Wound Healing, Anti-pancreatic Cancer, and α-amylase Inhibitory Potentials of the Edible Mushroom, Metacordyceps neogunnii

 

Abdu Ghalib ALKolaibe1, Waill A. Elkhateeb2*, Marwa O. Elnahas2,

El-Manawaty M.3, Deng CY4, Ting-Chi Wen5,6,7*, Ghoson M. Daba2

1Microbiology Department, Faculty of Science, Taiz University, Taiz, Yemen.

2Chemistry of Natural and Microbial Products Department, Pharmaceutical Industries Researches Division, National Research Centre, El Buhouth St., Dokki, 12311, Giza, Egypt.

3Drug Bioassay-Cell Culture Laboratory, Pharmacognosy Department, National Research Centre, Dokki, Giza, 12622, Egypt.

4Guizhou Institute of Biology, Guizhou Academy of Sciences, Guiyang, Guizhou 550009, China.

5State Key Laboratory Breeding Base of Green Pesticide and Agricultural Bioengineering, Key Laboratory of Green Pesticide and Agricultural Bioengineering, Ministry of Education, Guizhou University, Guiyang 550025, China. 6The Engineering Research Center of Southwest Bio–Pharmaceutical Resources Ministry of Education, Guizhou University, Guiyang 550025, Guizhou Province, China.

7The Mushroom Research Centre, Guizhou University, Guiyang 550025, China.

*Corresponding Author E-mail: tingchiwen@yahoo.com, waillahmed@yahoo.com

 

ABSTRACT:

Metacordyceps neogunnii is an edible mushroom that has been previously known as Cordyceps gunnii. Recently, it has been proven that Metacordyceps neogunnii differs from similar species by having longer asci and wider ascospores and is phylogenetically distinct to related species. Few studies are available to describe bioactivities exerted by Metacordyceps neogunnii extracts which is insufficient to evaluate the potency of this mushroom. Therefore, the current work was conducted to evaluate the in vitro α-Amylase inhibitory ability, wound healing, and anti-human pancreatic cancer activities of Metacordyceps neogunnii 80% methanolic extract. M. neogunnii extract showed no effect on human cancer pancreatic cell line (Paca2). However, the same extract exhibited slight wound healing effect. On the other hand, M. neogunnii extract exhibited good α-amylase inhibitory activity that reached 69.3±0.78%, and IC50 of 32.38±4.0.

 

KEYWORDS: Metacordyceps neogunnii, Mushroom, Wound healing, Anti-pancreatic cancer, α-amylase inhibitor.

 

 

1.       INTRODUCTION:

Cordyceps (Cordyceps sensu lato) is one of the famous edible and medicinal mushrooms worldwide. Moreover, it exhibited promising biological activities and have an important role in biological control1. Cordyceps species, which comprise an important group of entomopathogenic fungi, mainly grow on insects as well as on other arthropods and Elaphomyces Nees fungi2.

Various bioactive compounds belonging to different chemical classes have been reported from different Cordyceps species1,3,4. Cordyceps gunnii (berk.) Berk is a parasite on the larvae of Hepialidae, which belongs to Clavicipitaceae, Sphaeriales, Pyrenomycetes, and Ascomycota. It is widely distributed in Guizhou, Anhui and Hunan province of China5, and has various effects such as analgesia, sedation6, anti-tumor3, anti-aging7, improvement of human immunity8, hypocholesterolemic capability9, promoting sleep and enhancing memory10.

This mushroom is recognized as an alternative of Ophiocordyceps sinensis, and its price is about 2000 $/kg. Cordyceps species are ranked among the expensive natural alternatives used in traditional medicine due to their potent healing capabilities1.

