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Phytochemical proﬁling and larvicidal potential of common grass Pennisetum

polystachion against mosquito vectors
Article · December 2018
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© 2018 IJRAR December 2018, Volume 5, Issue 4 www.ijrar.org (E-ISSN 2348-1269, P- ISSN 2349-5138)
Phytochemical profiling and larvicidal potential of

common grass Pennisetum polystachion against
mosquito vectors
1
Babu. M, 2Kaleena P.K*, 3Janaki. A, 4Velu. K, 5Jeyanthi.R
1
Ph.D. Research Scholar,2Associate Professor, 3Ph.D. Research Scholar,4 Ph.D. Research Scholar,5Ph.D. Research Scholar
1
Department of Zoology, 1Presidency College (Autonomous), Chennai- 600005, Tamil Nadu, India.
Abstract
Mosquitoes are vectors for many diseases, causing millions of death every year throughout the world.
This necessitates the implementation of new methods to control and prevent mosquito – borne diseases and to
improve public health. The use of biopesticides have increased because of their high insecticidal properties and
eco-friendly nature. The aim of present study was to evaluate the larvicidal potential of the whole plant extracts
of a common grass, Pennisetum polystachion against fourth instar larvae of Aedes aegypti, Anopheles stephensi
and Culex quinquefasciatus and their larval mortality was recorded after 24 hrs of exposure. The preliminary
qualitative phytochemical analysis revealed the presence of various phytocompounds such as saponins,
flavonoids, alkaloids, cardiac glycosides, coumarins, and steroids etc. The ethanol extract ofP. Polystachion
showed potent larvicidal activity against all the three mosquito larvae, when compared to other extracts. TLC
and GC-GC-MS analysis of the ethanol extract of P. polystachion revealed the presence of phytocompounds
with insecticidal properties.
Keywords: Pennisetum polystachion, Phytochemicals, Mosquito larvicidal activity, Aedes aegypti, Anopheles
stephensi, Culex quinquefasciatus.
IJRAR1944680 International Journal of Research and Analytical Reviews (IJRAR) www.ijrar.org 103
1. Introduction
In the history of the world, more people would have died from diseases transmitted by mosquitoes
than from all the fighting in the wars. The world’s most dangerous creature is in fact the mosquito.
Mosquitoes can transmit more diseases than any other group of arthropods and affect millions of people
throughout the world [1]. WHO has declared the mosquitoes as “public enemy number one”. Several
mosquito species belonging to genera Anopheles, Culex, and Aedes are vectors for the pathogens of
various diseases like malaria, filariasis, Japanese encephalitis, dengue, yellow fever, chikungunya, etc.[2].
Every year, more than one billion people are infected and more than one million people die from vector-
borne diseases including malaria, dengue, yellow fever and lymphatic filariasis (WHO, 2014). Aedes
aegypti is known vector for arbo-viruses responsible for dengue fever, which is endemic to Southeast
Asia, the Pacific island area, Africa, and the America [3].
Anopheles stephensi is the primary vector for malaria in India and other west Asian countries. Every
year, an estimated 300–500 million new infections and 600,000 cases based on world malaria report
2013[4].
Culex quinquefasciatus, is a vector of lymphatic filiariasis which is widely distributed tropical disease
and there are nearly 1,100 million people living in areas endemic for lymphatic filiariasis and exposed to
the risk of infection; there are102 million cases of filiariasis, either having patent microfilarea or chronic
filarial disease[5].
Vector control remains the most effective measure and is often the only way to prevent disease
outbreaks because there are no vaccines for many vector-borne diseases and drug resistance is an
increasing threat. One of the methods to manage these diseases is to control the vectors for bringing about
interruption in disease transmission. The control of mosquitoes at larval stage is considered as an efficient
way in the integrated vector management. Worldwide all water sources are common habitats for the
immature stages of vector mosquito species and reducing mosquito-diseases morbidity in both urban and
rural areas where a sufficient proportion of larval habitats can be targeted [6].
Different mosquito control methods are being used including chemical method by targeting the adult
