M. Rashidi et al. IRJP 2012, 3 (11)
Page 134
INTERNATIONAL RESEARCH JOURNAL OF PHARMACY
www.irjponline.com
ISSN 2230
–
8407
Research Article
BIODETERIORATION OF CHEMICAL CONSTITUENTS IN DRUG
TRIBULUS TERRESTRIS
LINN. ROOTS
DUE TO SPOILAGE OF FUNGI
M. Rashidi*
1, S. S. Deokule
21
Ph. D. Student;
2Prof. and Head of Department of Botany, University of Pune, Pune-411007 India
Article Received on: 11/09/12 Revised on: 22/10/12 Approved for publication: 05/11/12
*
E- mail: Rashidi_129@yahoo.comABSTRACT
Total 15 fungi were obtained to be associated with the roots of Tribulus terrestris Linn. Roots stored at different relative humidities 30, 50, 75, 96 and 100% RH for 90 days. Quantitative estimation of main chemical constituents such as phenols, proteins, alkaloids, glycosides and sugars in relation to association of fungi were done. The drug stored at above 75% relative humidity showed maximum percentage incidence of fungi as well as deterioration of chemical constituents.
Key words: fungi, deterioration, chemical constituents, relative humidity
INTRODUCTION
Tribulus
terrestris
belongs to the family "Zygophyllaceae. It
is
called
commonly,
"Chota-gokhru",
"Punjabi",
"Gokhuru","Bakhra" and in English called "Calthrops" and
"Puncture vine"
15.
T. terrestris
is a procumbent, ascending or
sub erect herb. The roots are good for stomachic, appetiser,
diuretic and carminative
10. As
Tribulus terrestris
L.
medicinally very important multipurpose herb recently lots of
research reported on as chemical composition. The presence
of steroidal glycosides, steroidal saponins, flavonoids and
alkaloids in the plant has been reported
19. The roots are good
for stomachic, appetiser, diuretic and carminative
10.
Kianbakt and Johaniani (2003) and Abbasoglu and Tousun
1have been reported extracts of different parts of
T. terrestris
showed antibacterial activity. Kostova and Dinchev
11and
Iskenderov
8have studied steroidal saponins in this plant.
Al-Bayati and Al-Mola
2reported different part have
antibacterial and anti fungal activity. There are not any report
concentrating on the subject mycoflora associated with roots
of
Tribulus
terrestris
and changes in their chemical
constituents due to spoilage, therefore, a survey was carried
out to determine the percentage incidence of fungi associated
with the drug roots
of
T. terrestris
stored at different relative
humidities
and second part of this study involved
determination of quality and biodeterioration of chemical
constituent amounts in samples in relation to fungal
contamination.
MATERIAL AND METHODS
The crude root samples were collected from different places
and then were sent to the laboratory in polyethylene bags to
avoid aerial other contaminations as soon as and then were
dried in the shade. According International Seed Testing
Association
7, Moist Blotter test and Agar plate method were
done for isolation and identification of associated fungi. For
isolation of associated fungal roots, sterilized with 2%
sodium hypochlorite solution for some minutes and
thoroughly washed with sterilized distilled water. After
developing of colonies, were counted and average of 10 Petri
plates were calculated. Fungi were identified by using
references
3, 16, 17. Some part of root samples were stored in
small muslin clothes in desiccators at 30, 50, 75, 96 and 100
% RH for 90 days in the room temperature. The root samples
were taken out an internal 15, 30, 45, 60, 75 and 90 days,
thoroughly washed with distilled water and plated in Petri
plates. The isolation of mycoflora was recorded from first
day to 60
thday of storage. Some part of samples after drying
in oven, finely grinded for evaluation of changes in
biochemical constituents related to mycoflora. Quantitative
estimation of chemical constituents was carried out from first
day to 90
thday of incubation. Biochemical analysis were
estimated for total protein
13,
for total phenols
20, for total
alkaloids
6and for glycosides
12according standard procedure.
Anthrone methods for total sugars and Dinitrosalicilic acid
(DNSA) method for reducing sugar amount
18were also
followed. Simple correlation were run between selected
parameters using Statistical Package for Social Science
(SPSS) software in which statistical significance was
determined at 0.05 % probability levels.
