Pharmaco-Chemical characterization and Acaricidal Activity of Ethanolic Extract of Chassalia Curviflora ( Wall ex Kurz . ) Thwaites

Introduction: C. curviflora, an important ethno-medicinal plant used by the Kurichia local people in Western Ghats region of Wayanad is yet to be explored pharmacologically. It is used as paste on the body of cattle and birds for curing skin diseases. Objectives: To characterize the pharmacochemical features and to study the acaricidal effect of ethanolic extract of C. curviflora on engorged female ticks of R. (B.) annutatus. Methods: The pharmaco-chemical features such as physico-chemical, proximate, phytochemical, fluorescence, and HPTLC profiling were carried out using standard techniques. The pulverized leaves were subjected to soxhlet extraction using ethanol. The ethanolic extract at different concentrations (10% to 1.25%) was tested against ticks using adult immersion test (AIT). Result: The preliminary phytochemical investigation showed high contents of saponins, alkaloids and flavonoids. The HPTLC profiling of ethanolic extract showed the presence of 14 polyvalent components. Based on AIT, the extract at 10% revealed 43.76% of inhibition of fecundity (IF) and 29.16% of adult tick mortality. Conclusion: The results revealed that the extract has some active compounds that may influence in the reproductive system of female ticks.


INTRODUCTION
Plants produce an amazing array of organic chemicals with an enomerous diversity of structural types, essential for their growth.Medicines of herbal origin are preferred because of their fewer side effects compared to synthetic medicine. 1The medicinal plants continues to play a signifi cant role in providing valuable pharmaceutical products in health care system 2 in addition to its application in cosmetic, agricultural and food industry. 3icks are blood sucking heamatophagous ectoparasites, transmiting many diseases affecting livestock, humans and companion animals. 4hey cause significant loss in livestock industries through reduction of milk production, weight gain and reproductive efficacy 5 and also increa sed the incidence of life threatening diseases in human.Chemical acaricides are used for control of tick population throughout the world.However, development of acaricide resistant ticks, 6 toxicity to nontarget living organisms and environmental pollution 7 are some of their draw backs.Survey of published literature reveals many reports on the poten tial of plant extracts against ticks 8 .The species, Chassalia curviflora (Wall ex Kurz.)Thwaites is a flowering, tropical woody plant member of coffee family, Rubeacea.The different parts of the plant is used to cure ear and eye disease, headache, skin diseases, ulcers, phlegm, rheumatism, jaundice, pneumonia, wounds and sour throat. 9The phytochemical studies dealing with secondary metab olites are meager in C. curviflora, but some of the Chassalia species have proved to be a source of macrocyclic peptides, also known as cyclotids. 10 another study, Thenmozhi and coworkers (2013) detected presence of phenols, tannins and flavonoids in the fruits of C. curviflora.Recently, the GC/MS analysis of methanolic extract of C. curviflora revealed the presence of 69 phytoconstituents. 11Experimentally, the genus Chassalia was reported for its antihypertensive, 11 antibacterial 12 antimicrobial, insecticidal and cytotoxic activities. 13Currently, very limited reports exist on the chemical nature and pharmacological properties of this plant species.Therefore, the present investigation focuses on characteriza tion of the pharmacochemical features and acaricidal properties of the ethanolic extract of leaves of C. curviflora against R. (B).annulatus ticks.

Collection and identification of plant material
The taxonomically identified Chassalia curviflora (Wall.ex Kurz.)Thwaites plant leaves were collected from Western Ghats region of Kozhikode, Kerala, India.A herbarium for morphological studies was prepared, authenticated and a voucher specimen (No: CALI6806) deposited at the Department of Botany, Calicut University Herbarium, Kozhikode, Kerala.

Pharmaco-chemical characterization Evaluation of physiochemical parameters
The physicochemical parameters such as the percentage of loss on drying (LOD), total ash, foreign content, moisture content, acid soluble ash, water soluble ash and alcohol soluble ash were determined as per the Indian Pharmacopeia, (1996). 14

Determination of extractive values
Coarsely chopped 5 g of leaf powder was subjected to maceration for 24 h in a closed flask using 100 mL of solvent (such as alcohol, chloroform, ethyl acetate, hexane, petroleum ether and distilled water) frequently shaken during the first 6 h, allowed to stand for 18 h and filtered rapidly using Whatman's No: 42 filter paper.In a tared flat bottom shallow dish, 25 mL of filtrate was evaporated to dryness, dried at room temperature and weighed.Percentage of soluble extractive fraction was calculated with reference to the air dried powder.

