GJRMI - Volume 3, Issue 10, October 2014 by Dr. Hari Venkatesh.K.Rajaraman

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INDEX – GJRMI - Volume 3, Issue 10, October 2014 MEDICINAL PLANTS RESEARCH Biology CULTIVATION OF OYSTER MUSHROOM (PLEUROTUS OSTREATUS) ON WASTE PAPER WITH SUPPLEMENT OF WHEAT BRAN Asefa Keneni, Geda Kebede

370–380

Pharmacology WOUND HEALING ACTIVITY OF ALOCASIA MACRORRHIZOS (L.) G.Don PLANT – AN EXPERIMENTAL STUDY Santosh kumar singh, Sonia Thakur, Neha Shukla, Sanju Singh

381–388

COVER PAGE PHOTOGRAPHY: DR. HARI VENKATESH K R, PLANT ID – FRUIT OF ATI BALA – ABUTILON INDICUM (L.) SWEET. OF THE FAMILY MALVACEAE PLACE – KOPPA, CHIKKAMAGALUR DISTRICT, KARNATAKA, INDIA

Research Article CULTIVATION OF OYSTER MUSHROOM (PLEUROTUS OSTREATUS) ON WASTE PAPER WITH SUPPLEMENT OF WHEAT BRAN Asefa Keneni1*, Geda Kebede2 1,2

Department of Biology, College of Natural and Computational Sciences, Ambo University, Ethiopia. *Corresponding author: E-mail: asefa_keneni@yahoo.com

Received: 16/08/2014; Revised: 28/09/2014; Accepted: 30/10/2014

ABSTRACT The present study was under taken to evaluate the usability of waste paper as a major substrate with supplement of different ratio of wheat bran for cultivation of oyster mushroom. Five different treatments (T1-T5) were used in the study and these treatments showed significant (P≤0.05) variation. T2 showed the fastest mycelial extension (0.26 cm/day) and T4 and T5 showed slowest mycelial extension (0.14 and 0.15 cm/day). T2 and T3 showed shortest incubation periods (85 days) and T5 had longer (105 days) for overall cycle of the mushroom production. T4 showed shortest mean periods from pinning to maturation in the 2nd, 3rd and 4th harvests (8 to 5 days), while T1 took longer incubation periods 10 to 8 days in all the three harvests. T3 showed highest fresh weight in 1 st flush (900g) and T5 gave least fresh weight (150g). In T2 and T3 the forth harvest was lowest (130 and 124g) as compared to the former harvest while in the rest of the treatments it was absent. Maximum number (12) of bunches was recorded on T2 and the least on T5 (3). Pilus diameter was maximum from T3 (14 cm) and the minimum (8cm) was noticed from T5. However the stipe length of the mushroom from the different treatments did not vary considerably (2.5–3.0cm). The highest numbers of fruiting bodies were collected from T2 and T3 (72) and the least from T5 (15). Higher number of aborts was recorded on T2 (110) and the lowest on T5 (20). The highest total wet/fresh weight of matures and biological efficiency were recorded in T2, and T3 (2090–2214g, 104–110% respectively) and the least from T5 (370g, 6% respectively). The results obtained indicate that supplement of different ratio of wheat bran significantly resulted in the variability of growth, yield and biological efficiency of oyster mushroom and T3 and T2 may be used for commercial production of oyster mushroom in the areas where other substrates may be a limiting factor. KEY WORDS: Biological efficiency, Wheat bran, Oyster mushroom, Waste paper

Cite this article: Asefa Keneni, Geda Kebede (2014), CULTIVATION OF OYSTER MUSHROOM (PLEUROTUS OSTREATUS) ON WASTE PAPER WITH SUPPLEMENT OF WHEAT BRAN, Global J Res. Med. Plants & Indigen. Med., Volume 3(10): 370–380

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Global J Res. Med. Plants & Indigen. Med. | Volume 3, Issue 10 | October 2014 | 370–380

INTRODUCTION Mushrooms are the fungi that have been used as food since time immemorial. Nutritionally they are a valuable source of health food, which is low in calories, and rich in carbohydrates, essential amino acids, fibre, important vitamins and minerals. Mushrooms have also been used in medicine for centuries in the Orient but their potential as health potentiators and elicitors of immune system is recent (Chang and Miles, 1989; Synytsya et al., 2008). In addition, mushroom cultivation is considered as a possible option to alleviate poverty and develop the life style of the vulnerable people. In addition mushroom cultivation offers benefit to market garden when it is integrated in to the existing production system by producing nutritious food at a profit, while using materials that would otherwise be considered “waste” (Beetz and Kustida 2004; Sharma et al., 2013). This is because mushroom contains many essentials nutrients and they are found to solve dietary related health problems (Synytsya et al., 2008). Mushrooms are eaten as meat substitutes and flavoring agents. In addition to the nutritional and medicinal values, mushroom cultivation practices have a paramount importance in food self sufficiency; especially for low income country’s like Ethiopia. Mushroom can generate additional trade offering opportunities through processing enterprises. Mushroom cultivation is suitable for all job seeking groups including elders, disabled and youngsters. Besides, mushroom cultivation is labor intensive and creates job opportunities (Dawit, 1998). It also derives toward full uses of all materials in which nothing is left as waste, without any adverse impacts on the environment through sustainable utilization of lignocelluloses wastes available usually as byproducts form agriculture, forestry and households (Chang and Miles, 1989). Currently, mushroom are regarded as the most profitable and environment friendly method for recycling of the vast lignocelluloses waste substrates, which could otherwise be dropped