Table 1: Percentage incidence of fungal isolated from the roots of Tribulus terristeris stored at various relative humidity
Mycoflora con 30% 50% 75% 96% 100%
15 30 45 60 15 30 45 60 15 30 45 60 15 30 45 60 15 30 45 60
F. solani 0.43 0.43 0.70 0.98 1.26 0.56 0.98 1.26 2.11 0.43 1.26 1.55 2.11 0.56 0.70 1.83 2.11 1.26 2.39 2.96 3.94
F. acuminatum - - - 0.14 - - 0.14 0.28 - 0.14 0.43 0.56 0.14 0.70 0.98 1.26
F. semitectum - - - 0.14 - - 0.14 0.43 - 0.14 0.28 0.43 0.14 0.28 0.56 0.70
F. oxysporum - - - 0.14 0.14 - 0.14 0.14 0.28 0.14 0.14 0.28 0.43
F. lateritum - - - 0.14 0.14 - - 0.14 0.28
Papulaspora immerse 0.14 0.14 0.14 0.14 0.28 0.14 0.14 0.28 0.43 0.14 0.14 0.56 0.70 0.14 0.14 0.56 0.70 0.28 0.43 1.26 1.55
Aspergillus niger 0.14 0.14 0.14 0.14 0.14 0.14 0.14 0.14 0.14 0.14 0.14 0.28 0.43 0.28 0.14 0.28 0.43 0.14 0.56 0.70 0.98
A. fumigatus - - - 0.14 0.14 - 0.14 0.28 0.43 0.14 0.70 0.98 1.26
Trichoderma sp. 0.14 - - - 0.14 - 0.14 0.28 0.43 0.28 0.43 0.56 0.70 0.14 0.28 0.43 0.56 0.14 0.70 0.98 1.26
Chaetomium sp. - - - 0.14 - - 0.28 - 0.14 0.28 0.43
C. globosum - - - 0.14 0.14 - 0.14 0.28 0.43
Thraustotheca sp. 0.14 0.14 0.14 0.14 0.14 0.28 0.14 0.14 0.28 .14 0.28 0.43 0.56 0.14 0.14 0.28 0.43 0.14 0.43 0.70 0.98
Theilavia terricola 0.14 0.14 0.14 0.14 0.28 0.14 0.14 0.28 0.70 0.14 0.28 0.70 0.98 0.14 0.43 0.56 0.70 0.28 0.70 0.98 1.26
Neurospora sitophila 0.14 0.14 0.14 0.14 0.28 0.14 0.28 0.43 0.56 0.14 0.43 0.98 1.26 0.43 0.70 1.26 1.97 0.43 0.98 2.11 2.39
M. Rashidi et al. IRJP 2012, 3 (11)
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Table 2: Deterioration of Total sugar (TS) and reducing sugar (RS) content (mg/100mg) in root of Tribulus terrestris at different relative humidities
Incubation days Control 30% 50% 75% 96% 100%
TS RS TS RS TS RS TS RS TS RS TS RS
1 day 58.08±
1.48 16.74± 15.42 58.08± 1.48 16.74± 15.4 58.08± 1.48 16.74± 15.2 58.08± 1.48 16.74± 15.4 58.08± 1.48 16.74± 15.42 58.08± 1.48 16.74± 15.42
15 days 58.08±
1.57d 16.74± 15.29d 58.08± 1.57cd 15.83± 0.80cd 57.57± 1.53c 15.38± 15.9c 57.36± 1.49cb 14.47± 16.1cb 55.84± 0.49b
13.5±1 6.19ab
55.42±
0.12a
12.66±
16.32a
30 days 58.08±
1.48d 16.74± 15.43d 56.28± 1.62cd 15.38± 15.8cd 56.52± 1.36c 13.57± 15.6c 55.59± 0.07cb 13.57± 16.4cb 54.92± 0.55b
12.2±1 4.63ab
54.25±
1.07a
11.31±
14.89a
45 days 58.08±
1.46d 16.74± 15.55d 55.89± 1.96cd 14.02± 15.5d 54.62± 0.81c 12.21± 15.1c 53.15± 0.12cb 10.85± 14.6cb 52.56± 0.26ab
9.50±1 5.96a
52.22±
0.56a
10.40±
14.77a
60 days 58.08±
1.55d 16.74± 15.68d 55.42± 2.30c 12.21± 15.1c 53.49± 0.69c 10.85± 15.6c 52.35± 0.44cb 10.40± 0.59cb 50.92± 0.62ab
9.50±1 5.96a
50.12±
0.12a
9.049±
13.73a
75 days 58.08±
1.53d 16.74± 15.06d 53.57± 0.51c 10.40± 15.4c 52.69± 0.31c
9.50±1 5.73c 51.30± 0.69b 9.049± 0.0cb9 49.20± 0.29a
8.59±0. 20a
48.10±
0.12a
8.14±
13.61a
90 days 58.08±
1.57d
16.74±
15.08d
27.77±
1.75c
9.04±1 5.67c
27.52±
1.90c
8.59±1 4.43c
26.50±
1.90b
8.14±0. 25b
11.36±
0.12a
7.69±0. 46a
7.82±
0.66a
7.23±
12.20a Data are the mean of three replicates ± standard deviation. P- Value denoted the significance of differences between the mean by univariate comparison
statistics. The value followed by different letters differ significantly by Duncan’s multiple rang test at P=Sig= 0.05
Table 3: Deterioration of protein content (mg/100mg) in root of Tribulus terrestris at different relative humidities
Incubation days control 30% 50% 75% 96% 100%
