IJERD – International Journal of Environmental and Rural Development (2015) 6-2

Research article
erd

Efficiency of Crude Extract from Pummelo Peel onControlling the Growth of Colletotrichum gloeosporioides(Penz.)NUNCHANOK NANTHACHAI*
Faculty of Agricultural Technology, Rajamangala University of Technology Thanyaburi,
Phathum Thani, Thailand
Email: eaye22@yahoo.com

INTIRA LICHANPORN
Faculty of Agricultural Technology, Rajamangala University of Technology Thanyaburi,
Phathum Thani, Thailand

PALIDA TANGANURAT
Faculty of Agricultural Technology, Rajamangala University of Technology Thanyaburi,
Phathum Thani, Thailand

UNCHALIN SINGKHUM
Faculty of Agricultural Technology, Rajamangala University of Technology Thanyaburi,
Phathum Thani, Thailand
Received 15 December 2014

Accepted 1 April 2015(*Corresponding Author)

Abstract The objective of this research was to investigate the antifungal efficiency ofpummelo peel extracted with different solvents on Colletotrichum gloeosporioides. Crude
extracts from pummelo peels were eluted with four solvents, such as ethanol, hexane,
dichloromethane and ethyl acetate. Chemical compounds of the crude extracts wereanalyzed by GC-MS and HPLC and inhibition the growth of C. gloeosporioides was tested
by PDA culture. The result showed that limonene was the major compound in all extracts(79.55-89.91%). D-Phellandrene and 2, 6-octadien-1-ol, 3, 7-dimethyl were found only incrude extracts using ethyl acetate as a solvent. In addition, the extract was eluted with ethyl
acetate also had the amount of flavonoids: nobiletin and 3-hydroxy-7, 3’, 4’, 5’tetramethoxyflavone, higher than those eluted with other solvents. Inhibition the growth ofC. gloeosporioides of pummelo peel extracted with dichloromethane, ethanol, hexane andethyl acetate were 19.16, 52.81, 65.58, and 95.61%, respectively. Therefore, pummelo peel
extracted with ethyl acetate was the highest efficiency on controlling C. gloeosporioides (pd 0.05).
Keywords Crude extract, pummelo, C.gloeosporioides

INTRODUCTION
Mango is one of the most popular and best known tropical fruits (MacLeod and Troconis, 1982;
MacLeod and Pieris, 1984). It is a highly priced fruit due to its attractive color, delicious taste, andhigh nutritional values (Mitra and Baldwin, 1997; Lalel et al., 2003). Traditionally, the majormango exporting countries are the Philippines, Thailand, Mexico, and India (Jacobi et al., 2001).
Many different ripening conditions, postharvest treatments, and processing methods are used toprepare mangoes for marketing to consumers (Moore, 2003). Its short shelf life limits thecommercialization at long distances. It is also highly susceptible to damages caused by fungi,bacteria and fruit fly larvae (Diaz-Sobac et al., 1996).
Anthracnose is a major pre‐ and post‐harvest disease on mangoes, causing direct yield loss inthe field and packing plant, and quality and marketing issues (Ploetz, n.d.).Mango anthracnose isⒸ ISERD

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IJERD – International Journal of Environmental and Rural Development (2015) 6-2