in to the environment and cause pollution (Atikpo et al., 2008). Apart from their nutritional potentials, they are important medicinally for cholesterol reduction, immune enhancement, and cancer fighting, anti allergic activities, antimicrobial and cardiovascular treatments (Rajak et al., 2011). They also have a long history of use as traditional medicine in China. Their legendary effects on promoting good health and increasing adaptive abilities have been also supported by recent studies (Wasser, 2000). In addition to their edibility and health benefits, their mycelia can produce a group of complex extra cellular enzymes which can degrade and utilize the lignocelluloses wastes in order to reduce pollution (Atikpo et al., 2008). The major problem associated with the transfer of technology from mushroom cultivation is lack of technical knowledge for its cultivation. Studies conducted with the relation of cultivation of mushroom indicated that agricultural residues: rice husk, sorghums Stover, saw dust, cotton seed waste, cocoa bean shell, and sawdust-Gliricidia mixture were found to be suitable substrates (Rajak et al., 2011). Despite high diversity of wild edible mushrooms has been there in Africa especially in Ethiopia, its recognition very little. Cultivation and production of mushroom has not been practiced on commercial scale in most developing countries which has consequently affected commercial mushroom marketing which is yet to be embraced by most farmers (Abate, 1998; Atikpo et al., 2008). In developing countries, governmental and non-governmental organizations have not given due attention to mushrooms as an important crop that can fetch farmers substantial income to alleviate poverty (Atikpo et al., 2008). Similarly it is accepted that mushroom are not a luxury food but a national necessity to combat poverty and malnutrition (Chang, 2008). However, there is no mushroom cultivation practice in the country to fill the demands of people in tested in the mushroom consumption. Those very few mushroom farmers in Ethiopia

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Global J Res. Med. Plants & Indigen. Med. | Volume 3, Issue 10 | October 2014 | 370–380

are restricted to the capital city. Some research based practices in some parts of the country are still at the stage of trials. From all edible cultivated mushrooms; oyster mushroom (Pleurotus ostreatus) is considered as versatile fungal strain in its ability to use various organic waste materials to grow and give releasable yield. Besides, this mushroom strain is easier to grow by the beginner and also in a wide range of environmental conditions. Large amount of used waste papers has been produced from different governmental and non-governmental institutions as well as a pack for different goods. The well known methods of removal or reduction of these waste papers from the given area was by burning it. Burning of solid organic wastes including waste paper increases the emission of green house gases and not environmental friendly. Cultivation of mushroom on waste paper with supplement of different proportion of wheat bran was not reported from Ethiopia. Mintesnot et al. (2013) evaluated biomass of some invasive weed species as substrate for oyster mushroom and observed maximum biological efficiency on Parthenium hysterophorus; Beje Gume et al. (2013) evaluated of eight locally available substrates and substrate combinations for their productivity and biological efficiency (BE) for cultivation of commercial mushroom strain (Pleurotus ostreatus) and observed that the fastest mean value (0.69 cm/day) of mycelial extension was recorded from sdZcCh (combination of sawdust of Cordia africana and Pouteria adolfi-friederici, corncobs and coffee bean husks). However, mycelial growth in coffee bean husks was completely ceased after 15 days. The present study was undertaken mainly to assess the growth, yield and biological efficiency of oyster mushroom on substrates composed of different proportion of waste paper and wheat bran mixed on dry weight basis. MATERIALS AND METHODS Organism and culture conditions The fungal strain, Pleurotus ostreatus (Oyster mushroom) was obtained from

Mycology Laboratory, Department of Biology, Addis Ababa University, Addis Ababa, Ethiopia. The pure culture of Pleurotus ostreatus was transferred on to Potato Dextrose Agar (PDA) prepared in the laboratory using fresh potato 250 g; glucose (Dextrose) 20 g; agar 20 g and chloramphenicol 0.2 g in 1000 ml of water. The medium was poured into the Petri dishes and allowed to cool in under aseptic condition in laminar flow chamber. The cooled and solidified medium was inoculated by 1 cm×1 cm agar block of the fungal strain and incubated at 25 C. The growth of the culture and presence of contamination were visually inspected at three days interval. Grain Spawn production In this study, the spawn (mushroom seed) of Pleurotus ostreatus was produced on yellow colored sorghum grain, wheat bran and calcium sulfate (gypsum) in the ratio of 88:10:2 respectively (Dawit, 1998). The required amount of sorghum grain was weighed and soaked over night in sufficient amount of water. The grains were washed and drained to remove the dead and floating seeds with excess of water. After removing the excess water from the grain, the required amount of wheat bran and gypsum (CaSO4 2H20) were added and transferred to 1000 ml glass bottles (75% level) leaving a head space over the grain and autoclaved at 121°C temperature for 45 minutes. After cooling, each bottle was inoculated with 20 agar blocks (1 cm × 1 cm) of 15day old mushroom culture from the Petri dish and incubated for 21 days at 28 ± 2 C until the substrate were fully colonized and the mycelia invasion and contamination were inspected at five days interval. Waste paper was collected from different departments of the University and wheat bran was collected from the local market. Treatments Five treatments (T1–T5) comprising different proportions of waste paper and wheat bran (2000 g) along with lime stone (Calcium Carbonate 20 g) on dry weight basis were used as shown in Table 1.

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Global J Res. Med. Plants & Indigen. Med. | Volume 3, Issue 10 | October 2014 | 370–380

Table1: The composition of different treatments Treatment T1 T2 T3 T4 T5

waste paper (g) 1800 1600 1400 1200 1000

Preparation of the substrate The waste paper was cut into small pieces approximately (3–5 cm), weighed and soaked in sufficient amount of water immediately before use. Excess water present in the substrates was drained thoroughly and mixed with required amount of wheat bran and one percent calcium carbonate and filled in sterilizable yellow color polyethylene bags (Kurtu pestal). The substrates were autoclaved at 15Psi pressure at 121 C temperatures for 1h. After sterilization the substrates were transferred to transparent polyethylene cultivation bags for easy supervision of the growth of the mycelia and presence of contamination. Each substrate (2,000 g) with 70% moisture was mixed with 10% spawn (dry weight/wet weight basis) and the inoculated polythene bags were then tightly tied with string made from polyester/cotton cloth. Pin holes were made through the bags (1/100 cm2) for drainage and aeration. It was kept in a spawn running room at room temperature in the dark until primordia were formed. After primordial formation, large holes were made in the polythene bag to allow normal development of fruiting bodies. Bags were transferred to mushroom house under normal environmental conditions and relative humidity (the room maintained at 85–90%) by keeping water in open containers at different corners of the room. The cultivation bags were irrigated using tap water every morning and evening until all flushes of Pleurotus ostreatus fruiting bodies were harvested. Adequate ventilation was provided to prevent increased CO2 concentration in the room by opening the door and windows of the room for half an hour in the morning and in the evening. The