1 day 20.27±0.13 20.27±0.13 20.27±0.13 20.27±0.13 20.27±0.13 20.27±0.13 15days 20.27±0.21d 20.27±0.21cd 19.44±0.71c 19.44±0.08b 19.16±0.21a 18.19±0.68a 30days 20.27±0.13d 19.72±0.34cd 19.44±0.21c 18.75±0.69b 17.77±0.34a 16.80±0.21a 45 days 20.27±0.13d 19.16±0.65cd 17.77±1.39cb 16.38±0.28b 15.97±0.06ab2 15.27±0.13a 60 days 20.27±0d 18.05±0.71cd 16.66±0.56cb 15.27±0.56ab 14.02±0.21a 13.75±0.21a 75 days 20.27±0.13d 16.66±0.89c 16.25±0.71cb 13.61±0.92ab 12.08±0.55a 11.25±0.13a 90 days 20.27±0.13d 15.13±0.97c 14.02±0.13cb 12.63±0.92ab 10.68±0.34a 9.72±0.72a Data are the mean of three replicates ± standard deviation. P- Value denoted the significance of differences between the mean by univariate comparison
statistics. The value followed by different letters differ significantly by Duncan’s multiple rang test at P=Sig= 0.05
Table 4: Deterioration of total phenols content (mg/100mg) in root of Tribulus terrestris at different relative humidities
Incubation days
control 30% 50% 75% 96% 100%
1 day 4.62±0.063 4.62±0.063 4.62±0.063 4.62±0.063 4.62±0.063 4.62±0.063 15days 4.62±0.063d 4.60±0.052cd 4.52±0.022c 4.48±0.030bc 4.42±0.011a 4.33±0.019a 30days 4.62±0.063d 4.52±0.022c 4.48±0.039c 4.40±0.040bc 4.29±0.030a 4.27±0.022a 45 days 4.62±0.063d 4.46±0.034c 4.33±0.088c 4.15±0.081b 4.074±0.081a 3.91±0.030a 60 days 4.62±0.063d 4.33±0.11c 4.27±0.049c 3.93±0.040b 3.74±0.069a 3.54±0.040a 75 days 4.62±0.063d 4.27±0.049c 4.21±0.019c 3.95±0.015b 3.56±0.010a 3.32±0.19a 90 days 4.62±0.063d 3.95±0.019c 3.64±0.052c 3.79±0.079b 3.50±0.049a 3.18±0.049a Data are the mean of three replicates ± standard deviation. P- Value denoted the significance of differences between the mean by univariate comparison
statistics. The value followed by different letters differ significantly by Duncan’s multiple rang test at P=Sig= 0.05
Table 5: Deterioration of total alkaloids content (mg/100mg) in root of Tribulus terrestris at different relative humidities
Incubation days
control 30% 50% 75% 96% 100%
1 day 8.48±0.05 8.48±0.05 8.48±0.05 8.48±0.05 8.48±0.05 8.48±0.05 15days 8.48±0.05d 8.48±0.043cd 8.46±0.040bc 8.43±0.032b 8.41±0.041b 8.37±0.066a 30days 8.47±0.81d 8.39±0.017cd 8.31±0.01bc 8.20±0.0057b 8.13±0.041ab 8.10±0.045a 45 days 8.46±0.065d 8.32±0.10cd 8.18±0.03bc 8.11±0.030b 8.04±0.045a 7.95±0.055a 60 days 8.45±0.075d 8.27±0.22cd 8.08±0.07b 7.94±0.037b 7.84±0.058a 7.77±0.036a 75 days 8.44±0.069d 8.076±0.07cd 7.97±0.12b 7.83±0.047ab 7.74±0.049a 7.69±0.040a 90 days 8.43±0.064d 7.96±0.035cd 7.86±0.040b 7.68±0.014ab 7.36±0.075a 7.30±0.091a Data are the mean of three replicates ± standard deviation. P- Value denoted the significance of differences between the mean by univariate comparison
statistics. The value followed by different letters differ significantly by Duncan’s multiple rang test at P=Sig= 0.05
Table 6: Deterioration of total glycosides content (mg/100mg) in root of Tribulus teresteris at different relative humidities
Incubation days control 30% 50% 75% 96% 100%
1 day 9.52±0.21 9.52±0.21 9.52±0.21 9.52±0.21 9.52±0.21 9.52±0.21 15days 9.52±0.21d 9.52±0.070cd 9.52±0.21c 9.51±0.20cb 9.50±0.18ab 9.45±0.22a 30days 9.51±0.20d 9.51±0.21cd 9.51±0.021c 9.47±0.23cb 9.45±0.047ab 9.38±0.20a 45 days 9.51±0.19d 9.45±0.30cd 9.43±0.045c 9.36±0.18cb 9.32±0.22ab 9.25±0.49a 60 days 9.50±0.18d 9.43±0.18c 9.37±0.22c 9.29±0.021cb 9.25±0.19a 9.21±0.23a 75 days 9.49±0.21d 9.34±0.25c 9.32±0.24c 9.22±0.23cb 9.19±0.07a 8.94±0.20a 90 days 9.48±0.23d 9.25±0.011c 9.23±0.25cb 9.17±0.60ab 9.07±0.16a 8.97±1.021a Data are the mean of three replicates ± standard deviation. P- Value denoted the significance of differences between the mean by univariate comparison
M. Rashidi et al. IRJP 2012, 3 (11)
Page 136
RESULTS
Total 15 fungi from fresh roots of
T. terrestris
were isolated
which include
F. solani, F. acuminatum
,
F. semitectum
,
F.
