Evaluation of Antioxidant and Antidiabetic activity of White Oyster Mushroom (Pleurotus florida) cultivated in North Bengal
Evaluation of Antioxidant and Antidiabetic activity of White Oyster Mushroom (Pleurotus florida) cultivated in North Bengal
Abstract:
Oyster mushroom is one of the popular mushrooms cultivated in North Bengal. Pleurotus florida is one of the cultivated mushroom having high demand for its bright white colour, texture and good taste. Seasonal productivity of P. florida was estimated and it was observed that the production rate is higher in late rainy season to winter season as the fruiting body required moderately low temperature. Antioxidant activity of P. florida also evaluated in respect of DPPH scavenging activity and Free reducing antioxidant power (FRAP) activity which showed significant results. In vivo antidiabetic activity was also evaluated in using Streptozotocin induced albino rats and it was observed that the mushroom powder helps in lowering the blood glucose level and actively regain the body weight. P. florida also showed significant blood creatinine and urea activity in compare to control groups. Thus it is clear that P. florida mushroom showed both antidiabetic and antioxidant activity which may be considered to be good food for human health.
Keywords:
Pleurotus florida, Seasonal Productivity, DPPH, Antidiabetic activity, Creatinine, Urea.
Introduction:
Mushroom cultivation is now a days an appropriate way of agricultural waste management through the bio- conservation technique for the economic growth (Chang and Miles, 2004). Mushrooms are the fleshy fruiting body or the sporophyte normally grown on the decaying organic matter or tree trunks. About hundreds of species are known as edible but only 22 species are now being cultivated worldwide (Manzi et al, 1999). Syed et al., (2009) stated that the mushrooms are one of the healthy food consisting of rich source of proteins, vitamins and lower amount of calories and fat which improves human health. Krishnamoorthy and Sankaran (2014) suggested that Pleurotus sp consists of numerous nutritional compositions and various active components which show several medicinal properties like antidiabetic, anticancer, antibiotic, anti-cholestrolic and antioxidant activities. Pleurotus sp is also a very good source of different bioactive compounds and metabolites like terpinoids, phenolics and many polysaccharides which enhances the nutraceutical properties (Dundar et al., 2013 and Correa et al., 2016).
Materials and Methods:
A] Cultivation of mushroom
(i) Substrate preparation and spawning
Paddy straw was used for the cultivation of oyster mushroom. Chopped (2-4cm long) rice straw was washed and soaked in water for overnight. The straw was again cleaned and pasteurized at 55-650 C for 20-30 min. and it was allowed for cooling at room temperature. Layer spawning was done using the polypropylene bags. 100 gm of spawn was used for 1kg of substrate for spawning. The bags were then filled using the substrate and spawn and the bags were closed tightly. Small holes were made in each bottle and bag for aeration. The bags were then incubated at room temperature (25-300 C) for 10-12 days. After 10-12 days, the white mycelia covered the whole substrate. Then the plastic was removed and 80-90% moisture was maintained by spraying water on the substrate for 2-3times in a day for the initiation of pinhead (Sarker et. al. 2007).
(ii) Harvesting of Fruiting body
The pinhead appeared after 4-5 days of opening the bags. Fruiting body was developed at room temperature and 80-90% relative humidity. The fruiting body was harvested from the base carefully so that there should be no injury of the mycelia. Humidity was maintained again after the harvest for further flushes.
B] Antioxidant Activity
(i) DPPH Scavenging activity
10g of dried mushroom powder was taken and 100 ml of 95% ethanol was added and stirred for 24 hrs. at 250 C. Then the solution was filtered using Whatman No1 filter paper and the extract was evaporated using rotary evaporator at 400 C. Then the evaporated sample was redissolved in 95% ethanol. 100 µl sample was taken and 5µl DPPH solution was added to it and incubated for 30 min in dark. After incubation absorbance was taken at 517nm against a control. DPPH scavenging activity was measured using the following formula
Inhibition % = 100 (Acontrol– Asample)/Acontrol
(ii) Ferric Reducing Antioxidant Power Assay
2.5ml of (0.2 M) phosphate buffer (pH6.6) was taken in a test tube and to it 2.5 ml of 1% potassium ferricyanide, 1 ml of distilled water and 1 ml of ethanolic extract of the test sample was taken and incubated at 500C for 20 min in water bath. Then 2.5ml of 10% trichloro acetic acid was added and mixed well. Then it was centrifuged at 10000 rpm for 15 min. the upper layer of the mixture was collected. 2.5ml of reaction mixture was taken and to it 2.5 ml of distilled water and 300µl of 1% ferric chloride was added and mixed well. Then the absorbance was taken at 700 nm in spectrophotometer.
