In vitro acaricidal properties of extracts from Carica papaya seeds and Chrysanthemum roseum leaves against Rhipicephalus microplus

doi:10.21203/rs.3.rs-4443263/v1

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In vitro acaricidal properties of extracts from Carica papaya seeds and Chrysanthemum roseum leaves against Rhipicephalus microplus

https://doi.org/10.21203/rs.3.rs-4443263/v1

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The present study aimed to assess the adulticidal and larvicidal efficacy of chloroform, methanol, and hexane extracts obtained from the Chrysanthemum roseum (leaves) and the Carica papaya (seeds) against Rhipicephalus microplus ticks. The percentage of extractability for all the extracts varied between 1.5% and 25%. The hexane extract of C. roseum showed a mortality rate ranging from 6.66 ± 6.66% to 100.00 ± 0.00% at doses ranging from 0.62–5%. An impact on the oviposition capacity of the treated ticks, resulting in a significant reduction of 90.15 ± 6.460% in oviposition at a concentration of 2.25% was also observed. The methanol extract of C. roseum exhibited a higher concentration of anti-tick action and did not demonstrate a significant influence on the reproductive potential of the treated ticks. A mortality rate of 33.33 ± 17.63 to 93.33 ± 6.67% for treated ticks during 24 hrs. of treatment, was observed at concentration range of 5 to 12.5% of hexane extract of Carica papaya, Furthermore, the extract also resulted in the suppression of egg-laying (98.72 ± 1.27%) at 12.5%. Against larvae, the extract exhibited equivalent efficacy and LC₅₀ and LC₉₀ values of 0.003% and 0.012% for LIT, and 0.08% and 0.28% for LPT, respectively.

Chrysanthemum roseum

Carica papaya

Acaricidal

Rhipicephalus microplus

The limits of synthetic acaricides have strengthened the necessity and scientific drive for alternative methods, which are being expedited in order to find environmentally benevolent, cost-effective, biodegradable, and specifically targeted acaricides for ticks. Researchers worldwide have shown interest in the screening of natural products (Kebede et al. 2010). Plants play significant roles in ecosystems (Garcıa et al. 2007) and have the potential to be used as substitutes for current insect-control chemicals due to their diverse range of bioactive substances (Qin et al. 2010). These plant species are commonly found in various angiospermic families such as Lamiaceae, Rutaceae, Myrtaceae, Zingiberaceae, and Asteraceae. The acaricidal efficacy of several plant extracts (Juliet et al. 2012; Jadhav et al. 2021a, b; Ghosh et al. 2011, 2013) and seco Regitano ndary metabolites produced by plants are documented (Lopez et al., 2008).

Geevarghese et al. (1997) found 106 tick species in India that infest animals. Rhipicephalus microplus (Canestrini,1888) is the most important species due to its wide distribution, ability to transmit diseases, blood-feeding behaviour, and its impact on cow populations. The R. microplus tick transfers Babesia bigemina and Anaplasma marginale and is common in nearly every Indian state (Ghosh et al. 2007). The annual global economic impact of tick-borne infections and the expenses associated with tick control measures have been calculated to range from US$ 13.9 to 18.7 billion (De Castro 1997). Ticks not only spread diseases, but they also damage hide and skin, which costs US$500,000 every year (Bekele 2002). They also result in a financial loss of 57.2 million US$ due to concerns related to ticks (Minjauw and McLeod 2003), and a decrease in milk output by 23% (Regitano and Prayaga 2011).

The Chrysanthemum genus has around 300 species of herbaceous plants and shrubs. These plants are highly valued for their capacity to generate a widely recognized commercial insecticide called "Pyrethrum" (Kumar et al. 2018). The Carica papaya plant has been documented in published literature to have therapeutic benefits and a wide range of pharmacological activity. C. papaya has demonstrated efficacy in treating malaria and splenomegaly (Adjanohoun et al. 1996), combating intestinal parasites (Longuefosse and Nossin 1996), and repelling mosquitoes (Okolie 2006), indicating the presence of potent bioactive chemicals. Nevertheless, there is a lack of recorded data regarding the usefulness of C. roseum, and there is very limited evidence available regarding the efficacy of C. papaya against ticks.