 

 

Metacordyceps neogunnii, namely Cordyceps gunnii (Berk.) Berk, is a mushroom that has been wrongly regarded in China as ‘Cordyceps gunnii’ for more than 30 years. Recently, it has been proven that Metacordyceps neogunnii differs from similar species by having longer asci and wider ascospores and is phylogenetically distinct to related species11. Till now, studies describing the chemical composition and pharmacological potentials of Metacordyceps neogunnii are insufficient when compared to studies conducted on other Cordyceps species. Therefore, this study was conducted to describe the α-amylase inhibitory activity, wound healing, anti-pancreatic cancer, and anti- microbial activities of the crude extract of Metacordyceps neogunnii. Results from this study gave important information about the not well studied mushroom, Metacordyceps neogunnii which encourage for further studies to evaluate this interesting mushroom.

 

2.       MATERIAL AND METHODS:

2.1       Sample extraction

Metacordyceps neogunnii mushroom sample (Figure 1 a, b) was provided by Prof. Dr. Ting-Chi Wen. For extract preparation, two hundred and fifty grams of Metacordyceps neogunnii fruiting bodies were washed with distilled water, then air-dried. The cleaned fruiting bodies were cut into small pieces, placed and left soaked for 48h at room temperature in an Erlenmeyer flask containing 80% methanol. After that, filtration was conducted and filtrate was concentrated using a rotary evaporator (Heidolph rotary evaporator, Schwabach, Germany). Resulting extract was stored at 4ºC until further use.

 


Figure (1). Metacordyceps neogunnii mushroom sample, photograph taken by Dr. Waill Elkhateeb, Chemistry of natural and microbial products department, National Research Centre, Egypt.

2.2       In vitro wound healing activity of Metacordyceps neogunnii extract:

The wound healing activity of Metacordyceps neogunnii extract and the migration rates of BJ-1 cells were evaluated in the Bioassay-cell Culture Laboratory, National Research Center, Dokki, Egypt. Using the scratch assay method. BJ-1 cell line is human skin fibroblast that is derived from normal foreskin and was obtained from American Type Culture Collection, ATCC® CRL-2522™, Manassas, VI, USA). The cell density of 2×105 cells were seeded into each well of a 24-well plate and incubated with complete medium at 37

°C and 5% CO2. After 24h of incubation, the monolayer confluent cells were scrapped horizontally with a sterile P200 pipette tip. The debris was removed by washing with PBS. The cells were treated with Metacordyceps neogunnii 80% methanolic extract at concentration 25, 50, 100, 200µg/mL. The cells without treatment were used as negative control. The induced scratch that represented the wound was photographed at 0h using phase-contrast microscopy at ×40 magnification, before incubation with the samples. After 24h of incubation, the second set of images was photographed. To determine the migration rate, the images were analyzed using “image J” software, and percentage of the closed area (wound closure %) was measured as following:

Measurement at 0 h – Measurement at 24 h Wound closure % = ––––––––––––––––––––––––––––––––––– × 100

Measurement at 24 h

The increase in the percentage of the closed area was taken as an indication for the cell migration. Experiments were performed in the triplicate and the data were recorded and analyzed statistically using SPSS 11.

2.3       Cytotoxic effect of M. neogunnii extract on human pancreatic cancer cell line (Paca2):

Cell viability was assessed by the mitochondrial dependent reduction of yellow MTT (3-(4, 5- dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide) to purple formazan. Human pancreatic cancer cell lone (paca2) were suspended in DMEM-F12 medium, 1% antibiotic-antimycotic mixture (10,000U/mL Potassium Penicillin, 10,000µg/mL Streptomycin Sulfate and 25µg/mL Amphotericin B) and 1% L-glutamine at 37ºC under 5% CO2. Cells were batch cultured for 10 days, then seeded at concentration of 10x103 cells/well in fresh complete growth medium in 96-well microtiter plastic plates at 37ºC for 24h under 5% CO2 using a water jacketed Carbon dioxide incubator (Sheldon, TC2323, Cornelius, OR, USA). Media was aspirated, fresh medium (without serum) was added and cells were incubated either alone (negative control) or with different concentrations of sample to give a final concentration of (100-50-25-12.5-6.25-3.125-1.56 and 0.78μg/mL). After 48 h of incubation, medium was

 

aspirated, 40μL MTT salt (2.5μg/mL) were added to each well and incubated for further four hours at 37ºC under 5% CO2. To stop the reaction and dissolving the formed crystals, 200μL of 10% Sodium dodecyl sulphate (SDS) in deionized water was added to each well and incubated overnight at 37ºC. A positive control which composed of 100µg/mL was used as a known cytotoxic natural agent who gives 100% lethality under the same conditions.