Wheat bran (g) 200 400 600 800 1000

Total (g) 2000 2000 2000 2000 2000

mushrooms were manually harvested at maturity which was indicated by up ward curving of the edges of the cap. Biological efficiency was calculated and defined as the ratio of weight (g) of fresh mushrooms harvested to dry weight (g) of the substrate. Biological Efficiency = Weight of fresh fruiting bodies (g) × 100 Weight of dry substrate (g)

Data analysis The data were analyzed by comparing the mean weights and percent biological efficiency through one way ANOVA. The data groups were analyzed using Statistical Package for Social Sciences (SPSS) for windows 16.0. Treatment mean were compared using LSD. RESULTS Mycelia extension There were significant (P≤0.05) differences in the mycelial extension of oyster mushroom grown on different substrates. T2 showed the fastest mycelial extension followed by T3 while, T4 and T5 exhibited slowest mycelial extension on 7th and 14th days of incubation periods (Table 2). There were significant (P≤0.05) differences in the days required for complete invasion of the substrates receiving different treatments. The time required for complete invasion of the substrates was significantly (P≤0.05) less for T2 and T3 when compared to that of T1 and T5 (Table 2). Total days required to complete the production cycle was observed shortest for T2 and T3 while it took more days for T5.

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Global J Res. Med. Plants & Indigen. Med. | Volume 3, Issue 10 | October 2014 | 370–380

Table 2: Mycelial extension on the substrates at different treatments measured on 7th and 14th days of incubation Treatments Mycelia extension in (cm) 7th day 14th day T1 T2 T3 T4 T5

1.9 2.5 2.3 1.2 1.1

5.5 6.5 6.25 4.5 4.3

Mean values Number of days Total days (cm/day) required for required to complete invasion complete the cycle 0.16 26 90 0.26 22 85 0.24 23 85 0.14 25 95 0.15 27 105

Growth rate of mushroom (Flushes)

Yield of mushroom per flushes

Mean incubation periods of mushroom flushes showed highly significant differences (P≤0.05). T1 showed relatively shorter incubation to 1st flush while 1st–2nd flush and 2nd–3rd flush took more days than T2 and T3. T2 and T3 took moderate days from incubation to 1st flush and shortest for the remaining three consecutive harvests as compared to the other treatments. T5 took longer incubation periods at all harvest. Besides number of harvest significantly varied for different treatments; T2, T3 and T4 gave four harvests; T1 three harvests and T5 only two harvests (Table 3).

Yield of mushroom per flush (wet weight) showed significant variation between treatments (P≤0.05) (Table 5) as well as between flushes. T3 showed highest fresh weight in grams in 1st and 2nd flushes followed by T2; the result observed with T1 and T4 were not comparable with the highest yielding treatments, while T5 was poor in this regard. In 3rd flush, T2 and T3 gave relatively higher fresh weight of mushroom while all the remaining treatments showed least. In all the treatments the 4th flush not comparable with other harvest cycle (Table 5).

Pinning to maturation duration of oyster mushroom The mean periods taken from pinning to maturation of each treatment showed significant (P≤0.05) variation. T1 and T5 relatively took longer periods from pinning to maturation and T2, T3 and T4 took shorter periods from pinning to maturation in all flushes as compared to other treatments (Table 4).

Number of bunches, matures and aborts More number of bunches were recorded on T2 followed by T3 while all the remaining treatments showed least in number of bunches. The highest and equal numbers of fruiting bodies were collected from T2 and T3. T5 and T4 gave the least number of fruiting bodies. Higher number of aborts were recorded with treatments T2 followed by T3 and the least number of aborts were recorded in T1 (Fig1).

Table 3: Incubation periods of different harvests Treatments Incubation -1st flush 40 T1 45 T2 45 T3 50 T4 55 T5

1st–2nd flush 18 15 15 17 18

2nd–3rd flush 3rd–4th flush 16 − 13 12 23 12 15 13 − −

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Table 4: Pinning to maturation of the oyster mushroom under different treatments regimes Treatments st

1 Flush

Mean duration (days) 2 Flush 3nd Flush nd

4nd Flush

−

Mean values with in a column sharing the same superscript letter(s) are not significantly different by using LSD test at P≤0.05

Table 5: Mean yield per flush in the different treatments Treatments st

1 Flush T1 T2 T3 T4 T5

413 880 900 290 150

Mean fresh- weight of mushroom (g) 2nd Flush 3nd Flush 4nd Flush 251 560 765 210 130

150 520 515 150 90

− 130 125 75 −

Total 814 2090 2215 560 370

Mean values with in a column sharing the same superscript letter(s) are not significantly different by using LSD test at P≤0.05

Fig 1: The different stages of mushroom production in this experiment

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Pilus Diameter and Stipe length

Total yield and Biological efficiency

Pilus diameter was found to be the largest for the samples collected from T3 followed by T2, T4 and T1 respectively, while it was smallest for the sample collected from T5. The stipe length of the samples collected from different treatments did not show significant variation (Fig2).

The highest total wet/fresh weight of matures was recorded in T3, followed by T2. The least total fresh/wet weight was recorded in T5 (Table 5). The effect of different treatments on biological efficiency of oyster mushroom showed significant (Pâ&#x2030;¤0.05) differences. The highest biological efficiency was recorded with T3 followed with T2. The least was recorded with the treatment T5 (Fig 3).