caused by the fungus Colletotrichum gloeosporioides (Pitkethley and Conde, 2007). Ripe fruits
affected by anthracnose develop sunken, prominent, dark brown to black decay spots before orafter picking. The fruit spots can usually do coalesce and can eventually penetrate deep into thefruit, resulting in extensive fruit rotting. Most green fruit infections remain latent and largely
invisible until ripening (Nelson, 2008). Post-harvest treatments are available for control ofanthracnose in mango fruits. Prochloraz is used as a cold nonrecirculating spray. Hot water dips,used to control fruit flies, will also control anthracnose and stem end rots. Hot benomyl dips willcontrol anthracnose and are useful where stem end rots are a problem (Pitkethley and Conde,2007). However, there are many studies using plant extracts such as essential oils and purecompounds against plant pathogenic fungi, which have been conducted. Antifungal compounds
have been found in plants, derived from secondary metabolites such as tannins, terpenoids,
alkaloids and flavonoids (Arif et al., 2009).
Pummelo (Citrus grandis or Citrus maxima) is an original citrus fruit, with the look of a biggrapefruit, native to South and Southeast Asia. Citrus fruits contain several bioactive compounds
such as flavonoids, limonoids, and organic acid. Polymethoxyflavones are group of flavonoids that
present mainly in the fruit peel. There are some reports that show polymethoxyflavones, which areimportant components of plant defensive mechanism against various diseases causing pathogens
such as phytophthora and Colletotrichum gloeosporioides (Del Rio et al., 1998; Ortuño et al., 2006;
Uckoo et al., 2011). Thus, this research aimed to investigate the major volatile compounds,flavonoids and polymethoxyflavones as well as the antifungal efficiency of crude extract frompummelo peel on C. gloeosporioides.
OBJECTIVESThe study was conducted with the following objectives:
1. To determine the major volatile compounds and flavonoids of crude extract from pummelo peel;and2. To investigate the effect of crude extracts from pummelo peel on inhibition the growth ofColletotrichum gloeosporioides.
METHODOLOGY
Pummelo was purchased from a commercial market in Phathumtani province, Thailand. The
flavedo of the fruit was cut into small pieces and then immersed with a solvent for 24 hours and theratio of peel and solvent was 1:3. The four solvents were ethanol, dichloromethane, hexane andethyl acetate which were used in this experiment. The extracts were pooled and evaporated at 40 oC
using a rotary evaporator. Then the extracts were stored at freezer until used. Volatile compounds
and polymethoxyflavones in the extracts were analyzed by GC-MS and HPLC. Each sample wasanalyzed in triplicate.
C. gloeosporioides was isolated from infected mango fruits with typical symptoms andmaintained on potato dextrose agar (PDA) at 25 oC for seven days. The pathogen was inoculated
into mango and re-isolated on PDA before the experiment.
The effect of extracts from pummelo peel on mycelial growth of C. Gloeosporioides wasassayed by modifying the method of Liu et al., (2007). Mycelial disks (5 mm in diameter) fromone-week-old culture of C. gloeosporioides were placed in the center of Petri dishes containing 20
ml of PDA with the 10,000 ppm extracts eluted with different solvents, and then incubated at 25oC.
The plate without the extract served as the control. Mycelial growth was determined by measuringcolony diameter (mm) each day after inoculation. Each treatment was five times replicated, and theexperiment was repeated thrice. The inhibition rate on mycelial growth was calculated according to
the following formula: Inhibitory rate (%) = 100 x (colony diameter of the control − colony
diameter of the treatment) / (colony diameter of the control).

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RESULTS AND DISCUSSION
Pummelo peel in this study had the color L* a* and b*, which the values were between 62.8-67.6,
3.1-8.7 and 47.3-48.7, respectively. Chemical compounds of the crude extracts were analyzed by
GC-MS (Table 1) and HPLC (Table 2). The result showed that the extract from pummelo peel waseluted with ethyl acetate that could identify volatile components and flavonoids better than those
extracted with dichloromethane, hexane and ethanol (Table 1). Terpenes were the major
components in extracts of pummelo peel. Volatile compounds were always found in citrus such aspinene, sabinene, myrcene, phellandrene, limonene, ociemene, linalool, terineol, carvone,caryophyllene, and geraniol. Ester such as geranyl acetate was also found in the extracts. Limonene
was the major compound in all extracts (79.55 - 89.91%) and the result was consistent with Hosni
et al., (2010). However, this research could identify the compounds of the extracts less than anotherresearch. This might be due to the difference of solvents and extraction methods. Hosni et al.,(2010) used both of pummelo flavedos and leaves in their research as well. Linalool was also foundin all extracts; however, it was presented in very small amounts (0.20 - 2.22% peak area).
Jabalpurwala et al., (2009) found that linalool was the main compound in pummelo blossom. DPhellandrene and 2, 6-octadien-1-ol, 3, 7-dimethyl were found only in crude extracts using ethyl
acetate as a solvent.
Table1 Volatile compounds (% peak area) of extracts from pummelo peel eluted
with four solvents
Volatile Compounds
D-Pinene
Sabinene
E-Pinene
E-Myrcene
D-Phellandrene
Limonene
E-Ociemene
Linalool
D-Terpineol
2-Cyclohexen-1-ol, 2-methyl-5-(1methylethene)-cisD-Carvone
2,6-Octadien-1-ol, 3,7-dimethyl
Geraniol
Geranyl acetate
Caryophyllene
1,6-Cyclodecadiene, 1-methyl-5-methylene8-(1-methylethyl)nd: Not detected

Ethanolndnd
nd0.35
nd79.55
nd
2.22
nd
nd

Hexane
0.42
0.98
nd
1.49
nd
81.52
0.19
0.20
ndnd

Solvent
Dichloromethanendnd1.52
nd
nd
84.62
0.167
0.63
0.76
0.21

Ethyl acetate
0.72
nd2.262.540.20
89.91
0.15
0.28
nd
nd

nd
nd
nd
0.47
0.55
0.73

nd
nd
nd
nd
0.25
1.17

0.28
nd
0.44
nd
nd
nd

nd
0.58
0.30
0.12
0.10
0.71

Citrus peel was rich in flavanones and polymethoxlated flavones (Ortuño et al., 2006).
Nobiletin in the extracts were eluted with ethanol, dichloromethane, hexane, and ethyl acetate were4.87, 0.20, 0.24 and 0.68%, respectively. Tangeretin of pummelo peel extracted with ethanol,
dichloromethane, hexane and ethyl acetate were 7.39, 4.36, 0.10 and 7.80%, respectively.
Polymethoxyflavones were groups of flavonoids that presented mainly in the fruit peel. These
compounds had two or more methoxyl’s on their basic flavonoid structure (Uckoo et al., 2011).
Pummelo peel extracted with ethanol had very small amount of 3, 6, 3’, 4’-tetramethoxyflavone(0.66%), when compared to other solvent extractions. And 3-hydroxy-7,3’,4’,5’tetramethoxyflavonein the extract were eluted with hexane, and dichloromethane were found only
0.34 and 0.47%, respectively. Whereas extraction pummelo peel with ethanol and ethyl acetate had
3-hydroxy-7, 3’, 4’, 5’-tetramethoxyflavone higher than 7.00% (Table 2).