mosquito through spraying chemical insecticides or by killingthe mosquito larvae by using synthetic
larvicides[7] such as pyrethroids and organophosphorus compounds[8]. Extensive use of chemical
insecticides against vector mosquitoes for the control of mosquito borne diseases has caused development
of resistance in mosquitoes to these insecticides and hazards to the environment. In spite of the sustained
and prolonged use of chemical insecticides, these diseases are not onlystill prevalent but also outbreak
into epidemics[9]. For these reasons, researchers are increasingly focusing their attention on the
development of biodegradable phytopesticides. Biodegradable pesticides of plant origin mitigate the long
term environmental effects of pesticide use, and, furthermore, pests rarely develop resistance against
pesticides of plant origin [10,11].
Today, a number of pharmaceutical companies are investing a lot of money and time to devise cost
effective natural drugs from plant extracts[12]. Selection of the potent plants andthe solvent systems for
extraction purpose plays a vital role in the recovery of biomolecules with the desired properties[13].
According to the World Health Organization (WHO) approximately 65–80% of the developing
countries depend on traditional medicine for their health care due to difficulties of accessing modern
medicine or poverty [14].
India is one of the biodiversity nations, which embrace the indigenous knowledge of traditional
healers[15].Natural products are generally preferred due to being less harmful to non-target organisms,
and thanks to their biodegradability, efficiency and low cost [16]. The larvicidal properties of indigenous
plants have also been documented in many parts of India along with the repellent and anti-juvenile
hormones activities [17]. Almost all tropical regions of the world are experiencing the resurgence and
reoccurrence of one of the world’s most deadly diseases, i.e., malaria, filariasis, dengue, and chikungunya
in world and India is no exception. Traditionally, plants and their derivatives were used to kill mosquitoes
and other household and agricultural pests. In all probability, these plants used to control insects
contained insecticidal phytochemicals that were predominantly secondary compounds produced by plants
to protect themselves against herbivorous insects [18,19].
Kishore et al.,[20] reviewed the efficacy of phytochemicals against mosquito larvae according to their
chemical nature and described the mosquito larvicidal potentiality of several plant derived secondary
metabolites such as alkanes, alkenes and simple aromatics, lactones, essential oils and fatty acids,
terpenes, alkaloids, steroids, isoflavonoids, pterocarpans and lignans. Researchers have also documented
the isolation of several bioactive toxic principles from various plants and reported their toxicity against
different mosquito species.
The grasses that yield food and fodder are well known from times immemorial and considerable
amount of research work has been carried on them. But the grasses that yield therapeutically important
products are among the least studied in the Poaceae family. Phytochemicals are naturally occurring,
biologically active chemical compounds in plants. They act as a natural defense system for host plants
and provide colour, aroma and flavour. These are non-nutritive plant chemicals that have protective or
disease preventive properties[21].
Pennisetum polystachion (Mission grass) is a noxious weed that grows in the farmlands, grasslands,
upland tropical hills and croplands including perennial crops, especially after forests have been cleared.
The plant has been used in Nigeria as a traditional means of preventing bleeding, [22]. The present study
is aimed to investigate the larvicidal potential and to screen the phytocompounds of P. polystachion grass
extracts.
2.0. Materials and Methods
2.1. Selection of Plant
The grass P. polystachion (Fig.1) belonging to the family Poaceae was collected from the natural
population in the Presidency College campus, Chennai, Tamilnadu, India. The plant was identified and
authenticated by Prof. P. Jayaraman and deposited at the plant anatomy research center (PARC) West
Tambaram, Chennai-45, Tamil Nadu, India. The collected plants were washed with tap water, distilled
and then it dried for 14 days.
Fig 1. Pennisetum polystachion