Fig 2: Number of bunches, matures and aborts of different treatments Number of bunches, matures and aborts

120

100 80 60

Number of bunches Number of matures

Number of Aborts 20 0 1

Treatments

Pilus Diameter and Stipe length(cm)

Fig 3: Pilus diamter and stipe lengths of different treatmnets 16 14 12 10 8 Pilus Diameter

Stipe length

4 2 0 1

Treatments

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Global J Res. Med. Plants & Indigen. Med. | Volume 3, Issue 10 | October 2014 | 370–380

Fig4: The biological efficiency of different treatments 120

Biological Efficiency(%)

100 80 60 40 20 0 1

Treatments

DISCUSSION Selection and optimization of available substrates in order to obtain reasonable yield of mushroom could be considered as a priority target in mushroom research. The use of waste paper as a major substrate for the cultivation of oyster mushroom was not yet practiced in Ethiopia. In this experiment the complete colonization of the substrate took 22 days in the faster and 27 in the slower and then another 14 days were taken after complete invasion of the mycelium to first harvest in the fastest treatment and 28 days in the slower. In this study, the periods taken for spawn running on the different treatments showed relatively longer time as compared to results reported in the literature. Ashraf et al. (2013) reported shorter periods for the substrate completely colonized by mycelium of the different oyster species which also indicates differences among the different species and the substrates. According to these authors the minimum number of days 16.20 took by P. ostreatus 16.20 ± 0.59 while species P. sajor-caju and P. djmor showed same level of significance with 18.07 ± 0.69 and 18.67 ± 0.61. Oseni et al. (2012) reported periods of colonization to first harvest from 33 to 43 days on fermented saw dust supplemented with different proportions of wheat bran. In this study the different treatments showed significant variation periods

taken for primordial formation after the complete colonization of the substrate by the fungal strain. T1 took only four days for initiation for the primordial formation while T5 took another 19 days for initiation for the primordial formation. These longer days of initiation of primordial formatyion after mycelia running may be due to slow releasing of nutrients from waste paper as compared to other substrates, for example, wheat straw and rice straw on which much of research work has been done on this mushroom species. Ashraf et al.(2013) reported that all the treatments they tested showed 3.73 to 5.13 days for primordial initiation after mycelia running. In this study, in all the treatments, the successive pinning to harvest duration was shortened by at least a day. The shortest mean duration of pinning to maturation was 8 in the 1st, 7 in the 2nd, 6 in 3rd and 4th. And the longest mean duration of pinning to maturation was 10, in the 1st, 9 in the 2nd and 3rd. The duration observed in the present study was longer when compared with the reports in the literature (Gume et al. (2013) which was 3.3 in the shortest and 6.0 in the longest. Studies indicated that environmental factor affects the incubation periods of oyster mushroom. According to Zadrazil (1976) and Daba et al. (2008) longer period of incubation for oyster

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Global J Res. Med. Plants & Indigen. Med. | Volume 3, Issue 10 | October 2014 | 370–380

mushrooms was at lower temperatures and low relative humidity. In this study, in all the treatments the yield (fresh/wet weight) of the mushroom harvested in the first cycle was greater than the remaining successive harvests. In addition to the minimized yield in the next consecutive harvest, treatment T1 and T5 did not give harvest at the fourth cycle. Our observation on the different harvest is in line with reports in the literature. Ashraf et al. (2013) reported that the different treatments vary in the amount of mushroom yield harvest at different flushes and at each successive harvest, the amount of the yield declined. Number of bunches formed on different treatments were significantly different. T2 produced highest number of bunches, followed by T3 and all the remaining treatments gave least number of bunches. The highest and equal numbers of fruiting bodies were collected from T2 and T3 (72). T5 (15) and T4 (22) gave the least number of fruiting bodies. Higher number of aborts observed in treatments T2 (110) and T3 (92) and the least number of aborts T1(16), this may be due to the optimal proportion of waste paper and wheat bran which allowed maximum primordial formation from which some of them aborted. In this study more number of bunches result in more number of fruiting bodies. This observation was in line with the results reported by Gume et al. (2013) who reported that substrates that gave higher yield also contained higher number of propagating fruit bodies per bunch and highest variability among different treatments on the mean number of mature fruit bodies and aborts. In majority of the substrates, the number of pinhead abortions exceeded number of matures. Kimenju et al. (2009) reported that more than 50% of pinheads emerged did not grow into marketable products. Gume et al. (2013) observed high rate of pinhead abortion from low-yield substrates such as sd1C and ZcCh. The largest pilus diameter was measured with T (14 cm) and the smallest with T5 (8 cm); the rest of the treatments gave pilus diameter between the largest and the smallest. Largest pilus diameter significantly increased the total fresh/wet

weight of oyster mushroom. Oseni et al. (2012) reported highest mean pilus diameter 57.9 to 62.3 mm on sawdust supplemented with different levels of wheat bran. The largest pilus was obtained from sawdust substrate supplemented with 15% wheat bran (62.3 mm) and the smallest obtained on sawdust substrate supplemented with 5% wheat bran (57.9 mm). The pilus diameter obtained in the present study was greater than all those reported earlier, may be due to the varied proportions of the major substrate (waste paper) which can supplement the necessary nutrients for mushroom growth. The stipe length of all the 5 treatments did not vary significantly (2.5–3.0 cm), which is in agreement with the results of Gume et al. (2013). 1.4–1.9 cm. Oseni et al. (2012) observed stipe length of oyster mushrooms ranging from 39.4–59.5 mm (3.94–5.95cm) on fermented sawdust substrate supplemented with different wheat bran levels and highest stipe length (59.5 mm) (5.95 cm) was observed on substratum supplemented with 15% wheat bran. The mean number of mature fruit body and aborts were greatly varied among the different treatments in the study. The total fresh weight of the mushroom was highest in T3 followed by T2 (2214–2090 g per 2000 g of the dry substrate and their biological efficiency (110–104%). In all the parameter tested T3 and T2 (1400 g waste paper 600 g wheat bran and 1600 g waste paper 400g wheat bran) found to superior this may be due the proportion of the waste paper and wheat bran for bio-availability of the various nutrients contained in the substrates mixture. In this study the least total fresh/wet weight of the mushroom and biological efficiency was recorded in T5 (370 g per 2000 g dry substrate and 6% BE. In all the treatments yield of mushroom declined successively throughout the four cropping periods. Kimenju et al. (2009) reported that yields of mushroom in different substrates slightly declined from the first flush to the successive harvests. The crops of oyster mushroom were harvested in four flushes and the maximum yield was obtained in the first flush than the 2nd, 3rd and 4th flushes,