C] Antidiabetic activity
(i) Induction of Diabetes
Male Wister albino rats (120-150g) were used for the experimental purpose. The animals were kept into polypropylene cages and maintained in Antisera Reserves of Plant Pathogen under laboratory conditions (Niyonzima et. al 1993). Animals were maintained by feeding 50 gm bread and pea twice per day before starting the treatment.
Animals were allowed to fast for 15-18 hrs and freshly prepared solution of Streptozotocin (55mg/kg body weight) in 0.1M Sodium Citrate buffer pH4.5 was injected intra-peritoneally in a volume of 1ml/kg body weight. After 48 hrs. of injection, blood sugar level was observed and rats with more than 200 mg/dl glucose level was considered as diabetic and used for further experiment.
Chemicals used
Streptozotocin (N-Methylnitrosocarbamoyl-α-D-Glucosamine) was used for induction of blood sugar in white albino rats. The chemical procured from the HiMedia Laboratory Mumbai (Product code- CMS-1758-250MG).
Experimental Design
In this experiment, 20 rats were used and these were divided into four groups of 5 each.
Group-I untreated rats receiving distilled water designated as Normal control;
Group-II rats were induced with Streptozotocin (55mg/kg) was considered as Diabetic control or Negative control;
Group-III was induced with Streptozotocin and treated with a commercially available standard drug Metformin tablets and was designated as positive control and
Group-IV was Streptozotocin induced rats treated with aqueous suspension of P. florida powder administered orally was designated as Sample Treated. STZ treated rats were fed with aqueous suspension (250mg/kg b.w.) of P. florida powder for 20 days. The body weight and blood sugar level was observed in regular intervals of all the groups. After 20 days the blood were collected by puncturing heart of each rats for further experiments.
(ii) Determination of blood sugar
Blood sample was collected from the tail vein of two hours fasted rats and glucose level was estimated using the Accucheck Blood Sugar measuring kit.
(iii) Estimation of blood creatinine
Blood creatinine level was determined using the ERBA-CRE creatinine kit (Product code 120246). 100 µl sample or standard solution was added to 1000µl of working reagent and mixed well. Absorbance was taken after 20th second and 80th second at 505nm as described by Young et al (1975).
Blood creatinine = ΔA of test sample X Conc. of Standard/ ΔA of standard
(iv) Estimation of blood urea
Blood urea was determined using the ERBA-UREA (BUN) kit (Product code 120214). 20µl standard or the taste sample added to 1000µl working reagent and mixed well. Then the absorbance was taken at 340 nm and urea content calculated by following formula
Blood urea = ΔA of test sample X Conc. of Standard/ ΔA of standard
Results and Discussion:
A] Cultivation of Pleurotus florida
Growth in different substrates
Oyster mushroom is one of the major mushrooms cultivating widely in North Bengal and different species of oyster mushroom are now being cultivated by a large number of farmers in this region. P. florida commonly known as white oyster mushroom largely cultivated for its bright white fruiting body, texture and great taste (Figure 1). The results showed that the mycelial run period was about 14 days during the early winter season to early summer (Table 1). It was observed that the spawn run period reduces during November to April. Cultivation also depends on the environmental conditions and the results the production increased during December to April and the complete cropping period also decreased during this period. Results also revealed that for fruiting body initiation, lower temperature required with very low relative humidity.
