loss, abnormal tangles and plaques within nerve cells and deficiencies of several neurochemicals such as acetylcholine (ACh) and butyrylcholine (BCh), which are essential for the transmission of nerve messages. It was postulated that blocking the enzyme cholinesterase (ChE) induced hydrolysis of ACh and subsequent increase in Ach concentration in central synapses and enhancement of cholinergic functions provides the symptomatic improvement to AD patient (Schwarz et al., 1995;Pohanka et al.,2009).
ChE inhibitors were developed to improve the effectiveness of ACh by inhibiting its breakdown and increasing the levels in the brain or by strengthening the way nerve cells responds to it. Increased concentrations of ACh in the brain leads to increased communication between nerve cells and may temporarily improve or stabilize the symptoms of AD. These drugs appear to work best in the early and moderate stages of AD. It has been further suggested that dual inhibition of AChE and BChE enzymes should be one of the objectives in the treatment of cognitive dysfunction associated with AD (Pohanka et al., 2010).
The results obtained with the crude extract of C. argentea var. cristata, exhibits outstanding enzyme inhibitory activity against AChE (80.3%), & BChE (68.2%). The outstanding results obtained with the crude extracts of C. argentea var. cristata, indicate the need for further work on the isolation, purification and investigation of the active principles responsible for the extract inhibitory activity.

Conclusion
The crude extract of Celosia argentea var. cristata possesses inhibitory activity against tyrosinase, acetylcholine estrase and butrylcholineestrase enzymes which provide a base for its use in hyperpigmentation of skin, Parkinson's disease and other neurodegenerative disorders like Alzheimer's disease and dementia.