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Table2 Flavonoids and polymethoxyflavones (% peak area) of extracts
from pummelo peel eluted with four solvents
Flavonoids and Polymethoxyflavones
Nobiletin
3, 6, 3’, 4’-Tetramethoxyflavone
3-Hydroxy-7, 3’, 4’, 5’- tetramethoxyflavone
Tangeretin

Ethanol
4.87
0.66
8.35
7.39

Hexane
0.20
77.44
0.34
4.36

Solvent
Dichloromethane
0.24
70.59
0.47
0.10

Ethyl acetate
0.68
75.72
7.67
7.80

Figure 1 showed that mycelial growth of C. gloeosporioides on PDA plate without the extract
served as the control. Antifungal efficiency of crude extract from pummelo peel on C.
gloeosporioides was showed in table 3 and figure 2. Inhibition the growth of C. gloeosporioides ofpummelo peel extracted with dichloromethane, ethanol, hexane and ethyl acetate were 19.16, 52.81,
65.58 and 95.61%, respectively. Thus, pummelo peel extracted with ethyl acetate was the highest
efficiency on controlling C. gloeosporioides (p d 0.05). Polymethoxyflavones are importantcomponent of plant defensive mechanism against various disease causing pathogens such as
Phytophthora and C. gloeosporioides (Del Rio et al., 1998). The present study found two
compounds of polymethoxyflavones; 3, 6, 3’, 4’-tetramethoxyflavone and 3-hydroxy-7, 3’, 4’, 5’tetramethoxyflavone. Extraction of pummelo peel with hexane and dichloromethane had the highpeak area of 3, 6, 3’, 4’-tetramethoxyflavone (more than 70%), while 3-hydroxy-7, 3’, 4’, 5’tetramethoxyflavone were only 0.34 and 0.47%, respectively. On the contrary, the extract eluted
with ethanol had a higher 3-hydroxy-7, 3’, 4’, 5’- tetramethoxyflavone (8.35%), whereas 3, 6, 3',4'-tetramethoxyflavone was very low. Extraction of pummelo peel with ethyl acetate had the highpeak area of both 3, 6, 3’, 4’-tetramethoxyflavone and 3-hydroxy-7, 3’, 4’, 5’-tetramethoxyflavone
were 75.72 and 7.67%, respectively, which was different from the extracts eluted with othersolvents. Therefore, this might be the cause of effectiveness in inhibiting the growth of C.
gloeosporioides.
Table 3 Effect of crude extracts from pummelo peel eluted with four solventson the growth of C. gloeosporioides
Solvent

Inhibitory rate (%)
52.81b65.58b
19.16c95.61aEthanol
HexaneDichloromethane
Ethyl acetate

Mean of the same column with different superscripts indicating significantly differences (p ≤ 0.05)

Fig. 1 Mycelial growth of C. gloeosporioides (on plate without crude extractfrom pummelo; control) after incubated at 25oC for 7 days

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Fig. 2 Effect of extracts from pummelo peel eluted with ethanol (A), hexane (B),dichloromethane (C) and ethyl acetate (D) on mycelial growth of C. gloeosporioides
after incubated at 25oC for 7 days
CONCLUSION
The major volatile compound of crude extract from pummelo peel was limonene. The extract
eluted with ethyl acetate had the most volatile components, followed by the extraction withdichloromethane, hexane, and ethanol, respectively. Flavonoids (nobiletin and tangeretin) andpolymethoxyflavones (3, 6, 3’, 4’-tetramethoxyflavone and 3-hydroxy-7, 3’, 4’, 5’tetramethoxyflavone) were found in all solvent extractions. However, the extract eluted with ethyl
acetate had the amount of nobiletin and 3-hydroxy-7, 3’, 4’, 5’- tetramethoxyflavone, which werehigher than those eluted with other solvents.
Inhibition the growth of C. gloeosporioides of pummelo peel extracted with dichloromethane,
ethanol, hexane and ethyl acetate were 19.16, 52.81, 65.58, and 95.61%, respectively. The crude
extract of pummelo peel eluted with ethyl acetate was the highest efficiency on controllingmycelial growth of C. gloeosporioides.
ACKNOWLEDGEMENTS
This work was supported by National Research Council of Thailand (NRCT) and Faculty ofAgricultural Technology, Rajamangala University of Technology Thanyaburi (RMUTT).
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