INTRODUCTION:

Seed germination inhibition assay was previously shown to be a cost-effective approach for the rapid screening and identification of cytotoxic compounds with anticancer potential^1,2. Antiproliferative efficacy of HST-K drug (Patent No.GB2454875A) obtained from Asteracantha longifolia, was quantified by Murthy et al,2011 ^[2]in terms of reduction in seed weight gain owing to reduced water imbibition following drug treatment in the case of green gram seeds.

The biological potency of drugs could be evaluated, using assays that measure inhibition of specific enzymes elevated in disease states³.

Using seed germination approach, in our laboratory, we have previously quantified the HST-K drug efficacy in terms of dose-dependent reduction in specific activities of amylase and alkaline phosphatase in the seeds of horsegram⁴. Studies have revealed increased activities of these enzymes during the progression of certain cancers as well^5,6and were thus chosen by us for our present studies. Our earlier studies have indicated that the altered profiles of amylolytic and alkaline phosphatase activities following vinblastine and the HST K-drug treatments were comparable⁴. We thus considered it worthwhile to compare the efficacy of the patented HST-K drug with a commercially available Kokilaksha formulation from another manufacturer and quantify their half-maximal effective concentrations (EC-50) as equivalents of vinblastine. Our objectives include the following:

i) Comparison of the EC-50 values of the HST-K drug with the commercially available Kokilaksha formulation (termed VRS-K), using weight, amylolytic and alkaline phosphatase activities as study parameters.

ii)Quantifying half-maximal effective concentrations (EC-50) of both Kokilaksha formulations in terms of vinblastine equivalents.

MATERIALS AND METHODS:

All chemicals were of analytical/reagent grade and obtained from standard manufacturers. Vinblastine (commercial name: Cytoblastine of stock concentration = 1.0mg/ml) was purchased from the Kidwai Institute of Oncology, Bangalore. Horse gram (Macrotyloma uniflorum) seeds were purchased from the local markets.

Preparation of The Drug Formulations Used in Our Study:

The HST-K formulation (Patent No.GB2454875A) was obtained from the Herbal Science Trust Bangalore. A sample of the Kokilaksha formulation or ‘kashayam’ was purchased from the markets and used in our comparative studies (indicated as VRS-K hereafter).

Both HST-K and VRS-K formulations (50 ml) were dialyzed against 500ml of distilled water with 3 changes and the dialyzed permeate, which was reported as the therapeutically active fraction ²obtained.

Both the permeate fractions were concentrated by heating to 50ml, i.e. the original pre-dialysis volume of both formulations. The concentrated permeate fractions of both formulations was used in our studies as the test solutions. Both test solutions were fold-diluted to different concentrations with distilled water.

Likewise, the stock solution of vinblastine (1.0mg/ml) was also diluted with distilled water to different concentrations ranging from 0.01-0.12mg/ml.

Seed Treatment and Crude Enzyme Extract Preparation:

Seeds of horse gram were weighed and randomly distributed into petri plates (500±2mg/plate), excluding the selection of damaged seeds.

Table 1: Experimental design

S. No	Categories tested	Seed Treatments/Incubation	Drug dosage
Group 1	Negative Control*	Distilled water	Nil
Group 2	Positive Control (n=12)	Vinblastine	0.01-0.09 mg/ml **
Group 3	Test 1 (n=12)	VRS-K drug formulation	1:5-1:120 fold dilution **
Group 4	Test 2 (n=12)	HST-K drug formulation	1:5-1:120 fold dilution **

* For weight and amylolytic activity measurements, n = 30 and ALP measurements, n =22, in case of the negative control group respectively;

** All dilutions performed with distilled water

The control plates contained seeds incubated in a total volume of 6.0 ml distilled water alone, while the test groups consisted of seeds incubated with different drug concentrations as indicated in Table No.1.

The test and control plates were incubated for 120 h as previously described⁴, after which the fresh weights of seeds were recorded. Homogenization of the 120h old seedlings at 4⁰C followed by centrifugation at 10,000 X g, 4⁰C for 10 min yielded a clarified supernatant as previously reported by us ⁴.

Enzyme Assays and Protein Estimation:

Amylase activity was estimated, using starch as substrate, by the method of Zhizhuang et al, 2006⁷, while the chromogenic substrate method of Nigam and Aiyyagari, 2008⁸was used for the estimation of alkaline phosphatase activity. Controls with acid-denatured enzyme and devoid of starch were set up for comparison in the starch-iodine assay.

Similarly, buffered p-nitrophenyl phosphate with enzyme added after reaction termination with 0.5N NaOH constituted the control, for the alkaline phosphatase assay.

Protein estimation was carried out by the Coomassie G-250 dye –binding method of Bradford, 1976⁹, using bovine serum albumin as standard.