Livestock farmers in India use synthetic acaricides of various classes to manage tick infestation. However, persistent and unselective use of chemical acaricides led to resistance, making R. microplus the sixth most resistant arthropod worldwide (De León et al. 2020). Taking into account the limitations of chemical acaricides, the purpose of this study was to evaluate the effectiveness of extracts derived from Chrysanthemum roseum and Carica papaya against R. microplus ticks,

Collection of plant material

Piper longum dried fruits were acquired from the local market of Parbhani, Maharashtra. The leaves of Chrysanthemum roseum plants were collected from the botanical garden of Vasantrao Naik, Marathwada Agriculture University, as well as the nearby areas of the Parbhani region. The plant species was verified by a botanist. The seeds of Carica papaya were harvested from mature fruits. The seeds were dried until all moisture was depleted, and then the dry seed materials were coarsely pulverized using a grinder.

Collection of field isolates of engorged adult female ticks and maintenance of larvae

Adult female R. microplus ticks were hand collected either from naturally infected cattle or from the animal shed soon after they detached. The ticks were washed with tap water, dried off with absorbent tissue paper, and thereafter placed individually in glass vials for the purpose of egg laying. Subsequently, ticks were carefully positioned inside the desiccator to ensure a constant relative humidity of 85%. After the entire process of laying eggs (12–14 days after dropping), the eggs were moved to individual vials and wrapped with cotton clothing, and secured with rubber bands. The eggs were permitted to hatch for a period of ten days. The in vitro bioassay employed larvae that were 12–14 days post-hatching.

Production and preparation of solvent-guided extracts

Room-temperature air-drying of Carica papaya seeds and Chrysanthemum roseum leaves eliminated all moisture. 100g of powdered seeds and leaves were extracted in a round-bottom flask with 600ml of hexane in a soxhlation unit. The temperature was 70°C for 48 hours. After 48 hours, the extract along with solvent was removed and concentrated at 40⁰C using a rota evaporator. After the hexane fraction, chloroform solvent was used for 48 hours at 75°C soxhlation extraction. After replacing chloroform with methanol, soxhlation extraction continued for 48 hours at 65⁰C and all the extracts were reserved at 4°C. The solvents 10% ethanol and acetone were used to prepare 5%, 2.5%, 1.25%, 0.62%, and 0.31% hexane extracts from Carica papaya and hexane and chloroform leaf extracts from Chrysanthemum roseum. Concentrations of 5%, 7.5%, 10%, 12.5%, and 15% Chrysanthemum roseum methanolic extract were made in 10% methanol. The extracts' working concentration of solvents was kept below 10%.

Effectiveness of extracts using in-vitro bio assays

Adult Immersion Test

The adult immersion test (AIT) mentioned by Ghosh et al. (2013) was used to evaluate plant extracts' acaricidal efficacy against adult ticks. Three sets of trials with five ticks per concentration were done. Five minutes were spent immersing mature female ticks in various extract concentrations and control ticks were in solvents. After immersion, ticks were placed in petri dishes wrapped with filter paper. The petri plates were incubated in a BOD incubator at 28°C and 85 ± 5% moisture. Ticks that did not lay eggs up to 14 days of monitoring were counted as a dead. The absence of ticks' movement and pedal reaction after light exposure were commonly used. The surviving ticks were raised to test plant extracts' oviposition-inhibiting effects. The Stendel (1980) approach examined egg-laying suppression.

$$\text{R}\text{e}\text{p}\text{r}\text{o}\text{d}\text{u}\text{c}\text{t}\text{i}\text{v}\text{e} \text{i}\text{n}\text{d}\text{e}\text{x} \left(\text{R}\text{I}\right)=\frac{\text{E}\text{g}\text{g} \text{m}\text{a}\text{s}\text{s}\text{e}\text{s}}{\text{E}\text{n}\text{g}\text{o}\text{r}\text{g}\text{e}\text{d} \text{t}\text{i}\text{c}\text{k} \text{w}\text{e}\text{i}\text{g}\text{h}\text{t}}$$

Percentage inhibition of oviposition$\left(\%\text{I}\text{O}\right)=\frac{\text{R}\text{I} \left(\text{c}\text{o}\text{n}\text{t}\text{r}\text{o}\text{l}\right)-\text{R}\text{I}\left(\text{t}\text{r}\text{e}\text{a}\text{t}\text{e}\text{d}\right)}{\text{R}\text{I} \left(\text{c}\text{o}\text{n}\text{t}\text{r}\text{o}\text{l}\right)}\text{x}100$

The effectiveness of the extracts was assessed as suggested by Azhahianambi et al. (2009).