Table 1: Seasonal productivity of P. florida
S E A S O N S | Month | Environmental condition | Morphological features | Productivity | |||
Temperature (°C) | Relative humidity (%) | spawn run (day) | Initiation fruiting (day) | Production per bag (g/kg straw) | Biological Efficiency (%) | ||
S U M M E R | March | 26.1±2.5 | 42±5.3 | 12 | 17 | 520 | 52.0 |
April | 26.9±3.1 | 44±4.6 | 14 | 20 | 485 | 48.5 | |
May | 27.9±4.4 | 39±7.0 | 17 | 21 | 445 | 44.5 | |
June | 32.2±2.3 | 87±2.1 | 17 | 22 | 405 | 40.5 | |
R A I N Y | July | 30.7±2.6 | 88.7±2.3 | 19 | 24 | 387 | 38.7 |
August | 27.5±3.7 | 42±5.5 | 18 | 24 | 375 | 37.5 | |
September | 24.8±2.9 | 40±3.8 | 17 | 22 | 353 | 35.3 | |
October | 21.6±1.8 | 42±4.1 | 19 | 25 | 300 | 30.0 | |
W I N T E R | November | 20.4±1.3 | 90±1.5 | 14 | 18 | 465 | 46.5 |
December | 18.8±2.3 | 86±2.6 | 12 | 15 | 535 | 53.5 | |
January | 17.4±2.1 | 78±3.3 | 11 | 14 | 590 | 59.0 | |
February | 22.5±3.6 | 50±4.5 | 10 | 13 | 615 | 61.5 | |
Fig.1: (A-I) Cultivation of P. florida (A-C) pinhead stage, (D-G) early mature stage and (H&I) showing the mature stage.
In order to determine the antioxidant property of P. florida, DPPH free radical scavenging test was done. The ability of the ethanolic extract of to neutralize the free radicals such as DPPH radicals was studied and it was observed that the ethanolic extract of P. florida significantly in neutralizing the DPPH free radicals. The results also revealed that the increased concentration showed higher activity (Figure 2A). Highest DPPH scavenging activity was observed in 20mg/ml in compare to other concentrations. Khan et al 2010 and Zhang et al 2002 also reported significant DPPH scavenging activity in several species of Pleurotus, Grifola and Ganoderma. Simultaneously it was also observed that the higher concentration of ethanolic extract of P. florida showed higher reducing power activity (Figure 2B). The radical scavenging activity of P. ostreatus mushroom is reported to be higher (6 mg/ml) than those of other mushrooms like Agaricus bisporus, Volvariella volvaceae, Calocybe indica and Hybsizus ulmarius reported by Ramkumar et al (2010). Rao et al (2013) also reported that the Ethanolic extracts of the button mushrooms, showed higher antioxidant activity such as DPPH free radical scavenging, carotenoid activity, total phenolic compounds and total flavonoid concentration revealed antioxidant activity.
Fig 2: Antioxidant activity of P. florida; (A) DPPH scavenging activity and (B) Free radical antioxidant power activity
After induction of blood sugar, body weight were measured and it was observed that in case of control groups, there was no significant changes of normal control group, but in negative control group body weight drastically decreased and the rats were gradually become very sick. On the other hand, initially the body weight of the positive control rats was decreases but the rats were gained their body weight prior treatment with standard drug and become healthy again. In case of mushroom treated rats, it was observed that initially after the induction the body weight decreases which generally happened in diabetic patients. But by the treatment, it was observed that the rats were able to regain their weight (Figure 3A). Al-Shamaony (1994) stated that the decrease in body weight due to derangement is one of the most common phenomenon in diabetes. Blood sugar also measured in regular intervals and the results showed that the blood glucose increased rapidly after induction of Streptozotocin and the rats were able to lowering the blood glucose level after prior treatment with standard drug as well as oral administration of P. florida powder. Roy et al. (2018) reported that powder of Pleurotus djamor helps in lowering the blood glucose level and also effectively regain the bodyweight of STZ treated albino rats.
Fig. 3: Effect of diabetes on body weight (A) and blood glucose level on albino rats before and after treatment with P. florida .
As we know creatinine is a waste product formed within the muscle from the high energy storage compound creatinine phosphate and it is an important indicator of renal function. In the present study, it was observed that the creatinine level was high in case of negative control but it is quite lower in positive control as well as in mushroom treated animals (Figure 4A). Besides, blood urea level was also estimated which regulate mainly the liver and kidney function. It was observed that the blood urea level was lower in mushroom treated rats in compare to the control groups. (Figure 4B). Roy et al (2018) also reported that the oral administration of P. djamor helps in controlling the urea and creatinine level of albino rats.
Fig. 4 : Effect of P. florida powder on Blood creatinine (A) and Urea (B)
Conclusion:
Pleurotus florida is one of the popular mushroom cultivated throughout the year. The present study reveals that the winter season is very much suitable for the cultivation. In relation to this, P. florida found very effective on STZ treated albino rats and it can be concluded from the above study that P. florida is good for the human health.
Acknowledgement:
Author acknowledge the financial assistance received from University Grants Commission, Govt. of India under the UGC-BSR scheme.
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