Units Of Activity/Comparison:

a) Amylase activity: One unit of amylase activity = decrease in absorbance of the starch-iodine complex by 0.01A at 580nm at 37^oC under the conditions of the assay.

b) Alkaline phosphatase activity: One unit of activity = amount of enzyme required to produce 10µM of p-nitrophenol under the conditions of the assay.

c) Specific enzyme activity (Units/mg) calculated by dividing total activity by total protein

Total enzyme activity (units)

Specific enzyme = -----------------------------------------

activity Total protein content (mg)

d) Percentage inhibition of enzyme activity or seed weight at a given drug concentration was calculated:

Sp.Act(control)

% activity inhibition =---------------------- X 100

Sp. Act(test)

Wt.inc(control

% weight inhibition = ----------------------X 100

Wt. inc(test)

(Sp. Act : specific activity; Wt.inc : weight increase)

Where in weight increase = final weight (120h incubation) – initial weight of unsprouted seeds at 0h.

e) EC-50 or half maximal effective drug concentration was calculated as the concentration wherein 50% inhibition of weight and specific enzyme activities was achieved in the test samples vis-à-vis the control.

Statistical Tools:

Alterations in weight and specific activities of both enzymes were expressed as average of multiple trials ± standard error, after which they were plotted against drug concentration (which comprised fold-dilution in the case of VRS-K and HST-K formulations respectively). The number of trials (‘n’) performed is indicated within brackets wherever applicable. The EC-50 values in respect of weight and enzyme inhibition were calculated from the linear region of the plot. The F-test was performed for comparison of statistical differences between the various drug treatments. Statistical differences were defined as significant (or otherwise) and highly significant (or otherwise) at probability values of 0.05 and 0.01 respectively.

RESULTS:

Seed weight and specific enzyme activities were estimated in the drug treated seed samples and compared with one another and the controls at the end of the 120h incubation period.

i) Weight profile: Seeds weighing 0.5±0.01gm per plate were incubated with distilled water (controls) and the suitably diluted drugs (test samples) for 120hours at the end of which, their fresh weights were measured. At the end of 120h, the control group exhibited a fold increase of 5.0±0.1 with respect to fresh weight (n=30). Such an increase however was significantly retarded, following treatment with the Kokilaksha formulations (ranging from 5-120 fold dilution) when measured at the end of 120h (n =12). Comparison of inhibitory profiles revealed that HST-K formulation showed greater efficacy as evident by F_obs>F_crit (for a = 0.05) for a majority of the concentrations tested (n =12) VRS-K (Fig 1)

Half-maximal or 50% seed weight inhibition (EC-50) relative to the control was observed upon treatment with the 49.2-fold diluted HST-K formulation. Similarly, treatment with the 21.4-fold diluted VRS-K formulation also yielded half-maximal inhibition. Comparison of half-maximal inhibition values indicated that both formulations were equivalent to 0.07 mg/ml of vinblastine (Figure 2).

Figure No.1: Comparison of inhibition profiles of VRS-K and HST-K formulations in relation to weight increase of seeds:

Fresh weight of horse gram seeds germinated in distilled water (control) and different fold-dilutions of the VRS-K and HST-K formulations for 24-120 h, were estimated, vide. Materials and Methods (* : significant; **:highly significant)

Figure No.2: Effect of Vinblastine treatment on seed weight: Percent inhibition in fresh weight of seeds germinated using different test concentrations of vinblastine (n=12), were measured relative to control vide. Materials and Methods. EC-50 was calculated as 0.072 mg/ml. (*: significant; **:highly significant)

Amylolytic activity:

The specific amylolytic activity was equal to 1184.6± 99.2 Units/mg protein (n = 30) in the control. The VRS-K formulation exhibited a linear inhibition, over a 15-60 fold-dilution range, whereas in the HST-K drug, linearity of inhibition was observed at fold dilutions ranging from 15 – 180.

HST-K was observed to inhibit amylolytic activity by 75.7±2.1% at 60-fold dilution while VRS-K brought about 22.4±8.4% inhibition respectively (n=12) at the same fold-dilution. Thus, the difference between the two groups was found to be significant (F_obs>F_crit fora = 0.01). Even at 180-fold dilution the HST-K drug displayed 27±2.7% inhibitory activity, whereas the VRS-K drug failed to show any significant inhibition beyond 60-fold dilution. (Fig 3).

Figure No.3: Comparison of inhibition profiles of VRS-K and HST-K formulations with respect to specific amylolytic activity: Horse gram seeds germinated in distilled water (control) and different fold-dilutions of the VRS-K and HST-K formulations for 24-120h, were harvested and the clarified supernatant assayed for amylolytic activity vide. Materials and Methods. Percent inhibition relative to control was plotted against drug concentration. Readings represent the average of 12 trials±S.E. (*: significant;**: highly significant)

Half-maximal amylolytic inhibition occurred at fold-dilution values of 117.6 and 38.1 in the HST-K and VRS-K drugs respectively. Correspondingly half-maximal amylolytic inhibition occurred at 0.05 mg/ml of standard vinblastine (Fig 4).