 

The absorbance was then measured using a microplate multi-well reader (Bio-Rad Laboratories Inc., model 3350, Hercules, California, USA) at 595nm and a reference wavelength of 620nm. A statistical significance was tested between samples and negative control (cells with vehicle) using independent t-test by SPSS 11 program. DMSO is the vehicle used for dissolution of plant extracts and its final concentration on the cells was less than 0.2%.

 

The percentage of change in viability was calculated according to the formula:

(Reading of extract/Reading of negative control) -1) x 100

 

A probit analysis was carried for IC50 and IC90 determination using SPSS 11 program.

2.4          α-Amylase inhibitory effect of Metacordyceps neogunnii extract:

α-Amylase of concentration 4 U/mL was prepared in phosphate buffer saline (pH 6.8). After that, 15μL of sample at varying concentrations (0.6 to 220ppm final volume) were mixed with 60μL of 4 U/mL α-amylase and incubated for 15 min at 37°C in a 96 well plate. Then, 60μL of 0.2% soluble starch solution (dissolved by heating in a microwave and then filtered) were added to the mixture and incubated at 37°C for 10 min. The reaction is terminated by the addition of 30μL of 1M HCl, 150μL KI/I2 aqueous solution. α-Amylase activity was determined spectrophotometrically at 595nm by measuring the quantity of the released blue color. Negative control was prepared by using 15μL of buffer solution instead of the sample, while acarbose is used as a positive control. The calculation and analysis of data is performed using graphpad prism program.

 

3.       RESULTS:

3.1        In vitro wound healing activity of M. neogunnii

extract:

Investigating the effect of Metacordyceps neogunnii extract on the migration of fibroblast cells (BJ-1) was conducted to evaluate its wound healing ability.

 

Figure (2). Wound healing ability of Metacordyceps neogunnii on BJ-1 cells. Images were acquired at (a) 200 ppm 0h, and (b) 200 ppm after 24h of exposure to extract. (c) 100ppm 0h, and (d) 100ppm after 24h of exposure to extract. (e) 50 ppm 0h and (f) 50 ppm after 24h of exposure to extract. (g) 25ppm 0h and (h) 25 ppm after 24h of exposure to extract. Images were analyzed using “image J” software, and percentage of the closed area was measured and compared with the value obtained at 0h.

 

 

Figure (3). (a) Human cancer pancreatic cell line (Paca2) Untreated cells. (b) Human cancer pancreatic cell line (Paca2) treated cells with 200 ppm of M. neogunnii extract. (c) Human cancer pancreatic cell line (Paca2) treated cells with 100 ppm of Metacordyceps neogunnii extract.

Results shown in Figure 2, revealed that cells migration towards the provisional gap was slightly induced after 24h of exposure to Metacordyceps neogunnii crude extract. Migration analysis values showed that 50ppm of the Metacordyceps neogunnii crude extract was the concentration causing that slight effect in cell migration (41%). However, increase in extract concentration had no effect on cell migration.

3.2        Cytotoxic effect on human cancer pancreatic cell line (Paca2):

The cytotoxic activity of Metacordyceps neogunnii extract was evaluated against human cancer pancreatic cell line (Paca2) using the MTT method. The results showed that Metacordyceps neogunnii extract has no effect on human cancer pancreatic cell line (Paca2) (Figure, 3).