2.2. Plant extract preparation
The dried plants were sieved and powdered using electric blender. The grass powder was extracted
successively using solvents respectively aqueous, chloroform, ethanol, hexane, methanol. 50gm powder
was soaked in 500ml of each solvent. The extracted solvents were concentrated using Rotary evaporator
and stored it under 4⁰C in air tight bottles for further use.
2.3. Selection of mosquito species
The fourth instar larvae of Aedes aegypti, Anopheles stephensi and Culex quiquefasciatus were
selected for this present study. The larvae were procurred from the Entomology Research Institute (ERI),
Loyola College, Chennai, Tamilnadu, India.
2.4. Phytochemical screening
Phytochemical screening of the samples was carried out as described by [23,24]. The plant extract
samples were screened for secondary metabolites like carbohydrates, saponins, alkaloids, flavonoids,
glycosides and proteins etc.
2.5. Separation of bioactive compounds using TLC
2.6. Preparation of plant extract
10 mg / ml of the plant extract in ethanol solvent was used for TLC examination.
2.7. TLC Plate preparation

The silica gel 60o F 254 coated aluminum sheets were slice in size 1.5 x 6.5cm and the prepared
ethanol plant extract was loaded on silica plate and air dried.
2.8. Mobile phase preparation
The ethanol extracts were standardized in ethyl acetate with acetone and finally in hexane: ethyl
acetate (2:1) ratio [25].
2.9. GC-MS analysis

The sample was prepared in the concentration range of 0.2 - 0.5 mg/ml and injected by flow analysis at
a flow rate of 10 μl min-1. The recorded mass was in the range 100-500 m/z using the mass spectrum was
obtained using an Agilent technologies (6890 N) instrument (JEOL GCMATE II) fitted with an electron
spray ionization source. Software version 4.0 was used for data acquisition. The positive ion mode, using
a spray voltage at 3.5 kV, at a source temperature of 80oC, was employed for recording the spectra. Mass
spectra were recorded under electron impact ionization at 70 eV energy.
2.11. Mosquito Larvicidal Bioassay
The larval susceptibility tests were done according to standard procedures [26]. A laboratory reared
vector mosquitoes viz., Aedes ageypti Anopheles stephensi and Culex quinquefasciatus free of exposure
to insecticides and pathogens. Cyclic generations of vector mosquitoes were maintained at 25-29°C
insectariums. Larvae were fed on powdered dog biscuit and yeast in the ratio of 3:1 and adult mosquitoes
on 10 per cent sucrose solution.
Larval susceptibility test was carried out as described by [27]. A total of three trials with five
replicates per trial for each concentration was done against mosquito vectors. The crude plant extracts
were separately tested for its larvicidal potential against fourth instar larvae of A. ageypti, A. stephensi
and C. quinquefasciatus. The Stock solution (1000 ppm) of the extract was prepared by dissolving 100
mg of crude extract in 1 ml acetone and volume raised to 100 ml with distilled water. From the stock
solution different dilution of 50 ppm, 100 ppm, 150 ppm, 200 ppm and 250 ppm were prepared in 200 ml
distilled water and 20 fourth instar larvae were released and mortality was recorded after 24 h. The
beakers were kept in a temperature controlled room at 28°C ± 2°C and the larvae were exposed to 200 ml
water containing 0.1 ml of acetone which served as control.
2.12. Larval susceptibility tests
The different concentrated solutions of extracts was prepared and tested against the three mosquito
larvae A. aegypti, A. stephensi and C. quinquefasciatus were placed in each test solutions to observe the
larvicidal properties as per the following procedure.
Groups of 20 larvae were placed in glass beakers containing 200 ml of the grass extract solution.
Control experiments without extract were run in parallel. The larvae in each solution were then left for 24
h and the numbers of dead larvae were counted after 24 h of exposure, and the percentage mortality was
calculated from the average of five replicates and mortality in the control was corrected by Abbott’s [28]
formula.
Number of dead larvae
Percentage mortality = ---------------------------------------- X 100
Number of larvae introduced
2.13. Dose-response bioassay
From the stock solution, different concentrations ranging from 25 to 150 ppm were prepared. Based
on the preliminary screening results, the prepared grass extracts were subjected to dose-response bioassay
for larvicidal activity against A. aegypti, A. stephensi and C. quinquefasciatus. The numbers of dead
larvae were counted after 24 hours of exposure, and the percentage mortality was reported from the
average of five replicates.
2.14. Statistical analysis
The average larval mortality data were subjected to probit analysis for calculating LC50, LC90[29] as
described by [30] and other statistics at 95% fiducial limits of upper confidence limit and lower
confidence limit and chi-square values were calculated using the software (SPSS, 11.5).
3.0. Results and Discussion

