Comperative phyto toxicological and anti inflammatory effects of leaves extracts of holoptelea integrifolia

Original article: Comperative phyto toxicological and anti inflammatory effects of leaves extracts of holoptelea integrifolia Yasin H1, Khalid S2, Abrar H3, Rizwani GH4, Perveen R5, Fatima K6 Abstract Objective: Plants play valuable role in the new drug discovery and significantly used to treat different diseases. Nowadays scientists are investigating the therapeutically active phytochemical constituents that are safe and producing lesser side effect in comparative to other standard drugs. Methods: The plant Holoptelea integrifolia is medicinally important and this study was carried out to evaluate the anti inflammatory activity of aqueous extract of the leaves of Holoptelea integrifolia in male albino rats wistar stain treated with acetic acid to induced paw edema. Results: Result indicated the significant anti-inflammatory activity while compared with standard drug (diclofenac sodium).Brine shrimp bioassay (cytotoxicity), phytotoxicity, insecticidal and enzyme inhibition activity was performed in different extracts of the leaves of H. integrifolia. Results of brime shrimp bioassay indicating positive lethality at high dose in BuOH and H2O only. While the results of phytotoxicity in all crude extracts displayed mild phytotoxicity(46.3 μg/ml) in high concentrations (1000 μg/ml) except H2O extract showed no phytotoxicity. Result of insecticidal activity revealed that BuOH extract were found more effective against Rhyzoperthadominica, the EtOH extract expressed major while EtOAC extract showed mild activity against Callosobruchusanalis. Aqueous extract possessed no insecticidal activity. Conclusion: Results of Urease inhibition activity suggested that EtOAC and BuOH extracts of this plant expressed no activity while EtOH and H2O possessed mild inhibiting activity. Keywords; Holoptelea integrifolia; phytotoxicity; insecticidal activity; enzyme inhibition activity; Anti-inflammatory activity; Brine Shrimp Bioassay

Hexacosanol, octacosanol, β-sitosterol and β-amyrin have been obtained in alcoholic extract of leaves of H.integrifolia [17][18][19] .This research has been conducted for the anti-inflammatory activity with aqueous extract of plant H. integrifolia in compression to standard drug (Diclofenac sodium) 20 .While different extracts of H. integrifolia were selected to evaluate the cytotoxicity, phytotoxicity, insecticidal and enzyme inhibition activities of this medicinally valuable plant.

Plant collection;
The fresh leaves of H. integrifolia were collected from the Faculty of Pharmacy, University of Karachi, Pakistan and the plant material was dried (500 g), chopped and macerates with methanol (MeOH) for 15 days after identification.Solvent was evaporated at reduced pressure and controlled temperature to obtained MeOH extract.This extract was subjected for step by step extraction with n-hexane, ethanol (EtOH), ethyl acetate (EtOAC), butanal (BuOH) and H 2 O then concentrated in rotary evaporator to obtained different fractions.Brine shrimp bioassay, phytotoxicity 21 , insecticidal activity and enzyme inhibition activity (indophenol method) 22 were performed.For the determination of insecticide activity and toxicity different methods are being used on various surfaces (Insecticide impregnated dust on grain, direct spray on grain, impregnated filter paper test) [23][24][25] .Anti-inflammatory activity has performed with aqueous leave extract H. integrifolia [26][27][28] .% Inhibition of paw volume = (Control mean -treated mean) / Control mean×100.Ethical approval was taken prior the study.

Results and discussion
Anti-inflammatory activity; Aqueous extract of H.integrifolia has significant anti-inflammatory activity.Active constituents of plants play a major role for the discovery of new pharmaceutical products for the treatment of various ailments due to its efficacy and safety 29,30 .Inflammatory diseases are one of the most common causes of different health disorders 31 .Anti-inflammatory studies have been conducted using aqueous extract of the leaves of H.integrifolia and outcomes were analyzed in comparison with standard anti-inflammatory drug diclofenac sodium 20,28 .Paw edema was calculated at different time interval using plethysmometer (Table 2).New bould method was used to evaluate percentage inhibition of paw edema (Table 3) 32 .The statistically significant results has been produced in group that is treated with aqueous extract of the leaves of H. integrifolia after 3 hours in compression to group that treated with diclofenac sodium that shown % of inhibition after 4 hours.The significant (P< 0.05) anti-inflammatory activity of aqueous extract of the leaves of H. integrifolia markedly reduced the paw edema in compression to standard drug i.e.Diclofenac sodium against acetic acid induced paw edema in albino rats at a dose of 250mg/kg /body weight and outcomes have been mentioned in Fig. 1 and 2 (Table 1).The data were subjected to statistical analysis using one-way analysis of variance (ANOVA).P values < 0.05 were considered significant.