E%=100 [1- (CRT x CRO)]

The CRT (coefficient of reduction in engorged female ticks in the treatment/control group) was calculated by dividing the treated group's live ticks by the control groups.

CRO, represents the coefficient of reduced oviposition in treatment group in relation to control group, was determined by dividing the reproductive index (RI) of the treated ticks by the control.

The dose response results underwent probit analysis, as described by Finney (1962). The analysis involved applying a logarithmic transformation to the acaricide dose and then graphing it against the probit mortality values. The LC₅₀ and LC₉₀ values, represent the concentrations of acaricide that result in 50% and 90% death in ticks, respectively, were determined using a regression equation. These results were then compared at a 95% confidence level as below.

The equation Y = mx + Garcia c represents a mathematical relationship, m represents the slope of the curve, c represents the y-intercept, x represents the logarithmic value of the concentration of acaricide, and Y represents the probit value (mortality percentage).

Larval Immersion Test

Larvicidal efficacy of all extracts was tested by immersing larvae for 10 minutes. Over 100 larvae were placed in 1.5 ml centrifuge tubes and immersed in 200 µl extracts. The solution was pipetted after 10 minutes. After drying the tubes, rubber bands and cotton cloths wrapped the tubes. Immersion exclusively in solvents was used for the control group. Three replicates were done for each extract. After treatment, larvae were transferred in a BOD incubator at 28°C and 85 ± 5% relative humidity. The tubes were unsealed after 24 hours to count dead and living larvae to calculate mortality.

Larval Packet Test

The LPT was employed in accordance with FAO (1971). Extracts were applied on 3.75 × 8.5 cm filter paper rectangles (541 Whatsman, Maidstone, Kent, UK) using 0.6ml of varied concentrations of extracts. The filter papers impregnated with extracts of different concentration were incubated at 37°C to evaporate the solvent. After folding the dried impregnated filter sheets in half, adhesive tapes secured the sides to produce a packet with an open end for larvae. After adding over 100 larvae, each package was sealed and kept in a BOD incubator (28°C and 85 ± 5% RH). The number of larvae that died 24 hours after opening the packets was counted. Then, probit statistical analysis was performed.

The study found that using hexane as a solvent for C. papaya seed resulted in the highest yield of 25% and among the extracts derived from Chrysanthemum roseum leaves the highest yield was obtained using methanol (5.6%) followed by chloroform (2.8%) and hexane (1.5%). The chloroform extract obtained from the leaves of C. roseum was not dissolved in any solvent due to safety concerns, and its acaricidal activity was not investigated.

The hexane extract of C. roseum exhibited very strong acaricidal activity at lower concentration and mean per cent adult mortality was ranged from 6.66 ± 6.66 to 100.00 ± 0.00% at the dose range of 0.62–5% prepared in 10% acetone (Table 1). Additionally, the extract had an impact on the capacity to lay eggs, specifically resulting in a reduction of 90.15 ± 6.460% in oviposition at 2.25%. The LC₅₀ and LC₉₀ values were determined to be 1.43% and 2.53%, respectively. The methanol extract of C. roseum exhibited greater anti-tick action at higher concentrations compared to the hexane extract. However, it did not have a significant influence on reproductive potential, as shown in Table 2 and had low larvicidal activity. The LC₅₀ and LC₉₀ values obtained using methanol extract were 7.58% and 9.47% for LIT, and 13.32% and 18.04% for LPT, respectively. The anti-larval efficacy of the hexane extract of C. roseum was significantly stronger at very low concentration with 0.02 and 0.94%. %, LC₅₀ and LC₉₀ values.