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respectively as observed by Oseni, et al. (2012). CONCLUSION Production of edible mushroom has been considered as diversification of food production and also contribute in the struggle for food self sufficiency and attaining food security particularly in the developing world like Ethiopia. Testing the usability of waste paper as a major substrate with the supplement of different ratio of wheat bran was not yet tried for mushroom production in Ethiopia. The different treatments resulted in significant variation on growth, yield, yield parameters and biological efficiency of oyster mushroom. From all the treatments T3 and T2 observed to be considered as highest yielding with all the

parameters tested and recommended for commercial production of oyster mushroom. While the rest of the treatments performed below the acceptable yield and biological efficiency. The result of this study shows the possibility of mixing waste paper with wheat bran in different proportion and obtaining high yield and good quality mushroom fruiting bodies. The future research direction should focus on developing a substrate that will give highest yield of mushroom by mixing waste paper with other locally available organic wastes. ACKNOWLEDGEMENT The authors are greatly acknowledging Ambo University, Ethiopia for extending the financial support for this research work.

REFERENCES Ashraf J, Asif Ali M, Ahmad W,Muhammad Ayyub C, Shafi J (2013). Effect of Different Substrate Supplements on Oyster Mushroom (Pleurotus spp.) Production Food Science and Technology 1(3): 44–51, Atikpo M, Onokpise O, Abazinge M, Louime C, Dzomeku M, Boateng L Awumbilla (2008). Sustainable mushroom production in Africa A case study in Ghana. Afr. J. Biotechnol., 7: 249–253. Beetz, A. and M. Kustida, (2004). Mushroom Cultivation and Marketing. ATTRA Publication # IP 087, Retrieved from: http://attra.ncat.org/attrapub/ mushroom.html. Beje Gume, Diriba Mulata and Dawit Abate (2013). Evaluation of locally available substrates for cultivation of oyster mushroom (Pleurotus ostreatus) in Jimma, Ethiopia. African Journal of Microbiology Research.,7(20): 2228– 2237

Chang, S.T. AND P.G. Miles, 1989, Edible mushrooms and their cultivation vol.1, CRC Press Boca Raton, FL; USA, ISBN-13: 9780849367588, PAGES: 345. Chang, S.T., 2008. Overview of Mushroom Cultivation and Utilization as Functional Foods. Retrieved from: http:// media. wiley. com/ product_ data/ excerpt/69/04700540/0470054069. pdf, (Accessed on: January 19, 2009). Daba AS, Kabeil SS, Botros WA, El-Saadani MA (2008). Production of mushroom (Pleurotus ostreatus) in Egypt as a source of nutritional and medicinal food. World J. Agric. Sci., 4:630–634. Dawit A (1998). Mushroom Cultivation: A practical approach, Berhanena Selam Printing Enterprise, Addis Ababa Ethiopia.

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Kimenju JW, Odero OM, Mutitu EW, Wachra PM, Narla RD, Muiru WM (2009). Suitability of locally available substrates for oyster mushroom (Pleurotus ostreatus) cultivation in Kenya. Asian J. Plant Sci., 8:510–514. Lqbal SM, Rauf CA,Sheikh M (2005). Yield performance of oyster mushroom on different substrates. Int.J.Agric bio.7 :900–903. Mintesnot B, Ayalew A, Kebede A (2013). Evaluation of Some invasive weed species as substrate for oyster mushroom (Pleurotus spp.) cultivation. Pak. J. Biol. Sci. _: 1-7 DOI 10.3923 Oseni T O, Dube S S, Wahome, P K, Masarirambi, M T, and Earnshaw D M (2012). Effect of Wheat Bran Supplement on Growth and Yield of Oyster Mushroom (Pleurotus Ostreatus) on Fermented Pine Sawdust Substrate. Experimental Agriculture & Horticulture :V-30–40 Park, G. and Kwang, H.O. (2001).Nutritional value of a variety of Mushrooms.www.Mushworld.com/suben.html. Pathmashini L, Arulnandhy V, Wijeratnam SW (2008) cultivation of oyster mushroom (Pleurotus ostreatus ) on saw dust J. Biol. Sci., 37: 177–182.

Source of Support: Ambo University, Ethiopia

Rajak S, Mahapatra S.C. and Basu M 2011. Yield , Fruit body diameter and cropping duration of oyster mushroom ( Pleurotus sajor caju ) grown on different grasses and paddy straw as substrate. European Journal of Medicinal plants. 1(1): 10–17. S.R mondal, M.J. Rehana, M.S. noman and S.K.Adhikary 2010. Comparative study on yield performance of oyster mushroom (Pleurotus florida) on different substrates Agrotechnology discipline, Khulna University, Khulna. 9208, Bangladesh. Sharma S, Kailash R, Yadav P., Pokhre C P (2013). Growth and Yield of Oyster mushroom (Pleurotus ostreatus) on differentSubstrates. J. on New Bol.l Rep. 2(1): 03–08 Synytsya A., Mickova, K, Jablonsky I, Slukova M, Copikova J (2008). Mushrooms of genus Pleurotus as a source of dietary fibers and glucans for food supplements. Czech. J. Food Sci., 26:441–446. Wasser, S. P. (2002). Nutraceuticals and bio pharmaceuticals from edible and medicinal mushrooms’. Int. Med. Mushrooms. 8: 1–17. Zadrazil F (1976). The ecology and industrial production of Pleurotus ostreatus, P. florida, P. cornucopiae and P. eryngii. Mush. Sci., 9:621–652.