3.1. Phytochemical Screening
The preliminary phytochemical analysis of P. polystachion confirmed the strong presence of
carbohydrates, saponins, alkaloids, anthocyanin and coumarins, phenols, tanins, flavonoids, cardiac
glycosides, acids and steroids were present in the ethanol extract of the P. polystachion.(Table.1). Since
ethanol extract showed the strong presence of various phytocompounds therefore it was further subjected
to larvicidal assay.
Table 1. Qualitative phytochemical profiling of Pennisetum polystachion grass extracts
S. No Secondary metabolites Aqueous Chloroform Ethanol Methanol Hexane
1 Carbohydrate +++ +++ +++ +++ +++
2 Tannins - + + + -
3 Saponins +++ +++ ++ ++ +++
4 Flavonoids +++ +++ ++ ++ +++
5 Alkaloids - +++ +++ - +
6 Anthocyanin +++ +++ +++ + ++
7 Quinones - - - - -
8 Glycosides + - +++ ++ -
9 Cardiac glycosides - +++ ++ ++ ++
10 Terpenoids +++ - - - -
11 Triterpenoids - - - ++ ++
12 Phenols - - ++ ++ -
13 Coumarins +++ +++ +++ ++ ++
14 Acids ++ + ++ + +
15 Protein - - - - -
16 Steroids +++ +++ ++ - ++
++ Strongly positive ++ Positive + Trace - Not detected
3.2. Larvicidal Bioassay

The ethanol extract of P. polystachion, exhibited potent larvicidal activity at very low concentrations
against the fourth instar larva of all three mosquito species tested. Ethanol extracts of P. polystachion
showed 100% mortality at 200 ppm against the fourth instar larvae of A. stephensi, A. aegypti and C.
quinquefasciatus. Ethanol extract at low concentration of 100 ppm showed 55 to 60% mortality, when
exposed for 24 h. Ethanol plant extracts were also found to be equally effective showing 80 to 95%
activity against the fourth instar larvae of the three mosquito species tested (Fig 3 a, b and c).Based on the
probit analysis between the concentrations of plant extract against the fourth instar larvae of A. aegypti, A.
stephensi and C. quinquefasciatus after 24 h exposure, the results are presented in Tables 2, 3 and 4. The
ethanol extract of P. polystachion was found to be more toxic against C. quinquefasciatus and A.
stephensi with LC50 value of 87.146 ppm and 88.229 ppm respectively and LC90 value of 183.04 ppm
and 179.428 ppm respectively, when compared to its toxicity against A. aegypti with LC50 value of
95.178 ppm and LC90 value of 205.033 ppm respectively. Methanol extracts of Pennisetum polystachion
showed LC50 and LC90 values of 94.346 ppm and 202.913 ppm respectively against C. quinquefasciatus
when compared to A. stephensi (98.011 ppm and 216.596 ppm) and A. aegypti (103.069 ppm and 236.107
ppm) respectively. All the extracts (aqueous, hexane and chloroform) also showed mosquito larvicidal
activity at a relatively high concentration when compared to ethanol and methanol plant extracts. Since
the ethanol extract showed potent larvicidal activity, when compared to the other extracts.
Table 2. Larvicidal activity of P. polystachion extracts against fourth instar larvae of A. aegypti
Concentration 24hr% LC50 LC90

Extracts
(ppm) Mortality (LCL–UCL) (LCL–UCL) Chi-Sq
(ppm) (ppm)
50 16
Aqueous 34 122.738 316.199
100
81.847±167.846 214.463±1032.402 11.748
150 53
200 72
250 92
50 17
Chloroform 100 38
111.561 265.876 13.165
150 58
71.547 ±150.400 187.08 ±725.71
200 80
250 96
50 21
100 44
Ethanol 95.178 205.033 19.816
150 68
49.182 ±133.946 143.857 ± 622.390
200 93
250 100
50 14
100 27
Hexane 156.311 491.041 8.343
150 42
114.533 ±232.622 296.880 ±2583.746
200 57
250 79
50 19
Methanol 100 41
103.069 236.107 14.538
150 63
62.868 ±139.403 168.146 ±609.635
200 86
250 98
Fig.3(a).Lavicidal activity of ethanol extract of Pennisetum polystachion against A. aegypti
Table 3. Larvicidal activity of P. polystachion extracts against fourth instar larvae of A. stephensi
24hr LC50 LC90