Table 1

Results of AIT on adult female ticks treated with a hexane extract of *C. roseum*
Concen. (Dose) (%)	Mean tick weight (mg)/replicate (Mean ± SE) N = 5	Mortality (%) (Mean ± SE)	Mean egg mass (mg) (Mean ± SE)	Reproductive index (RI) (Mean ± SE)	Inhibition of Oviposition (IO%) (Mean ± SE)
0.62	569 ± 27.89	6.66 ± 6.66	215.16 ± 9.14	0.37 ± 0.013	10.27 ± 2.67
1.25	546.9 ± 15.94	24.00 ± 9.79	169.7 ± 30.12	0.30 ± 0.05	34.68 ± 10.20
2.25	556.81 ± 18.16	85.45 ± 7.18	29.18 ± 20.98	0.05 ± 0.03	90.15 ± 6.460
5	553.75 ± 14.53	100.00 ± 0.00	0.00 ± 0.00	0.00 ± 0.00	100.00 ± 0.00
Control	772.00 ± 53.90	0.00	333.5 ± 14.01	0.46 ± 0.02	0.00 ± 0.00
LC₅₀: 1.43, LC ₉₀: 2.53, R²: 0.9687, Slope: 0.18683

Table 2

Results of AIT on adult female ticks treated with a methanol extract of *C. roseum*
Concentration (Dose) (%)	Mean tick weight (mg)/replicate (Mean ± SE) N = 5	Mortality (%) (Mean ± SE)	Mean egg mass (mg) (Mean ± SE)	Reproductive index (RI) (Mean ± SE)	Inhibition of Oviposition (IO%) (Mean ± SE)
7.5	568.50 ± 22.30	5.00 ± 5.00	217.25 ± 6.5	0.383 ± 0.00	9.423 ± 3.82
10	580.00 ± 12.25	10.00 ± 5.77	237.25 ± 26.4	0.385 ± 0.02	13.70 ± 1.92
12.5	566.75 ± 22.69	20.00 ± 8.1	192.5 ± 17.06	0.338 ± 0.01	19.900 ± 5.80
15	539.75 ± 17.22	35.00 ± 5.0	159.25 ± 11.22	0.294 ± 0.00	30.49 ± 3.67
Control	772.0 ± 53.90	0	326.75 ± 24.03	0.423 ± 0.00	0.00 ± 0.00
LC ₅₀:19.43%, LC₉₀: 39.10% R²: 0.977, Slope:0.238

Observing the effects of a hexane extract from C. papaya, a mortality rate of 33.33 ± 17.63 to 93.33 ± 6.67% was reported in treated ticks within 24 hours of treatment at concentrations ranging from 5 to 12.5% (Table 3). Furthermore, the extract suppressed egg-laying, with a maximal inhibition rate of 98.72 ± 1.27% at 12.5%. At very low concentrations, the extract exhibited equivalent efficacy against the larvae and calculated LC₅₀ and LC₉₀ values were 0.003% and 0.012% for LIT, and 0.08% and 0.28% for LPT, respectively.

Table 3

Results of AIT on adult female ticks treated with a hexane extract of the *Carica papaya*
Concentration (Dose) (%)	Mean tick weight (mg)/replicate (Mean ± SE) N = 5	Mortality (%) (Mean ± SE)	Mean egg mass (mg) (Mean ± SE)	Reproductive index (RI) (Mean ± SE)	Inhibition of Oviposition (IO%) (Mean ± SE)
5	654.66 ± 63.39	33.33 ± 17.63	181.66 ± 33.19	0.281 ± 0.05	60.03 ± 6.75
7.5	564.33 ± 16.89	46.66 ± 6.66	101.33 ± 14.65	0.181 ± 0.03	74.05 ± 4.67
10	679.66 ± 16.79	86.667 ± 6.66	16.66 ± 8.35	0.025 ± 0.01	96.46 ± 1.77
12.5	656.00 ± 43.92	93.33 ± 6.67	6.00 ± 6.00	0.008 ± 0.00	98.72 ± 1.27
Control	620.00 ± 14.57	0	434.66 ± 5.23	0.701 ± 0.00	0.00 ± 0.00
LC ₅₀: 6.56%, LC ₉₀: 11.61%, R²: 0.9187, Slope: 0.17827

The extractability of a specific extract is vital for the manufacture of extracts as it directly affects the effectiveness of use and the manufacturing expenses. The most productive outcome was achieved when hexane was utilized as a solvent for C. papaya seed, resulting in a yield of 25%. Conversely, the extraction efficiency was notably reduced for the leaves of C. roseum when extracted using hexane (1.5%). Noticeable variations were noted in the extraction yields achieved using various solvent systems. The variations can be ascribed to the solubility of extractable bioactive constituents and the disparity in solvent polarities, which have a crucial role in resolvability of phytochemical substances (Silva et al. 2014; Naima et al. 2015).

