INTRODUCTION

The control of gastric diseases such as gastric irritation, ulcer, lack of appetite, gastritis etc, is one of the major problems faced by people. The use of medicinal seeds to treat various gastric diseases has been in practice in the Indo – Pak Subcontinent and other eastern countries, for centuries. At present, these treatments are the major tools of eastern style medicine known as Unani Hikmat. Plant seeds, being effective in gastric troubles, scientists particularly those in the field of Chemistry have been interested in their chemical analysis and their therapeutic action. A number of studies have been undertaken on medicinal plants in the said context^1-3. As a result, a number of important medicinal seeds prescribed by Hakims have been investigated from digestion point of view. Their chemical composition has been determined and these have been successfully tested on patients^4-6. To establish the efficacy of the drug the therapeutic action of medicinal seeds in the alimentary canal may be due to the presence of proteases, which are capable of hydrolyzing proteins, medicinal seeds were investigated for their kinetic activity.

MATERIALS AND METHODS

Preparation of Samples

                The samples included in the experimental work for the determination of protease activity were selected on the basis of their medicinal value particularly as the treatments of gastrointestinal disorders on empirical basis and regular use by the Hakims (Oriental physicians). The samples were easily obtained from Hamdard Dawa Khana Wahdat Road Lahore. These were ground to a fine powder, using an electric grinder and preserved in sample bottles.

Assay of Protease Activity

The protease was assayed by the method of McDonald and Chen⁷. 100mg of the powdered seed sample was incubated with 4ml of the substrate (1% casein in citrate phosphate buffer of pH 7.0) for one hour at 30°C. Adding 5ml of the 5% trichloroacetic acid precipitated the residual protein. The precipitate was allowed to settle for 30min. The contents of the tubes were filtered through Whitman No.40 filter paper. After filtration, 1 ml aliquot of filtrate was mixed with 5ml alkaline reagent mixture prepared by mixing 100ml of 2% sodium carbonate, 1ml of 2.7% sodium potassium tartrate and 1ml of 1% copper sulphate, Then, 2ml of 1 N NaOH was added to make contents of the tube alkaline. After at least 10 min, 0.5ml of Folin and Ciocalteu Phenol Reagent was added and the contents were mixed. The absorbance of the blue color produced, was measured at 660nm after exactly 30 min.

                Unit of protease activity defined as the amount of enzyme required to produce an increase in optical density at 660nm of 0.1 per hour at 30°C and pH 7.0 under the assay condition defined.

                The activity was reported as a mean of three determinations.

Effect of pH

                The effect of pH on the enzyme samples was studies within the pH range 1.5 to 10 using casein substrates made in buffers of different pH; 100mg of each sample was incubated with 4ml of casein substrate of each pH for one hour at 30°C. The protease activity was assayed by the method of MacDonald and Chen. Protease activity was plotted as a function of pH.

Effect of Temperature

                The effect of temperature on protease activity was studied in the range of 30°C to 80°C. 100mg of each sample was taken and incubated with 4ml of casein substrate for one hour at temperatures 30°C, 40°C, 50°C, 60°C, 70°C and 80°C respectively. Protease activity was plotted as a function of temperature.

Effect of Prolonged Heating

                The effect of prolonged heating in boiling water bath (100°C) on protease activity was studied from 30 to 120 minutes. 100mg of each sample was taken in different test tubes. The tubes were then heated in boiling water bath from 30 to 120 minutes. The substrate was subsequently added and assay was carried out by the method of McDonald and Chen. The protease activity was plotted as function of time.

RESULTS

The protease activities (units/g) of the medicinal seeds have been reported in Tables 1–3. The seeds have been placed in the following categories on the basis of the presence of relative amount of enzymes:

·         High activity medicinal seeds

·         Medium activity medicinal seeds

·         Low activity medicinal seeds

Table 1:  Medicinal seeds with high protease activity

Table 2: Medicinal seeds with medium protease activity

Table 3: Medicinal seeds with low protease activity

                The results presented in Tables 1–3 indicate that all samples of dry seeds analyzed are associated with significant amount of protease activity. The comparison of protease activity reveals that Carum copticum (Ajwain) is associated with the highest amount of protease activity out of the samples analyzed, while Punica glantum (Anardana) is next in this potential. Trigonella foenum graecum contains the lowest protease activity. Allium sepa though associated with medium protease activity but being a parallel subject of recent research studies with Carum copticum, these two seeds were subjected to further investigation for their pH and temperature profiles and study of effect of prolonged heating on the protease activity of powdered seeds, etc.