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Research Article WOUND HEALING ACTIVITY OF ALOCASIA MACRORRHIZOS (L.) G.Don PLANT – AN EXPERIMENTAL STUDY Santosh kumar singh1, Sonia Thakur2*, Neha Shukla3, Sanju Singh4 1,2,3,4

Assistant Professor, Mittal Institute of Pharmacy, Opp. Bhopal Memorial Hospital and Research Centre, By Pass, Nabibagh, Bhopal- 462038, Madhya Pradesh, India. *Corresponding Author: Email: soniathakur92@gmail.com; Mobile: +91-9425452815

Received: 18/07/2014; Revised: 28/08/2014; Accepted: 20/09/2014

ABSTRACT A tissue injury is invariably followed by varying degree of inflammatory changes in lipid peroxidation levels during different stages of cutaneous wound repair have been investigated earlier. It is observed that any drug that reduces the generation of free radicals by a drug (herbal or otherwise) during proliferative phase is a wound healing agent. The wound healing potency of methanolic extract of leaf of Alocasia macrorrhizos was evaluated by excision, incision and histopathological wound model on albino mice the wound healing activity was assessed for wound contraction, period of epithelialization and skin breaking strength of granulation tissue. The result obtained in the study revealed that methanolic leaf extract has significant wound healing potency as compared to standard. KEYWORDS: Wound healing, Alocasia macrorrhizos, Excision, Incision, Histopathological, etc.

Cite this article: Santosh kumar singh, Sonia Thakur, Neha Shukla, Sanju Singh (2014), WOUND HEALING ACTIVITY OF ALOCASIA MACRORRHIZOS (L.) G. Don PLANT – AN EXPERIMENTAL STUDY, Global J Res. Med. Plants & Indigen. Med., Volume 3(10): 381–388

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INTRODUCTION India has a rich flora that is widely distributed throughout the country. Herbal medicines have been the basis of treatment and cure for various diseases and physiological conditions in traditional methods practiced such as Ayurved, Unani and Siddha. Medicinal components from plants play an important role in conventional as well as western medicine. (Perumal S.R., et al., 2008, Fabricant D.S., et al., 2001, Priya K.S. et al., 2002, Steenkamp V. et al., 2004, Principe P., 2005). A wound may be defined as a break in the epithelial integrity of the skin or may also be defined as a loss or breaking of cellular and anatomic or functional continuity of living tissue. According to the Wound Healing Society, wounds are physical injuries that result in an opening or break of the skin that cause disturbance in the normal skin anatomy and function. They result in the loss of continuity of epithelium with or without the loss of underlying connective tissue (Ramzi S.C. et al., 1994, Strodtbeck F., 2001). Wounds are classified as open and closed wound on the underlying cause of wound creation and acute and chronic wounds on the basis of physiology of wound healing. In this case blood escapes the body and bleeding is clearly visible. It is further classified as: Incised wound, Laceration or tear wound, Abrasions or superficial wounds, Puncture wounds, Penetration wounds and gunshot wounds (Schultz G.S. 1999). In closed wounds blood escapes the circulatory system but remains in the body. It includes Contusion or bruises, hematomas or blood tumor, Crush injury etc. Acute wound is a tissue injury that normally precedes through an orderly and timely reparative process those results in sustained restoration of anatomic and functional integrity. Acute wounds are usually caused by cuts or surgical incisions and complete the wound healing process within the expected time frame (Lazarus G.S. et al., 1998).

Chronic wounds are wounds that have failed to progress require a prolonged time to heal or recur frequently. Local infection, hypoxia, trauma, foreign bodies and systemic problems such as diabetes mellitus, malnutrition, immunodeficiency or medications are the most frequent causes of chronic wounds (Menke N.B. et al., 2007, Krishnan P., 2006). The wound healing activities of plants have since been explored in folklore. Many Ayurvedic herbal plants have a very important role in the process of wound healing. Plants are more potent healers because they promote the repair mechanisms in the natural way. Extensive research has been carried out in the area of wound healing management through medicinal plants. Herbal medicines in wound management involve disinfection, debridement and providing a moist environment to encourage the establishment of the suitable environment for natural healing process (Purna S.K.et al., 2000). Alocasia macrorrhizos, (L.) G. Don., elephant ear Taro plant is a large evergreen, mainly rhizomatous, sometime tuberous rooted perennials. The plant, which belongs to the family Araceae, is found in tropical forests and sunny open or shaded, usually damp sites and marshes in south East Asia. This plant is known for many medicinal properties & hence the present study was undertaken to study the wound healing activity of Alocasia macrorrhizos on excision, incision & histopathological wound models on Albino mice. MATERIALS AND METHODS Plant Aunthentication: The leaf part of A. macrorrhizos was collected in the month of October in Sanjivini Ayurvedic Nursery in Bhopal, Madhya Pradesh, India. Plants were identified by Dr. Pradeep Tiwari, Botanist in the department of Botany Dr. Hari Sing Gour, Vishwavidyalaya (Sagar, Madhya Pradesh, India). A voucher specimen has been deposited in our library for further reference (no.1166).

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Global J Res. Med. Plants & Indigen. Med. | Volume 3, Issue 10 | October 2014 | 381–388

Preparation of leaf extract:

Excision wound model:

Fresh leaf of A. macrorrhizos were separated from the plant and allowed to shade dry for 15 days and then homogenized to get a coarse powder. Powder (250 g) was extracted with hydroalcoholic mixture (ethanol 45% and water in 1:1 proportion) at room temperature by cold maceration method. It was also extracted with methanol by soxhlet method. The filterate was collected and concentrated on a heating mantle at 45°c till a syrupy mass was obtained. The percentage yield was found to be 48% and 38.5% with respect to the initial dried plant material.

In the excision wound model, rats were depilated by removing hairs at the dorsal thoracic region before wounding. Rats were anaesthetized by diethyl ether prior to excision. Circular wound of about 2.5 cm diameter was made on depilated dorsal thoracic region of rats under aseptic conditions and were observed throughout the study. The areas of the wound were measured (in mm2) immediately by placing a transparent polythene graph paper over the wound and then tracing the area of the wound on it (approx. area 100 MM2) this was taken an initial wound area reading.