Percentage Inhibition of Paw edema (on 5 th day);
the results of percentage inhibition of paw edema with aqueous extract of H.integrifolia and standard drug (diclofenac sodium) compared with acetic acid (Table 2).(Finney, 1971).While EtOH and EtOAC extracts were not showed such toxicity against brine shrimps although at high concentration.Permethrin was employed as standard sample with 235.9 µg/cm 2 concentration and experiment were performed with sample in 1019.10 µg/cm 2 concentration.Some activity against Rhyzoperthadominica was observed in EtOH extract, while major activity was shown against Callosobruchusanalis. EtOAC extract was found to be only moderately activated against C. analis and significant activity was examined in BuOH extract against Rhyzoperthadominica; while in similar concentration of both extracts other pests were found ineffective.Aqueous extract not displayed effect against all pests or no insecticidal activity was observed (Table 11).

Enzyme inhibition activity:
The results showed that crude extracts of H. integrifolia (EtOH and H 2 O) possessed very weak urease inhibiting activities while EtOAC and BuOH extracts of plant were found ineffective (Table12). .Activity of urease has employed majorly as virulence determinant in the pathogenesis of various diseases in human being and animal health and for agriculture 38,39 .So far, designed based on urease inhibition is studied as tool first time, for evaluation of infections originated by using urease producing bacteria.This result indicated that EtOAC and BuOH extracts of this plant showed no activity while EtOH and H 2 O exhibited mild inhibiting activity.

Conclusion
This aqueous extract of Holoptelea integrifolia has shown potent anti inflammatory activity without any liver and kidney damage.This study showed that H. integrifolia leaves extracts of EtOH, EtOAC and BuOH could be useful as natural herbicides in comparison with other related herbal species at high concentration and could be deliberated as a source of bioactive agrochemical.

Table 2 : Percentage Inhibition of Paw Edema.
33xicity StudiesBrine Shrimps Bioassay (LD 50 ); Beside the therapeutic activity of plants and their constituents, some of these are detrimental effects to humans and other living organisms.Shrimp larvae (Artemiasolina) are susceptible to various harmful constituents and are used to evaluate the toxicity.Different extracts (EtOH, EtOAC, BuOH and H 2 O) of the plant H.integrifolia were subjected for the detremination of LD 50 activity using brine-shrimps (Artemiasolani).Brine shrimp bioassay was conducted in triplicate in three different concentrations i.e. 10, 100, 1000 µg/ ml of EtOH, EtOAC, BuOH and H 2 O extracts of H.integrifolia and Etoposide was selected as control (Table3,4,5 and 6).Results revealed positive lethality at high dose with LD 50 629.5723and100,000 µg/ ml in BuOH and H 2 O extract respectively and compared with the standard drug Etoposide at high concentration dose33

Table 6 : Brine shrimp toxicity bioassay of H 2 O extract of H. integrifolia (Roxb) Planch.
Different plants material, their extracts and / or their isolated active compounds may use as allele chemicals to other plants and be utilize for cultivation35,36.The results obtained from the this study showed that H. integrifolia leaves extracts of EtOH, EtOAC and BuOH could be useful as natural herbicides in comparison with other related herbal species at high concentration and could be deliberated as a source of bioactive agrochemical.

Table 12 .
In-vitro urease inhibition activity of different crude extracts of H. integrifoliaThis urea was used as control to perform the enzyme inhibition activity with the leaves extracts of H. integrifolia against Jack bean.For the discovery of new antiulcer drugs urease inhibitors have shown effectiveness nowadays (-No activity, + Low activity, ++ Moderate, +++ High)