                The pH profiles of the protease of Carum copticum and Allium sepa are shown in Fig.1 and 2. The profile of Carum copticum indicates that its protease is active in both acidic and alkaline media (maximum activity at pH 3 and 7) respectively. The protease activity of Allium sepa, on the other hand, is active only in the alkaline medium (maximum activity at pH 10)

 Fig. 1: The pH profile of Allium sepa

 Fig.2: The pH profile of protease of Carum copticum:

Fig.3: The temperature profile of Allium sepa.

Fig. 4: The temperature profile of Carum copticum

The temperature profiles of Carum copticum and Allium sepa are shown in Fig 3 and 4. The profiles indicate that the temperature optimum of both enzymes is 70°C.

The effect of prolonged heating on protease activity of Carum copticum and Allium sepa are shown in Fig 5 and 6.

Fig. 5: Effect of prolonged heating on protease activity of Allium sepa.

Fig. 6: Effect of prolonged heating on protease activity of Carum copticum

DISCUSSION

                The major objective of the work reported here was the rationalization of the empirical and medicinal use of seeds examined here as digestive aids to the humans on the basis of their protease activity, as the proteases are capable of degrading proteins (meat, milk and egg etc.) in stomach as well as in intestine. The results in Tables 1-3 show that out of eighteen samples of medicinal seeds analyzed, two have high protease activity and can be effectively used to cure the gastrointestinal disorders as these act, as digestive aids. Nine out of eighteen samples have moderate protease activity. So these can also be used in the gastrointestinal disorders. Remaining samples show very low protease activity and thus these may not be significant in this context.

                The proteases are digestive in stomach where the medium is acidic and also in small intestine where the medium is alkaline⁸. The protease present in Carum copticum is a mixture of acidic and alkaline proteases, pH optima 3 & 7. It may be effective both in stomach where the medium is acidic and in small intestine where the medium is alkaline. The Allium sepa, on the other hand, contains only alkaline protease (Fig 2), which significantly differs from the protease referred above, as its pH optimum is around 10. It may not be effective digestive aid in alimentary canal at sites where medium is acidic.

                The alkaline protease of Carum copticum compares well with the normal plant enzymes i.e. papain, solanam etc. whose pH optimum is 7 to 7.5 as reported by Greenberg and Winnick⁹, with the crude protease of Calotropis procera (Aak) with optimum pH 7 to 7.5 reported by Khan, et al.¹⁰ and with isolated proteases of Calotropis procera, pH optimum 7 to 7.5 reported by Jilani and Khan¹¹.

                The acidic protease with optimum pH 3 is a different enzyme. It seems that it has not been reported before.

                From the temperature profiles (Fig 3 & 4), it is evident that Allium sepa as well as Carum copticum show higher temperature optimum at 70°C. Most of plant proteases generally denature between 40 - 50°C as described by Khan et al. (10) and Jilani and Khan (11).

                The proteases of Carum copticum and Allium sepa exhibit relatively higher temperature optimum. This may be due to their binding with the cells in powder form as this binding increases thermostability. It is also confirmed by the effect of prolonged heating on the activity of protease of Carum copticum and Allium sepa (Fig 5 & 6), which shows that both proteases are thermostable even at 100°C and denature completely after the prolonged heating of about two hours. 

REFERENCES

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2.        Pant, R and Srivastava, S C.  Protolytic Activity of Some Plant Latex- Effect of Time Variation (C. A. 64): 10085 (1996).

3.        Basha, SM and Beevers, L The development of proteolytic activity and protein degradation during the germination of Pisum sativum. Planta 124: 77-87(1975)

4.        Harrach, T., Eckert, K., Maurer, H.R., Machleidt, I., Machleidt, W. & Nuck, R. Isolation and characterization of two forms of an acidic bromelain stem                proteinase. J Protein Chem. 17, 351-361(1998).

5.        Khan, MR, Amira, R and Khalid, M. Antibacterial Activity of some Medicinal Plants and Seeds. Pakistan Journal of Biochemistry.23 (2): 55-62 (1990)

6.        Maurer, H.R. Bromelain: biochemistry, pharmacology and medical use. Cell Mol Life Sci. 58, 1234-245 (2001)-

7.        McDonald, C. E. and Chen, I. L. The Lowry modification of the Folin reagent for determination of proteinase activity. Anal. Biochem. 10.175 -77(1965)

8.        Khan, M. R. Biochemistry, vol. 1. Publishers, Caravan Book House, 3rd edition. 1988. 292 –6.

9.        Reenberg, DM, Winnick T.  Plant proteases. I. Activation-inhibition reactions. J Biol. Chem, 1940; 135.761 – 73.

10.     Khan MR, Nasreen K. Parveen Z. J Natural Sci Mathematics 1981; 21(2). 199 – 208.

11.    Jilani, Khan MR, Masood S. Extraction and partial purification of the protease of calotropis procera. J Pure App Sci 1986; 5 (1): 13-7.