Extracts Concentration % (LCL–UCL) (LCL–UCL) Chi-Sq
(ppm) Mortality (ppm) (ppm)
50 17
100 34
Aqueous 119.864 304.579 13.791
150 53
74.995 ± 168.022 204.209 ±1165.120
200 75
250 93
50 18
100 37
112.007 276.758 12.117
Chloroform 150 59
72.781 ± 150.477 193.588 ± 759.767
200 78
250 95
50 23 27.020
100 47
Ethanol 88.229 179.428
150 74
34.103 ± 131.142 122.146 ± 780.537
200 100
250 100
50 15
100 29
Hexane 139.462 391.485 12.920
150 46
91.122 ± 212.017 242.636 ±2650.262
200 63
250 87
50 20
Methanol 43 15.895
100
98.011 216.596
150 68
57.494 ± 133.327 155.089 ± 546.331
200 88
250 100
Fig.3(b). Lavicidal activity of ethanol extract of Pennisetum polystachion against A. stephensi
Table 4. Larvicidal activity of P. polystachion extracts against fourth instar larvae of C. quinquefasciatus
Concentration 24hr LC50 LC90

Extracts (ppm) % (LCL–UCL) (LCL–UCL) Chi-Sq
Mortality (ppm) (ppm)
50 18
100 37
Aqueous 112.915 281.690 12.758
150 58
71.799 ± 153.630 194.567 ± 847.923
200 77
250 95
50 20
100 41
102.225 237.536 15.340
Chloroform 150 63
59.872 ±140.127 167.052 ±671.257
200 86
250 98
50 24
100 49
Ethanol 87.146 183.046
150 74
41.359 ± 123.970 128.087 ±554.842 21.218
200 98
250 100
50 17
100 35
Hexane 122.778 335.877 8.347
150 53
88.160 ± 160.861 231.930 ± 877.319
200 72
250 89
50 21
Methanol 100 45
94.346 202.913 18.247
150 69
51.412 ± 130.684 144.160 ±548.331
200 93
250 100
Fig. 3(c). Lavicidal activity of ethanol extract of Pennisetum polystachion against C. quinquefasciatus
3.3. Thin Layer Chromatography analysis

The phytocompounds present in the ethanol extracts were further analysed by TLC (Fig.2).
Ethanol extract of P. polystachion showed 7 bands with Rf values of 0.30, 0.40, 0.51, 0.58, 0.64, 0.72 and
0.80.
Fig 2. Thin layer Chromatography analysis of Pennisetum polystachion ethanol extract
3.4. GC- MS analysis

The spectral data of GC-MS analysis of the extract is shown in Fig. 4. The retention time names,
molecular weight of the some of the components of the test extract were ascertained. Ten compounds
were identified by GC-MS. Some of the compounds observed were 9,12,15-Octadecatrienoic acid, 2,3-
bis, n-Hexadecanoic acid, pentamethyldisilyl, 17-(1,5-Dimethylhexyl)-10,13-dimethyl-3-, 6,6'-Diacetyl-
7,7'-dihydroxy-2,2',4,4',5, Oxazepam ditms, Acetic acid, 1,1',4'-triacet, Acetamide, N-[5-(diethylamin,
Normorphine, N-trimethylsily, Echinenone, 3-Phorbinepropanoic acid, 9- (Table. 5).
Reducing mosquito-borne diseases remains a big challenge even at the most advancement of
modern sciences [31]. Mosquitoes in the larval stage are attractive targets for pesticides because they are
confined to water and their habitat is easily treatable. Eliminating the larval stage is advantageous
because the mosquitoes cannot disperse or acquire human pathogens[32]. Thus, this has necessitated the
exploration of natural products for the control of vector insects in general and mosquitoes in particular
[33,34]. [35] reported that plants derived extracts using different solvents crude extracts have potential
larvicidal activity. To evaluate the potential larvicidal activity of the plant preliminary screening is a good
measure (Alireza et al. 2012). This study is an attempt to highlight the need for research and development
in biological larvicides. Phytochemical results clearly indicates the present of various phytocompounds,
according to Jeffrey et al., 1976, P. polystachion plant extracts confirmed the presence of glycosides and
flavonoids and also revealed the presence of Saponins, tannins and alkaloids in Pennisetum sp. Present
study briefly presents the mosquito larvicidal potential of the whole plant extracts of P. polystachion.
This is the first report of mosquito larvicidal activity on P. polystachion plant extracts. Ethanol extract
exhibits good larvicidal activity against C. quinquefasciatus and A. stephensi with LC50 value of 87.146
ppm and 88.229 ppm respectively and LC90 value of 183.04 ppm and 179.428 ppm . The methanol,
Aqueous, hexane and chloroform grass extracts also exhibits pronounced larvicidal activity against
mosquito larvae at very low ppm concentrations.
Fig 4. GC-MS analysis of Pennisetum polystachion grass ethanol extract extracts
Table 5. GC-MS analysis of Pennisetum polystachion grass ethanol extract extracts
S. No RT Name of the compound Peak Area (%) Amount
1. 41.946 9,12,15-Octadecatrienoic acid, 2,3-bis 61390 0.098
2. 42.337 n-Hexadecanoic acid, pentamethyldisilyl 85148 0.135
3. 45.521 17-(1,5-Dimethylhexyl)-10,13-dimethyl-3- 26389 0.042
4. 47.551 6,6'-Diacetyl-7,7'-dihydroxy-2,2',4,4',5 17899 0.028
5. 47.822 Oxazepam ditms 11953 0.019
6. 48.069 Acetic acid, 1,1',4'-triacet 12075 0.019
7. 50.502 Acetamide, N-[5-(diethylamin 8758 0.014
8. 53.052 Normorphine, N-trimethylsily 67139 0.107
9 53.634 Echinenone 133023 0.212
10 54.146 3-Phorbinepropanoic acid, 9- 170862 0.272
4.0. Conclusion
It is evident that the plant products are emerging as a potential source of mosquito control. Crude
extract or isolated bioactive compounds from the plant P. polystachion could be used in dormant water
bodies which are known to be the breeding grounds for the mosquitoes. The grass P. polystachion
extracts showed promising activity in mosquito control and its commercial utilization is very much
feasible.
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Phytochemical proﬁling and larvicidal potential of common grass Pennisetum