Proximate analysis
The proximate analysis of ash, crude fibre, crude protein, carbohydrate, crude fat, dry matter and moisture content of the powdered leaves of C. curviflora were done using standard proximate analysis techniques. 15

Preparation of ethanolic extract
The collected plant leaves were cleaned by washing in running water.The leaves were dried at room temperature for two weeks.The dried leaves (100 g) were powdered in a plant sample grinder at controlled temperature and used for extraction using ethanol in a soxhlet extraction apparatus attached with a rotary vacuum evaporator (Buchi, Switzerland).Solvents were removed using rotary vacuum evaporator at 175 mbar at a temperature ranging from 40 0 C to 60 0 C.

Preliminary phytochemical screening
Preliminary phytochemical screening of crude drug powder was done as per standard procedure described by Harbone, (1991) 16 for various phytoconstituents such as steroids, terpenoids, alkaloids, tannins, phenolic compounds, flavanoids, carbohydrates and amino acids.

High performance thin layer chromatography (HPTLC)
High performance thin layer chromatography (HPTLC) analysis was carried out on a HPTLC (Camag, Switzerland) system with ethanolic extract of C. curviflora.Chromatographic separation was performed on Merck TLC plates precoated with silica gel 60 F 254 (20×10 cm with 200 μm layer thicknesses) from E. Merck, Germany.The 10 μL of extract (2 mg/mL) was applied onto the plates as a band with 6 mm width using Camag 100 μL sample syringe (Hamilton, Switzerland) using Camag Linomat 5 applicator (Camag, Switzerland).Linear ascending devel opment was carried out in a twin trough glass chamber hexane: ethyl acetate (8:2).Scanning was performed using Camag TLC scanner 3 at 254 nm and 366 nm through fluorescence mode and operated by win CATS software (version 1.4.1,Camag).Plates were visualized under UV 254, UV 366 nm and in visible light.

Bioassay test Adult immersion test (AIT)
Adult immersion test was performed based on Drummond et al.  (1973). 17Fully engorged adult female R. (B.) annulatus were collected from the naturally infested calves, washed with distilled water, dried on an absorbent paper and was used for adult immersion test (AIT).The different concentrations of ethanolic extract (10% to 1.25%) were prepared in methanol.Four replicates, each with six ticks, were used for each concentration.The groups of six ticks selected randomly based on the size were weighed before the experiments and were immersed for two minutes in the respective dilution in a 50 mL beaker containing 10 mL extract.Ticks were recovered from the solution, dried using absorbent paper and placed in a separate plastic specimen tube (25×50 mm).The tubes were incubated at 28 ± 2 o C and 80 per cent relative humidity in a BOD incubator.Methanol was used as control.Adult tick mortality was observed up to 15 th day posttreatment.After oviposition, the eggs laid by the female ticks were collected and weighed.Eggs were kept under the same incubation conditions in a BOD incubator for the next 30 days.

The index of egg laying (IE) and percentage inhibition of fecundity (IF)
were calculated as follows.
Index of egg laying/fecundity (IE)=weight of eggs laid (g)/weight of females (g) Per cent inhibition of fecundity (% IF)=[(IE control group-IE treated)× 100]/IE control group.Hatching of eggs was observed visually.

Statistical analysis
Analysis of data was performed. 18Data were expressed as the mean ± SEM.Groups were compared using oneway ANOVA for repeated measurements using SPSS software.Duncan's test was used for posthoc analysis.A value of P<0.05 was considered significant.
The ethanolic extracts of leaves of C. curviflora, when subjected to HPTLC profiling revealed 14 polyvalent compounds (Figure 1) with the solvent system hexane: ethyl acetate (8:2).Out of these polyvalent compounds with Rf values 0.

DISCUSSION
During last few decades there has been an increasing surge in the study of medicinal plants and their traditional uses in different parts of the world.Herbal remedies are considered as one of the oldest form of health care known to mankind on this earth.The species C. curviflora is an important ethnomedicinal plant.The Kurichia local people in Western Ghats region of Wayanad, used the plant paste on the body of cattle and birds for curing skin diseases, 19 and the plant is also used to treat wound healing and snake bite.This plant has been confused with other species of similar plants due to their relative similarities.Therefore, it is necessary to establish the pharmacochemical parameters and standards in order to properly identify the plant.
In the present study, we evaluated the pharmacochemical characteristic and acaricidal activity of the ethanolic extracts of leaves of C. curviflora.

Table 2 : Effects of ethanolic leaves extract of C. curviflora against R. (B.) annulatus Acaricide Mean ticks weight per replicate ± SEM (g) Mean % adult mortality within 15 days ± SEM Mean eggs mass per replicate ± SEM (g) Index of fecundity ± SEM Percentage Inhibition of Fecundity (%)
, values are Mean ± SEM, means bearing different superscripts a, b or c (P<0.05), indicates significant difference when compared with control.