3.3       α-Amylase Inhibitory Activity:

The in vitro inhibitory effect of Metacordyceps neogunnii crude extract on α-amylase was investigated and the results are shown in table 1. Metacordyceps neogunnii extract have good α-Amylase activity (69.3±0.78%), and IC50 of 32.38±4.0ppm. in comparison with the positive control (acarbose), which caused 100% inhibition, and IC50 of 20.8±3.5, r2=0.92. This result suggested that Metacordyceps neogunnii has promising α-Amylase inhibitory activity.

Table 1. Inhibition of α-Amylase by Metacordyceps neogunnii

extract at 55 ppm

Sample

α-amylase inhibition (%)

IC50 (ppm)

Metacordyceps neogunnii extract

69.3 ± 0.78

32.38 ± 4.0, r2=0.96

Acarbose (Positive control)

100

20.8 ± 3.5, r2=0.92

The results are expressed as average inhibition of three results ± Standard deviation; IC50 results are expressed as average IC50 ± Standard deviation, coefficient of determination (r2).

4.       DISCUSSION:

Current spread of metabolic syndromes including type 2 diabetes and obesity has forced researchers to screen for sources capable of exerting activities against such diseases. On the other hand, cancer diseases represent serious threaten to human life and health in today's world

and have a very high incidence. It is a major cause of human deaths worldwide, and this mortality rate is still rising. Experts estimate that the number of deaths caused by cancer may exceed 13.1 million by 203012. Mushrooms are among natural and promising sources of bioactive compounds that keep attracting researchers’ attentions13-24. One of the mushrooms that still under investigation is M. neogunnii, and available data describing its biological activities are relatively insufficient. For more than 3 decades, M. neogunnii has been incorrectly known under the name Cordyceps gunnii. Recently, it has been proven that M. neogunnii has longer asci and wider ascospores and is phylogenetically distinct to related species11. Hence, this study investigated the in vitro biological activities of the

M. neogunnii extract concerning its wound healing enhancing ability, anti-pancreatic cancer activity, and α- amylase inhibiting action.

In order to evaluate the wound healing potential of M. neogunnii extract, cell migration was tested after treatment with the extract as cell migration plays a key role in wound repair and healing, M. neogunnii extract has slightly improve the migration of fibroblast cells (BJ-1). On the other hand, pancreatic cancer is one of the most aggressive malignancies and is characterized by a 5-year survival rate, a broad genetic diversity and a high resistance to conventional therapies. As a result, novel therapeutic agents to improve the current situation are needed urgently. According to reports, the mycelium of Metacordyceps neogunnii has an inhibitory effect on the growth of tumor cells3,9,25. However, in our study Metacordyceps neogunnii extract couldn’t inhibit human pancreatic cancer cell line (Paca2).

α-Amylase is one of the main enzymes in the process of starch digestion as it helps in starch digestion and can cause postprandial blood sugar increase in diabetic patients26,27. Inhibiting α-Amylase can hinder the hydrolysis and digestion of carbohydrates in food, reduce the digestion of sugars, effectively control the increase in blood sugar after meals, and achieve the purpose of anti-diabetic28-37. In this study, Metacordyceps neogunnii 80% methanolic extract

 

showed good α-inhibitory activity in comparison with the positive control. Hence, Metacordyceps neogunnii can be nominated for further investigations for the prevention and treatment of diabetes.

 

CONCLUSION:

The wound healing, anti-tumor, and α-amylase inhibitory activities of Metacordyceps neogunnii were investigated in this study. Results nominate Metacordyceps neogunnii for further investigations in order to evaluate its application prospect in the prevention and treatment of diabetes which could be developed as a therapeutic product for diabetes.

 

CONFLICT OF INTEREST:

The author(s) declare (s) that there are no conflicts of interest to disclose.

 

ACKNOWLEDGEMENTS:

This work was supported by the Biodiversity Survey and Assessment Project of the Ministry of Ecology and Environment, China (No. 2019HJ2096001006), and the Science and Technology Foundations of Guizhou Province (No. [2019]2333).

 

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