In in-vitro bioassays, plant-based extracts or fractions must be dissolved in polar or non-polar solvent or detergent. The present investigation dissolved methanolic, chloroform, and hexane extracts in methanol, ethanol, and acetone by solubility. Compared to n-butanol, Ravindran et al. (2011) found that acetone causes instantaneous mortality, 100% fecundity inhibition, and total eclosion blocking, while methanol has little effect on adult tick mortality. Gonçalves et al. (2007) employed acetone, methanol, ethanol, DMSO, triton X100, and tween 80 and found that ethanol with 1% DMSO, triton X100, or tween 80 was adequate for in-vitro bioassays against R. (B.) microplus. No deleterious effects were reported on adult tick mortality, fecundity, or egg hatching in present study using 10% of all solvents to prepare varied extract concentrations. Each solvent had a final concentration below 10%. Ghosh et al. (2012) observed that solvent polarity influences chemical compounds recovered from complicated natural matrices. According to Lima et al. (2014), acaricide efficacy varies on extraction process, solvent polarity, and plant component used to extract.

While the anti-plasmodial activity of C. indicum and the insecticidal capabilities of several Chrysanthemum spp. have been documented in published literature, no information regarding the acaricidal efficacy of C. roseum has been recorded to date. The hexane extract demonstrated potent adulticidal effect at a lower dose. Furthermore, it was observed that a complete prevention of egg-laying occurred at a concentration of 5%, and a concentration of 0.94% resulted in a 90% death rate of the larvae. When comparing the methanol and hexane extracts, methanol extract exhibited low adulticidal and larvicidal efficacy. In contrast, Haouas et al. (2008) documented the potent adulticidal and larvicidal effects of a 1% methanolic extract derived from the flowers of C. fuscatum and C. grandiflorum against Tribolium confusum insects.

In India, the practice of using C. papaya seeds in conjunction with honey to prevent roundworms in people has been observed (Kapoor 1990). Prior research has indicated the effectiveness of C. papaya extracts in treating parasitic worms in animals, such as chickens, as well as its ability to act as an antimalarial agent. The study found a death rate of 93.33 ± 6.67% in the treated ticks when exposed to hexane extract of C. papaya (seeds) at a 12.5%. Additionally, the LC₉₀ value was revealed to be 11.61%. The impact of seed extract was also noted on the oviposition capacity of ticks, with rates of 60.03 ± 6.75% and 98.72 ± 1.27% at doses of 5 to 10%, respectively.

According to the findings of our investigation, Shyma et al. (2014) likewise observed that the seed extracts of C. papaya completely prevented egg laying and resulted in a mortality rate of 93.33% for adult R. microplus ticks when used at a dose of 100mg/ml. Nevertheless, the extract exhibited larvicidal efficacy at greater concentrations, with estimated LC90 values of 12.11% and 13.48% using LIT and LPT, respectively. Earlier study by Sudharani et al. (2018), demonstrated that the methanolic extracts of C. papaya seeds (100mg/ml) had a complete adulticidal action, affected the reproductive potential of treated ticks, and exhibited 82% larvicidal efficacy.

The positive outcomes achieved in this investigation by utilizing the hexane extract of C. roseum can be ascribed to the presence of biologically active ingredients. This excerpt exhibits promising prospects for future research in control of field tick infestation. It can serve as a substitute for chemical acaricides and can be incorporated into a comprehensive approach for managing ticks, following validations and in vivo research.

Acknowledgements

The authors express gratitude to the Associate Dean of the College of Veterinary and Animal Sciences, Maharashtra Animal and Fishery Sciences University, Parbhani for providing the required resources to conduct the research.

Declaration of competing interest

Conflict of interest

The authors declare that none of the work reported in this study could have been influenced by any known competing financial interests or personal relationships.

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In vitro acaricidal properties of extracts from Carica papaya seeds and Chrysanthemum roseum leaves against Rhipicephalus microplus

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Abstract

Introduction

Materials and Methods

Collection of plant material

Collection of field isolates of engorged adult female ticks and maintenance of larvae

Production and preparation of solvent-guided extracts

Adult Immersion Test

Larval Immersion Test

Larval Packet Test

Results

Discussion

Conclusion

Declarations

References

Status:

Version 1