Wound healing model

The mice are categorized in to four groups (n=6). The animal of group I treated as control and only ointment base applied topically. The animal of group II treated as standard drug and III group treated as polyhedral applied topically, respected. All the samples were applied once daily for 16 days, starting from the day of wounding. The observations of percentage wound closure were made on 4th, 7th, 10th, and 13th, post wounding days. The wound area of each animal was measured by using tracing paper methods. The percentage of wound contraction was calculated from the days of measurements of wound area (Shirwaikar A. et al., 2003).

Selection of model: Excision, incision and histopathological wound model, using albino mice was selected for assessing the wound healing activity. This model was employed to study the rate of wound contraction, time required for full epithelization and tensile strength. These parameters were selected because of easy availability of albino mice and simplicity in handling then. Selection and procurement of animals: After taking permission for animal studies, albino mice were procured and mice of either sex weighting 150–200gm were selected, maintained at 24–28°C, housed individually with free access to food and water. The animals were left for 48hr. to acclimatize to the animal room conditions. They were fed with standard diet. To perform the experiment, the mice were divided in to three groups (n=6) Group I – kept as control group which received simple vehicles. Group II – kept as which received extract of leaf part of A. macrorrhizos formulation. Group III – kept as standard group which received betadine ointment.

Wound contraction: The wound contraction was calculated as percentage reduction in wound area with respect to initial wound area while the epithelization time was noted as the number of days after wounding required for scar to fall off leaving no raw wound behind. Incision wound model: In the incision wound model, mice depilated by removing hairs at the dorsal thoracic region before wounding. Mice were anaesthetized by diethyl ether prior to incision. Six centimeter long paravertebral incisions were made through full thickness of skin on either side of vertebral column of the mice. The wounds were closed with interrupted sutures of one centimeter apart.

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Global J Res. Med. Plants & Indigen. Med. | Volume 3, Issue 10 | October 2014 | 381–388

The mice are categorized in to four groups (n=6).the animals of group l treated as control and only ointment base applied topically. The animal of group II treated as standard drug and third group treated as polyherbal applied topically, respectively. All the samples were applied once daily for 13 days, starting from the day of wounding. The sutures were removed on 8th post wounding day. The tensile strength of wounds was measured on 10th day following continuous water flow technique (Shirwaikar A. et al., 2003). Tensile strength in incision wound model: The tensile strength was calculated in incision wound model. On 10th day the mice were again anesthetized and each mice is placed on a stack of paper towel on the middle of the board. The amount of the towel could be adjusted in such a way so that the board. The amount of the towel could be adjusted in such a way so that the wound is on the same level of tips of the arms. The clamps are then carefully clamped on the skin of the opposite side of the skin of wound at a distance of 0.5 cm away from the wound. The longer pieces of the finishing line are placed on the pulley and finally to the polyethylene bottle and the position of the board is adjusted so that the bottle receive a rapid and constant mice of water from the large reservoir,until the wound began to open. The amount of water in polyethylene bag is weighted and consider as tensile strength of the wound (Kamano et al., 1994). Histopathology study On 13th day some of animals under each group were sacrificed and wounds were excised together with surrounding skin. The 1µ thin paraffin section of wounds bed material were fixed in 10% neutral buffer formalin and histological evaluation was performed on heamotoxylin and eosin stain. After complete staining the slides, microscopic photographs of collagen tissue were taken as were shown in figure for control, standard and treated.

Histological studies of granulation tissue of the methanolic extract treated animals showed significant increase in collagen deposition with macrophages, fibroblast, and blood vessels as compare to control. Statistical analysis All the data are expressed as mean ± SD. The values obtained for the extracts were compared with control group using one way ANOVA followed by tukey’s test. The values of P≤0.001 were considered to indicate a significant difference between the groups. RESULTS The methanolic extracts obtained by soxhlet solvent extraction were subjected to various qualitative tests to detect the presence of plant constituents. Alkaloids, Glycosides, Carbohydrates, Phytosterols, Saponins, and Tannins, phenolic compounds, Proteins, free amino acids and Flavonoids are shown in table 1. Excision wound heal by contraction process closure and epithelization the percentage of wound closure or closure rate include by recording the changes in wound area at fixed intervals of time 4th, 7th, 10th and 13th days in fig.1 after treating with methanolic extract the percentage of wound closure on the 13th day was 1.67 ± 0.573 mm2 (table 2). Incision wounds heals by granulation and collagenation the mean wound breaking strength or tensile strength of wound in control group was 172.2 ± 1.7049 gm while in the case of methanolic extract treated group it was 381.22 ± 0.572 gm. The granuloma tissue dissected out was subjected to histopathological examination in control group which revealed presence of chronic inflammatory cells, edema cells. and blood vessels were under developed. Collagen appeared to be incomplete and improper in growth. In methanolic extract treated group a bulk of collagen was seen with fewer amounts of inflammatory cells. Collagen maturity was better than the control group blood vessels were developed (Figure 2).

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Global J Res. Med. Plants & Indigen. Med. | Volume 3, Issue 10 | October 2014 | 381–388

TABLE 1. Phytochemical screening of various extract of A. macrorrhizos S.No

Alocasia macrorrhizos (Methanolic Extract)

Test

Test for steroids (1. Salkowaski test)

Test for glycosides (1.bontrager 2.kellar-killiani test 3.legal’s test Test for saponins (foam test)

+++

Test for carbohydrate (1.molisch’s test 2. Barfoard’s test 3. Fehling test 4.tollen’s test Test for alkaloids(1.mayer’s test 2.wagner test 3.dragondroff’s test 4.hagger’s test) Test for flavonoids (ferric chloride) Test for tannins (1.ferric chloride 2. Gelatin test) Test for protein (1.precipitation 2.xanthoproteic)

−

3. 4. 5. 6. 7. 8.