Article · December 2018

Babu Muthu P.K. Kaleena

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Janaki Arumugam Kuppan Velu

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Phytochemical profiling and larvicidal potential of

Fig 1. Pennisetum polystachion

2.7. TLC Plate preparation

2.9. GC-MS analysis

3.0. Results and Discussion

Table 1. Qualitative phytochemical profiling of Pennisetum polystachion grass extracts

S. No Secondary metabolites Aqueous Chloroform Ethanol Methanol Hexane

1 Carbohydrate +++ +++ +++ +++ +++

3 Saponins +++ +++ ++ ++ +++

4 Flavonoids +++ +++ ++ ++ +++

5 Alkaloids - +++ +++ - +

6 Anthocyanin +++ +++ +++ + ++

9 Cardiac glycosides - +++ ++ ++ ++

13 Coumarins +++ +++ +++ ++ ++

16 Steroids +++ +++ ++ - ++

++ Strongly positive ++ Positive + Trace - Not detected

3.2. Larvicidal Bioassay

Concentration 24hr% LC50 LC90

Fig.3(a).Lavicidal activity of ethanol extract of Pennisetum polystachion against A. aegypti

24hr LC50 LC90

Fig.3(b). Lavicidal activity of ethanol extract of Pennisetum polystachion against A. stephensi

Concentration 24hr LC50 LC90

3.3. Thin Layer Chromatography analysis

Fig 2. Thin layer Chromatography analysis of Pennisetum polystachion ethanol extract

3.4. GC- MS analysis

Fig 4. GC-MS analysis of Pennisetum polystachion grass ethanol extract extracts

Table 5. GC-MS analysis of Pennisetum polystachion grass ethanol extract extracts

S. No RT Name of the compound Peak Area (%) Amount

1. 41.946 9,12,15-Octadecatrienoic acid, 2,3-bis 61390 0.098

2. 42.337 n-Hexadecanoic acid, pentamethyldisilyl 85148 0.135

3. 45.521 17-(1,5-Dimethylhexyl)-10,13-dimethyl-3- 26389 0.042

4. 47.551 6,6'-Diacetyl-7,7'-dihydroxy-2,2',4,4',5 17899 0.028

5. 47.822 Oxazepam ditms 11953 0.019

6. 48.069 Acetic acid, 1,1',4'-triacet 12075 0.019

7. 50.502 Acetamide, N-[5-(diethylamin 8758 0.014

8. 53.052 Normorphine, N-trimethylsily 67139 0.107

9 53.634 Echinenone 133023 0.212

10 54.146 3-Phorbinepropanoic acid, 9- 170862 0.272

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