Figure No.4: Effect of Vinblastine treatment on specific amylolytic activity: Seeds germinated under control (distilled water) and with vinblastine concentrations ranging from 0.01-0.09 mg/ml for different periods of time ranging from 24-120h, as indicated under Materials and Methods. Readings represent the average of 12 trials ±S.E. (*: significant; **: highly significant)

Alkaline phosphatase activity: Specific activity of alkaline phosphatase (ALP) in seeds germinated in distilled water (control) was equal to 247.7±15.1 Units/mg protein (n=22). In this case, the VRS-K formulation failed to show any significant inhibition of ALP beyond 90 fold-dilution, while the HST-K formulation exhibited 33.2 ± 3.3% inhibition (n =12) at higher dilutions e.g 120-fold (Fig 5).

Figure No.5: Comparison of inhibition profiles of VRS-K and HST-K formulations with respect to specific activity of alkaline phosphatase: Horse gram seeds germinated in distilled water(control) and different fold-dilutions of the VRS-K and HST-K formulations for 24-120 h, were harvested and the clarified supernatant assayed for alkaline phosphatase activity vide Materials and Methods. Readings represent average of 12 trials ±S.E.

Half maximal ALP inhibition occurred at 55.2 and 88.7- fold dilutions of VRS-K and HST-K respectively, which was equivalent to 0.04 mg/ml of standard vinblastine (Fig.6).

Figure No.6: Effect of Vinblastine treatment on specific alkaline phosphatase activity: Seeds germinated in distilled water(control) and with vinblastine concentrations ranging from 0.01-0.06 mg/ml for 24-120h vide. Materials and Methods. Readings represent the average of 12 trials ± S.E.

Statistical analysis indicated that significant differences between the inhibitory profiles of the two formulations were absent beyond a 30- fold dilution (F_obs>F_crit for a = 0.05).

DISCUSSION:

Our findings in the present study maybe summarized thus:-

· The HST-K formulation at 88.7 and 117.6-fold dilutions brought about 50% inhibition of ALP and amylolytic activity, which were equivalent to 0.04 mg/ml and 0.05mg/ml of vinblastine respectively (n =12).

· Comparison of EC-50 values has indicated that, of the two Kokilaksha formulations, HST-K was more efficacious than VRS-K.

Significance of these observations maybe noted since the adverse effects of vinblastine with respect to hematological parameters as well as organ toxicities have been reported^10,11. The efficacy of the HST-K formulation along with reports on its hepatoprotective properties¹²and restoration/stabilization of haematological parameters^13,14leads us to believe that preclinical/clinical studies using HST-K along with vinblastine could be addressed with the objective of exploring better treatment outcomes –namely equal or better efficacy and reduced adverse reactions. Considering that chemotherapeutic approaches presently rely upon combination therapy, such a formulation having a reduced dosage of vinblastine supplemented with HST-K deserves to be explored. Confining oneself to the single-molecule concept of drug discovery may also not be the apt approach while dealing with complex disorders such as cancer, as observed by Spudich and Menon¹⁵

Observations indicating hepatoprotective properties of Asteracantha longifolia aqueous extract¹² are especially of significance, considering that elevated alkaline phosphatase are indicative of liver damage¹⁶ as well as liver malignancies. Hepatic alkaline phosphatase levels (as well as disease progression) in response to HST-K and/or vinblastine treatment would thus need to be monitored as a means of testing the aforesaid hypothesis. Similarly, comparative and synergistic studies using HST-K/vinblastine would offer newer insights into hyperamylasemic cancers as well.⁴

Enzyme inhibition based approaches have been previously performed with the objective of identifying or delineating mechanisms of therapeutic action in plant extracts with potentially antidiabetic^17-20anti-inflammatory, antioxidant and antimicrobial properties ^21-22. Authors have also suggested the use of seed germination bioassay approach as an alternative to animal models, especially considering the ease of statistical measurements “as the number of samples used can be increased without any restrictions”^23.Studies comparing the two approaches wherein the extent of extrapolation is determined, would be essential prior to complete replacement of animal models with those of seeds.

CONCLUSION:

Based upon our observations we wish to state that the seed germination assay method could serve as a means for ascertaining the comparative antiproliferative efficacy of newer compounds, using known cytotoxic drugs as positive controls.

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

ACKNOWLEDGMENT:

We owe our grateful thanks to the Visvesvaraya Technological University (VTU), Belgaum for the award of the VTU Research Grant that enabled these investigations. We gratefully acknowledge Prof. H.G Nagendra (Prof and Head, Dept.of Biotechnology, Sir MVIT) and Dr.V.R Manjunath, Former Principal Sir MVIT for facilities extended. MM also wishes to thank Dr.M.S Indira, presently Professor & Dean, MSR University of Applied Sciences for the kind support and encouragement extended towards these studies during her tenure as Principal, Sir MVIT. Our thanks are also due to our Principal Prof. S.G Rakesh for his encouragement and support.

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Received on 03.11.2021 Modified on 20.12.2022

Research J. Pharm. and Tech 2023; 16(12):5732-5737.

DOI: 10.52711/0974-360X.2023.00927