− + ++ +

TABLE 2. Percentage wound contraction in excision wound model Area of wound closure (sq mm ± S.D) 7th day 10th day 13th day

Group

4 day

I (control)

78.5 ± 0.54 (10.75%) 54.6 ± 1.502 (22.7%) 56.04 ± 1.50 (21.98%)

II(standard) III(treated)

69.2 ± 0.469 (15.4%) 28.8 ± 0.59 (35.6%) 39.8 ± 1.03 (30.1%)

38.2 ± 1.97 (30.9%) 10.1 ± 1.068 (44.95%) 21.9 ± 1.00 (39.05%)

26.3 ± 1.97 (36.85%) 0.62 ± 0.51 (49.69%) 1.67 ± 0.573 (49.16%)

p≤ 0.01 v5 control p≤ 0.01 indicates significant when compared with control. ¥ Figure in parenthesis indicates percent wound contraction.

TABLE 3. Tensile strength in incision model Group Control (I) Standard (II) Treated (III)

Tensile strength (in gram) 172.2 ±1.704 405.32 ± 134.94 381.22 ± 0.572 P≤0.05v5 control

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Epithelization period (days) 22 12 13

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Figure 1. Photography of the wound contraction of 7 days old wound

Wound contraction of 7 days old wound in Control group (A), 7 days old wound in Standard group (B), 7 days old wound in Treated group (C), 13 days old wound in Control group (D), 13 days old wound in Standard group (E), 13 days old wound in Treated group (F).

Figure 2.: Histopathological photography

Histopathological photography of Standard group (A), Treated group (B), Control group (C)

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Global J Res. Med. Plants & Indigen. Med. | Volume 3, Issue 10 | October 2014 | 381–388

DISCUSSION Wound healing process consists of different phases such as granulation, collagenation, collagen maturation and scar maturation which are concurrent but independent to each other. Hence in the study three different models were used to assess the effect of herbal ointment on various phases. The result showed that methanolic extract possesses a definite prohealing action. This was demonstrated by significant increase in the rate of wound contraction and by enhanced epithelialization. Significant increase (P<0.001) in tensile strength and collagen levels were observed, which was further supported by histopathological studies and gain in granuloma breaking strength. The effect of methanolic extract of A. macrorrhizos were screened on excision, incision and histopathological wound models concurrently with the control and reference standard. betadine treated animals showed more pronounced wound healing activity than methanolic extract treated animals. The rate of wound contraction was faster in these and complete epithelialization of the excision wound was observed on 13th day. In standard

betadine treated animals complete epithelialization was noticed on 12th day. The result of percentage wound contraction and period of complete epithelialization has been depicted in table 2. In incision model significant increase in tensile strength of healed wounds was observed in methanolic extract treated group (381.22 ± 0.572). Histological studies of granulation tissue on control group of animals showed accumulation of more macrophages and very few collagen fibers. In methanolic extract treated group, histological section of granulation tissue showed very few macrophages and showed complete epithelialization and collagen where in standard group showed complete collagen. CONCLUSION In conclusion, the significant increase in tensile strength and the prominent haemostatic activity exhibited by the methanolic extract of A. macrorrhizos shows the potent wound healing property of plant. Further more detailed investigation on the actives responsible for the activity are to be identified and further more clinical researches should be conducted to bring the effects of the drug out to the scientific world.

REFERENCES Fabricant D.S., Farnsworth N.R. (2001). The value of plants used in traditional medicine for drugdiscovery, Environ Health Pers, 109 (Suppl 1), p. 69–75. Krishnan P. (2006). The scientific study of herbal wound healing therapies: Current state of play. Curr.Anaesthesia Crit. Care.17, p.21–27. Lazarus G.S. Cooper D.M. KInghton D.R. Margolis D.J. Pecoraro R.E. Rodeheaver G. Robson M.C. (1998). Defination and guidelines for assessment of wounds and evaluation of healing.Arch. Dermatol.; 130, p. 49– 493.

Chen J. Kirsener R. (2007). Pathophysiology of acute wound healing.Clin. Dermatol. 25, p. 9–18.

Madden J.W. Peacock E.E. (1968). Studies on the biology of collagen during wound healing. I. Rate of collagen synthesis and deposition in cutaneous wounds of the rat. Surgery, 64, p. 288–294. Menke N.B. Ward K.R. Witten T.M. Bonchev D.G. Diegelmann R.F. (2007). Impaired wound healing Clin.Dermatol. 25, p. 19–25.

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Perumal S.R., Ignacimuthu S., Patric R.D. (2008). Preliminary screening of ethnomedicinal plants from India. Eur Rev Med Pharmacol Sci.12, p. 1–7. Principe P. (2005).Monetising the pharmacological benefits of plants. US Environmental protection Agency,Washington, D.C. 1991. Priya K.S. Gnanamani A., Radhakrishnan N., Babu M. (2002). Healing potential of Datura Alba on burn wounds in albino rats. J. Ethnopharmacol. 83, p.193–199. Prockop D.J. Kivirikko K.I. Tuderman L. Guzman N.A. (1979). The biosynthesis of collagen and its disorders.N.Engl. J. Med. 301, p. 13–23. Purna S.K. Babu M. (2000). Collagen based dressings/a review. Burns. 26, p. 54–62. Ramzi

S.C., Vinay K., Stanley R. (1994).Pathologic Basis of Diseases.5th edition, WB Saunders Company, Philadelphia, p. 86.

Source of Support: NIL

Schultz G.S. (1999). Molecular Regulation of Wound Healing In: Acute and Chronic Wounds: Nursing management. Bryant R.A., 2nd Edition, WB Saunders Publisher, USA, p.413–429. Shirwaikar A., Shenoy R., Udupa A.L., Udupa S.L., Shetty S. (2003). Activity of wound healing. Indian journal of Experimental Bilogy, 41, p. 238−241. Stadelmalmann W.K. Digenis A.G. Tobin G.R. (1998).Physiology and healing dynamics of chronic cutaneous wounds.Am. J. Surg. 176, p. 26S–38S. Steenkamp V., Mathivha E., Gouws M.C., Rensburg C.E.J. (2004). Studies on antibacterial, antioxidant and fibroblast growth stimulation of wound healing remedies from South AfricaJ. Ethnopharmacol.95, p.353–357. Strodtbeck F. 9 (2001).Physiology of wound healing.Newborn Infant Nurs. Rev. 1, p. 43–45. Conflict of Interest: None Declared

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