oxysporum
,
F. lateritum
,
Papulaspora immerse
,
Aspergillus
niger
,
A. fumigatus
,
Trichoderma
sp.,
Chaetomium
sp.,
Ch.
globosum
,
Thraustotheca
sp.,
Theilavia
terricola,
Neurospora sitophila
and
Rhizoctonia solani. F. solani
observed in maximum percentage incidence;
F. lateritum
and
Rhizoctonia solani
occurred in less percentage incidence.
Percentage incidence and growth of fungi are correlated with
high relative humidity and increased incubation days (Table
1). The growth and developing of fungi effect on chemical
constituents amounts such as sugars, alkaloids, glycosides,
phenols and proteins. The fresh roots of
T. terrestris
stored
under high relative humidity 75, 96 and 100% RH and after
60 days of incubation period showed maximum reduction in
chemical constituents.
Genuine samples of this drug contained 58.08 and 16.74% TS
(Total sugar) and RS (Reducing sugar). Under different
relative humidity and different incubation days, deterioration
of TS and RS values observed while after 90 days of
incubation and 100 % RH observed maximum reduction in
TS and RS amounts; 7.82 and 7.23% (Table 2).
The drug
T. terrestris
showed 20.27% total protein in
samples and reduction in total protein amount after different
incubation days and under different RH observed. While after
90 days of incubation under 30, 50, 96 and 100% RH total
protein amount recorded 15.13, 14.02, 12.63, 10.68 and 9.72
% respectively (Table 3).
Control samples of drug
T. terrestris
contained 4.62 % total
phenols, this amount gradually deteriorated, and maximum
deterioration observed under 75, 96 and 100% RH; 3.79,
3.50, 3.18% (Table 4). In case of total alkaloids samples
contained 8.48% total alkaloids at the first day but after 15
days and under 96 and 100% RH, showed 8.41 and 8.37 %
deterioration in total alkaloids, these amounts more
deteriorated to 7.36 and 7.30 % after 90 days of incubation
in same RH (Table 5).
Control samples of drug
T. terrestris
contained 9.52% total
glycosides, maximum deterioration observed under 75, 96
and 100% RH; 9.17, 9.07, 8.97% (Table 6).
DISCUSSION
From the result, it seems to be definite correlation between
growth and sporulation of fungi and the loss of chemical
constituents, analysis of variance also, showed that relative
humidities and incubation days on the deterioration of
chemical
constituent
’
s
contents
were
significant.
Biodeterioration of selected chemical constituents might be
due to enzymatic degradation into simpler components which
are subsequently utilized by mycoflora for their growth
4, 5, 14.
From the result observed maximum storage period and high
relative humidity proliferate the growth of fungi and they
influenced on chemical constituent
’
s contents considerably.
Maximum reduction in total proteins, phenols, glycosides,
alkaloids and sugars (TS and RS) contents was recorded after
60
thof storage where percentage incidence of fungi was also
maximum. Significant reduction of all selected chemical
constituents contents were noticed at above 75% RH and
prolonged incubation periods (after 45 days). The result
indicates that the quality of herbal drugs may be retained by
storage condition at lower RH (below 50% RH) and short
storage period.
ACKNOWLEDGMENT
Authors would like to express a sincere thank to Head,
Department of Botany, University of Pune, Pune-411 007 for
encouragement and necessary laboratory facilities.
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Source of support: Nil, Conflict of interest: None Declared
